The Mono and Combined Anti-Proliferative Activity of D - Mannoheptulose and Newcastle Disease Virus Against Breast Cancer Cell Lines Targeting Glycolysis Inhibition

Ministry of Higher Education
& Scientific Research

Al-Nahrain University
College of Medicine
The Mono and Combined Anti-Proliferative Activity of

D- Mannoheptulose and Newcastle Disease Virus Against
Breast Cancer Cell Lines Targeting Glycolysis Inhibition
A Thesis
Submitted to the Council of College of Medicine, Al-Nahrain
University in Partial Fulfillment of the Requirements for the Degree
of Doctor of Philosophy (PhD) in Medical Chemistry
By
Ahmed Ghdhban Theiban Al-Ziaydi
BSc. Chemistry, University of Al-Qadisiyah, 1993
PGDip.Sc. Chemistry- Chemical and Pharmaceutical
Analyses, Kerbala University, 2008
MSc. Biochemistry, Osmania University, India, 2015
Supervised by
Assistant Professor Assistant Professor

Dr. Mohammed Imran Hamzah Dr. Ahmed Majeed Al-Shammari
PhD. Clinical Biochemistry PhD. Pathology
2020 A.D 1441 A.H

DEDICATION
I dedicate this humble work…..To
My dear Father….
My beloved Mother….
My dear and beloved wife ……
My dear son and daughters…
My dear brothers and sisters….
My dear teachers…
To my lovely friends…
Ahmed Ghdhban Al-Ziaydi

30 / 6 /2020
ACKNOWLEDGEMENT
((Thanks to God for His entire blessing during
the pursuit of my academic and career goals))
I would like to express my sincere thanks and gratitude and appreciation to my

supervisor ((Asst. Professor Dr. Mohammed Imran Hamzah, College of Medicine,
Al-Nahrain University)), Co-supervisor ((Asst. Professor, Dr. Ahmed Majeed
Al-Shammari, Iraqi Center for Cancer and Medical Genetic Research,
Al-Mustansiriyah University)), Consultant ((Professor Dr. Haider Sabah Kadhim,
College of Medicine, Al-Nahrain University)) and ((Dr. Majid S. Jabir, Department
of Applied Sciences, Technology University)) and ((Professor Dr. Ali Abed Al-
lateef, University of Basrah)) for their valuable guidance and advice throughout this
research.
A special thanks and deep gratitude to ((Iraqi Center for Cancer and Medical
Genetic Research, Al-Mustansiriyah University)), ((Department of Medical
Chemistry, College of Medicine, Al-Qadisiyah University)) and ((Iraqi Company for
Biotechnology)) for their support and help in finishing my work. Special thanks and
deep gratitude to the discussion committee for their great efforts.
Ahmed Ghdhban Al-Ziaydi

30 /6 /2020
Abstract………………………………………………..…………………………………………………….
ABSTRACT
Recently, there have been several new approaches aimed at treating cancer,
breast cancer in particular, by reducing the side effects of conventional therapies,
other novel approaches such as targeted therapies, virotherapy and combinations of
several of these new treatments. Synergism therapy reduce the side effects and
toxicity, and overcome resistance of the cancer cells to the treatment.
Most cancer cells exhibit increased glycolysis, using this metabolic pathway to
generate Adenosine tri phosphate for cell growth and proliferation. The Warburg
effect illustrates that inhibition of glycolysis might have a therapeutic value in anti-
tumor treatment. Newcastle disease virus (Iraqi strain) and enzyme inhibitor can
modulate differentiation and induce apoptosis in breast cancer cell lines.
D-Mannoheptulose functions biologically by inhibiting glycolysis, thus, inhibiting
tumor growth.
Aim: To assess the synergistic effects of Newcastle disease virus as viral

therapy and D-Mannoheptulose as a Hexokinase inhibitor, on the inhibition of the
glycolysis pathway and eventually metabolism, to prevent proliferation and induce
apoptosis in breast cancer cell lines, in comparison with normal Rat embryo
fibroblast cell lines.
Method: Newcastle disease virus is propagated in embryonated chicken eggs

and quantified using Hemagglutination assay and Median tissue culture infectious
dose on the Vero cell line. The human breast cancer Ahmed Mortada jabria 2013 and
Michigan cancer foundation-7, and normal Rat embryo fibroblast cell line were used.
The cytotoxicity ratio of virus and inhibitor was measured against Ahmed Mortadha
Jabria 2013, Michigan cancer foundation-7, and Rat embryo fibroblast cell lines for
72 hrs using the Methyl thiazolyl tetrazolium assay, to determine half maximal
inhibitory concentration at different Multiplicity of infections (0.1-12.8 MOI) and
diluted concentrations (13.125-1680 μg/ml) for virus and inhibitor, respectively, and
Page I
Abstract………………………………………………..…………………………………………………….
the Half maximal inhibitory concentration values were used for combination study.
Using the CompuSyn software, the Combination index was measured to identify the
effective doses of virus and inhibitor. Crystal violate/morphological changes,
Acridine orange/Propidium iodide/apoptosis, Hexokinase activity, pyruvate,
Adenosine tri phosphate concentration, and pH (acidity) were measured in treated
and untreated breast cancer and normal cell lines, to investigate the effect on the
glycolysis pathway.
Results: This study revealed that virus and inhibitor individually inhibited
proliferation and growth, reduced cell viability, and induced morphological changes
and apoptosis in breast cancer cell lines. Through glycolysis inhibition and apoptosis
induction, there were no noticeable percentages of cytotoxicity for
D-Mannoheptulose against normal Rat embryo fibroblast cells as the cytotoxicity
ratio ranged from (1.67%, to 24.72%) at higher concentrations. While there was
higher cytotoxicity against breast cancer cells ranged from (27.29% to 58.64%) for
Ahmed Mortada jabria 2013 cell line; and (26.26% to 60.49%) for Michigan cancer
foundation-7 cell line after inhibitor treatment. Virus did not induce cytotoxic effect
against normal cell line as the cytotoxicity ratio ranged from (4.31% to 28.75%) for
embryo fibroblast cells. Breast cancer cells were more sensitive to virus as the
cytotoxicity ratio ranged from (24.69% to 64.26%) for Ahmed Mortada jabria 2013;
and (23.95% to 62.02%) for Michigan cancer foundation-7 cell line after virus
treatment. Glycolysis inhibition was observed by decreased Hexokinase activity
from (3.2 U/ml as control to 2.0 U/ml with virus, 1.4 U/ml with inhibitor and 1.1
U/ml with combination in Ahmed Mortada jabria 2013 and from 3.0 U/ml as control
to 1.9 U/ml with virus, 1.4 U/ml with inhibitor and 1.2 U/ml with combination in
Michigan cancer foundation-7), pyruvate concentration from (0.48 μmol/ml as
control to 0.28 μmol/ml with virus, 0.25 μmol/ml with inhibitor and 0.14 μmol/ml
with combination in Ahmed Mortada jabria 2013 and from 0.5 μmol/ml as control to
0.23 μmol/ml with virus, 0.23 μmol/ml with inhibitor and 0.15 μmol/ml with
Page II
Abstract………………………………………………..…………………………………………………….
combination in Michigan cancer foundation-7) and Adenosine tri phosphate

concentration from (1.2 mmol/L as control to 0.58 mmol/L with virus, 0.61 mmol/L
with inhibitor and 0.39 mmol/L with combination in Ahmed Mortada jabria 2013 and
from 1.3 mmol/L as control to 0.65 mmol/L with virus, 0.69 mmol/L with inhibitor
and 0.35 mmol/L with combination in Michigan cancer foundation-7). Also, the
decreased acidity was noticed by increasing pH from (6.3 as control to 7.3 with virus,
7.3 with inhibitor and 8.2 with combination in Ahmed Mortada jabria 2013 and from
6.3 as control to 7.4 with virus, 7.4 with inhibitor and 8.2 with combination in
Michigan cancer foundation-7).
Combination therapy results revealed that the synergism between virus and
inhibitor produced a significant anti-tumor effect than those of monotherapy with
either. Their combination resulted in higher inhibition of cell viability, increase
cytopathic morphological changes, and higher apoptosis induction in the treated
cancer cell lines, via glycolysis pathway inhibition, as shown by decreased
Hexokinase activity, pyruvate and Adenosine tri phosphate concentrations, and
decreased environmental acidity which due to reduced lactate concentration,
compared with normal cell lines. Moreover, this therapy was relatively safe in normal
cells.
Conclusion: Synergism between Newcastle disease virus and

D-Mannoheptulose increase the cytotoxicity and induce significant growth inhibition
by glycolysis inhibition and promote apoptosis in breast cancer cells but not in
normal cells in vitro, also it is better than individual treatment. A significant
reduction was identified in the Hexokinase activity, Pyruvate, Adenosine tri
phosphate concentrations, and acidity in all the treatment modalities in the breast
cancer cells but not in the normal cells. Synergism of virus and inhibitor can be
considered as a future treatment of cancer cells and a new strategic way to be used
as an effective treatment.
Page III
List of Contents, Figures, Tables, Schemes, Appendix and Abbreviations …..…………………..
LIST OF CONTENTS
Item Title Page

Abstract I
List of Contents IV
List of Figures VIII
List of Tables X
List of Schemes XI
list of Appendixes XI
List of Abbreviations XII
Chapter one
1. Introduction 1
1.1 The aim of the study 3
Chapter Two
2. Literatures Review 4
2.1 Tumor (Aneoplasia) 4
2.2 Carcinogenesis 6
2.3 Cancer and Cell death 6
2.4 Breast Cancer 6
2.4.1 Etiology of breast cancer 7
2.4.2 Signs and symptoms of breast cancer 8
2.4.3 Genetics and breast cancer 9
2.4.4 Breast Cancer Spread and metastasis 9
2.4.5 Types of breast cancer 9
2.4.5.1 Non-invasive breast cancer or in situ carcinoma 10
2.4.5.2 Invasive Carcinoma 11
2.5 Glycolysis Pathway 12
2.5.1 Glycolysis in cancer cells 13
2.5.2 Metabolizing pathways in cancer 13
2.5.3 Glycolysis and differences between cancer and normal cells 14
2.5.4 Warburg effect and the glycolytic inhibitors 16
2.6 Programmed Cell Death (Apoptosis) 19
2.7 Cancer Therapy 21
2.7.1 Conventional cancer therapeutics 21
2.7.1.1 Surgical Therapy 21
2.7.1.2 Radiotherapy 21
2.7.1.3 Chemotherapy 21
2.7.1.4 Hormone therapy 22
Page IV
Item Title Page

2.7.1.5 Immunotherapy 22
2.7.2 Novel therapeutics 23
2.7.2.1 Anti- Angiogenesis Therapy 23
2.7.2.2 Biological Therapy 23
2.7.2.3 Gene Therapy 24
2.7.2.4 Virotherapy 24
2.7.2.4.1 Newcastle Disease Virus (NDV) 25
2.7.2.4.2 Molecular Biology of NDV 25
2.7.2.4.3 Mechanisms of NDV-induced Oncolysis 26
2.7.2.4.4 Safety of NDV Administration as Anticancer Agent 27
2.7.2.4.5 Replication of NDV 28
2.7.2.4.6 Clinical usage of NDV in cancer therapy 29
2.7.2.5 Combination therapy 30
Chapter Three
3. Material and Methods 32
3. Subject 32
3.1 Materials 33
3.1.1 Instruments 33
3.1.2 Chemical and biological materials 34
3.1.3 Reagents for cell culture 35
3.1.3.1 Penicillin - Streptomycin (Pen-Strep) (Antibiotics) 35
3.1.3.2 Sodium Bicarbonate 36
3.1.3.3 Phosphate buffer saline (PBS), without Ca+2 and Mg+2 36
3.1.3.4 Trypsin 36
3.1.3.5 Stain 36
3.1.3.5.1 Methyl Thiazolyl Tetrazolium (MTT) Solution 36
3.1.3.5.2 Crystal Violates stain 37
3.1.3.5.3 Acridine Orange- Propidium Iodide (AO/PI) 37
3.1.3.6 Tissue culture media 37
3.1.3.6.1 Preparation of MEM 37
3.1.3.6.2 Preparation of RPMI-1640 Medium 38
3.1.4 Cell Lines 39
3.1.4.1 Ahmed Mortadha Jabria 2013 (AMJ13 cell line) 39
3.1.4.2 Michigan Cancer Foundation-7 (MCF7 cell line) 40
3.1.4.3 Vero cell line 40
3.1.4.4 Rat Embryo Fibroblast (REF) Cell Line 40
3.1.5 Requirements for Virus Propagation and HA Test 41
3.1.5.1 NDV 41
3.1.5.2 Embryonated Chicken Eggs (ECE) 41
Page V
Item Title Page

3.1.5.3 Chicken Red Blood Cells 41
3.1.6 Colorimetric Assays 42
3.1.6.1 Hexokinase Assay Kit 42
3.1.6.2 Pyruvate Assay Kit 42
3.1.6.3 ATP Assay Kit 43
3.2 Methods 45
3.2.1 The Propagation of the Newcastle Disease Virus 49
3.2.1.1 Virus Preparation 49
3.2.1.2 The Propagation of NDV in ECE 49
3.2.2 Hemagglutination assay (HA) 50
3.2.2.1 Preparation of chicken RBC solution 50
3.2.2.2 Titration of NDV 51
3.2.3 Cell lines 52
3.2.3.1 Maintenance of Cell Lines in Vitro 53
3.2.3.2 Cell culture of Vero cell line 53
3.2.4 Preparation of diluted virus (NDV) 55
3.2.5 Exposure of NDV to Vero cells line 55
3.2.6 Determination of TCID50 of NDV 56
3.2.7 Exposure stages of the cell lines 58
3.2.7.1 Treatment of cell lines with NDV, MH, and DXL 58
3.2.7.1.1 Preparation of stock and diluted solutions 58
3.2.7.1.2 Exposure of NDV, MH, and DXL to cell lines 59
3.2.7.1.3 Calculation of the number and volume of virus 59
3.2.8 MTT Cytotoxicity Assay 60
3.2.9 Synergistic cytotoxicity assay and Chou-Talalay analysis 62
3.2.10 Morphological analysis 63
3.2.11 Assessment of apoptosis (PI/AO staining) 63
3.2.12 Glycolysis parameters determination 64
3.2.12.1 Determination of Hexokinase 64
3.2.12.2 Determination of Pyruvate 65
3.2.12.3 Determination of ATP 66
3.2.13 PH measurements 68
3.2.14 Statistical Analysis. 68
Chapter Four
4. Results 69
4.1 Virus propagation in Embryonated Chicken Eggs ECE 69
4.1.1 Propagation of virus (NDV) in chicken embryo 69
4.1.2 Harvest of NDV and measurement of HAU 69
4.1.3 TCID50 70
Page VI
Item Title Page

4.2 Cytotoxicity assay 73
4.2.1 Cytotoxicity of NDV and MH against cell lines 79
4.3 Determination of IC50 80
4.4 Study the synergistic effect of NDV and MH 86
4.5 Combination cytotoxicity assays and Chou–Talalay analysis of cells 93
4.6 Morphological changes 97
4.7 Apoptosis assay 104
4.7.1 Relationship of apoptosis and morphological changes 108
4.8 Glycolysis pathway and its products inhibition 109
4.8.1 Hexokinase assay 109
4.8.2 Pyruvate assay 111
4.8.3 ATP assay 113
4.8.4 PH measurements 115
4.8.5 Comparison between testes 118
Chapter Five
5. Discussion 120
5.1 Newcastle disease Virus Preparation and Collection 120
5.2 TCID50 of NDV in Vero cell line 122
5.3 Cytotoxicity and IC50 of NDV, MH, and DXL in cell lines 123
5.4 Synergistic effect of NDV and MH on cells lines 124
5.5 Study of morphological changes and apoptosis in cell lines 128
5.6 Glycolysis inhibition 130
Conclusion and Recommendations
Conclusion 138
Recommendations 139
References
References 140
Appendix
Page VII
LIST OF FIFURES
Figure Title Page
Chapter Two (Literatures Review)
2-1 Scheme of molecular basis of cancer 4
2-2 Structure of the normal female breast 7
2-3 Ductal carcinoma in situ (DCIS). 10
2-4 Lobular carcinoma in situ (LCIS). 11
2-5 Glycolysis in tumor cell 15
Differences between Oxidative phosphorylation, anaerobic
2-6 16
and aerobic glycolysis
2-7 Programmed Cell Death (Apoptosis) 20
2-8 Structure of Newcastle Disease Virus 26
2-9 Oncolytic virus function 27
2-10 Replication of Newcastle Disease virus 29
Chapter Three (Material and Methods)
3-1 Calculation of titer of NDV in 96 -well microplate 52
3-2 Trypsinization to detached cells from wall in tissue culture 53
3-3 Vero cell confluent monolayer after storage at -86°C 55
3-4 Calculation number of cells in sample by hemocytometer 55
Exposure of NDV to Vero cells line and calculation of
3-5 57
TCID50
Chapter Four (Results)
4-1 Inoculation of NDV in embryonated chicken eggs 69
4-2 Hemagglutination test 70
4-3 Vero cells treated with NDV 71
4-4 Exposure NDV to Vero cell line to measure TCDI50 72
4-5 Cytotoxicity effect after treatment of cell lines with NDV 73
Comparison of cytotoxicity between different MOI after
4-6 74
treatment of cell lines with NDV
4-7 Cytotoxicity effect after treatment of cell lines with MH 75
Comparison of cytotoxicity between different concentrations
4-8 76
(μg/ml) after treatment of cell lines with MH
4-9 Cytotoxicity effect after treatment of cell lines with DXL 77
Comparison of cytotoxicity between different concentrations
4-10-I 78
(μg/ml) after treatment of cell lines with DXL
4-10-I I MH and NDV are cytotoxic against breast cancer cells 79
4-11 IC50 to exposure of NDV to cell lines 81
4-12 IC50 to exposure of MH to cell lines 82
4-13 IC50 to exposure of DXL to cell lines 83
Page VIII
4-14 Comparison of IC50 between cell lines 84

4-15 Comparison of CT% between cell lines 85
CT% of REF cell line after treated with MH, NDV and their
4-16 87
combination
CT% of AMJ13 cell line after treated with MH, NDV and
4-17 88
their combination
CT% of MCF7 cell line after treated with MH, NDV and
4-18 89
their combination
4-19 CT% of NDV against cell lines by using 3 different doses 90
4-20 CT% of MH against cell lines by using 3 different doses 91
4-21 CT% of DXL against cell lines by using 3 different doses 92
4-22 Comparison between the effect of DXL and Combination
93
therapy in cell lines
4-23 Isobologram analysis and combination cytotoxicity assay via
95
Chou–Talalay analysis of cell lines
4-24 Effect of NDV and MH and their combination on unstained
98
AMJ13 cell lines
99
MCF-7 cell lines
100
REF cell lines
4-27 Morphological changes in stained AMJ13 cell line 101
4-28 Morphological changes in stained MCF7 cell line 102
4-29 Morphological changes in stained REF cell line 103
4-30 Apoptosis effect in stained AMJ13 cell lines 105
4-31 Apoptosis effect in stained MCF7 cell lines 106
4-32 Apoptosis effect in stained REF cell lines 107
4-32 a Fluorescent intensity: Investigation combined therapy 108
4-33 Effect of NDV and MH on the activity of hexokinase (U/ml) 110
4-34 Effect of NDV and MH on the pyruvate concentration
112
(μmol/ml) after treatment of cell
4-35 Effect of NDV and MH on the ATP concentration (mmol/L)
114
after treatment of cell lines with NDV, MH and combination
4-36 Effect of NDV and MH on the acidity (based on PH values)
116
after treatment of cell lines
Normalization (Nor): effect of treatment with NDV, MH and
4-37 117
their combination on the acidity of cell lines
4-38 Comparison between tests: (HK, Pyruvate, ATP and pH) 119
Page IX
LIST OF TABLES
Table Title Page

3-1 list: The instruments and glassware used in this study 33
3-2 chemical and biological materials 34
3-3 Kit components for determine pyruvate in sample 43
Calculation method of TCID50 of the virus after treating
3-4 57
Vero cell line with NDV
Preparation of diluted MOI of NDV solutions depend on
3-5 60
TCID50
3-6 Operation steps to determine pyruvate in sample 65
3-7 Operation steps to determine ATP in sample 67
Chapter Four (Results)
4-1 Results of Vero cells treated with NDV 72
4-2 Final value for TCID50/ml for exposure NDV to Vero cells 73
4-3 CT% after treatment of cell lines with NDV 74
4-4 CT% after treatment of cell lines with MH 76
4-5 CT% after treatment of cell lines with DXL 78
Statistical data for comparison IC50 in cell lines after
4-6 84
exposure to NDV, MH and DXL
Statistical data for comparison of CT% in cell lines after
4-7 85
treated with NDV, MH, and DXL.
4-8 Statistical data for cytotoxicity% of NDV against cell lines 90
4-9 Statistical data for cytotoxicity% of MH against cell lines 91
Combination index (CI) data for non-constant combination
4-10 96
(NDV+MH) of cell lines
Statistical data of Inhibition of hexokinase enzyme after
4-11 109
treatment of cell lines with NDV, MH and their combination
Statistical data of inhibition of pyruvate after treatment of
4-12 111
cell lines with NDV, MH and their combination
Statistical data of inhibition of ATP after treatment of cell
4-13 113
lines with NDV, MH and their combination
Statistical data of acidity after treatment of cell lines with
4-14 115
NDV, MH and their combination
Page X
LIST OF SCHEMES
Table Title Page

3-1 Outline of laboratory experiments of the study (Study Design) 47
LIST OF APPENDIXES
App. Title Page

Appendixes
A-1 Preparation of completed culture media by using powder i
A-2 Preparation of completed culture media by using solution i
A-3 Preparation of SFM (without FBS) by using powder ii
A-4 Preparation of SFM (without FBS) by using solution ii
A-5 Propagation of NDV in Chicken Embryo iii
A-6 Preparation of RBC Solution iv
A-7 Determination of Titer of NDV by HA Test iv
A-8 Cell culture of Vero cell line v
A-9 Preparation of the diluted virus solution from stock virus vi
A-10 Exposure NDV to Vero cell line to measure TCDI50 vii
A-11 Preparation of different MOI for NDV depend on TCID50
viii
and exposure of NDV to cell lines
A-12 Preparation of MH stock solution and diluted MH solution ix
A-13 Exposure of MH to cell lines x
A-14 Preparation of stock and diluted DXL solution xi
A-15 Exposure of different concentrations for DXL to breast
xii
cancer cell line
A-16 MTT Cytotoxicity Assay xiii
A-17 HK Kit components and preparation of reagents xiv
A-18 Transillumination of eggs by candling lamp xiv
A-19 ATP Kit components and preparation of reagent xv
A-20 Save Eppendorf tubes in ice xvi
Embryonated chicken eggs (9-days-old) were incubated in a
A-21 xvi
conventional incubator at 37oC
Allantoic fluid of infected embryo was harvested after
A-22 xvi
calculating TCID50.
A-23 cold centrifugation at 3000 rpm for 30 min at 4°C xvii
Page XI
A-24 Inverted and fluorescent Microscope xvii

A-25 Calculation of titer of NDV by HA test xix
A-26 Outline of the programs were used in the study xx
A-27 HA (the clumping together of red blood cells) by NDV xx
ABBREVIATIONS
Symbol Description
A Absorbance
AMJ13 Ahmed Mortadha Jabria 2013 Cell line
AO Acridine Orange
AO/PI Acridine Orange- Propidium Iodide
Apoptosis Programmed Cell Death
ATP Adenosine Tri Phosphate
BRCA Breast Cancer
BSH Biosafety Hood
Caspases Cysteine aspartyl-specific proteases
CEF Chicken Embryo Fibroblast
CI Combination Index
CM Complete Media (Media contain PSS, TDW and Serum)
Conc. Concentration
CRBC Chicken Red Blood Cells
CT% Cytotoxicity Ratio
CTMH Cytotoxicity MH
CV Coefficient of variation
D.W Distilled water
DCIS Ductal Carcinomas In Situ
DXL Docetaxel
DMSO Dimethyl Sulfoxide
DNA Deoxyribonucleic Acid
ECE Embryonated Chicken Eggs
ELISA Enzyme-Linked Immunosorbent Analysis
ER Endoplasmic Reticulum
FBS Fetal Bovine Serum
GLUT Glucose Transporter
HA Hemagglutination Test
HAU Hemagglutination Unit
HEPES Hydroxyethyl Piperazine ethane sulfonic acid
Page XII
Symbol Description
HK Hexokinase
HKI Hexokinase Inhibitor
IC50 The half maximal Inhibitory Concentration
ICCMGR Iraqi Center of Cancer and Medical Genetics Research
IDC Invasive Ductal Carcinomas
ILC Invasive Lobular Carcinoma
LCIS Lobular Carcinoma In Situ
LDH Lactate Dehydrogenase
LFH Laminar Flow Hood
MCF-7 Michigan Cancer Foundation-7 Cell line
MEM Minimum Essential Medium
MH D - Mannoheptulose
MOI Multiplicity of Infection
MTT Methyl Thiazolyl Tetrazolium
NDV Newcastle Disease Virus
OD Optical Density
OXPHOS Oxidative Phosphorylation
PBS Phosphate Buffer Saline
Pen-Strep Penicillin-Streptomycin Solution
PSS Penicillin - Streptomycin
R Reagent
RBC Red Blood Cell
REF Rat Embryo Fibroblast
RIF Rat Embryonic Fibroblast
RNA Ribonucleic acid
RPMI Rosswell Park Memorial Institute
SD Standard Deviation
SEM Standard Error Mean
SFM Serum - Free Media
TCA Tricarboxylic Acid Cycle
TCF Tissue Culture Flask (Falcon).
TCID50 50% Tissue Culture Infective Dose
TCM Tissue Culture Media (CM &cells)
TCP Tissue Culture Plate (96 wells).
TDW Trebled Distilled Water
VAF Virus Allantoic Fluids
96WMP 96 wall Micro Plate
Page XIII
CHAPTER-ONE
Chapter one ………………………………………………..………………………… Introduction
1- Introduction
Cancer is a major public health problem worldwide and it is the second

common cause of death. Cancer is a group of diseases characterized by
uncontrolled growth and spread of abnormal cells, If the spread is not controlled, it
can result in death (Siegel et al., 2019). Cancer results from the accumulation of
genetic and epigenetic changes in tumor suppressor genes (Wang et al., 2018).
Cancer therapy aims at killing cancer cells while minimizing harm on normal cells
(Fournier, 2013). Cancer treatment includes localized and/or systemic therapies or
supportive therapies reduce side effects (Miller et al., 2019).
Europe accounts for 23.4% of the global cancer cases and 20.3% of the cancer
deaths. America has 13.3% of the global population and accounts for 21.0% of the
incidence and 14.4% of the mortality worldwide. The proportions of cancer deaths
in Asia and Africa (57.3% and 7.3%, respectively). Female breast cancer (6.6%).
Breast cancer is the most commonly diagnosed cancer in women (24.2%). In Iraq,
breast cancer is the most common tumor in females, number of new cases in 2018,
females, all ages (rank 1, 36.7%) of all malignant diseases, (rank 2, death 11.9%)
and one-third of registered cancers in females (Bray et al., 2018). The rate of breast
cancer is 30%, and the death rate is 15% of female for 2019 in USA according to
World Health Organization (WHO).
This increment in the incidence rate of breast cancer may be due to exposure
to environmental factors and many other factors (Al-Shammari, 2016). The
methanolic extracts of Avocado fruit and leaves are highly promising for use in
cancer therapy (Paul et al., 2011). D-Mannoheptulose (MH) accumulates in
avocado leaves and fruit. Phytochemicals extracted from avocado induces
apoptosis in human oral epithelial cancer cell lines, but not normal cells (Ding et
al., 2009). MH is a hexokinase inhibitor that blocks the enzyme hexokinase, which
prevents glucose phosphorylation and breakdown (Tesfay et al. ,2012). Newcastle
Page 1
Chapter one ………………………………………………..………………………… Introduction
disease virus (NDV) is a member of the genus avulavirus, in the family of

Paramyxovirus (Jin et al., 2017). NDV proved to have promising anti-tumor
activity and great safety in lab animals (Costa-Hurtado et al., 2015). It is endemic
in poultry, resulting in huge economic losses (Abdisa and Tagesu, 2017). Increased
glycolysis is the main source of energy in cancer cells that use this metabolic
pathway for ATP generation (Gill et al., 2016). The breakdown of glucose via
glycolysis yields 2 molecules of pyruvate and 2 net molecules of ATP (Chaudhry &
Varacallo, 2019). In the presence of oxygen, normal cells generate energy from
glycolysis coupled with oxidative phosphorylation (Dhup et al., 2012). Cancer cells
mainly produce energy by increasing the rate of glycolysis compared with normal
cells, even if oxygen is plentiful (Alfarouk et al. 2014). Relative to normal cells,
cancer cells are highly proliferative and thus require increased ATP to meet their
metabolic demand (Cairns et al., 2011). The Warburg effect promotes cancer cell
proliferation. Cancer cells need high energy for continuous growth, and therefore
rely on the Warburg effect (Koppenol et al., 2011).
Inhibiting the activity of glycolytic key enzymes, results in ATP depletion and
decreased lactate generation (Guo et al., 2016). Cancer cells are disrupted by
decreased lactate production (Lu et al., 2011). Starvation leads to a decrease in
glucose uptake and lactate production (Gonin-Giraud et al., 2002). Glucose enters
the cell and generates pyruvate after a series of enzymatic reactions (Lu et al.,
2015). In cancer cells, pyruvate is preferentially converted into lactate even in the
presence of oxygen. Depletion of ATP by glycolytic inhibition potently induces
apoptosis. Starvation induces apoptosis of breast cancer cells (Fu et al., 2007).
Current strategies for cancer therapy are focused on apoptosis. Oncolytic
virotherapy alone is not effective for tumor eradication (Chu et al., 2004).
Combination therapies attack tumor cells and preventing treatment resistance
development. The combination of chemotherapy and virotherapy is promising in
cancer treatment. (Al-Shammari et al, 2016).
Page 2
Chapter one …………………………………………………….………………… Aim of the study
Aim of the study
1- To determine the anti-proliferative activity of NDV, D-Mannoheptulose (MH) and

their combination.
2- Determination of TCID50 of NDV and IC50 values of NDV and MH.
3- Determination of synergism, or antagonism between NDV and MH.
4- Study the morphological changes of cancer and normal cells after treatment with
either NDV or MH, and their combination.
5- Investigate the apoptosis induction by mono- and combination therapies in cancer

and normal cells.
6- To compare the effect of mono- and combination therapy on cancer cell glycolysis,
by studying the effect on levels of glycolysis products such as hexokinase,
[pyruvate], [ATP], and acidity (that represent lactic acid).
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CHAPTER-TWO
Chapter Two ……………………………………..…………………………………Literatures Review
2. LITERATURES REVIEW
2.1 Tumor (Neoplasia)

A tumor is a pathologic disturbance of cell growth, characterized by excessive
and abnormal cell proliferation (Sinha, 2018). Tumor are classified as benign or
malignant. Benign tumors are noninvasive, remain localized, and grow away from
the surface because they cannot invade (Vinik et al., 2018). Malignant tumors are
invasive, capable of spreading directly or by metastasis, and have relatively rapid
growth rates (Strayer et al., 2015; Davies, 2014). Most human tumors do not display
pure logarithmic growth patterns, as not all cells within the tumor are actively
dividing. (Grimes et al., 2016) (Figure (2-1)).
Benign neoplasms
typically have smooth
borders, are sharply
demarcated from the normal
tissue at the tumor site, and
are encapsulated by a fibrous
capsule that forms a barrier
between the neoplastic cells
and the host tissue (Coleman,
2018). Benign neoplasms
typically grow slowly such as
lipomas, whereas malignant
neoplasms often demonstrate
rapid growth. (Studdiford &
Trayes, 2018).
Figure (2-1) Scheme of molecular basis of cancer (Stricker and Kumar, 2007)
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Cancer is a group of diseases characterized by uncontrolled growth and spread

of abnormal cells, which if not controlled can result in death. Cancer is caused by
external factors and internal factors. Cancer cells are generally hungrier for nutrients
than normal cells, in order to sustain their high proliferative rates. This is shown by
their higher consumption of glucose, due to the lower efficiency in energy production
by anaerobic glycolysis, and increased extracellular acidosis, (Tarver, 2012). Many
factors contribute to the cell changes that lead to cancer. (Fernandes et al., 2015).
The interplay between the intrinsic and extrinsic factors is the major determinant of
an individual’s cancer risk (Wang and Karin, 2015). Risk factors vary widely
worldwide based on differences in lifestyle and in social, economic, and political
development (Wu et al., 2016). The most important modifiable risk factors are
infection with cancer-causing viruses or bacteria (Pacheco et al., 2016).
As normal cells divide, mature, and die, abnormal cells that do not follow this
progression lead to cancer development (Kim et al., 2015). Cancer begins when
damage is caused to 1 or more genes in a single cell. This harm will cause the cell to
divide incorrectly, producing abnormal cells (Marzouk and Schofield, 2011). Cancer
cells divide more rapidly than normal cells (El Mesallamy et al., 2011). During
early stages of cancer, a person may have just 1 small cancerous tumor. More
advanced stages may involve a larger tumor (Hong et al., 2014). Cancer is caused by
alterations in oncogenes, tumor-suppressor genes, and microRNA genes. (Croce,
2008).
Nutrition and physical activity directly affect cancer risk. Physical activity
reduces the risk of breast. Eating a diet high in fruits and vegetables is associated
with lower risk of cancers (Smith et al., 2018). The goal of cancer therapy is to push
the death of cancer cells while minimizing the harm on normal cells. (Fournier and
Schirrmacher, 2013). When cancer spreads from its original location to another part
of the body, the new tumor has the same kind of abnormal cells and name as the
primary tumor. (Santa-Maria, 2015).
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2.2 Carcinogenesis
Carcinogenesis or neoplastic pathogenesis is a process caused by a series of
mutations in the genetic material of normal cells (Dermadi Bebek, 2014). Neoplastic
pathogenesis can be described in 3 stages: initiation, promotion, and progression
(Trosko, 2001). During initiation and promotion, apoptosis and cell proliferation can
occur at various rates; this balance is modified during progression so that malignancy
can arise (Hanahan and Weinberg, 2011).
2.3 Cancer and cell death

Abnormalities in cell death regulation can be a significant component of
diseases such as cancer. regulated cell death can occur in the absence of any
exogenous environmental perturbation (Galluzzi et al., 2016). Classify cell death into
three different forms: type I cell death or apoptosis, exhibiting cytoplasmic
shrinkage, chromatin condensation, nuclear fragmentation , and plasma membrane
blebbing with the formation of apoptotic bodies that are efficiently taken up by
neighboring cells with phagocytic activity, type II cell death or autophagy,
manifesting with extensive cytoplasmic vacuolization with phagocytic uptake and
consequent lysosomal degradation; and type III cell death or necrosis, displaying no
distinctive features of type I or II cell death and terminating with the disposal of cell
corpses in the absence of obvious phagocytic (Lorenzo et al., 2017).
2.4 Breast cancer

Breast cancer is the most important and frequently diagnosed cancer in women
worldwide (24.2%, i.e. about 1 in 4 of all new cancer cases diagnosed in women
worldwide are breast cancer), and it is the leading cause of cancer-related deaths in
developing countries and women (15.0%), (Bray et al., 2018). In Iraq, breast cancer
is the most common tumor in female, number of new cases in 2018, females, all ages
(rank 1, 36.7%) of all malignant diseases, (rank 2, death 11.9%) and one-third of
registered cancers in females. Number of new cases in 2018, both sexes, all ages
Page 6
(rank 1, 20.3%) (Bray et al., 2018). This increment in the incidence rate of breast
cancer may be due to exposure to environmental and other factors such as life style,
chemicals, and radioactive pollution (Hassan et al., 2014). The poor survival is a
consequence of the late stage at presentation reflecting the limited access to
screening, diagnostic, and treatment facilities together with the lack of awareness on
the significance of early detection of breast cancer among the population (Sankar et
al., 2013) (Figure (2-2)).
A B
Figure (2-2): A- Structure of the normal female breast, cross-section scheme of the mammary
gland. Chest wall, 2- Pectoralis muscles, 3- Lobules, 4- Nipple, 5- Areola, 6- Milk duct, 7- Fatty
tissue and 8- Skin (Love, 2015). B- Breast cancer
2.4.1 Etiology of breast cancer

In Iraq, breast cancer is the most common cancer in women, accounting for
36.7% of all malignant diseases (Bray et al., 2018). Breast cancer can be diagnosed
in women of all ages, but the risk of developing breast cancer increases with age, and
Page 7
most cases (80%) occur in women over the age of 50 years. Several factors that
increase a woman’s risk of developing breast cancer (Parkin et al., 2011). Breast
cancer is a complex, heterogeneous, and multifactorial disease. Although some
genetic factors have a strong and well-defined impact, such as mutations in the breast
cancer 1 (BRCA1) and BRCA2 genes, only 5–10% of all breast cancers are
considered to be due to mutations in inherited high penetrance genes (Di Sibio et al.,
2016). The risk of breast cancer has been consistently associated with age, a family
or personal history of breast cancer, reproductive and hormonal factors, hormone
replacement therapy, obesity, alcohol consumption, physical inactivity, exposure to
ionizing radiation, and genetic predisposition (Lauby-Secretan et al., 2015).
Evidence indicates that breastfeeding reduces the risk of developing breast

carcinoma (Zhou et al., 2015). Epidemiological evidence has suggested that the use
of contraceptives is associated with an increased risk of breast cancer among carriers
of BRCA1 or BRCA2 mutations (Friebel et al., 2014). It was found that a 5 kg/m2
increase in BMI increases the risk of postmenopausal breast cancer by 12% (Arnold,
M., et al., 2015), and that physical inactivity increase the risk of breast cancer by
33% (Pena et al., 2014).
2.4.2 Signs and symptoms of breast cancer

The first sign of breast cancer is a new lump or mass in the breast. Breast cancers
show up as red or thickened skin rather than a lump. The most common physical sign
is a painless lump. Sometimes it can spread to underarm lymph nodes and cause a
lump or swelling. It is important to note that pain does not indicate the presence or
the absence of breast cancer. Less common signs and symptoms include breast pain
or heaviness; persistent changes to the breast, such as swelling, thickening, or redness
of the breast’s skin; and nipple abnormalities such as spontaneous discharge, erosion,
inversion, or tenderness, irritation and nipple discharge other than breast milk,
including blood, or dimpling of breast skin (Berry Khatri, 2019).
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2.4.3 Genetics and breast cancer

Cancer is fundamentally a disease of tissue growth regulation. In order for a
normal cell to transform into a cancer cell, the genes that regulate cell growth and
differentiation must be altered (Croce, 2008). Oncogenes are genes that promote cell
growth and reproduction. The first major gene associated with hereditary breast
cancer is BRCA1, or BRCA2 confers an increased risk of breast cancer. Female
carriers of mutations in BRCA1 or BRCA2 have a lifetime risk (50–85%) of breast
cancer. Male carriers of BRCA1 have an increased risk of breast cancer (Shiovitz
and Korde, 2015).
Genetic changes can occur at different levels and by different mechanisms.

(Colotta et al., 2009). Disruption of a single gene may also result from integration of
genomic material from a DNA virus or retrovirus, leading to the expression of viral
oncogenes in the affected cell and its descendants (Borrello et al., 2008).
2.4.4 Breast cancer Spread and metastasis

Breast cancer can spread through the lymph system, which includes the lymph
nodes, lymph vessels, and lymph fluid found throughout the body. Lymph nodes are
small, bean-shaped collections of immune system cells that are connected by lymph
vessels. Lymph vessels are like small veins, except that they carry a clear fluid called
lymph away from the breast. Lymph contains tissue fluid and waste products, as well
as immune system cells. Breast cancer cells can enter lymph vessels and grow in
lymph nodes. Most of the lymph vessels of the breast drain into lymph nodes under
the arm, around the collar bone, and inside the chest near the breast bone (Smith et
al., 2011).
2.4.5 Types of breast cancer

Breast cancer is often classified into 2 types: non-invasive and invasive
(infiltrating).
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2.4.5.1 Non-invasive breast cancer or in situ (in the same place) carcinoma:
In situ carcinoma is defined by the clonal proliferation of neoplastic epithelial cells
within the ducts. It is located in the ducts and lobules of the breast. They do not grow
into or invade normal tissues within or beyond the breast. Tumor cells of in situ
carcinomas are confined to the breast epithelial cells where they started (Phuc Van
Pham, 2017).
A. Ductal Carcinomas in Situ (DCIS): It is the cells that line the milk ducts of the
breast have become cancer, but they have not spread into surrounding breast tissue.
It is the most common type of non-invasive breast cancer and attributed about 15-
20% of new breast cancer cases (Figure (2-3)) (Phuc Van Pham, 2017).
Figure (2-3): Ductal carcinoma in situ (DCIS). (Phuc Van Pham, 2017).
B. Lobular Carcinoma in Situ (LCIS):

It is an uncommon condition in which abnormal cells. It arises inside the milk-
producing glands of the breast and constitutes about 12% of female in situ breast
cancers. Therefore, Patients diagnosed with LCIS have an increased risk of
developing invasive cancer at a later point in time. It is an area of abnormal cell
growth that increases a person's risk of developing invasive breast (Figure (2-4)),
(Phuc Van Pham, 2017).
Page 10
2.4.5.2 Invasive Carcinoma

The breast cancer epithelial cells spread outside the basal membrane that covers
a duct or alveoli and invade the surrounding breast cancer tissues (Sharma et al.,
2010). Most breast
cancers are invasive
and are of different
types. The 2 most
common are invasive
ductal carcinoma and
invasive lobular
carcinoma (Visser et
al., 2019).
Figure (2-4): Lobular carcinoma in situ (LCIS). (Phuc Van Pham, 2017).
A. Invasive Ductal Carcinomas (IDC)

This is the most common type of breast cancer. IDC arises in the epithelial cells
within the ducts and starts in the cells lining the milk duct in the breast. It may be
able to spread to other organs such as liver or lungs through the lymph system and
blood stream (Dillon et al., 2014). IDC often presents alone or with a co-existing
ductal carcinoma in situ component (IDC + DCIS) (Kole et al., 2019). Treatment of
invasive breast cancer depends on how advanced the cancer and the other factors.
(Overmoyer and Pierce, 2015).
B. Invasive Lobular Carcinoma (ILC)

The invasive tumor cells of this cancer type are originated in lobules and
inevitably metastasize to other body parts. It is the second most frequent invasive
carcinoma of breast cancer, contributing to about 10-15% of invasive cases (Watheq,
2016).
Page 11
C. Less Common Types of Invasive Breast cancer

Special types of breast cancer that are sub-types of invasive carcinoma, each
typically makes up less than 5% of all breast cancers. They include tubular, papillary,
adenocystic, mucinous, and medullary carcinomas. Some sub-types, which include
metaplastic, micropapillary, and mixed carcinomas have the same prognosis as IDC
(Jagsi et al., 2019).
2.5 Glycolysis Pathway

Glycolysis (Embden-Meyerhoff Pathway (EMP)) is a metabolic pathway and
an anaerobic source of energy. In glycolysis, 1 glucose molecule utilizes 2ATP
molecules to produce 4ATP, 2NADH, and 2 pyruvates. The pyruvate can be used in
the citric acid cycle, or serve as a precursor for other reactions (Chaudhry and
Varacallo, 2019). Glycolysis is an inefficient means of energy production, producing
only 2 net molecules of ATP per molecule of glucose (Melkonian and Schury, 2019).
Glycolysis is an oxygen independent metabolic pathway, that occurs in the cellular
cytosol of most organisms (Keller, et al., 2014). If a cell has mitochondria and
oxygen, glycolysis is aerobic. If either mitochondria or oxygen is lacking, glycolysis
may occur anaerobically although some of the available energy is lost (Sam Turco,
et al., 2016).
Under certain conditions, especially under oxygen deprivation, OXPHOS

cannot take place, so pyruvate is instead converted to lactate by LDH. Lactate is then
ejected out of the cell by monocarboxylate transporter 4 (MCT4), in order to maintain
the intracellular pH within acceptable levels (Knobloch et al., 2019). Disposal of
lactate from the cell is one of the main causes of extracellular acidosis. Anaerobic
glycolytic pathway is much less efficient than OXPHOS in producing energy, as only
2 molecules of ATP are produced per glucose molecule, versus the ~36 ATP units
usually produced in OXPHOS. Glycolysis generates ATP more rapidly than
OXPHOS, and this offers a selective advantage to rapidly growing tumor cells.
Page 12
Glycolytic inhibitors are being designed that target the enzymes involved in the
glycolysis pathway (Fadaka et al., 2017). D-Mannoheptulose, a heptose
(monosaccharide), is a hexokinase inhibitor found in avocados. By blocking the
enzyme hexokinase, it prevents glucose phosphorylation, inhibiting the breakdown
of glucose. It behaves as a competitive inhibitor of hexokinase, an enzyme that
phosphorylates hexoses such as glucose. The inhibition of hexokinase leads to the
prevention of glycolysis (Liu et al, 2002).
2.5.1 Glycolysis in cancer cells

Cancer cells mainly produce energy by an increased rate of glycolysis (200
times more as compared to normal tissues of origin) followed by fermentation of
lactate in the cytosol of the cell, even if oxygen is plentiful (Alfarouk et al., 2014).
Increased glycolysis is the main source of energy supply in cancer cells that use this
metabolic pathway for ATP generation. The immune system can prevent tumor
growth by eliminating cancer cells (Kheshwant et al., 2016).
Lactate dehydrogenase (LDH) is a tetrameric enzyme with 2 subtypes: LDH-A

and LDH-B. In hypoxic cells, the conversion of pyruvate into lactate is catalyzed by
LDH-A, whilst LDH-B catalyzes the conversion of lactate into pyruvate. Lactate
accumulation substantially reduces intracellular pH which is detrimental to the cell.
Production of NAD+ from the oxidation of cofactor NADH is necessary at the
reduction step of pyruvate into lactate, to continue glycolysis. Evidence suggests that
LDH-A, which is upregulated in invasive glycolytic cancers, plays a critical role in
cell proliferation. This allows the tumors to survive even in low oxygen levels (Jin
et al, 2013).
2.5.2 Metabolic pathways in cancer

Cancer metabolism refers to the alterations in cellular metabolism pathways. The
metabolism of cancer cells is glycolysis dependent. Cancer cells continuously rely
on glycolysis rather than oxidative phosphorylation even under aerobic conditions,
Page 13
which require high quantities of glucose to produce energy and support the metabolic
function (i.e., the Warburg effect) (Warburg, 1956). This effect shows that glucose
uptake of cancer cells is higher than that of normal tissues. Thus, the glucose
metabolism could be targeted as a site for chemotherapeutic intervention by agents
(Al-Shammari et al., 2019).
Cancer is a disease condition arising from uncontrolled division of cells in the

body to form lumps of tissues called tumors. There are many different types of cell
in the body, and many different types of cancer which arise from different types of
cell. Some are more easily treated than others, particularly if diagnosed at an early
stage (Fadaka et al., 2017).
2.5.3. Glycolysis and differences between cancer cells and normal cells
Aerobic glycolysis occurs when oxygen is plentiful, and the final product is
pyruvate along with the production of 8 ATP molecules. Anaerobic glycolysis occurs
when oxygen is scarce, and the final product is lactate along with the production of
2 ATP molecules (Chaudhry and Varacallo, 2019). In normal cells, the last step in
the breakdown of glucose is called oxidative phosphorylation (OXPHOS). It takes
place in the mitochondria, produces a large amount of ATP, and requires oxygen for
completion (Urry et al., 2017).
Aerobic breakdown of glucose consists of four stages: glycolysis, pyruvate

oxidation, Krebs cycle and Electron transport chain (ETC). Increased glycolysis is
the main source of energy supply in cancer cells that use this metabolic pathway for
ATP generation (Kheshwant et al., 2016). Cancer cells only partially break down
sugar molecules unlike healthy cells that burn the entire molecule of sugar to capture
a large amount of energy as ATP. They overuse the first step of respiration,
glycolysis, and frequently do not complete the second step, oxidative
phosphorylation. As a result, cancer cells need to use a lot more sugar molecules to
grow (Elf and Chen, 2014).
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Figure (2-5): Glycolysis in tumor cell (Fadaka et al., 2017).
This produces far less energy than oxidative phosphorylation and as cancer cells
require a lot of energy to grow, this seems paradoxical. The Warburg effect may be
beneficial to cancer cells because it provides precursors for many biosynthetic
pathways. These precursors include amino acid precursors and NADPH and ribose
sugars for DNA and RNA synthesis. The Warburg effect may be caused by impaired
oxygen sensing in cancer cells. Glycolytic enzymes such as Glucose transporter 1
(GLUT1(, lactate dehydrogenase, pyruvate kinase and the lactate exporter are
unregulated in cancer cells, whilst pyruvate dehydrogenase is inhibited leading to
increased glycolytic flux and impaired ability of pyruvate to enter oxidative
phosphorylation (Fadaka et al., 2017) (Figure (2-5)).
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2.5.4 Warburg effect and the glycolytic inhibitors

Warburg effect phenomenon, is considered one of the most fundamental
metabolic alterations during malignant transformation. One hallmark of cancer is the
shift from aerobic to anaerobic metabolism seen within tumor cells, referred to as the
Warburg Effect (Melkonian and Schury, 2019). The increased dependence of cancer
cells on glycolytic pathway for ATP generation provides a biochemical basis for the
design of therapeutic strategies to kill cancer cells by pharmacological inhibition of
glycolysis (Kheshwant, et al., 2016). In the presence of oxygen, nonproliferation
(differentiated) tissues first metabolize glucose to pyruvate via glycolysis and then
completely oxidize most of that pyruvate in the mitochondria to CO2 during the
process of oxidative phosphorylation, (Figure (2-6)).
Figure (2-6): Schematic representation of the differences between oxidative phosphorylation,

anaerobic glycolysis, and aerobic glycolysis (Warburg effect) (Vander Heiden et al, 2009).
Oxygen is essential for this process as it is the final electron acceptor to

completely oxidize the glucose. When oxygen is limited, cells can redirect the
pyruvate generated by glycolysis away from mitochondrial oxidative
phosphorylation by generating lactate (anaerobic glycolysis). This generation of
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lactate during anaerobic glycolysis allows glycolysis to continue (by cycling NADH
back to NAD+), but results in minimal ATP production when compared with
oxidative phosphorylation. Warburg originally proposed that the aerobic glycolysis
in cancer cells was due to a permanent impairment of mitochondrial oxidative
phosphorylation (Kroemer and Pouyssegur, 2008). Malignant cells exhibit
significantly elevated glycolytic activity even in the presence of sufficient oxygen,
and this phenomenon is considered as the most fundamental metabolic alteration in
malignant transformation, or ‘the origin of cancer cells’ (Hammoudi et al., 2011).
Warburg observed that cancer cells tend to convert most glucose to lactate
regardless of whether oxygen is present (aerobic glycolysis). This property is shared
by normal proliferative tissues. The mitochondria remain functional and some
oxidative phosphorylation continues in both cancer cells and normal proliferating
cells. Nevertheless, aerobic glycolysis is less efficient than oxidative
phosphorylation for generating ATP. Under anaerobic conditions, pyruvate has a
different fate i.e., instead of entering the mitochondria, the cytosolic enzyme lactate
dehydrogenase converts it to lactate. Although lactate itself is not utilized by the cell
as a direct energy source, this reaction allows the regeneration of NAD+, an
oxidizing cofactor necessary to maintain the flow of glucose through glycolysis, from
NADH. Glycolysis produces 2 ATP molecules per glucose molecule, providing a
direct means of producing energy in the absence of oxygen. This process of breaking
down glucose in the absence of oxygen is aptly named anaerobic glycolysis (Chen et
al., 2017).
In proliferating cells, ~10% of the glucose is diverted into biosynthetic

pathways upstream of pyruvate production. Cancer cells produce large amounts of
lactate regardless of the availability of oxygen; hence their metabolism is often
referred to as aerobic glycolysis. Warburg originally hypothesized that cancer cells
develop a defect in the mitochondria that leads to impaired aerobic respiration and a
subsequent reliance on glycolytic metabolism. There is an advantage to oxidative
Page 17
metabolism during nutrient limitation and non-oxidative metabolism during cell

proliferation (Vander Heiden et al., 2009). The glycolytic inhibitors are particularly
effective against cancer cells with mitochondrial defects. Because increased aerobic
glycolysis is commonly seen in human cancers and hypoxia is present in most tumor
microenvironment, development of novel glycolytic inhibitors as a new class of
anticancer agents is likely to have broad therapeutic applications. The biological
differences between normal and cancer cells are essential for the design and
development of anticancer drugs with selective anticancer activity. Each reaction in
the glycolytic pathway is catalyzed by a specific enzyme or enzyme complex.
(Pelicano et al., 2006).
This effect means higher glucose uptake by cancer cells compared to normal
tissues, which can be used as target for selective therapy by targeting glucose
metabolism as a site for chemotherapeutic intervention. Targeting cellular
metabolism can improve cancer therapeutics (Zhao et al., 2013). Warburg hypothesis
explains that cancer cells mainly produce energy by an increased rate of glycolysis
followed by fermentation of lactate in the cytosol of the cell, even if oxygen is
plentiful (Alfarouk et al., 2014).
Hexokinase is a tissue-specific isoenzyme that catalyzes the phosphorylation of

glucose to glucose-6-phosphate (G6P), after glucose enters the cell via glucose
transporters (GLUT). This ATP-dependent reaction is the first step in glycolysis, and
it is also a rate limiting step as hexokinase possesses the ability to transfer an
inorganic phosphate group from ATP to a substrate. (Kheshwant et al., 2016). ATP
is a key indicator of cellular activity and has been utilized as a measure of cell
viability and cytotoxicity in research and drug discovery (Schwarzer et al, 2008).
The electron transport chain requires oxygen as the final electron acceptor,
inadequate tissue oxygenation inhibits the process of oxidative phosphorylation
(Melkonian and Schury, 2019). Anaerobic glycolysis serves as a means of energy
production in cells that cannot produce adequate energy through oxidative
Page 18
phosphorylation (Chen et al., 2017). As anaerobic glycolysis allows more rapid

production of ATP, glycolysis is approximately 100 times faster than oxidative
phosphorylation (Melkonian and Schury, 2019).
2.6 Programmed cell Death (Apoptosis)

Cell death was once believed to be the result of one of two distinct processes,
apoptosis (programmed cell death) or necrosis (uncontrolled cell death); in recent
years, several other forms of cell death have been discovered highlighting that a cell
can die via a number of differing pathways. Apoptosis is characterized by a number
of characteristic morphological changes in the structure of the cell, together with a
number of enzymes‐dependent biochemical processes. Failure of apoptosis and the
accumulation of damaged cells in the body can result in various forms of cancer. An
understanding of the pathways is therefore important in developing efficient
therapeutics (D’Arcy, 2019). The increased understanding of how environmental
exposures cause diseases through apoptosis disruptions could ultimately lead to the
discovery of therapeutic strategies (Rager, 2015).
Programmed cell death is a natural process of eliminating cells that have been
produced redundantly, developed improperly or have genetic damages. Apoptosis
can be initiated by 3 alternative pathways: the extrinsic pathway, which is mediated
by death receptors on cell surface; the intrinsic pathway, which is mediated by
permeabilization of the mitochondria and the stress signaling pathway of
endoplasmic reticulum (ER) and mitochondria (Kuwana et al., 2016). Apoptosis, the
cell’s natural mechanism of death, is a promising target for anticancer therapy. Both
the intrinsic and extrinsic pathways use caspases to carry out apoptosis through the
cleavage of hundreds of proteins. In cancer, the apoptotic pathway is typically
inhibited through a wide variety of means. Many of these changes cause intrinsic
resistance to the most common anticancer therapy (Pfeffer and Singh, 2018).
Initiation of apoptosis occurs principally by signals from 2 distinct but convergent
Page 19
pathways; the extrinsic (receptor-initiated) and intrinsic (mitochondrial) pathway

(Pistritto et al., 2016). The last phase of apoptosis (Execution Phase) is mediated by
a proteolytic cascade of proteases that belongs to the caspase (cysteine-aspartic
proteases) family (Salvesen et al., 2016). At early stages of apoptosis, dying cells
secrete soluble factors that induct phagocytes and facilitate clearance of apoptotic
cells before they undergo secondary necrosis and release their cellular contents that
can result in inflammation. Also, apoptosis may be essential for the prevention of
tumor formation (Poon et al., 2014).
The apoptotic bodies are gulped by macrophages and discarded from the tissue
unless they give rise to an inflammatory response. Apoptotic cells can be recognized
by the following stereotypical morphological changes. The cell shrinks, shows
deformation, and loses contact with its neighboring cells, its chromatin condenses
and marginates at the nuclear membrane, the plasma membrane is budding, and
finally the cell is fragmented into compact membrane-enclosed structures called
'apoptotic bodies' which contain cytosol, the condensed chromatin, and organelles
(Salvesen et al., 2016) (Figure (2-7)).
Figure (2-7) Programmed cell Death (Apoptosis) (Srivastava, 2007).
Page 20
2.7 Cancer Therapy
2.7.1 Conventional cancer therapeutics

There are widely accepted therapies and used by most healthcare professionals
(NCI., 2016). The spectrum of the conventional cancer treatment includes 3 basic
methods: operations, chemotherapy, and radiotherapy (Damyanov et al., 2018).
2.7.1.1 Surgical Therapy

Surgical therapy is considered the oldest type of cancer treatment. It had been
an effective treatment to cure patients from cancer (Tai, 2013). Randomized trials
have shown that the addition of preoperative chemotherapy to surgery significantly
improves survival (Ajani et al., 2019). The primary goals of breast cancer surgery
are to remove the cancer from the breast and determine the stage of the disease.
Surgical treatment for breast cancer involves breast-conserving surgery, a surgery in
which only the part of the breast containing the cancer is removed, or mastectomy.
The presence of cancer cells in the lymph nodes will help in determination the need
for subsequent therapy (Miller et al., 2019). Surgical therapy is very unlikely to cure
breast cancer that has spread to other parts of the body (Harris et al.,2014).
2.7.1.2 Radiotherapy
Radiotherapy utilizes ionizing radiation to control or kill malignant cells.
Ionizing radiation works by damaging the DNA of cancerous tissue leading to
cellular death (Borrego-Soto et al., 2015). Despite the fact that radiation damages
both cancer and normal cells. The goal of radiation therapy is to damage as many
cancer cells as possible. (McIntyre and Harris, 2015). It is mainly applied to primarily
treat solid tumors when combined with the other conventional methods. It is used as
a palliative treatment to reduce the disease symptoms. (Christo et al., 2018).
2.7.1.3 Chemotherapy
Chemotherapy agents can be divided into many types: Alkylating agents such
as cyclophosphamide and antibiotics (Chock et al., 2010), and platinum compounds
Page 21
such as cisplatin and antimetabolites that have an effect on nucleic acids (Torrence,
2012).The benefit of chemotherapy depends on multiple factors, including the size
of the cancer, the number of lymph nodes involved, and the presence of estrogen or
progesterone receptors (Cortazar et al., 2014). Drug combinations are more effective
than a single drug for breast cancer treatment, and many combinations are being used
(Moura et al., 2015).
One problem with chemotherapy is that they are toxic to normal dividing cells
as their high growth fraction is as that of cancer cells (Mitchison, 2012).
Chemotherapy can be given before surgery (neoadjuvant) or after surgery (adjuvant).
In most cases, chemotherapy is most effective when drug combinations are used.
Drugs for breast cancer that has spread include taxanes, such as paclitaxel, docetaxel
and albumin-bound paclitaxel, anthracyclines, platinum agents (cisplatin and
carboplatin), vinorelbine and capecitabine. Although drug combinations are often
used to treat early breast cancer, advanced breast cancer more often treated with
single chemo drugs. (Smith et al., 2018).
2.7.1.4 Hormone therapy

Hormone therapy is significantly less applicable additional method to treat
cancer (Damyanov et al., 2018). Hormone therapy is often used after surgery to help
reduce the risk of cancer recurrence. Sometimes it is started before surgery (Miller
et al., 2019). Women whose breast cancers test positive for estrogen or progesterone
receptors can be given hormone therapy to lower estrogen levels or to block the
effects of estrogen on the growth of breast cancer cells. Tamoxifen and toremifene
are drugs that prevent the binding of estrogen to breast cancer cells and are effective
in both postmenopausal and premenopausal women (Nicholas, 2019).
2.7.1.5 Immunotherapy
Immunotherapy is the treatment of disease by activating or suppressing the
immune system. Immune system plays a major role in rejecting transformed cells
Page 22
(Smyth et al., 2006). The disadvantages of surgery such as recurrence of tumor or

toxicity of radiotherapy or chemotherapy can be substantially reduced by
immunotherapy when used in combination with these treatment modalities (Mohan
et al., 2019). Immunotherapy is a significantly less applicable additional method.
This application is more in the field of the experimental research (Damyanov et al.,
2018). Immunotherapy involves manufacturing antitumor effects primarily through
the action of natural defense processes (Smith and Andreansky, 2014). The work
with IL-2 in treating cancer substantiated the important role of adaptive immunity in
cancer therapy (Guo, 2018).
Normal antibodies bind to external pathogens, but the modified immunotherapy

antibodies bind to the tumor antigens marking and identifying the cancer cells for the
immune system to inhibit or kill (Nielsen and Hawkes, 2019). Immunotherapies can
be categorized as active, passive, or hybrid. Active immunotherapy directs the
immune system to attack tumor cells by targeting tumor antigens. Passive
immunotherapies enhance existing anti-tumor responses (Korneev et al., 2017).
Passive antibody therapies involve targeting of cell surface receptors (Espinoza-
Sánchez and Götte, 2019).
2.7.2 Novel therapeutics

Alternative or complementary therapies, which are not as widely used (NCI., 2016)
2.7.2.1 Anti-Angiogenesis Therapy

Angiogenesis permits growth of cancer mass. Once the mutated cell growth
starts, the body recognizes this by cellular communication and starts transmitting
new micro-blood vessels to the region. Anti-angiogenesis will inhibit the expansion
of these new blood vessels and inhibit the growth of the cancer (Negro, 2015).
2.7.2.2 Biological Therapy

Biological therapy has a very important modality for cancer treatment that
produces antitumor effects primarily through the action of natural host defense
Page 23
mechanisms (Devita et al., 2015). A type of treatment that uses substances made
from living organisms to treat disease. Some biological therapies stimulate or
suppress the immune system to help the body fight cancer, infection, and other
diseases. They may also lessen certain side effects caused by some cancer treatments
(NCI, 2016). Biological therapy is an effective treatment for a range of immune-
mediated inflammatory diseases (Baumgart et al., 2019).
2.7.2.3 Gene Therapy

Gene therapy can be broadly defined as the transfer of genetic material to cure
a disease or at least to improve the clinical status of a patient. One of the basic
concepts of gene therapy is to transform viruses into genetic shuttles, which will
deliver the gene of interest into the target cells (Verma and Mishra, 2016). Gene
therapy is a replacement of a deficient gene product or a correction of an abnormal
gene. It can be done either in vitro, or in vivo, where the cells cannot be cultured, or
are replaced in the affected individual (Mueller and Young, 2012).
2.7.2.4 Virotherapy
The use of viruses for cancer treatment began before the 1950s (Kelly and
Russell, 2007). It has been identified for over 70 years that some viruses like
Newcastle disease virus (NDV) have the ability to destroy cancer cells. These viruses
are units either used with non-genetic manipulation or biotechnology for increasing
properties in animal models and human clinical trials (Delwar et al., 2016; Kaufman
et al., 2016).
NDV has been classified beside alternative viruses like reovirus and parvovirus
as viruses with inherent oncolytic effects; these viruses were manipulated in to how
they are attenuated in normal cells while not losing their ability to lyse tumor cells.
(Zimmermann et al., 2014). Numerous viruses are tested in various animals and
human models for their anti-cancer efficiency and tumor specificity (Addisu et al.,
2016). These viruses are used for cancer treatment either with or without genetic
Page 24
manipulation (Wong, et al., 2010). Oncolytic virotherapy divided in two main

groups, oncolytic wild natural occurring viruses with preferential replication in
human cancer cells and gene-modified viruses engineered to achieve selective
oncolysis (Garcia et al, 2008).
2.7.2.4.1 Newcastle Disease Virus (NDV)

NDV, a member of the Avulavirus genus of the Paramyxoviridae family (Meng
et al., 2019), infects a number of avian species. An oncolytic virus selectively kills
tumor cells, and it is not a natural human pathogen (Bai et al., 2015). NDV causes
highly infectious and economically significant Newcastle disease in birds of various
species worldwide (Costa-Hurtado et al., 2015).
Pathogenic NDV causes an economically necessary animal disease in various

bird species worldwide (Miller and Koch, 2013). Based on the virulence in infected
birds, NDV has been categorized into 3 pathotypes: Lentogenic which causes
moderate respiratory disease, Mesogenic which causes output respiratory and
nervous signs and the velogenic which causes severe gastrointestinal lesions and
neurological disease (Pantin-Jackwood et al., 2015). NDV selectively replicates in
human cancerous cells stinting normal cells and it activates apoptosis programmer
in cancerous cells (Biswas et al., 2012). The anti-cancer effect of NDV has been
tested on various human cancers (Farkona et al., 2016).
2.7.2.4.2 Molecular Biology of NDV

NDV is spherically shaped and surrounded with a lipid bilayer underneath
which is the matrix protein that forms the inner layer of virus (Battisti et al., 2012),
(Figure (2-8)). The genomic RNA in NDV consists of 6 genes encoding at least 8
proteins (Jin et al., 2016). An interesting aspect of NDV is its ability to selectively
replicate in tumor cells. site-specific alterations can be happened in the genomes of
RNA-virus by mutagenesis of single nucleotides or insertion of foreign genes. (Bai
et al., 2015).
Page 25
Figure (2-8): Structure of Newcastle Disease Virus (Battisti et al., 2012).
2.7.2.4.3 Mechanisms of NDV-induced Oncolysis

When infecting a cancer cell, it replicates speedily and infects neighboring
tumor cells through the discharge of progeny virions which are noticeable 3 hrs post-
inoculation, and plaques develop within 2 days post-inoculation (Elankumaran et al.,
2010). Several mechanisms for its anti-tumor effect have been proposed in humans.
Firstly, the lytic strains of the virus may artlessly kill tumor cells by a direct route
and initiate effectiveness in preventing tumors. Secondly, the non-lytic strains
injected into the tumor cell membrane after infection by the viral protein may
develop an immune response. Finally, the virus itself may stimulate the host to
produce effector cytokines such as interferons (IFN-γ) or tumor necrosis factors α
(TNF-α), which activate natural killer (NK) cells, macrophages, and sensitized T-
cells. (Sweiss et al., 2011). Apoptosis is susceptible to disruption by mutations as it
is a gene-controlled process (Hassan et al., 2014). Critical linkage between apoptosis
and cancer indicates that any curative planning aimed at specifically triggering
apoptosis in cancer cells will have a potential therapeutic effect (Su et al., 2015). It
was reported that NDV induces expression of varied cytokines which may activate
the host cell death pathway inflicting oncolysis (Lam et al., 2014).
Page 26
NDV might activate programmed cell death through the extrinsic and intrinsic
death pathway. Additionally, there are reports that NDV augments the expression of
evoked nitric oxide synthase in infected cells (Hrabak et al., 2006). Viruses are
utilized as possible anticancer therapeutics for cancer treatment (Assi, 2013), (Figure
(2-9)).
Figure (2-9): Oncolytic virus function (Assi et al., 2013)
2.7.2.4.4 Safety of NDV Administration as Anticancer Agent

Scientists are interested in the therapeutic effect of NDV because of its tumor
selectivity (Lam et al., 2014). NDV strains can selectively replicate up to 10000
times better in tumor cells, but not in normal cells (Alamares et al., 2010). Numerous
reports had shown the inability of the virus to replicate in non-transformed cells, such
as fibroblast, resting T lymphocytes, and normal primary culture (Fábián et al.,
2007). NDV is an immunostimulatory agent, as it can induce anti-tumor activities of
a variety of effector cells, including NK cells, macrophages, and cytotoxic T
lymphocyte (CTL) (Assayaghi et al., 2016).
Page 27
Iraqi strain of Newcastle disease virus (NDV) is a novel anti-tumor therapy.

Virulent strains of NDV are being used in gene therapy as possess a number of
desirable oncolytic properties. NDV Iraqi strain can reduce tumor cell infiltration,
invasion, and metastasis (Al-Shammari et al., 2013). Virotherapy is a promising
agent against cancer because of its safety and selectivity. NDV is safe, it selectively
targets tumor cells, and it could be used to augment other chemotherapeutic agents
and reduce their toxicity by halving the administered dose and replacing the
eliminated chemotherapeutic agents with it (Al-Shammari et al., 2016).
2.7.2.4.5 Replication of NDV

NDV selectively infects growing cells to produce more viral copies. The
infection process starts when the virus interacts with a target receptor on the host
cell, after which the initial steps of virus entry begin. The whole replication process
of NDV occurs in the cytoplasm. The replication process can be divided into
phases:1-initiation attachment, penetration 2- Replication genome synthesis, RNA
production with protein synthesis and finally 3-Release assembly, maturation exit
from the cell. NDV generates dsRNA during replication (Chu et al., 2019).
Replication of Newcastle disease virus explain the NDV‐infection and cellular
responses. NDV attaches via the hemagglutinin‐neuraminidase (HN) protein to sialic
acid containing host cell surface receptors. The viral fusion protein (F) initiates
fusion of viral and host cell membranes. The viral RNA polymerase starts replication
in the cytoplasm of the target cell by transcription of viral negative single‐strand
RNA into positive single‐strand RNA necessary as template for mRNA and protein
synthesis. The viral proteins undergo processing in the rough endoplasmic reticulum
and the Golgi apparatus. In tumor cells they are assembled at the host cell membrane
where budding leads to newly produced virions. In nontumorigenic cells,
endocytosis of viral components bound to Toll‐like receptors (TLR) and the
appearance of intermediate double strand RNA during the process of viral replication
induces danger signals to the host cell, triggering the transcription of various
Page 28
protective genes. DsRNA is recognized by RNA helicase (Fiola et al., 2006). (Figure
(2-10)).
Figure (2-10) Replication of Newcastle Disease virus (Fiola et al., 2006)
2.7.2.4.6 Clinical usage of NDV in cancer therapy

NDV infects the host cells and then replicates itself. Scientists are interested in
NDV because it can replicate more quickly in human tumor cells than in normal cells
and has oncolytic eﬀects (Lam et al., 2011). Newcastle disease virus (NDV) is an
oncolytic virus may interact with radiotherapy to enhance treatment against cancer
cells. (Vijayakumar et al., 2019). NDV is an avian virus that causes deadly infection
to over 250 species of birds. Many reports have demonstrated effect of the oncolytic
virus towards human tumor cells. The interesting aspect of NDV is its ability to
selectively replicate in cancer cells. NDV strains can be potential therapeutic agent
in cancer therapy (Ginting et al., 2017). NDV induces both extrinsic and intrinsic
apoptosis of malignant cells. NDV infection elicits both innate and adaptive antiviral
immunity, resulting in cross-activated anti-tumor immune responses (Schwaiger et
al., 2017). The majority of tumor cells could be infected by NDV, and the viral
Page 29
replication was detected into cells by the increase of viral antigens on the cell surface
(Fiola et al., 2006). The first report of the application of NDV to treat human cancers
was in the early 1950s, when NDV were injected directly into uterine carcinoma,
resulting in partial necrosis, but followed by regrowth. Many reports showed the
possibility of NDV as a therapeutic agent in cancer treatment, many studies both in
mouse models and in human clinical trials which showed favorable results (Lam et
al., 2011). Localized therapy with oncolytic NDV induces an inflammatory response
which leads to lymphocyte infiltration and an antitumor effect in distant tumors
without dissemination of the virus (Meng et al., 2019).
2.7.2.5 Combination therapy

Combination therapy, a treatment modality that combines two or more
therapeutic agents, is a cornerstone of cancer therapy. The amalgamation of anti-
cancer drugs enhances efficacy compared to the mono-therapy approach because it
targets key pathways in a characteristically synergistic or an additive manner. This
approach potentially reduces drug resistance, while simultaneously providing
therapeutic anti-cancer benefits, such as reducing tumor growth and inducing
apoptosis (Mokhtari et al., 2017). Targeting cancer cells metabolism is a promising
strategy in inhibiting the progression of cancer cells. The NDV was used as a
combination virotherapy to enhance the anti-tumor effect. Human and mouse-breast
cancer cells were treated with NDV and/or 2-Deoxy glucose, and the combination
treatment showed significant tumor growth inhibition compared to single treatments
in vivo (Al-Shammari et al., 2019). The therapeutic challenge is not only going to be
maximizing the effectiveness of individual agents, but also to integrate these drugs
into the best combination therapy (Mishra et al., 2013). Oncolytic virus has a better
level of tumor specificity relative to chemotherapy drugs attributable to each
associate innate preference for tumor cells (Ottolino-Perry et al., 2010). The theory
supported that combination chemotherapy assaulting tumor cells by using different
mechanisms of action can prevent tumor cell from having enough time to improve
Page 30
resistant to treatment (Post et al., 2003). Synergistic antitumor combination of NDV

and 2-Deoxy glucose had a greater effect on cancer cells, than their individual effects
in breast cancer cell lines (Al-Shammari et al., 2019). The combination of the 2-
targeted drugs was more effective. There is a certain synergistic effect in tumor cell
growth inhibition. This will be a theoretical foundation for treatment of cancer in
clinical applications (Zhang et al., 2013). The apoptosis induction in triple
combination had a better effect in comparison with double combination which had a
better effect than their individual effect (Dalirsani et al., 2012).
The combined use of 2 or more agents is often advantageous as it may permit

the lowering of the clinical dosages thereby decreasing the overall toxicity and thus
providing the potential for synergistic effects between agents. NDV is the only
oncolytic virus that showed no serious side effect in the clinical field (Al-shammari
et al., 2012). Oncolytic virus will by selection infect, replicate in, and kill tumor cells
with least impact on normal tissue. A virus ability to recognize and enter specific
receptors on the surface of tumor cells, additionally aids in the disposition of the
tumor cell towards viral replication (Pol, 2012).
The strategy of combination therapy is by attacking tumor cells through

different mechanisms of action to prevent cancer cells from developing resistance to
therapy (Kumar, 2014). The Iraqi oncolytic strain of NDV kills tumor cells by several
mechanisms, such as generation of specific immune response against infected tumor
cells (Al-Shammari et al., 2014). NDV induces apoptosis in the infected cells (Al-
Shammari et al., 2015). Phytotherapy has been used to treat a different type of
diseases including cancer for a long time, and it was a source for different active anti-
tumor agents. NDV-based monotherapeutics have not been very useful to some
resistant tumors. Thus, the efficiency of oncolytic NDV must enhance by combining
NDV with other novel therapies , the possibility of improving the oncolytic effect
induced by NDV. The novel combination recommended for clinical application for
cancer therapy (Al-Shammari et al., 2020).
Page 31
CHAPTER-THREE
Chapter Three………………………………..………………………… Material and Methods
3. Material and Methods

Subject:
The research project approval committee was formed by the Council of the
College of Medicine / Al-Nahrain University on 4/7/2018. The research project was
approved by the scientific committee in the Department of Chemistry and
Biochemistry / College of Medicine on 11/7/2018. The approval of the Institutional
Review Board (IRB) at the College of Medicine on the research project was obtained
on 15/7/2018.
The research project was carried out at the Iraqi Center for Cancer and Medical
Genetics Research / Al-Mustansiriyah University, with the support and assistance of
the Department of Chemistry and Biochemistry / College of Medicine / Al-Nahrain
University and the Department of Medical Chemistry / College of Medicine /
University of Al-Qadisiyah. The research project was completed and the thesis
submitted on 9/1/2020.
The research project included propagating of Newcastle disease virus (NDV)

(as Virotherapy) in Embryonated chicken eggs (ECE) and then it was isolated and
purified. The virulence of the Virus (Titer) was measured by Hemagglutination
assay (HA). Cell culture of Vero cell line was prepared, then it was treated with
NDV to measure TCID50 value. Cell culture of AMJ13 and MCF-7 cell lines were
prepared, then the effect of Virus and Hexokinase inhibitor (D-Mannoheptulose) (as
Phytotherapy) were studied separately on breast cancer cell lines (AMJ13 and MCF-
7) and studying their effect together (Synergistically) on cell lines at 72 hr, and
comparing their effect with the effect of Docetaxel drug (Positive control) as
(Chemotherapy).
The effect of these factors was determined by measuring cytotoxicity ratio by

MTT assay. The IC50 and Combination Index (CI) values were measured by Chou-
Talalay analysis. The morphological changes and induction of apoptosis were
Page 32
assessed by using the Crystal violet dye and Acridine Orange dye / Propidium
Iodide) respectively to determine dead and viable cells and comparing their effect
(Mono and Combination) on normal cell line (Rat Embryo Fibroblast) (REF). Also,
the effect of these factors (virus, inhibitor and their combination) was studied on the
glycolysis inhibition pathway after treated with cell lines at 72 hr. Hexokinase
activity, concentration of (Pyruvate, ATP) and acidity (pH) (which represent lactate
acid) were measured. The thesis has been written, designed, directed and performed
the statistical analysis by the researcher under the supervision and review of the
supervisors.
3.1 Materials
The reagents, stock, working solutions, equipment and apparatus used in the
present study are shown in (Table (3-1), (3-2)).
3.1.1 Instruments
Table (3-1): The instruments and glassware used in this study
NO. Instruments & Equipment Company Origin

1 Autoclave Lab. tech Korea
2 Beaker 100,50,25 ml Santa Cruze USA
3 Biological hood ESCO UK
4 Cell Culture plate flat bottom (96) well Nunc Denmark
5 Cell Culture plate U bottom (96) well Nunc Denmark
6 Centrifuge Hetttich Germany
7 Cooled Centrifuge Hetttich Germany
8 Cooler box Coleman China
9 Cover slips (22*22 mm). Apel China
10 Deep Freeze (-86 oC) Lab. tech Korea
11 Disposable syringes 20 ,10,5, 1 ml Medeco UAE
12 Disposable Tip (1000,10) μl Cypress Belgium
13 Distillation Apparatus Running waters USA
14 Elisa micro well system microplate reader Asyshitech UK
15 Elisa Plate reader, D-63303, Dreieich Karl Kolb Germany
17 Falcon cell culture flask (25,50 and 75) ml Santa Cruze USA
18 Fluorescent Microscope Olympus Japan
Page 33
19 Forceps Putex India

20 Glass culture bottle Santa Cruze USA
21 Graduated cylinder Santa Cruze USA
22 Hemocytometer HBG Germany
23 Incubator Memmert Germany
24 Incubator with CO2 Lab. tech Korea
25 Inverted Microscope MEIJI Techno Japan
26 Lab Spatula Scienceware China
27 Laminar air flow Cabinet K&K Korea
28 Light Microscope Olympus Japan
29 Light Microscope with digital camera Scopetek USA
30 Micro-pipette( 2-20, 10-100 ,100-1000,) μl Slamed Germany
31 Multichannel micropipette (0 -10 μl) Slamed Germany
32 Multichannel pipette (10- 250 μl) Slamed Germany
33 Nalgene filter syringe (0.22.0.45) Nalgen USA
34 Oven KENWOOD Turkey
35 pH meter tester Santec China
36 Refrigerator LG. Korea
37 Refrigerator Concorde Korea
38 screw-capped bottles, 100 ml, 250 ml Brand India
39 Analytical balance Stanton UK
40 Sterile 10 ml centrifuge tubes. Interlab China
41 Surgical scissors Putex India
42 Surgical tong Putex India
43 Tissue culture flasks size (25,50 and 75) ml Nunc Denmark
44 Vortex mixer Stuart UK
45 Volumetric flask (50-250) ml Santa Cruze USA
46 Water bath Memmert Germany
3.1.2 Chemical and biological materials

Table (3-2): The chemical and biological materials used in the study
NO. Chemical and biological materials Company Origin

1 Acridine Orange Sigma –Aldrich USA
2 AMJ13 human breast cancer cell line ICCMGR IQ
3 ATP determination kit ElabScience USA
4 crystal violet Santa Cruz USA
5 D- Mannoheptulose Santa Cruz USA
Page 34
6 DMSO (Dimethyl Sulphoxide) (99.9%) Santa Cruz USA

7 Docetaxel drug Santa Cruz USA
8 Ethanol absolute (96%) Hayman England
9 Ethylene diaminetetra acetic acid (EDTA) Merck USA
10 Fetal bovine serum Bio west USA
11 HEPES buffer Santa Cruz USA
12 Hexokinase Assay Kit ElabScience USA
13 MCF7 human breast cancer cell line ICCMGR USA
14 Methyl Thiaozolyl Tetrazolium (MTT) Bio west USA
15 Minimum Essential Medium (MEM) U.S Biological USA
16 Monkey kidney (Vero) cell line ICCMGR USA
17 NDV ICCMGR IQ
18 Penicillin - Streptomycin CAPRICORN Germany
19 Phosphate buffer saline (PBS) Sigma –Aldrich USA
20 Propidium Iodide Sigma –Aldrich USA
21 Potassium hydroxide Santa Cruz USA
22 Potassium chloride Santa Cruz USA
23 Pyruvate determination kit ElabScience USA
24 RPMI -1640 media Gibco USA
25 REF cell line ICCMGR USA
26 Sodium Bicarbonate (99%) Santa Cruz USA
27 Trypsin -Versene Santa Cruz USA
28 Formaldehyde (37%) Sigma –Aldrich USA
3.1.3 Reagents for cell culture

All instruments have been sterilized by Autoclave and all reagents and buffers
were prepared according to (Freshney, 2015).
3.1.3.1 Penicillin-Streptomycin (Pen-Strep) (Antibiotics)

Penicillin 500 mg powder and streptomycin 1 g powder were each dissolved in
5 ml Distilled Water (D.W) and stored separately at -20°C as stock solutions. Then,
1ml Penicillin and 0.5 ml streptomycin were removed from their stock solutions and
added to each 1 L of culture media after sterilization with 0.22 filter syringe. Also,
Penicillin–Streptomycin solution ready for use was bought from Sigma-Aldrich
(Freshney, 2015).
Page 35
3.1.3.2 Sodium Bicarbonate

It was made by dissolving 2.2 g of sodium bicarbonate (99% purity) powder in
1 L of culture media as suggested by the manufacturing company.
3.1.3.3 Phosphate buffer saline (PBS), without Ca 2+ and Mg 2+

It was prepared by mixing Sodium chloride (NaCl) 8 gm, Potassium chloride
(KCl) 0.2 gm, disodium hydrogen phosphate (Na2HPO4) 0.9 gm, and Potassium
dihydrogen phosphate (KH2PO4) 0.2 gm. The volume was diluted to the mark
(1L) using trebled distilled water (T.D.W).
In addition, dry powdered phosphate-buffered saline was used to prepare 1L of

phosphate-buffered Saline: 10.8 g of PBS powder was dissolved in 1000 ml T.D.W,
0.5 ml ampicillin and 0.25 ml streptomycin were added and filtered by Nalgene filter
units (pore size, 0.22 μm) in sterilized bottles and stored in the fridge at 4°C until
use. The pH was 7.2 (Freshney, 2015).
3.1.3.4 Trypsin
Trypsin solution for primary cultures was prepared by dissolving 2.5 g of
trypsin in 100 ml of PBS (25mg/ml) at the pH of 7.2, and stirred continuously on a
magnetic stirrer at room temperature. Then they were sterilized by Nalgene filter
0.22 μm, and stored at 4 °C (Freshney, 2015).
3.1.3.5 Stain
3.1.3.5.1 Methyl Thiazolyl Tetrazolium (MTT) Solution
Methyl Thiazolyl Tetrazolium (0.2 g) was dissolved in 100 ml of PBS in order
to prepare 2 mg/ml of the dye (Betancur-Galvis et al., 2002). The solution was
filtered through 0.2 μm syringe filter to remove any blue Formosan product as
recommended by (Russel, 1998), and then stored in sterile dark, screw-capped bottles
at 4°C. The solution was used within 2 weeks of preparation (Betancur-Galvis et al.,
2002).
Page 36
3.1.3.5.2 Crystal Violet stain

It was prepared by dissolving 5 g of crystal violet powder in 200 ml methanol.
The solvent was filtered using Whatman No.1 filter paper, 50 ml of formaldehyde
(37%) was added, the volume of mixture was completed to 1000 ml by distilled
water, and excess solid residue was filtered off using Whatman No.1 filter paper
(Yaseen, 1990).
3.1.3.5.3 Acridine Orange-Propidium Iodide (AO/PI)

Acridine Orange/Propidium Iodide Stain is a cell viability dye that causes viable
nucleated cells to fluoresce green and nonviable nucleated cells to fluoresce red. It
can be used to assess cell viability with the automated fluorescence cell counters.
Acridine orange permeates viable cells and binds to nucleic acids. Binding to dsDNA
causes Acridine orange to fluoresce green and binding to ssDNA or RNA causes it
to fluoresce red.
Propidium iodide binds to nucleic acids. Not being able to permeate intact cell
membranes, it is taken up by nonviable cells and cells with compromised
membranes. Once bound to nucleic acids, its fluorescence increases 20-30-fold
causing the cell to fluoresce red. Here, 1µl of 10 µg/ml Acridine orange/Propidium
iodide (AO/PI) by PBS reagent was used (Paramasivam et al. 2012).
3.1.3.6 Tissue culture media

All media were prepared as the manufacturer's recommendation.
3.1.3.6. 1 Preparation of Minimum Essential Medium (MEM)

It was prepared by dissolving 11g of MEM (with L-glutamine) (US biological,
USA) powder in ~600 ml of TDW and adding Sodium bicarbonate powder (2.2 g),
10 ml of 1 M HEPES buffer, Ampicillin 100 μg/ml, Streptomycin 50 μg/ml, and
Fetal Bovine Serum (FBS) 100 ml (10%). The volume was diluted to the mark
(1 L) with TDW, and the medium was sterilized using Nalgene 0.2 μm filter unit
(Freshney, 2015).
Page 37
3.1.3.6.2 Preparation of RPMI-1640 Medium

Manufacture protocol according to Gibco Company was used as follow:
A- RPMI-1640 medium powder (with HEPES buffer and L-glutamine)

RPMI-1640 medium powder (10.4 g) was dissolved in ~600 ml of TDW
(usbiologi, USA). Sodium bicarbonate powder 2.2 g, 1 ml/L Ampicillin 100μg/ml,
0.5 ml/L Streptomycin 505 μg/ml, and FBS 100 ml. (10%) were added. The volume
was completed to 1 L with TDW, and medium was sterilized using Nalgene filter
using 0.22 filter unit as recommended by the manufacturing company (Freshney,
2015).
B- RPMI-1640 medium powder (with HEPES buffer, L-glutamine, and Sodium

bicarbonate)
1- Preparation of completed culture media (with FBS) (100 ml)

a- RPMI-1640 medium (powder)
100 ml of TDW was added in a scroll bottle sterilized by autoclave and UV. 1.6
gm Media RPMI-1640 (powder) was added. 10 ml Fetal Bovine Serum (FBS) (10%)
was added and antibiotic (Penicillin - Streptomycin Solution) (Pen-Strep) 1 ml was
added and mixes gently till a yellow color (acidic) appeared. KOH (1N) drops were
added to neutral solution (PH=7) to change the color to reddish-orange. The solution
was filtered using syringe filter 0.22 to remove debris, and cooled at 4°C, (Appendix
(1)).
b- RPMI-1640 medium (solution)

89 ml Media RPMI-1640 solution (ready to use) was added in a scroll bottle
sterilized by autoclave and UV, and 10 ml FBS (10%) was added. 1 ml antibiotic
(Penicillin - Streptomycin Solution) (Pen-Strep) was added and mixed gently till a
yellow color (acidic use solution) appeared. KOH (1N) drops were added to
neutralize the solution (pH=7) and the color was changed to reddish-orange, the
Page 38
solution was filtered using syringe filter 0.22 to remove debris, and cooled at 4°C,
(Appendix (2)).
2- Preparation of Serum Free Media (SFM) (without FBS) (100 ml)

A- RPMI-1640 medium (Powder)
100 ml TDW was added in a scroll bottle sterilized by autoclave and UV . 1.6
gm Media RPMI-1640 (powder) was added and 1 ml antibiotic (Penicillin -
Streptomycin Solution) (Pen-Strep) was added and mixed gently till a yellow color
(acidic) appeared. KOH (1N) drops were added to neutralize the solution (pH=7) and
the color was changed to reddish-orange, the solution was filtered by syringe filter
0.22 to remove debris, and cooled at 4°C, (Appendix (3)).
B- RPMI-1640 medium (Solution) ready for use

100 ml Media RPMI-1640 Solution was added in sterilized scroll bottle. 1 ml
antibiotic (Penicillin-Streptomycin Solution) (Pen-Strep) was added and mixed
gently, (Appendix (4)).
3.1.4 Cell Lines

Four cell lines (AMJ13, MCF-7, Vero, and REF) were supplied from cell bank
unit, experimental therapy department, Iraqi Center of Cancer, and Medical Genetics
Research (ICCMGR).
3.1.4.1 Ahmed Mortadha Jabria 2013 (AMJ13 cell line)

This new breast cancer cell line (Ahmed Mortadha Jabria 2013) was primarily
established by Ahmed Majeed Al-Shammari, at the Iraqi Center for Cancer and
Medical Genetic Research at Mustansiriyah University in 2013, from the primary
tumor of ductal carcinoma, with a histological diagnosis of infiltrating ductal
carcinoma of the breast of a 70-year-old Iraqi woman, mother of 6 children, named
Jabria, weighed 70 Kg and 160 cm in height, from the city of Al-Amen in Baghdad.
It is considered unique because it is the first breast cancer cell line from an Iraqi
Page 39
population, and it is expected to be a useful tool in breast cancer research. The cells
were morphologically characterized by light and scanning electron microscopy, and
found to be elongated multipolar epithelial-like cells with a population doubling time
of 22 hrs (Al-Shammari et al, 2015).
3.1.4.2 Michigan cancer Foundation-7 (MCF-7 cell line)

MCF-7 is a breast cancer cell line isolated in 1970 from a 69-year-old Caucasian
woman. MCF-7 is the acronym of Michigan Cancer Foundation-7, referring to the
institute in Detroit where the cell line was established in 1973 by Herbert Soule and
co-workers (Lee et al., 2015).
Human breast cancer cell line AMJ13 (Iraq) and MCF-7 (USA) were cultured
in RPMI-1640 medium (US Biological, USA) supplemented with 10% Fetal Bovine
Serum (FBS) (Capricorn-Scientific, Germany), 100 unit/ml penicillin, and 100 μg/ml
streptomycin (Capricorn-Scientific, Germany), and incubated at 37°C.
3.1.4.3 Vero cell line

Vero cells are a lineage of cells used in cell cultures. The 'Vero' lineage was
isolated from kidney epithelial cells extracted from an African green monkey. The
lineage was developed on 27 March 1962 by Yasumura and Kawakita at the Chiba
University in Chiba, Japan. The original cell line was named Vero after an
abbreviation of Verda Reno, which means green kidney in Esperanto, while Vero
itself means truth in Esperanto (Sheets, 2000). These cells are used to calculate virus
titer in TCID50 assay.
3.1.4.4 Rat Embryo Fibroblast (REF) cell Line

It is a normal fibroblast endothelial cell of rat embryos. It was established by A.
M. Al-Shammari experimental therapy department in Iraqi Center for Cancer and
Medical Genetic Research (ICCMGR). The morphological change and apoptosis of
REF, MCF-7, and AMJ13 cells were observed with inverted and fluorescent
Page 40
microscopes, respectively. REF, MCF‑7, and AMJ13 cells were seeded in a 96‑well
plate at 2 × 105 cells per well. The cells were treated with different concentrations of
NDV and MH (Ali et al., 2019).
3.1.5. Requirements for Virus Propagation and Hemagglutination Test
3.1.5.1 Newcastle Disease Virus

The Iraqi attenuated strain of NDV (Iraq/Najaf/ICCMGR/2013) (Al-Shammari
et al., 2014) is supplied by ICCMGR, Experimental Therapy Department, Cell Bank
Unit, Mustansiriyah University. A stock of infectious virus is propagated in
embryonated chicken eggs (Al-Khaleel Company) for producing embryonated
chicken eggs, (Baghdad, Iraq), harvested from allantoic fluid, and purified from
debris by centrifugation (3000 rpm, 30 min, 4°C).
NDV was quantified by a hemagglutination test (HA) in which 1

hemagglutination unit (HAU) is defined as the smallest virus concentration leading
to visible chicken erythrocyte agglutination. The virus was stored at −86°C. Viral
titer was determined by 50% tissue culture infective dose (TCID50) titration on
monkey kidney (Vero) cells.
3.1.5.2 Embryonated Chicken Eggs (ECE)

Fertilized eggs (180 eggs) obtained from Al-Khalil hatchery, located in the Abu
Ghraib area, were used in propagating the virus and isolating it from a pathological
state.
3.1.5.3 Chicken Red Blood Cells (CRBC)

They were collected from brachial veins under the right wing of chicken by
using a heparinized syringe to draw about 2 ml of blood which was then used in
carrying out hemagglutination test (HA) to determine the titer of the virus.
Page 41
3.1.6 Colorimetric Assays

3.1.6.1 Hexokinase Assay Kit
Sample: cell lines. Method: Colorimetric method. Specification: 30 tubes/28
samples according to manufacturing protocol (ElabScience Biotechnology Inc.).
Application: This kit can measure Hexokinase (HK) activity in serum, plasma, tissue,
cells, and other samples. Detection principle: By utilizing the coupling reaction with
glucose-6-phosphate dehydrogenase (G-6-PD), and providing a sufficient amount of
substrate, HK activity was calculated by measuring the increase value of absorbance
at 340 nm.
a- Kit components and preparation of reagents

Reagent 1: liquid, 8 ml x I vial was stored at 4oC. Reagent 2: Stock solution, 4
ml x I vial was stored at 4oC. Reagent 2 was prepared with double distilled water at
a ratio of 1:9. Fresh solution was prepared before use. Reagent 3: Powder 2 vials was
stored at - 20oC. Preparation of reagent 3: a vial of powder was dissolved with 1 ml
double distilled water. Fresh solution was prepared before use. Reagent 4: powder 7
vials were stored at - 20oC. Preparation of reagent 4: a vial of powder was dissolved
with 1 ml double distilled water. Needed amount was prepared before use. Reagent
5: liquid, 330 μl x 1 vial was stored at 4oC. Appendix (17).
b- Working solution and instruments

Working solution was prepared by the reagent 1, 2, 3, 4, 5 and double distilled
water was thoroughly mixed at the ratio of 20:10:5:20:1:40. Fresh solution was
prepared before use. Experimental instruments and glassware: Test tube,
Micropipette, Vortex mixer, water bath, Centrifuge, ELISA plate reader (340nm).
3.1.6.2 Pyruvate Assay Kit

Sample: cell lines. Method: Colorimetric method. Catalog No: E-BC-K130
according to manufacturing protocol (ElabScience Biotechnology Inc.).
Experimental instruments: Test tube, Micropipette, Vortex mixer, 37℃ water
Page 42
bath/gas bath, and ELISA plate reader (505 nm). Application: This kit measures the
pyruvate content of serum, plasma, culture cells, cell culture supernatant, and other
samples. Detection principle: Pyruvic acid can react with a chromogenic agent to
give a product that is reddish brown in alkaline solution. The depth of the color is
directly proportional to the pyruvate content which was calculated by measuring the
OD value at 505 nm. Kit components can be seen in (Table (3-3)).
Table (3-3): Kit components for determine pyruvate in sample
Component Specification Storage

Reagent 1 Clarificant 10 mL × 1 vial 4℃, 6 months
4℃, 6 months, shading
Reagent 2 Chromogenic agent 50 mL ×1 vial
light
Reagent 3 Alkaline solution 50 mL × 3 vial 4℃, 6 months
2 μmol/mL Sodium
Reagent4 1 mL × 1 vial 4℃, 6 months
pyruvate standard
Preparation of 0.2 μmol/ml sodium pyruvate standard solution: Reagent 4

was diluted with double distilled water for 10 times.
3.1.6.3 ATP Assay Kit

Sample: cell lines. Method: Colorimetric method. Specification: 100 tubes/48
samples according to manufacture protocol (ElabScience Biotechnology Inc.).
Detection principle: Phosphokinase catalyzes the reaction between adenosine
triphosphate and creatine to produce creatine phosphate which was then detected by
phosphomolybdic acid colorimetry. Experimental instruments and glassware: Test
tube, Micropipette, Vortex mixer, water bath, and ELISA plate reader (636nm). This
kit measures the ATP content of serum, plasma, cells and cell culture supernatant,
and other samples.
Page 43
a- Kit components and preparation of reagents

Reagent 1: Substrate Ⅰ solution was dissolved in 10 ml of boiled double distilled
water and was allowed to stand in a boiling water bath until it completely dissolved.
If it appeared crystal before assay, it was allowed to stand in a boiling water bath
until it completely dissolved, and then was stored at 37℃ for assay. Reagent 2:
Substrate Ⅱ, 20 ml × 1 vial at 4℃.
Reagent 3: Accelerant: Powder × 2 vials at -20℃, diluent, 760 μl x 2 vials, 4℃.

Reagent 3 application solutions: 1 vial of Reagent 3 diluent was added to 1 vial of
Reagent 3 powder and was dissolved fully before use. Reagent 4: Precipitant, 5.5 ml
× 1 vial at 4℃. Reagent 5: Developer, A solution, 7 ml × 4 vials at 4℃. B solution,
6 ml × 4 vials 4℃.
Developer application solution: 1 vial of Reagent 5 A solution was added to 1

vial of Reagent 5 B solution, was dissolved fully, and was stored at 4℃. Fresh
solution was prepared before use. Reagent 6: Stop Solution, 50 ml ×1 vial at Room
Temperature. Reagent 7: ATP Standard, Powder × 2 vials at 4℃. 5 mmol/L ATP
standard stock solution: before assay, double distilled water was added into 1vial of
ATP standard powder to make a final volume of 1 ml. Fresh solution was prepared
before use. 1 mmol/L ATP standard application solution: 5 mmol/L ATP standard
stock solutions were diluted with double distilled water 5 times, appendix (18).
Page 44
3.2. Methods
1. Propagation (injection, collection) of NDV in embryonated chicken eggs (ECE)
(180 eggs), purification, filtration, and distribution of NDV.
2. Determination of Titer of NDV by Hemagglutination Test (HA)
3. Cell culture of Vero cell line and preparation of diluted NDV.
4. Determination of Tissue Culture Infective Dose (TCID50) of virus by using Vero

cell line to determine the number of NDV in 1 ml of allantoic fluid of embryonated
chicken eggs.
5. Determination of the cytotoxicity ratio (CT%) for NDV, D-Mannoheptulose (MH)

(HKI) and Docetaxel (DXL) (positive control) by MTT cytotoxicity assay after
treatment with NDV, MH and DXL separately by using different drug
concentrations (μg/ml), different multiplicity of infection (MOI) for virus depend
on TCID50 and different inhibitor concentrations (μg/ml) to normal rat embryo
fibroblast (REF), and human breast cancer cell lines (AMJ13 and MCF-7).
6. Determination of IC50 for NDV, MH and DXL through measurements of CT%

for NDV, MH and DXL by using GraphPad Prism.
7. Choosing of the most suitable MOI of virus, and concentrations of inhibitor and
DXL depending on IC50 values, and then exposure NDV, MH and DXL
separately, then NDV and MH synergistically to REF, AMJ13, and MCF-7 cell
lines with comparison to the control (untreated cell).
8. Measurement of cytotoxicity ratio (proliferation inhibition), by using the MTT

cytotoxicity assay, for REF, AMJ13, and MCF-7 cell lines after their treatment
with NDV (MOI 0.3, 1 and 2), MH (62.5, 125 and 250 μg/ml) and DXL (1.25, 2.5
and 5 μg/ml) separately and synergistically for (NDV and MH) at 37oC for 72 hrs,
depending on the MOI of NDV, and concentration of MH and DXL that have been
selected.
Page 45
9. Determination of synergism (combination) between NDV and MH, by

combination cytotoxicity assays, Combination Index (CI), or Chou-Talalay
equation through Isobologram, GraphPad prism, and CompuSyn program, for
REF, AMJ13 and MCF-7 cell lines.
10. Choosing of the most suitable the most suitable MOI of virus and
concentration of MH depending on combination index, and then exposure NDV
and MH separately and synergistically to REF, AMJ13 and MCF-7 cell lines at
37oC for 72 hrs with comparison to the control (untreated cell).
11. Studying of morphological changes using crystal violate after treating REF,
AMJ13, and MCF-7 cell lines with NDV (MOI=2) and MH (62.5μg/ml) depends
on CI, separately and synergistically at 37oC for 72 hrs, to explain the
morphological changes in treated and untreated control cells.
12. Studying of apoptosis pathway using Propidium Iodide (PI)/ Acridine Orange
(AO) after treating REF, AMJ13, and MCF-7 cell lines with NDV (MOI=2) and
MH (62.5μg/ml) depends on CI, separately and synergistically at 37oC for 72 hrs,
to explain live and dead cells in treated and untreated control cells.
13. Determination of hexokinase (HK) enzyme activity, Pyruvate, ATP

concentration, and acidity (pH values) after treating REF, AMJ13, and MCF-7 cell
lines with NDV (MOI=2) and MH (62.5μg/ml) depends on CI, separately and
synergistically at 37oC for 72 hrs, and with comparison to control using
colorimetric methods by ELISA plate reader and litmus papers to determine pH
values, Scheme (3-1) and (3-2).
Page 46
Study Design:
NDV NDV Injection / ECE NDV Harvest NDV Purification
Preparation
NDV Storage -86Co NDV NDV Filtration
ECE Distribution
NDV Propagation MH Stock Preparation
NDV
Hemagglutination Test
Chicken RBC solution Determination of NDV Titer
Cell Culture of Vero Cells Exposure of Virus to Vero cells
Preparation of diluted NDV Determination of TCID50
Calculation of number
Cell culture of REF, AMJ13 and MCF7 cell
and volume of NDV in
well lines
Preparation of different MOI Preparation of different

for NDV depend on TCID50 concentrations for MH
Exposure of NDV to normal and Exposure of MH to normal and

breast cancer cell lines for 72hr breast cancer cell lines for 72hr
Determination of Cytotoxicity%
by MTT assay
Preparation of different Exposure of DXL to normal and

concentrations for DXL breast cancer cell lines for 72hr
Page 47
Cytotoxicity% of NDV, MH and DXL at different concentrations
Determination of IC50 of NDV, MH and DXL by GraphPad prism
Determination of Cytotoxicity% of NDV, MH and DXL after selected three

concentrations depending on IC50 separately to cell lines
Determination of CT% of NDV and MH synergistically to cell lines
Study the synergistic effect of NDV and MH on cell lines

by combination MTT cytotoxicity assay
Study a Through combination cytotoxicity assays

Morphological and Chou–Talalay analysis of cell lines
change of NDV,
MH and Com in
unstained cells Determination of Combination Index (CI)
for 72hr by CompuSyn Isobologram
Treatment of NDV (MOI 2), MH (62.5μg/ml) and their

combination (depending on combination index) to cell
lines
Study a Morphological change of NDV, Study Apoptosis of NDV, MH and

MH and Com by Crystal violet for 72hr Com by (AO/PI) staining for 72hr
Determination of Glycolysis parameters of NDV,

MH and Com at MH (62.5μg/ml) and NDV MOI (2)
Determination of HK activity
Determination of Pyruvate concentration Determination of Acidity (lactic acid)

of NDV, MH and Com by pH
Determination of ATP concentration
Scheme (3-1): Outline of laboratory experiments of the study
Page 48
3.2.1 The Propagation of the Newcastle Disease Virus (NDV)

3.2.1.1 Virus Preparation
NDV Iraqi strain was provided as a frozen culture by the experimental therapy
department at ICCMGR. It was directly thawed and the antibiotics penicillin 100
unit/ml and Streptomycin 100 μg/ml were added to the virus sample. The virus was
prepared by the ratio (1:10) with Phosphate buffer saline (PBS) (sterilized and
cooled). Then, 6 ml (6000 µl) was prepared for 60 eggs by adding (600 µl Virus in
buffer + 5400 µl PBS), or 1ml (1000 µl) was prepared for 10 eggs by adding (100 µl
Virus + 900 µl PBS) in cold test tube and placing it on ice.
The sample was centrifuged at 3000 rpm for 30 min at 4oC to remove any debris
and large particular matter, then the supernatant was extracted and the virus sample
was injected into 9 or 10 days embryonated chicken eggs. Allantoic fluid of infected
embryo was harvested in a small tube and stored at -86oC App (22) (Allan et al.,
1978).
3.2.1.2 The Propagation of NDV in Embryonated Chicken Eggs (ECE)

ECE (9-days-old) (180 eggs) were incubated in a conventional incubator at
37oC and on suitable day humidity appendix (21). The eggs were turned twice daily,
and after 1 day of incubation, the eggs were transilluminated with an egg-candling
lamp to insure the viability of the embryo appendix (18). All non-fertile eggs and
those containing a dead embryo were discarded and live eggs (embryo) were saved
in an incubator for the second candling before inoculation, the air sac was marked
off and a suitable site of injection was marked on the eggshell.
Where on important blood vessels were running away from the embryo, the egg
was rinsed with 70% ethanol to sterilize the outer surface, and then the egg shell was
sterilized with iodine at the inoculation site where a small hole was drilled about 0.4
cm above the air sac border (Haas et al, 1998). After that, 0.1 ml (100 μl) of prepared
virus solution was injected into each (9 or 10-days-old) embryonated chicken egg
Page 49
with a fine needle (0.3mm), and the puncture holes were covered with paraffin. The
eggs were incubated at 37oC in a humidified incubator for 24 hrs. The embryos were
observed daily every 3 hrs for viability. Up to 24-hr dead embryos were discarded
and immediately transferred into the refrigerator (4 oC) if 24–48 hrs after their death.
Live embryos were saved in an incubator. Then they were harvested (Collected)
after 3-6 hrs in refrigerator at 4oC., the egg shell was cut by scissors and the allantoic
fluid was harvested using sterile syringes, (unclear or bloody allantoic fluids were
rejected) purified from debris by cold centrifugation at 3000 rpm for 30 min at 4°C,
(Appendix (23)) filtered by syringe filter (0.45μm), and distributed in eppendorf
tubes (Appendix (5)). Then, it was measured by HA to find the titer of virus (Figure
(3-1)), (Appendix (7)) (Appendix (27)). The TCID50 of NDV was calculated (Figure
(3-6)), and it was then stored at -86°C. The virus was further propagated in
embryonated eggs in which high titers of virus can be recorded from the infected
eggs unit (Alexander and Senne, 1998).
3.2.2 Hemagglutination assay (HA)

NDV was quantified using the hemagglutination assay in which 1
hemagglutination unit (HAU) is defined as the smallest virus concentration leading
to visible chicken erythrocyte agglutination (Allan et al., 1978).
3.2.2.1 Preparation of chicken RBC solution

The chicken RBC solution was prepared by collecting blood from a healthy bird
into a heparinized tube. Chicken blood (2ml) was added into an anticoagulant test
tube (heparinized tube) and then transferred into a centrifuge test tube where 7 ml
cold PBS was added and mixed using a centrifuge (1000 rpm, 4oC, and 10 min) to
wash the blood, washing it 3 times with PBS.
Supernatant was discarded, the pellet (RBC) was saved, and then taking 0.5 ml
of the washed RBCs, which was completed to 50 ml with PBS to achieve 1% RBC
solution, or RBC solution (100 ml) was prepared with PBS in a ratio 1:100 (1%): 1
Page 50
ml RBC + 99 ml PBS, 250 µl RBC + 25 ml PBS or 100 µl RBC + 10 ml PBS for the
Hemagglutination test (Versteeg, 1985), (appendix (6)).
3.2.2.2 Titration of NDV

PBS (50 µl) was added to all wells in a 96-well microplate u-shape (7 lines for
7 eggs from A-G and H was the control). Virus allantoic fluids (VAF) (50 µl) was
taken from each tube and added to the tissue culture plate (U-shape), it was added to
well1 and mixed with PBS, then 50 µl was taken from the mixture (well 1) and added
to well2 and also mixed with PBS.
50 µl was taken from the mixture (well2) and added to well3 and so on till well12
(2 fold serial dilution was done) where 50 µl was discarded from it (The tips were
replaced between each addition). Adding was limited to 7 lines because the 8 line
was the control (Appendix (7)). RBC solution (50 µl) (1%) was added to all wells in
the 96-well micro plate U-shape (8 lines) and mixed by inverting, not by vortex.
The 96-well micro plate was saved in an incubator for 30 min to get the virus
titer for all lines. NDV titer was calculated for all virus allantoic fluids tubes by HA
test (Appendix (7)) (Allan et al., 1978)
The VAF tubes (Known titer) were separated by cooling, centrifuged and
collected, those with high titer values (7-12) were collected together and those with
titer values (5-6) were also collected together in tubes (25 ml). VAF tubes with titer
values (7-12) and (5-6) were filtrated using syringe filters (0.45 µm) and distributed
to several eppendorf tubes and were stored at deep freeze (-86 oC) (Appendix (5)).
Agglutination is a positive result while no agglutination is a negative result, and

the titer value was determined by the number of positive results as compared to the
control (Figure (3-1)), (Appendix (25)) and (Appendix (27)) (Haas, et al., 1988).
Page 51
Figure (3-1): Calculation of titer of NDV by HA test in 96-well microplate
3.2.3 Cell lines

The AMJ13 human breast cancer cell line (Al-Shammari et al., 2015), MCF7
human breast cancer cell line and REF cell line were cultured in RPMI-1640 medium
with 10% fetal bovine serum (FBS), 100 unit’s/ml penicillin, and 100 μg/ml
streptomycin, and incubated at 37°C. Monkey kidney Vero cell line was cultured in
MEM medium with 10% fetal bovine serum (FBS), 100 unit’s/ml penicillin, and 100
μg/ml streptomycin, and incubated at 37°C.
Different cell lines (Vero, REF, AMJ13 and MCF-7) were detached from their
flasks when they reached the subconfluent monolayer by trypsinization. Culture
medium (20 ml) and 10% serum were added to the falcons and mixed gently with
cells to prepare cell suspension. The cell suspension in the culture flask was poured
aseptically into a sterile beaker. Using a multi micropipette, 200μ1 of the cell
suspension was transferred into each well in a 96-well microplate, and the plate was
covered with a sterile adhesive film, lid placed on and rocked gently, and incubated
Page 52
in a 5% CO2 incubator at 37oC for 24 hrs to allow cell attachment, proliferation, and
confluent monolayer achievement. These cells were regularly assessed for standard
growth characteristics and regularly authenticated (Freshney, 2015).
3.2.3.1. Maintenance of cell lines in Vitro

Cell lines were subcultured when they were confluence monolayers. The growth
medium was decanted off and the cell culture was washed once with 2 ml trypsin-
versene solution. Trypsin (3 ml) was added to the cell culture, the flask rocked gently,
and part of it was decanted again to obtain about 1 ml of trypsin solution covering
the cell surface. Then, the cells were incubated at 37°C until they were detached from
the flask. The cells were dispensed in 5 ml growth medium and then redistributed at
the required concentration into culture. The cells were incubated at 37oC under a
humidified atmosphere containing 5% CO2 (Al-Shammari and Yaseen, 2012).
3.2.3.2 Cell culture of Vero cell line

Tissue culture falcon (TCF) contains complete media (CM) and Vero cell line
saved in an incubator. After the cell became a monolayer (Figure (3-3)), complete
media was discarded from TCF, this means the Vero cells remained adhesive on the
walls. Also, 3 ml trypsin 0.25% was added
to TCF to detach the cells from the wall or
surface (Trypsinization), after which they
were incubated for 3 min (Appendix (8)).
A change of solution from pure to turbid
is indicated by indicators on the detached
cells. Detached cells should be round
shaped and free floating in the trypsin
solution as soon they detach (Figure (3-
2)). Complete media was added to TCF Figure (3-2): Trypsinization to detach
cells from the wall in cell culture.
(contains Vero cells + Trypsin). Trypsin
Page 53
was inhibited by FBS to remove or to end trypsin. Solution (10 µl) was taken from
TCF (Vero cells + CM) solution to a hemocytometer to calculate number of the cells
by light microscope, must be at least 40 cells in 4 squares (Figure (3-4)).
A. Calculation of cells in 1ml and 1 well

The number of cells in each square (4 squares) was calculated, and then the
average was calculated. The average was multiplied by 104 to find the number of
cells in 1 ml (1000 µl). For example, if the average number of cells is 10 cells, 10
cells × 104 = 100000 cells, implying that 1 ml contain 105 cells. The cell suspension
(Vero cells + CM solution) (X) volume in each well was calculated (at least 7000
cells in a well) through number of cells in 1 ml as follow:
No. of cells 100000 1000 µl cell suspension

No. of cells in well 7000 X
--------------------------------------------------------------------------------
X= 7000 cell × 1000 µl / 100000 cell = 70 µl cell suspension
Each well must contain 100 µl cell suspension; therefore, 70 µl was completed to 100
µl by adding 30 µl CM for each well.
70 µl cells + 30 µl CM …………… 100 µl for one well

7000 µl cells + 3000µl CM ……... 10000 µl (10ml) for 100 wells
This implies 10 ml is required to complete the 100 µl cell suspension for all wells
in 96-well microplate. Then, 10 ml (cell suspension 7 ml + CM 3 ml) was added into
a petri dish and distributed into all wells in 96-well microplate to complete the 100 µl
cell suspension, after which 96- well microplate was incubated for (24-48) hrs. The
cell culture was examined by an inverted microscope to confirm that the cells were
monolayer, after which the media was discarded leaving the Vero cell ready for use.
(Figure (3-4)). Then TCID50 was calculated.
Page 54
Figure (3-3): Vero cells confluent monolayer Figure (3-4): Calculation number of cells in
after storage at -86°C for 4 months sample by hemocytometer under microscope
3.2.4 Preparation of diluted NDV

Ten sterile eppendorf tubes (1.5ml) were formatted and numbered in sequence,
50 ml of SFM was prepared, and 900 µl SFM was added in sequence to all eppendorf
tubes, 100µl stock virus suspension was added serially tenfold diluted after replacing
the tip each time. Diluted allantoic virus suspension was prepared (10-1-10-10).
(Appendix (9)) (Al-Shammari, 2003).
3.2.5 Exposure of NDV to Vero cells line

SFM (100 µl) was added to column 1 and 12 in 96- well microplate as a control,
and then 50 µl SFM and 50 µl diluted virus were added to column 2-11, taking into
account the eppendorf tubes sequence when adding, after which the 96- well
microplate was wrapped with parafilm (Appendix (10)).
Each column has the same concentration, but each row has different
concentrations. Any well with syncytia or cytopathic effect is considered a positive
result. After complete addition of diluted virus, 96- well microplate was incubated at
37°C for 5 days, after which the titer value of the virus was read (TCID50) (Figure
(3-5)).
Page 55
3.2.6 Determination of Tissue Culture Infective Dose 50% (TCID50) of NDV

TCID50 which means the amount of a pathogenic agent that will cause infection
in 50 % of cell cultures inoculated. Vero cell lines were seeded at 104 cell/well in 96-
well microplate flat bottom, and after 24 hr or when confluent monolayer was
achieved, growth media was discarded and virus suspension (ten-fold serial dilution)
was prepared, 1/10, 1/102, 1/103, 1/104, 1/105, 1/106, 1/107, 1/108, 1/109, and 1/1010.
Then, the cells were inoculated with 50 μl/well of NDV with 8 replicates for each
dilution, control treated with SFM (100 µl) only. The plate was covered with a sterile
adhesive paper and incubated for 2 hrs at room temperature to allow virus
penetration, (Appendix (10)).
After that, the cells were washed with PBS and 200 μl of serum free medium
was added into each well. The plate was covered again and incubated at 37°C for 5
days. NDV titer was calculated on Vero cell lines in comparison with the control to
determine infections dose of 50% in tissue cultures as described by (Flint et al.,
2009), in which the titer was determined as the dilution that cause 50% cytopathic
effect in the incubated well. The following formula is used to calculate the TCID50
used (Table (3-4) and (Figure (3-5)).
log10 (TCID50 /ml) = L + d (S - 0.5) + log (1/v)
L = negative log10 of the most concentrated virus dilution tested, in which all wells
are positive (no. of positive columns completely). L = 5
d = log10 of dilution factor. d = log 10= 1 (1:10 dilution)
S= sum of individual proportions (pi) Dilution Factor
Pi = calculated proportion of an individual dilution = V total/ V Sample

v = volume of inoculum (ml/well)
Pi = Amount of positive wells / total amount of wells per dilution
V= volume of inoculum (ml/well) = 50 µl /ml = 50 µl / 1000µl = 0.05
Page 56
Table (3-4): Calculation method of TCID50 of the virus after treating Vero cell line with NDV
log10 (TCID50 /ml) = L + d (S - 0.5) + log (1/v) log10 virus ratio of Proportion
S =1+ 0.75+ 0.375+ 0.125 + 0 = 2.25, dilution infection (pi)
d= 1, L= 5, v = 50 µl = 0.05ml -1 8/8 1
-2 8/8 1
log10 (TCID50/ml) = 5+1(2.25 - 0.5) + log10
-3 8/8 1
(1/0.05)
-4 8/8 1
log10 (TCID50/ml) = 5+1 (2.25 - 0.5) +1.3
-5 8/8 1
log10 (TCID50/ml) = 5+2.25-0.5+1.3 = 8.05
-6 6/8 0.75
TCID50/ml = 108.05 = 1.12 x 108
-7 3/8 0.375
TCID50/ml =1.12 x 108/ml -8 1/8 0.125
TCID50/ml =112.2 x 106/ml -9 0/8 0.0
-10 0/8 0.0
Negative Positive
Control
Control X X -1 X -2 X-3 X-4 X-5 X -6 X-7 X-8 X-9 X-10 X
Figure (3-5): Exposure of NDV to Vero cells line and calculation of TCID50
Page 57
3.2.7 Exposure stages of the cell lines

In the present study, cell lines were exposed to 3 different agents of exposure:
1- NDV as an agent (Virotherapy) to induce apoptosis
2- D-Mannoheptulose as an agent (phytotherapy) to inhibit of glycolysis
3- Docetaxel drug as an agent (Chemotherapy) as positive control
In addition, cell lines were treated with combination between (NDV+MH)
(Synergistically).
3.2.7.1 Treatment of cell lines with NDV, MH, and DXL

3.2.7.1.1 Preparation of stock and diluted solutions
Sterile eppendorf tubes (1.5 ml) were formatted and numbered in sequence.
NDV stock solution (TCID50=124.6 × 106 virus/ml), 25 ml SFM, and 250 µl
antibiotic (Pen-Streptomycin) (1%) were used to prepare diluted NDV solutions (0.1,
0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 MOI) after adding 500µl cold SFM and NDV
stock solution (0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, and 5.12 µl, respectively), in
accordance with calculations of MOI NDV (Appendix (11)) and (Table (3-5)).
MH Stock solution (14.87 ml) was prepared by dissolving 50 mg MH in

14.87ml PBS, and then used in preparing diluted MH solution [(8, 4, 2, 1, 0.5, 0.25,
0.125, and 0.0625 mM) or (1680, 840, 420, 210, 105, 52.5, 26.25, and 13.125 µg/ml)]
after adding 300 µl SFM and 300 µl MH stock solution (3360 µg/ml)(16mM),
serially eightfold dilution (Appendix (12)).
DMSO (dimethyl sulfoxide) (1%) 25 ml was prepared. Docetaxel Stock

Solution (DXLSS) [2000 µg/ml or (2 mg/ml, 2475 µM, and 2.475 mM)] was
prepared by dissolving 20 mg docetaxel in 10 ml DMSO solution. Diluted stock
docetaxel solution (DSDXLS) (20µg/ml) was prepared using 50µl DXLSS in 5ml
DMSO (1%) which was used to prepare diluted docetaxel solutions [(10, 5, 2.5, 1.25,
0.625, and 0.313 µg/ml) or (12.378, 6.189, 3.09, 1.547, 0.773, and 0.386 µM)] after
Page 58
adding 600 µl cold SFM and 600µl DSDXLS (20µg/ml), 6-fold serial dilution in
sterile tubes (Appendix (13)) (Freshney, 2015).
3.2.7.1.2 Exposure of NDV, MH, and DXL to cell lines

Cell culture of REF, AMJ13, and MCF-7 cell lines were done in the same way
as that of Vero cell line (3.2.3.2), with the replacement of Vero cell line with others
cell lines. 96- well microplate cell line after incubation, the media was aspirated from
96-well microplate using a multipipette and 100µl diluted MOI NDV solution or
diluted MH concentration or diluted docetaxel drug solution was added to wells in
each column in accordance with the number of eppendorf tubes, after which 100 µl
SFM was added to another wells as control. Three replicates were used for each
concentration. The plate was covered with a sterile adhesive parafilm and incubated
at 37°C for 72 hrs (Appendix (11, 14 and 15)), after which the optical density (OD)
was read by MTT assay (Appendix (16)) and cytotoxicity ratio was calculated
(Freshney, 2015).
3.2.7.1.3 Calculation of the number and the volume of virus in a well in

accordance with TCID50 of NDV:
1- Calculation of the number of virus in a well:

Each MOI of virus was calculated as follow:
0.1MOI …………1 virus to each 10 cells or 1000 virus to each 10000 cells or 1000
virus to 1 well (1 well = 10000 cells).
2- Calculation of the volume of virus solution in a well

TCID50 =124.6 × 106 virus/ml = 124.6 × 106 virus/1000 µl (stock solution)
If MOI= 0.1
Stock: 124.6 × 106 virus ………….…… 1000 µl
Diluted: 1000 virus ……………… ……. X (volume of virus solution in 1 well)
Page 59
X= (1000 virus ×1000 µl)/ (124,600,000 virus) =0.008 µl (volume of virus solution
in 1 well) or 0.04 µl (volume of virus solution for 5 wells) was taken from stock
NDV solution + 500 µl SFM= 500 µl diluted NDV solution (0.1 MOI).
If MOI = 12.8
12.8 MOI …………12.8 virus to each 1 cell or 128000 virus to each 10000 cells
or 128000 virus to 1 well.
Stock: 124.6 × 106 virus ……….……… 1000 µl
Diluted: 128000 virus …………….……. X (volume of virus solution in 1 well)
X= (128000 virus ×1000 µl) / (124,600,000 virus) = 1.027 µl (volume of virus
solution in 1 well) or 5.135µl (volume of virus solution for 5 wells) + 500 µl SFM =
500 µl diluted NDV solution (12.8 MOI). Different MOI NDV was prepared (Table
(3-5)).
Table (3-5) Preparation of different MOI for NDV depend on TCID50
No. of No. of NDV/104 Volume of Volume of NDV

MOI/NDV
NDV/10 cells cells (1well) NDV µl /1 well µl /5 well
0.1 1 1000 0.008 0.04

0.2 2 2000 0.016 0.08
0.4 4 4000 0.032 0.16
0.8 8 8000 0.064 0.32
1.6 16 16000 0.128 0.64
3.2 32 32000 0.256 1.28
6.4 64 64000 0.512 2.56
12.8 128 128000 1.024 5.12
3.2.8. MTT Cytotoxicity Assay

Cell culture of REF, AMJ13, and MCF-7
cell lines were done as in the same way as that of
cell line (3.2.3.2). The cytotoxicity was
investigated using MTT
MTT, Methyl thiazolyl diphenyl-tetrazolium. Sigma Aldrich
bromide Page 60
[3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. After cell

lines were treatment with NDV, MH, and Docetaxel, the cytotoxicity ratio was
determined to measure the concentration of inhibition that kills 50% of cells (IC50).
The IC50 was calculated using GraphPad Prism (version 7) for NDV, MH and DXL.
The CompuSyn computer software compared the IC50 of MH and NDV

separately and the IC50 of them combined, on different cell lines (Gao et al., 2003).
After IC50 was measured, the cells were treated with selected different
concentrations of D-Mannoheptulose (Santa Cruz, USA) (250,125, and 62.5 μg/ml)
and Docetaxel (1.25, 2.5 and 5 μg/ml), and MOI of NDV (0.3, 1, and 2) depending
on the IC50 values, after which they were incubated at 37°C for 72 hrs. Triplicates
were used for each concentration of each treatment modality. (Al-shammari et al.,
2016)
After that, the media in 96- well microplate was discarded and 50 μl of serum
free media (SFM) and 50 μl of MTT dye (yellow solution 2 mg/ml) were added into
each well and incubated at 37°C for 3 hrs. MTT dye solution was discarded, the
crystals remaining in the wells were solubilized by the addition of 100 µl of Dimethyl
Sulphoxide (DMSO) (1%) (Santa Cruz Biotechnology, USA) into each well to
dissolve the MTT-Formosan crystals, and the plate was wrapped with parafilm,
rocked, and incubated for 15 min.
The optical density value for treated and untreated cells was measured at 492
nm using the ELISA plate reader (Freshney, 2015), (Appendix (16)). The endpoint
parameter that was calculated for each cell line included percentage of cell growth,
or percentage of cell proliferation (PR) = mean of treatment / mean of control (Van
Meerloo et al., 2011), and the inhibition of cell growth (G.I) or percentage of
cytotoxicity (CT %) which was calculated as:
Cytotoxicity % = (OD Control – OD sample) / OD Control × 100
Page 61
Where OD control is the mean optical density of untreated wells, and OD Sample is
the optical density of treated wells (Jabir et al., 2019). MTT was used to determine
the effect of NDV, MH, and their combination in a study on the viability of tumor
cells. This assay is based on metabolic reduction of colorless tetrazolium salt by the
mitochondrial enzyme activity in viable cells. It is particularly useful for assaying
cell suspensions because of its specificity for living cells (Mosmann, 1983).
3.2.9. Synergistic cytotoxicity assay and Chou-Talalay analysis

Cell lines were seeded at 1 × 104 cells/well into 96-well microplates and
incubated overnight. NDV was exposed first at MOI of 2, 1 and 0.3, then MH was
exposed at the indicated concentration (250,125 and 62.5 μg/ml) as serial dilutions
to test growth inhibition. After 72 hrs of incubation, cell cytotoxicity assay, or growth
inhibition was performed to assess the cytotoxicity rate or inhibition rate of cells by
MTT cytotoxicity assay. For synergism determination, NDV and MH were studied
as non-constant ratios. To analyze the combination of NDV and MH, Chou–Talalay
combination index (CI) was calculated using the CompuSyn software (CombuSyn
Inc., Paramus, NJ, USA). Unfixed ratios of NDV and MH and mutually exclusive
equations were used to determine the CI. A combination index between 0.9 and 1.1
is considered additive, whereas CI< 0.9 and > 1.1 indicates synergism and
antagonism, respectively (Al-shammari et al., 2016). The results were also analyzed
by isobologram technique using CombuSyn software. The axes on Isobologram
represented the doses of each treated. Two points on the x and y axes were chosen
that corresponded to the doses of each agent necessary to generate that particular
combination index (CI) called the fraction affected (Fa) value. The straight line or
hypotenuse drawn between these 2 points on the x and y axes corresponded to a
strictly additive interaction between 2 therapeutic agents. If the point was located on
the lower left of the hypotenuse, the effect was synergistic and if the point was
located on the upper right of the hypotenuse, the effect was antagonistic (Tallarida,
2011).
Page 62
3.2.10. Morphological analysis

Cell culture was done as described in (3.2.3.2). To visualize the shape of the
cells under an inverted microscope, 100 µl of cell suspension for cell lines were
seeded in 96- well microplate at a density of 1x104 cells, and incubated for 24-48 hrs
at 37oC.
After confluence of cells to become monolayered, the media was aspirated, and
then the cells were exposed to MH (62.5µg/ml) and NDV MOI 2 in accordance with
combination index separately and synergistically at 37oC for 72 hrs. The media was
discarded, the plates were stained with 50 µl crystal violet and incubated at 37 oC for
15 min, and the stain was washed off gently with tap water until the dye was
removed. The cell was observed under an inverted microscope at 40x magnification
and photographed with a digital camera (Ali et al., 2019).
3.2.11 Assessment of apoptosis (PI/AO staining)

The Propidium iodide/ Acridine Orange (PI/AO) dual staining method was
used to measure apoptosis of the REF, AMJ13 and MCF-7 cell lines. Cell culture
was done as described in (3.2.3.2). It was seeded 10000 cells/well, 100 μl of cell
suspension is transferred to a 96-well microplate for modified PI/AO staining. Cells
were treated with MH (62.5µg/ml) and NDV MOI (2) in the 37°C incubator for 72
hrs before they were subjected to PI/AO staining. Then, 1 ml of cell suspension was
used for conventional PI/AO staining, (Ribble et al., 2005).
Acridine orange (AO) (10 μg/ml) prepared using PBS (10 μl AO + 1ml PBS)
and Propidium iodide (PI) (10 μg/ml) prepared using PBS (10 μl PI + 1ml PBS) were
used after mixing them at a ratio of 1:1. The stain mixture (AO/PI) was used to add
50 μl to all 96-well microplate, after which it was allowed for 2 min at room
temperature (RT) before it was discarded. PBS (100 μl) was added to all 96-well
microplate for washing, it was discarded, and photographs were taken directly by
fluorescent microscope 40X magnification (Jabir et al., 2019; Ali et al., 2019).
Page 63
3.2.12 Glycolysis parameters determination

Hexokinase Assay Kit: Catalog No: E-BC-K121, Pyruvate Assay Kit: Catalog
No: E-BC-K130, and ATP Assay Kit: Catalog No: E-BC-K157, Method:
Colorimetric methods are supplied from ElabScience, USA. ELISA plate reader.
Operation procedures according to manufacturing protocol (ElabScience
Biotechnology Inc.).
3.2.12.1 Determination of Hexokinase

Operation procedures: Cells culture samples:
Cell culture was done as described in (3.2.3.2) but by using REF, AMJ13 and
MCF-7 cell lines in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine
serum (FBS) and 1% (v/v) penicillin-streptomycin, and incubated in a humidified
atmosphere of 5% CO2 at 37oC. The cell lines were treated with MH (62.5 μg/ml)
and NDV (MOI 2) separately and synergistically, incubator at 37℃ for 72 hrs. Cell
suspension was collected by thawing to prepare cells samples.
Operation steps to determine hexokinase in sample:

The Spectrophotometer or ELISA plate reader was preheated for 30 min, the
wavelength was adjusted at 340 nm, and it was set to zero with distilled water. The
prepared working solution was incubated at 37℃ for 10 min before detection.
Operation table as follow:
The solution was fully mixed and the time was recorded immediately, the tube
or plate was incubated in 37oC, and the absorbance was measured at 340 nm at 30 s
(A1) and 150 s (A2), respectively by using ELISA plate reader. 𝛥A=A2 - A1
Calculation of results for cell sample:
Operation steps Sample tube

Cell sample (μl) 50
Working solution (μl) 960
Page 64
𝛥𝐴
HK activity (U/ml) = ×
𝑚𝑚𝑜𝑙𝑒 𝐸𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡
1
×
𝐶𝑜𝑙𝑜𝑟𝑖𝑚𝑒𝑡𝑟𝑖𝑐 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 ×𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
1.01
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑚𝑢𝑙𝑡𝑖𝑝𝑙𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑟𝑒𝑎𝑐𝑡 𝑠𝑦𝑠𝑡𝑒𝑚 ( )
0.05
𝛥𝐴 1 1.01
HK activity (U/ml) = × 0.5×2 × 0.05
6.22
3.2.12.2 Determination of Pyruvate

Operation steps to determine Pyruvate in sample: Cells culture samples:
MCF-7 cell lines. The cell lines were treated with MH (62.5 μg/ml) and NDV (MOI
2) separately and synergistically, incubator at 37℃ for 72 hrs. Cell suspension was
collected by thawing to prepare cells samples. Blank tube: 0.1 ml of double-distilled
water (DDW), 0.1 ml of Reagent (R) 1 and 0.5 ml of R 2 were added. Standard tube:
0.1 ml of 0.2 μmol/ml Sodium pyruvate standard solution R4, 0.1 ml of R 1 and 0.5
ml of R 2 were added. Sample tube: 0.1 ml of sample, 0.1 ml of R 1 and 0.5 ml of R
2 were added. The content of each tube was mixed fully using a vortex mixer for 5s,
the tubes were incubated at 37℃ for 10 min, R 3 (2.5 ml) was added into each tube
and mixed fully, the tubes were incubated at room temperature for 5 min, ELISA
plate reader was set to zero with DDW. The OD value was measured of each tube or
well at 505 nm (Table (3-6)).
Table (3-6): Operation steps to determine pyruvate in sample

Operation steps Blank tube Standard tube Sample tube
Double-distilled water (ml) 0.1
0.2 μmol/ml Sodium pyruvate
0.1
standard solution (ml) (R4)
Cells sample (ml) 0.1
Page 65
Reagent 1 (ml) 0.1 0.1 0.1

Reagent 2 (ml) 0.5 0.5 0.5
The solutions were fully mixed and incubated at 37℃ for 10 min
Reagent 3 (ml) 2.5 2.5 2.5
Calculation of results for cell sample:

Pyruvate Concentration (𝜇𝑚𝑜𝑙/𝑚𝑙)
𝑂𝐷 𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷 𝐵𝑙𝑎𝑛𝑘
= × Concentration of Standard (0.2 μmol/ml)
𝑂𝐷 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 − 𝑂𝐷 𝐵𝑙𝑎𝑛𝑘
3.2.12.3 Determination of ATP

a. Sample preparation: Cells culture samples:
MCF-7 cell lines. The cell lines were treated with MH (62.5 μg/ml) and NDV (MOI
2) separately and their combination, incubator at 37℃ for 72 hrs. Cells suspension
was collected by thawing to prepare cells samples.
b. Operation steps to determine ATP in sample:

Blank tube: 30 μl standard solution, 100 μl R1, 200 μl R2, and 30 μl double
distilled water were added, standard tube: 30 μl standard solution, 100 μl R1, 200 μl
R2, and 30 μl R3 were added, sample tube: 30 μl sample, 100 μl R1, 200 μl R2, and
30 μl R3 were added, control tube: 30 μl sample, 100 μl R1, 200 μl R2, and 30 μl
double distilled water were added, the tubes were mixed and place in a water bath at
37℃ for 30 min, 50 μl R4 was added to all tubes and mixed fully, the content of each
tube was centrifuge at 4000 r/m for 5 minutes. 300 μl of supernatant samples were
taken and were added to another tubes, 500 μl R5 was added to all new tubes, mixed
and left at room temperature for 2 min, after which 500 μl R6 was added to all tubes.
The solutions were mixed and left at room temperature for 5 min, and their OD or
absorbance values were measured at 636 nm using ELISA plate reader (Table (3-7)).
Page 66
Table (3-7) Operation steps to determine ATP in sample
Operation steps Blank Standard Sample Control

1 mmol/l ATP standard
application solution (μl) 30 30
Cells sample(μl) 30 30
Reagent 1: substrate Ⅰ (μl) 100 100 100 100
Reagent 2: substrate Ⅱ(μl) 200 200 200 200
Reagent 3: accelerant (μl) 30 30
Double distilled water (μl) 30 30
The solutions were mixed and incubated for 30 min in water bath at 37℃
Reagent 4: precipitant (μl) 50 50 50 50
Centrifuge for 5 minutes at 4000 rpm after mixing fully
The solution was mixed, still for 5 minutes at room temperature. The OD values were
y Supernatant sample (μl) 300 300 300 300
Reagent 5: developer (μl)
500 500 500 500
The solutions were mixed and still for 2 minutes at room temperature
Reagent 6: stop solution (μl) 500 500 500 500

measured of each tube or microplate at 636nm wavelength using ELISA plate reader.
c. Calculation of results
Computational formula for cell:
Concentration of ATP (μmol /L)

𝑂𝐷 𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷 𝐶𝑜𝑛𝑡𝑟𝑜𝑙
= × Concentration of Standard (1000 μmol/L)
𝑂𝐷 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 − 𝑂𝐷 𝐵𝑙𝑎𝑛𝑘
Page 67
3.2.13. pH measurements
The cell lines (REF, AMJ13 and MCF-7) were cultured in RPMI-1640 medium
supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-
streptomycin, and incubated in a humidified atmosphere of 5% CO2 at 37oC,
overnight before the treatment. A of 96-well microplate was used to seed 1×104 cell
lines, and then incubated at 37oC for 24-48 hrs until monolayer was achieved as
observed by an inverted microscope (Cell culture was done as described in (3.2.3.2)).
The cell lines were treated with D-Mannoheptulose (62.5 μg/ml) and NDV
(MOI 2) separately and synergistically, incubated at 37℃ for 72 hr, cell suspension
was collected by thawing, and then the pH was measured using litmus papers for
AMJ13, MCF7, and REF cell lines with comparison to the control (Al-Shammari et
al., 2015).
3.2.14. Statistical Analysis.

All results were presented as, means ± SD, t-test was carried out and analyzed
using the statistical software Excel version 19, SoftMax program 6.4, SPSS version
16 and GraphPad prism version 7. Isobologram version 1 were performed to compare
the difference between groups under different conditions, P values >0.05 were
considered significant. CompuSyn software program algorithm assessed the
combination index CI. Combined dose-response curves were fitted on Chou-Talalay
lines. CI <1 indicates synergistic interaction, whereas CI >1.1 was antagonistic and
CI between 1 and 1.1 was considered additive (Chou, 2010).
Page 68
CHAPTER-FOUR
Chapter Four…………………………..…………………..………………………… Results
4. Results
4.1. Virus propagation in Embryonated Chicken Eggs (ECE)

4.1.1. Propagation of virus (NDV) in chicken embryo
Embryonated chicken eggs (ECE) were used in virus propagation. The
infected embryos were characterized with hemorrhage in comparison to control
embryos (Figure (4-1)). The results showed that NDV Iraqi virulent strain was able
to kill all embryos (9 or 10 days old) up to 48 hrs of inoculation in the ECE. Some
embryonated chicken eggs were killed before 24 hrs, this type of ECE were
canceled and save after 24 hrs to aggregate the virus.
A B
Figure: (4-1) Inoculation of NDV in ECE: (A) Skin hemorrhage in ECE after inoculated
with NDV (B) ECE control.
4.1.2. Harvest of NDV and measurement of Hemagglutination Unit (HAU)

The results showed that harvested Iraqi strain of NDV has the ability to kill all
the chicken embryos through 24-72 hrs after the inoculation of the chicken
embryonated eggs with marked hemorrhage in the infected embryos as compared to
the control uninfected embryos. Virus was aggregated, purified, tittered, and
quantified by hemagglutination test. Titer and HAU of NDV was measured
(Titer = 8), (HAU = 256). It showed a positive result as a typical hemagglutination
mesh pattern of chicken red blood cell at 256 HAU (Figure (4-2)).
Page 69
Titer: 1 2 3 4 5 6 7 8 9 10 11 12
HAU: 2 4 8 16 32 64 128 256 512 1024 2048 4096
Figures (4-2) Hemagglutination test, show the agglutination (positive result) of RBC forms
a distinctive mesh at the bottom (titer 8). The negative hemagglutination is characterized by
sharp red dot in comparison to the control.
4.1.3. Tissue Culture Infectious Dose (TCID 50)

The NDV titer was measured on Vero cell line to determine the TCID50 of the
virus. TCID50 was measured (124.6 x 106 virus/ ml) (Table (4-2)). It showed that
NDV formed plaques on infected Vero cells line compared with uninfected control
cells (Figure (4-3)).
Page 70
B B
B B
B B
B B
Figure (4-3) Vero cells treated with NDV: A- Vero cells untreated as control, B- Vero cells
treated with NDV showed hemolysis of cells (apoptosis) and plaques formation between cells,
(Red arrows refer to plaques formation).
Page 71
Table (4-1) Results of Vero cells were treated with NDV to determine TCID50
A B C
Proportion
Proportion
Proportion
log10 log10 log10
Ratio of Ratio of Ratio of
(pi)
(pi)
(pi)
virus virus virus
infection infection infection
dilution dilution dilution
-1 8/8 1 -1 8/8 1 -1 8/8 1

-2 8/8 1 -2 8/8 1 -2 8/8 1
-3 8/8 1 -3 8/8 1 -3 8/8 1
-4 8/8 1 -4 8/8 1 -4 8/8 1
-5 8/8 1 -5 8/8 1 -5 8/8 1
-6 6/8 0.750 -6 5/8 0.625 -6 5/8 0.625
-7 3/8 0.375 -7 3/8 0.375 -7 4/8 0.500
-8 1/8 0.125 -8 1/8 0.125 -8 2/8 0.250
-9 0/8 0.0 -9 1/8 0.125 -9 0/8 0.0
-10 0/8 0.0 -10 0/8 0.0 -10 0/8 0.0
A B
Positive
C
(Plaques formation)
Negative
(No plaques formation)
Figure (4-4) Exposure of NDV to Vero cell line to measure TCDI50: A- exp 1, B- exp 2, and C- exp 3
Page 72
Vero cell lines were treated with the virus showed plaques formation. The
positive result represents plaques formation between cells were explained in the
first experiment (Table (4-1-A)) and (Figure (4-4-A)), the second experiment
(Table (4-1-B)) and figure (4-B), and the third experiment (Table (4-1-C)) and
(Figure (4-4-C)). The final values for TCID50/ml for exposure of NDV to Vero cell
line in 96-well microplate which represents the number of viruses for each 1ml are
shown in (Table (4-2)).
Table (4-2) Final value for TCID50/ml for exposure of NDV to Vero cells
No. of Exp TCID50/ml Average TCID50/ml
1 1.122 x 108/ml 1.246 x 108 /ml

2 1.122 x 108/ml
3 1.496 x 108/ ml 124.6 x 106/ml
4.2 Cytotoxicity assay

The effect of cytotoxicity was evaluated using different MOI of NDV (Table
(4-3)) by MTT cytotoxicity assay. Through our findings, increased MOI of the
virus was shown to increase cytotoxicity or improve growth inhibition. There is a
statistically significant difference between inhibition of the virus for breast cancer
cell lines compared to normal REF cell line, where effect of NDV seen in breast
cancer cells was more than that seen in normal cells (Figure (4-5 and 6)).
REF AMJ13 MCF-7
Figure (4-5) Cytotoxicity effect (CT %) after treatment of cell lines with NDV: A- NDV to REF,
B- NDV to AMJ13, and C- NDV to MCF7 by linear regression.
Page 73
100
A 100 B
REF AMJ13
80 80
C y to to x ic ity %
60 60
40 40
20 20
0 0
0 .1 0 .2 0 .4 0 .8 1 .6 3 .2 6 .4 1 2 .8 0 .1 0 .2 0 .4 0 .8 1 .6 3 .2 6 .4 .8
12
MOI NDV MOI NDV
Table (4-3) CT% after treatment of cell lines with NDV

100
C
MCF-7
80
60
40
20
0 .1 0 .2 0 .4 0 .8 1 .6 3 .2 6 .4 12
.8
MOI NDV
Figure (4-6) Comparison of cytotoxicity between different MOI after treatment of cell lines with
NDV: A- CT % - NDV to REF, B- CT % - NDV to AMJ13, and C- CT % - NDV to MCF7 by
column bar graph
The effect of cytotoxicity was evaluated using different concentrations of MH

(μg/ml) (Table (4-4)) and by MTT cytotoxicity assay. From these results, increased
concentration of the inhibitor has been shown to increase cytotoxicity, or enhance
growth inhibition. There is a statistically significant difference between inhibition
Page 74
by MH for breast cancer cell lines compared to normal REF cell line, where a
greater effect of MH was seen in breast cancer cells than in normal cells (Figure (4-
7 and 8)).
REF AMJ13
100
C MCF-7
80
C y t o t o x i c it y %
60
40
20
0
0 500 1000 1500 2000
C o n c e n tr a tio n M H µ g /m l
Figure (4-7) Cytotoxicity effect (CT %) after treating cell lines with MH: A - MH to REF, B
- MH to AMJ13, and C - MH to MCF7 by linear regression.
Page 75
100
A B 100
REF AMJ13
80 80
60 60
40 40
20 20
0 0
5 5 2 .5 5 0 0 0 0
.1 2 6 .2 5 10 21 42 84 1 68 .1 2
5 .2 5 52
.5 10
5
21
0
42
0
84
0 80
13 2
13 26 16
C o n c e n tr a tio n M H C o n c e n tr a tio n M H
C 100
MCF-7 Table (4-4) CT% after treatment of cell lines with MH

80
.
60
40
20
5 .2 5 .5 5 0 0 0 80
.1 2 26 52 10 21 42 84 16
13
C o n c e n tr a tio n M H
Figure (4-8) Comparison of cytotoxicity between different concentrations (μg/ml) after cell
lines were treated with MH: A- CT % - MH to REF, B- CT % - MH to AMJ13, and
C- CT % - MH to MCF7 by column bar graph
The effect of cytotoxicity was evaluated using different concentrations of

DXL (μg/ml) (Table (4-5)) and by MTT cytotoxicity assay. From these results,
increased concentration of the drug has been shown to increase cytotoxicity or
Page 76
induce proliferation inhibition. There is a significant difference between inhibition

by drug for breast cancer cell lines compared to normal REF cell line, where a
greater effect of the drug was seen in breast cancer cells than normal cells (Figure
(4-9 and 10-I)).
C o r r e l a ti o n C o e ff ic i e n t = 0 .8 7
100
A B 100
REF AMJ13
80
80
C y to t o x ic ity %
C y t o t o x ic it y %
60
60
40
40
20 20
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
C o n c e n t r a t io n D X L (  g /m l) C o n c e n t r a t io n D X L (  g /m l)
100
C MCF-7
80
60
40
20
0
0 2 4 6 8 10 12
C o n c e n t r a t io n D X L (  g /m l)
Figure (4-9) Cytotoxicity effect (CT %) after cell lines were treated with Docetaxel (DXL)
(μg/ml): A- DXL to REF, B- DXL to AMJ13, and C- DXL to MCF7 by linear regression.
.
Page 77
REF AMJ13
Table (4-5) CT% after treatment of cell lines with DXL
MCF-7 .
C/DXL
Figure (4-10-I) Comparison of cytotoxicity between different concentrations (μg/ml) after cell
lines were treated with DXL: A- CT % - DXL to REF, B- CT % - DXL to AMJ13, and
C- CT % - DXL to MCF7 by column bar graph. DXL
.
Docetaxel is a chemotherapy drug that was used as a positive control for

comparison with the effect of D-Mannoheptulose as a phytotherapeutic, NDV as a
virotherapy, and their combination on normal and breast cancer cell lines.
Page 78
4.2.1 Cytotoxicity of NDV and MH against breast cancer and normal cell lines
MTT cytotoxicity assay was used to evaluate the effect against cancer and
normal cells of different concentrations of MH and over a range of MOI of NDV
(Figure 4-10-II). There were no noticeable percentages of cytotoxicity (CT%) for
MH against normal REF cells as the CT% ranged from 1.67%, to 24.72% at higher
concentrations. While there was higher cytotoxicity against breast cancer cells
ranged from 27.29% to 58.64% for AMJ13 cells; and 26.26% to 60.49% for MCF-
7 after MH treatment (Figures 4-10-II A, B, and C). NDV virotherapy did not
induce cytotoxic effect against normal embryonic REF cells as the CT% ranged
from 4.31% to 28.75% for REF (Figure 4-10-II F). Breast cancer cells were more
sensitive to NDV virotherapy as the CT% ranged from 24.69% to 64.26% for
AMJ13; and 23.95% to 62.02% for MCF-7 cell line after NDV treatment (Figures
4-10-II D and E).
Figure (4-10- II): MH and oncolytic AMHA1 NDV are cytotoxic against human AMJ13 and
MCF-7 breast cancer cells, but not cytotoxic to normal embryonic REF cells. The cells were
treated with (A, B, C) D-Mannoheptulose (MH) (13.125, 26.25, 52.5, 105, 210, 420, 840, and
1680 μg/ml) or (D, E, F) NDV (MOI 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8) for 72h.
cytotoxicity was investigated using MTT assay. All data shown are means ± SD from three
independent experiments.
Page 79
4.3. Determination of IC50

To evaluate the effect of each treatment on cell proliferation, IC50 value was
measured in normal and breast cancer cell lines. IC50 was measured for NDV and
it showed that 57.5 MOI infectious virus were needed to kill REF normal cells,
while only about 2 virus (1.648 MOI) were needed to kill AMJ13 Iraqi breast
cancer cells and (1.561 MOI) MCF7 breast cancer cells (Figure 4-11 A, B, and C)
with different MOI of NDV (Table (4-3)).
IC50 value for the hexokinase inhibitor MH in normal REF cells was 486.9
µg/ml while for breast cancer cells is was 75% less required to kill them, (124.7
μg/ml for AMJ13 and 122.6 μg/ml for MCF-7), (Figure 4-12 A, B, and C) with
different concentrations of MH (Table (4-4)).
IC50 of DXL was twice higher in normal cells (6.072 μg/ml) than in cancer
cells, (3.118 μg/ml for AMJ13 and 3.221 μg/ml for MCF-7), (figure 13- A, B, and
C) with different concentrations of DXL (Table (4-5)).
Statistical data of IC50 for comparison of cell lines after treatment with NDV,
MH, and DXL can be seen in (Table (4-6)). IC50 is a measure of the potency of a
virus, drug, and inhibitor in inhibiting breast cancer cell lines. It is a quantitative
measure that indicates the amount of a particular inhibitory substance (DXL, NDV,
and MH) needed to inhibit proliferation of breast cancer. It expresses the drug
concentration at which 50% of cells are inhibited which explains why the value of
IC50 that appeared in the breast cancer cells was less than that in the normal cell
lines.
The results showed that there are significant differences in IC50 between
breast cancer cell lines and REF cell line after treatment with virus, inhibitor, and
drug, while no significant differences were found between MCF-7 and AMJ13 cell
lines (Figure (4-14)). Cytotoxicity was measured between REF, AMJ13, and MCF-
7 cell lines at specific concentrations after treatment with 2 MOI NDV, 62.5 μg/ml
Page 80
MH, and, 1.25 μg/ml DXL depend on combination index for 72 hrs. The effect of
NDV, MH, and DXL on breast cancer cell lines was very clear compared to normal
REF cell line where a significant difference was found between them (Figure (4-
15)) and (Table (4-7)).
REF AMJ13
A B
C MCF-7
IC50 was estimated by plotting x-y and fit the data with
a straight line. IC50 value was then estimated using the
fitted line, i.e., Y = a * X + b, IC50 = (0.5 - b)/a
Or by Y=Bottom + (Top-Bottom)/(1+10^((X-LogIC50))
Figure (4-11): IC50 to exposure of NDV to cell lines: IC50 values after REF, AMJ13, and MCF-
7 cell lines were treated with NDV, by GraphPad Prism software. A- IC50 - NDV to REF, B-
IC50 - NDV to AMJ13, and C - IC50 - NDV to MCF-7.
Page 81
A REF
B 5000
AMJ13
4000
3000
2000
1000
0
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0
C o n c e n t r a t io n o f M H lo g µ g /m l
IC50 124.7
C
MCF-7
Figure (4-12): IC50 to exposure of MH to cell lines: IC50 values after REF, AMJ13, and MCF-7
cell lines were treated with MH, by GraphPad Prism software. A- IC50 - MH to REF, B- IC50 -
MH to AMJ13, and C - IC50 - MH to MCF-7.
The cytotoxicity assay analysis showed that IC50 values for NDV MOI (57.5
REF, 1.648 AMJ13 and 1.561 MCF-7), IC50 values of MH (486.9µg REF,
124.7µg AMJ13, and 122.6µg MCF7). IC50 values of DXL (6.072µg REF,
3.118µg AMJ13, and 3.221µg MCF7). Therefore, it was chosen IC50 related doses
of the MH and NDV for the combination study, (0.3, 1, 2 MOI) for NDV and (62.5,
125, and 250 μg/ml) for MH. (1.25, 2.5 and 5 μg/ml) for DXL as positive control.
Page 82
REF AMJ13
MCF-7
Figure (4-13): IC50 to exposure of DXL (μg/ml ) to cell lines: IC50 values after REF, AMJ13,
and MCF-7 cell lines were treated with DXL by GraphPad Prism software. A- IC50 - DXL to
REF, B- IC50 - DXL to AMJ13, and C - IC50 - DXL to MCF-7.
Page 83
Table (4-6): Statistical data for comparison of IC50 in cell lines after treated with NDV, MH, and
DXL.
IC50 – NDV Mean ± SD IC50 – MH Mean ± SD IC50 – DXL Mean ± SD

REF 57.5 ± 3.7 REF 487 ± 5.8 REF 6.1 ± 0.11
Lines
Lines
Lines
Cell
Cell
Cell
AMJ13 1.65 ± 0.06 AMJ13 125 ± 5.9 AMJ13 3.1 ± 0.23
MCF-7 1.56 ± 0.06 MCF-7 123 ± 5.5 MCF-7 3.2 ± 0.09
P value (T-test) P value (T-test) P value (T-test)
REF MCF7 REF MCF7 REF MCF7
****S, P > 0.0001 ****S, P > 0.0001 ****S, P > 0.0001
AMJ13 MCF7 AMJ13 MCF7 AMJ13 MCF7
ns, P = 0.162 (P <0.05) ns, P = 0.675 (P <0.05) ns, P = 0.515 (P <0.05)
REF AMJ13 REF AMJ13 REF AMJ13
****S, P > 0.0001 ****S, P > 0.0001 ****S, P > 0.0001
NDV
DXL
MH
Figure (4-14): Comparison of IC50 between REF, AMJ13, and MCF-7 cell lines after treatment
with NDV, MH, and DXL for 72 hrs, A-IC50-NDV, B- IC50 –MH, and C- IC50-DXL.
.
Page 84
Table (4-7): Statistical data for comparison of CT% in cell lines after treated with NDV, MH,
and DXL.
CT% – NDV Mean ± SD CT% – MH Mean ± SD CT% – DXL Mean ± SD

REF 16 ± 1.8 REF 7.3 ± 0.71 REF 5.8 ± 0.62
Lines
Lines
Lines
Cell
Cell
Cell
AMJ13 58 ± 2.8 AMJ13 43 ± 3.2 AMJ13 57.3 ± 4.04
MCF-7 67 ± 2.5 MCF-7 40 ± 1.0 MCF-7 59.6 ± 3.31
REF MCF7 REF MCF7 REF MCF7
****S, P > 0.0001 ****S, P > 0.0001 ****S, P > 0.0001
AMJ13 MCF7 AMJ13 MCF7 AMJ13 MCF7
*s, P = 0.014 (P > 0.05) ns, P = 0.208 (P < 0.05) ns, P = 0.492 (P < 0.05)
REF AMJ13 REF AMJ13 REF AMJ13
****S, P > 0.0001 ****S, P > 0.0001 ****S, P > 0.0001
NDV
MH DXL
Figure (4-15): Comparison of CT% between REF, AMJ13, and MCF-7 cell lines after treatment
with specific concentrations of NDV, MH, and DXL, at 2 MOI NDV, 62.5 μg/ml MH, and 1.25
μg/ml DXL, for 72 hrs. A-CT% -NDV, B- CT% –MH and C- CT%-DXL. Values represent the
(mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
.
Page 85
4.4. Study the synergistic effect of NDV and D-Mannoheptulose

Three specific MOI based on IC50 values (0.3, 1, and 2 MOI) of NDV with
three specific concentrations also based on IC50 value (250,125, and 62.5 μg/ml) of
MH were combined in MTT cytotoxicity assay to study the synergism in cancer
cells (Figure 4-16, 17 and 18). The results of cytotoxicity of NDV, MH, and their
combination, in REF, AMJ13 and MCF-7 cell line were explained in (Figure 4-16,
17, and 18). The virus (0.3, 1, and 2 MOI) and inhibitor (250, 125, and 62.5 µg/ml)
induced cytotoxicity (growth inhibition) significantly when compared between
them, but insignificantly as shown in (Figure 4-17 A, E and I), (Figure 4-18, C),
and significantly with combination in breast cancer cell line. The combination of
MH and virus was a significantly better promoter and enhancer of growth
inhibition when compared with their separate treatment in breast cancer cell lines
(Figure 4-17 and 18)).
While in normal REF cell line, the effect of virus, inhibitor, and their
combination was insignificant and induced cytotoxicity less than in breast cancer
cell (Figure (4-16)). A significant induction was identified in growth inhibition in
all the treatment modalities in the breast cancer cell lines but not in the normal cells
line. (Figure (4-19)) showed in vitro inhibition of growth induced by high MOI of
virus, also treatment with NDV showed enhancement of the growth inhibition
properties in breast cancer cell lines more than normal cell line. Dose response
evaluation of treatments differed significantly between REF and breast cancer cell
lines. Statistical data for CT% of NDV for comparison of cell lines can be seen in
(Table (4-8)). Figure (20) showed in vitro inhibition of growth induced by high
concentrations of MH, as well as treatment with MH showed enhancement of the
growth inhibition properties in breast cancer cell lines more than normal cell line.
Dose response evaluation of treatments differed significantly between REF and
breast cancer cell lines. Statistical data for CT% of MH for comparison of cell lines
can be seen in (Table (4-9)).
Page 86
REF cell line
Figure (4-16): Cytotoxicity ratio of normal (REF) cell line after treatment with MH (250, 125,
and 62.5 μg/ml), NDV (0.3, 1, and 2 MOI), and combination between them. Values represent the
(mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 87
AMJ13 cell line
Figure (4-17): Cytotoxicity ratio of breast cancer (AMJ13) cell line after treatment with MH
(250, 125, and 62.5 μg/ml), NDV (0.3, 1, and 2 MOI), and combination between them. Values
represent the (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 88
MCF-7 cell line
Figure (4-18): Cytotoxicity ratio of breast cancer (MCF-7) cell line after treatment with MH (250,
125, and 62.5 μg/ml), NDV (0.3, 1, and 2 MOI), and combination between them. Values
represent the (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 89
REF AMJ13
MCF-7
Figure (4-19): Cytotoxicity% of NDV against normal REF and breast cancer cell lines by using 3
different exposure doses (0.3, 1, and 2 MOI) of NDV depends on the IC50 value, A- CT % of
REF, B- CT % of AMJ13, and C-CT % of MCF-7 after treatment with NDV. The values
represent the (Mean ± SD) at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Table (4-8): Statistical data for cytotoxicity% of NDV against cell lines
CT% – NDV- CT% – NDV - CT% – NDV-

Mean ± SD Mean ± SD Mean ± SD
REF . AMJ13 MCF-7
0.3 9.3 ± 2.1 0.3 42 ± 2.3 0.3 51 ± 1.8
MOI
MOI
MOI
1 12 ± 2.8 1 51 ± 1.7 1 60 ± 1.6

2 16 ± 1.8 2 58 ± 2.8 2 67 ± 2.5
MOI 0.3 MOI 1 MOI 0.3 MOI 1 MOI 0.3 MOI 1
ns, P < 0.05 **S, P > 0.01 **S, P > 0.01
*s, P = (P > 0.05) **S, P > 0.01 ***S, P > 0.001
MOI 1 MOI 2 MOI 1 MOI 2 MOI 1 MOI 2
ns, P < 0.05 *s, P = (P > 0.05) *s, P = (P > 0.05)
Page 90
REF AMJ13
MCF-7
Figure (4-20): Cytotoxicity% of MH against normal REF and breast cancer cell lines by using
3 different exposure doses (250,125, and 62.5 μg/ml) of MH depends on the IC50 value, A-
CT % of REF, B- CT % of AMJ13, and C-CT % of MCF-7 after treatment with MH. The
values represent the (Mean ± SD) at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
.
Table (4-9): Statistical data for cytotoxicity% of MH against cell lines
.
CT% – MH- CT% – MH - CT% – MH-
Mean ± SD Mean ± SD Mean ± SD
REF AMJ13 MCF-7
Conc. MH
Conc. MH
Conc. MH
62.5 7.3 ± 0.7 62.5 43 ± 3.2 62.5 40 ± 1.0

μg/ml
μg/ml
μg/ml
125 9.4 ± 1.7 125 50 ± 2.1 125 46 ± 1.8

250 13 ± 1.7 250 57 ± 2.4 250 53 ± 1.9
ns, P < 0.05 **S, P > 0.01 **S, P > 0.01
*s, P = (P > 0.05) **S, P > 0.01 ***S, P > 0.001
MOI 1 MOI 2 MOI 1 MOI 2 MOI 1 MOI 2
ns, P < 0.05 *s, P = (P > 0.05) *s, P = (P > 0.05)
Page 91
Cytotoxicity was determined by MTT cytotoxicity assay in normal REF and

breast cancer (AMJ13 and MCF-7) cell lines, in accordance with specific
concentrations (5, 2.5, and 1.25 μg/ml) of DXL (Figure (4-21)). The best effect was
noticed at 5μg/ml of DXL. Inhibition of growth induced by high concentrations of
DXL was seen, and also, treatment with DXL showed enhancement of the growth
inhibition properties in breast cancer cell lines more than normal cell line.
Dose response evaluation of treatments differed significantly between REF

and breast cancer cell lines (Figure (4-21)). There is a significant difference
between the effect of DXL at 1.25 μg/ml and combination at (2 MOI-NDV and
62.5 μg/ml MH), to growth inhibition (cytotoxicity) for normal and breast cancer
cell lines (Figure (4-22)).
REF AMJ13 MCF-7
Figure (4-21): Cytotoxicity% of DXL against normal REF and breast cancer cell lines by using 3
different exposure doses (1.25,2.5, and 5 μg/ml) of DXL depends on the IC50 value, A- CT % of
REF, B- CT % of AMJ13, and C-CT % of MCF-7 after treatment with DXL. The values
represent the (Mean ± SD) at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 92
REF AMJ13 MCF-7
Figure (4-22) Comparison between the effect of DXL and Combination therapy in cell lines.
A- in REF, B- in AMJ13 and C- in MCF-7
4.5 Combination cytotoxicity assays and Chou–Talalay analysis of cell lines

In order to investigate the effects of oncolytic NDV and MH combination
therapy, it was examined the cytotoxicity ratio of the NDV (0.3, 1, and 2 MOI) and
for the MH (62.5, 125, and 250 μg/ml). Synergism were observed in all combined
doses against both breast cancer (AMJ13 and MCF7) cell lines (Figure 4-23 A, B,
G, H and C, D, I, J), but with the exception of the ninth point which indicated
additive as shown in (Table (4-10 A)) (Figure (4-23 A)). Whereas no synergism
relationships were observed among treatments against the non-cancerous REF cell
line (Table (4-10 C)) (Figure (4-23 E, F, K and L)).
The CI was estimated from the dose-effect data of single and combined
treatments by using CompuSyn Isobologram. CI < 1 indicates synergism; CI = 1 to
1.1 indicates an additive effect; and CI > 1.1 indicates antagonism. The AMJ13 cell
line had CI < 1 in eight combination points, indicating a synergistic effect or
interaction between NDV and MH. Additive was observed in one combination
point (Table (4-10 A) and (Figure (4-23 A and B)). The combination points for the
combined treatment to MCF-7 cell line indicated a synergistic effect or interaction
between NDV and MH at all points (Table (4-10 B)) and (Figure (4-23 C and D)).
Page 93
In the normal REF cell line, all combination points were CI > 1, indicating
antagonism and additive effect which is neglected effect as there was no killing
effect reached 50% at all tested concentrations (Table (4-10 C) and (Figure (4-23 E
and F)).
Isobologram Analysis
Page 94
Dose-effect curve Median-effect plot
G-AMJ13 H-AMJ13
I-MCF-7 J-MCF-7
K-REF L-REF
Figure (4-23) Combination of NDV and MH showed superior anticancer activity in compare to
monotherapies in both AMJ13 and MCF-7 breast cancer cells. However, there was no enhanced
toxicity against non-cancerous REF cells. (A, C and E) AMJ13, MCF-7 and REF cells were
treated with NDV (0.3, 1, and 2 MOI) and with MH (62.5, 125, and 250 μg/ml), then cell
viability was measured by MTT assay. Illustrations of normalized isobologram of non-constant
combination ratios (B, D and F), Dose-effect curve (G, I and K) and Median-effect plot (H, J and
L) for AMJ13, MCF-7 and REF respectively were measured by Chou-Talalay method where CI
value quantitatively defines synergism. (CI < 1), additive effect (CI = 1-1.1) and antagonism
(CI > 1.1). All data shown are means ± SEM (*P < 0.05 compared to mono-treatments) data from
three different experiments. The red line represents phytotherapy (MH) single treatment, blue line
represents the single treatment of NDV and green triangle is the combination treatment using
ComboSyn software. The ratio of the fraction affected (fa) vs the fraction unaffected (fu) is equal
to the dose (D) vs the median-effect dose.
Page 95
Table (4-10): Cytotoxicity of NDV and MH combination or alone against AMJ13, MCF-7 and
REF cells. Combination index (CI) was measured by CompuSyn software. CI data for non-
constant combination (NDV+MH) of cell lines showing synergism effect between NDV and MH
in breast cancer and normal cell lines. (A) CI values from treatment of AMJ13 cancer cells; (B)
CI values from treatment in MCF-7 cancer cells; (C) CI values from treatment of non-cancer cells
(REF).
A- AMJ13
Points NDV (MOI) MH (µg/ml) Effect CT CI Effect
1 2 250 0.80 0.10595 Synergism
2 2 125 0.72 0.24635 Synergism
3 2 62.5 0.65 0.49989 Synergism
4 1 250 0.74 0.20755 Synergism
5 1 125 0.65 0.41359 Synergism
6 1 62.5 0.61 0.49220 Synergism
7 0.3 250 0.68 0.35256 Synergism
8 0.3 125 0.63 0.34603 Synergism
9 0.3 62.5 0.48 1.07844 Additive
B- MCF-7
1 2 250 0.85 0.06841 Synergism
2 2 125 0.79 0.20042 Synergism
3 2 62.5 0.72 0.51245 Synergism
4 1 250 0.80 0.10016 Synergism
5 1 125 0.77 0.15299 Synergism
6 1 62.5 0.70 0.34948 Synergism
7 0.3 250 0.77 0.09957 Synergism
8 0.3 125 0.72 0.13597 Synergism
9 0.3 62.5 0.69 0.22648 Synergism
C- REF
1 2 250 0.17 1.01291 Additive
2 2 125 0.16 1.07549 Additive
3 2 62.5 0.16 1.03328 Additive
4 1 250 0.15 1.10258 Antagonism
5 1 125 0.14 1.04811 Additive
6 1 62.5 0.12 1.38593 Antagonism
7 0.3 250 0.14 1.03584 Additive
8 0.3 125 0.10 1.56003 Antagonism
9 0.3 62.5 0.09 1.51414 Antagonism
Page 96
4.6. Morphological changes

Morphological changes were showed after treatment with MH (62.5µg/ml) and
NDV (MOI = 2) to depend on combination index that has been measured for NDV
and MH for 72hrs. In unstained cells and stained cells, by crystal violate, was
noticed significant cytopathic effect, a large number of cramped cells with
granulation, formation of syncytial, and shrinkage of cells which results in opaque
foci of cells which leads to separation and floating of the treated cells in the
cultured media compared with control.
It was revealed that synergism between the inhibitor and the virus increased
morphological changes in unstained AMJ13 cell line and the following were
observed: cytopathic effect, large number of cramped cells, and empty plaque
spaces between cells compared with the control with granulation and shrinkage. It
was noticed differences in morphological changes after treated with MH, NDV and
their combination (Figure (4-24)), the yellow arrows in (Figure (4-24)) refer to
cytopathic effect areas. It was noticed that synergism between inhibitor and virus
increased morphological changes in unstained MCF-7 cell line and it was showed
cytopathic effect, and large number of cramped cells with granulation and
shrinkage. Differences in morphological changes were seen after treatment with
MH, NDV, and their combination in comparison with the control (Figure (4-25)),
The yellow arrows in (Figure (4-25)) refer to cytopathic effect areas. But there
were no differences in morphological changes or it was showed insignificant
cytopathic effect in unstained REF cell line (Figure (4-26)).
In AMJ13 and MCF-7 cells lines, proliferation of tumor cells has been
inhibited by MH and NDV. NDV and MH effectively induced morphological
changes in AMJ13 and MCF-7 treated cells compared with untreated cells (control)
in unstained cells (Figure (4-24 and 25)) and stained cells (Figure (4-27 and 28)).
Morphological changes were observed more in cells after treatment with NDV and
MH in comparison with cells treated with NDV and MH separately, and in
Page 97
comparison with control, morphological changes in treated cells showed significant

cytopathic effect within 72 hrs, also similar to incomplete confluence of cell culture
(Figure (4-27, 28)), but in stained REF cell lines, minimal morphological changes
showed insignificant cytopathic effect (Figure (4-29)).
Control NDV
Unstained cells - AMJ13
MH Combination
Unstained cells - AMJ13
Figure (4-24): Effect of NDV (MOI = 2), MH (62.5µg/ml), and their combination on unstained
AMJ13 cell lines to explain proliferation inhibition after treatment with different doses for 72
hrs by MTT cytotoxicity assay and by using inverted microscope 40X. The yellow arrows refer
to cytopathic effect areas.
Page 98
Control NDV
Unstained cells
MCF7
MH Combination
Unstained cells
MCF7
MCF-7 cell lines to explain proliferation inhibition after treatment with different doses for 72
hrs by MTT cytotoxicity assay and by using inverted microscope 40X. The yellow arrows refer
to cytopathic effect areas.
Page 99
Control NDV
Unstained cells
REF
MH Combination
Unstained cells
REF
REF cell lines to explain proliferation inhibition after treatment with different doses for 72 hrs
by MTT cytotoxicity assay and by using inverted microscope 40X. It was noticed insignificant
cytopathic effect in unstained REF cell line.
Page 100
Control NDV
Stained cells – Crystal Violate
AMJ13
MH Combination
AMJ13
Figure (4-27): Morphological changes in AMJ13 cell line after treatment with NDV (MOI =
2), MH (62.5µg/ml), and their combination: untreated cells (control), treated cells with
NDV, MH, and combination. All cells were stained by crystal violate and used inverted
microscope 40X to explain morphological changes. A-Control, B- NDV, C- MH and D-
Combination. The blue arrows refer to cytopathic effect areas.
Page 101
Control NDV

MCF-7
MH Combination
MCF-7
Figure (4-28): Morphological changes in MCF-7 cell line after treatment with NDV (MOI =
2), MH (62.5µg/ml), and their combination: untreated cells (control), treated cells with
NDV, MH, and combination. All cells were stained by crystal violate and used inverted
microscope 40X to explain morphological changes. E-Control, F- NDV, G- MH and H-
Combination. The blue arrows refer to cytopathic effect areas.
Page 102
Control NDV

REF
MH Combination
REF
Figure (4-29): Morphological changes in REF cell line after treatment with NDV (MOI = 2),
MH (62.5µg/ml), and combination: untreated cells (control), treated cells with NDV, MH,
and combination. All cells were stained by crystal violate and used inverted microscope 40X
to explain morphological changes. It was noticed insignificant cytopathic effect in stained
REF cell line.
I-Control, J- NDV, K- MH and L- Combination.
Page 103
4.7. Apoptosis assay

An apoptosis assay detects and quantifies the cellular events associated with
programmed cell death, during apoptosis, the cell shrinks and pulls away from its
neighbors. Apoptosis was measured using acridine orange/propidium iodide assay,
the control untreated viable cells appear green, and apoptotic cells in MH and NDV
treated cells appear stain yellow, or orange cells (dead cells) as shown by
fluorescent microscope (Figures (4-30 and 31)).
When the cells were exposed to the virus and MH, the results revealed
synergism between inhibitor and virus which increased apoptosis. It was noticed
more effect from combination than from NDV and MH alone.
In the figure (Figures (4-30 and 31)), red arrows explain viable cells (control)
(green cells) and blue arrows show dead cells (yellow or orange cells) after
treatment with dose of MH and NDV (MH (62.5µg/ml) and NDV (MOI = 2)) for
72 hrs (Figure (4-30 and 31)).
In AMJ13 and MCF-7 cell lines, yellow cells were seen on exposure to NDV
and MH separately while orange cells were seen on exposure to synergism between
them, in comparison to the control which showed green cells that are considered
untreated cells.
NDV induced apoptosis in the infected cells, the results showed that NDV
induced apoptosis in MCF-7 and AMJ13 cells. Based on current results in two
different cell lines (MCF-7 and AMJ13), NDV exhibited oncolytic activity on two
tumor cell lines and to a lower degree on normal cell line (REF).
Synergistic activity for the combination of MH with NDV batter than MH and
NDV in AMJ13 and MCF-7 cell lines with compared to normal REF cell line.
NDV may augment the anti-tumor activity of MH by increasing the cellular
sensitivity to phytotherapy agent (inhibitor) (MH), and this enhanced sensitivity is
partially caused by the induction of apoptosis in response to virulent NDV strain.
Page 104
Control NDV
Stained cells- AO/PI

AMJ13
MH Combination
AMJ13
Figure (4-30): Apoptosis effect in AMJ13 cell lines after treated with NDV (MOI = 2),
MH (62.5µg/ml), and their combination: (B, C and D) treated cells and A untreated cells
(control) in AMJ13. All cells (A-D) were stained by AO/PI and used fluorescent microscope
40X to explain apoptosis effects. The red arrows explain viable cells and blue arrows show
dead cells after treatment with a dose of MH and NDV for 72 hrs.
NDV and MH induced apoptosis which was viewed under a fluorescence

microscope 40X (Figure (4-30 and 31)). Fluorescent microscope, as shown in
Page 105
(Figure (4-30 and 31)), revealed that apoptosis occurred in cells after treatment
with NDV, MH, and their combination, especially cell shrinkage and condensation
of cytoplasm in a dose-dependent manner within 72 hrs of treatment. In stained
REF cell lines, minimal apoptosis showed and insignificant cytopathic effect
(Figure (4-32)).
Control NDV
MCF-7
MH Combination
MCF-7
Figure (4-31): Apoptosis effect in MCF-7 cell lines after treatment with NDV (MOI = 2),
MH (62.5µg/ml), and their combination: (F, G and H) treated cells and E untreated cells
(control) in MCF-7. All cells (E-H) were stained by AO/PI and used fluorescent microscope
40X to explain apoptosis effects. The red arrows explain viable cells and blue arrows show
dead cells after treatment with a dose of MH and NDV for 72 hrs.
Page 106
Control NDV

REF
MH Combination
REF
Figure (4-32): Apoptosis effect in REF cell lines after treatment with NDV (MOI = 2), MH
(62.5µg/ml), and their combination: (J, K, and L) treated cells and I untreated cells (control)
in REF. All cells (I-L) were stained by AO/PI and used fluorescent microscope 40X to
explain apoptosis effects. The red arrows explain viable cells after treatment with a dose of
MH and NDV for 72 hrs. It was noticed insignificant cytopathic effect in stained REF cell.
line.
Page 107
4.7.1 Relationship of apoptosis and morphological changes of cell lines

The morphological changes exhibited by treated cells after 72h of treatment were
attributed to an intense cytopathic effect for the combination therapy. Morphological
changes and apoptosis were more intense in the cells treated by combined NDV and MH
than in those treated with NDV or MH alone. This result indicated that the synergism
between the inhibitor and virus enhanced the morphological changes and percentage of
apoptosis in cancer cells (Figure (4-32. a diagram J and K)). It was noticed weaker or less
intense morphological changes and apoptosis in the normal REF cell line (Figure (4-32. a
M)) than in the breast cancer cell lines treated with MH, NDV, and their combination.
Figure (4-32.a). Fluorescent intensity: Investigation the MH-NDV combined therapy to induce
apoptosis in treated cells using AO/PI: (A-D) AMJ13 cancer cells indicated that MH-NDV
induce apoptosis as evidenced red stained cells, Untreated control cells emitting green
fluorescence (A, E and I). (E-H) MCF-7 cells showing that number apoptotic cells are higher in
the combination treatment. (I – L) There were no effect against REF cells by combination therapy
using MH-NDV. AMJ13 and MCF-7 cells (J, K) expressed significant apoptotic cell death in
compare to monotherapies and the control untreated cells. REF (M) no significant changes in all
treatment modalities. Values represent the (mean ± SD). *P < 0.05, **P < 0.01 and ***P < 0.001.
Page 108
4.8. Glycolysis pathway and its products inhibition
4.8.1. Hexokinase assay

The results revealed deficiency of hexokinase activity due to the effect of MH
(62.5µg/ml) and NDV (MOI = 2). The concentration of MH and MOI of NDV
were selected depending on the values of the combination index (CI). The
statistical analysis data of HK inhibition is shown in (Table (4-11)). The HK
enzyme was evaluated for the comparison of treated and untreated cells at 72 hrs.
Table (4-11): Statistical data of inhibition of hexokinase enzyme after treatment of

cell lines with NDV, MH, and their combination, in comparison with control.
HK (U/ml) - REF Mean ± SD HK (U/ml) – AMJ13 Mean ± SD

Control The group Control
The group
3.3 ± 0.24 3.2 ± 0.24

NDV 3 ± 0.11 NDV 2 ± 0.16
MH 3 ± 0.09 MH 1.4 ± 0.01
Com 2.9 ± 0.21 Com 1.1 ± 0.05
P value (T-test) P value (T-test)
Control NDV Control MH Control NDV Control MH
ns, P= 0.08 ns, P= 0.051 ***S, P= 0.0004 ***S, P= 0.0002
(P <0.05) (P <0.05) (P >0.0005) (P >0.0005)
Control Com NDV Com Control Com NDV Com
ns, P= 0.057 ns, P= 0.438 ***S, P= 0.0003 ***S, P= 0.001
(P <0.05) (P <0.05) (P >0.0005) (P > 0.005)
MH Com NDV MH MH Com NDV MH
ns, P= 0.623 ns, P= 0.614 *S, P= 0.01 **S, P= 0.001
(P <0.05) (P <0.05) (P >0.05) (P >0.005)
P value (T-test)
Control NDV Control MH
**S, P= 0.004 ***S, P= 0.0002
(P >0.01 (P >0.001)
Control Com NDV Com
HK (U/ml) – MCF-7 Mean ± SD
***S, P= 0.0002 **S, P= 0.003
Control 3.0 ± 0.27 (P >0.001) (P > 0.01)
The group
NDV 1.9 ± 0.25 MH Com NDV MH

MH 1.4 ± 0.09 *S, P= 0.0057 *S, P= 0.005
Com 1.2 ± 0.10 (P >0.05) (P >0.05)
The inhibition of HK’s effectiveness by the virus was significant when compared
with the control, or less than MH. MH also inhibited HK significantly in
comparison with the control or more than the virus. The combination of MH and
Page 109
virus was a significantly better inhibitor and enhancer of growth inhibition when
compared with separate treatment or with control of breast cancer cell lines, while
the effect was insignificant in normal cell line. Results showed insignificant
differences between (NDV - control), (NDV - Com), (NDV - MH), (MH - control),
(Com - control) and (Com - MH) in normal REF cell line whereas the difference
between them was significant in breast cancer cell lines (Figure (4-33)). It was
identified a significant reduction in the HK enzyme activity in all the treatment
modalities in the cancer cells but not in the normal cells. MH–NDV combination
efficiently inhibits glycolysis products in the treated breast cancer cells but not in
normal cells.
REF
AMJ13
MCF-7
Figure (4-33) Effect of NDV and MH on the Activity of hexokinase (HK) in glycolysis
inhibition represented in (U/ml). A) in REF, B) AMJ13 and C) MCF7 cell lines after treatment
with NDV at MOI 2, MH at 62.5 µg/ml, and their combination, and comparison with control at
*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 110
4.8.2. Pyruvate assay

From the results shown in (Figure (4-34)), it was observed reduction of
pyruvate concentration after 72 hrs of treatment with MH (62.5µg/ml) and NDV
(MOI = 2). The statistical analysis data of pyruvate inhibition is shown in (Table
(4-12)). The virus reduced the concentration of pyruvate significantly when
compared with control and less than MH. MH also reduced pyruvate significantly
in comparison with control and more than the virus in AMJ13 and almost equal to
the virus in MCF-7. The combination of MH and virus was a significantly better
inhibitor and enhancer of growth inhibition when compared with separate treatment
or with control of breast cancer cell lines.
Table (4-12): Statistical data of inhibition of pyruvate after treatment of cell lines
with NDV, MH, and their combination, and comparison with control.
Pyruvate Pyruvate
(µmol/ml) - REF Mean ± SD (µmol/ml) –AMJ13 Mean ± SD
Control Control
The group
The group
0.45 ± 0.013 0.48 ± 0.03

NDV 0.42 ± 0.027 NDV 0.28 ± 0.016
MH 0.43 ± 0.016 MH 0.25 ± 0.011
Com 0.40 ± 0.034 Com 0.14 ± 0.022
ns, P= 0.2 ns, P= 0.175 ***S, P= 0.0005 ***S, P= 0.0002
(P <0.05) (P <0.05) (P >0.001) (P >0.001)
ns, P= 0.0825 ns, P= 0.436 ***S, P= 0.0005 ***S, P= 0.003
(P <0.05) (P <0.05) (P >0.001) (P >0.001)
ns, P= 0.26 ns, P= 0.735 **S, P= 0.019 *S, P= 0.046
(P <0.05) (P <0.05) (P >0.01) (P >0.05)
P value (T-test)
***S, P= 0.0008 ***S, P= 0.0003
(P >0.001) (P >0.001)
Pyruvate
Mean ± SD Control Com NDV Com
(µmol/ml) – MCF-7
****S, *S, P= 0.017
Control 0.5 ± 0.035
(P >0.0001) (P > 0.05)
The group
NDV 0.23 ± 0.36 MH Com NDV MH

MH 0.23 ± 0.01 **S, P= 0.003 ns, P= 0.893
Com 0.15 ± 0.01 (P >0.01) (P <0.05)
Page 111
While, the effect was insignificant in normal REF cell line. The results showed
insignificant differences between (NDV -control), (NDV - Com), (NDV - MH),
(MH - control), (Com - control) and (Com -MH) in normal REF cell line, and
(NDV - MH) in MCF-7 cell line, whereas the difference between them was
significant in breast cancer cell lines (Figure (4-34)). It was identified a significant
reduction in the pyruvate concentration in all the treatment modalities in the cancer
cells but not in the normal cells.
REF AMJ13
MCF-7
Figure (4-34) Effect of NDV and MH on the Pyruvate concentration in glycolysis inhibition
represented in (μmol/ml). A) in REF, B) AMJ13 and C) MCF7 cell lines after treatment with
NDV at MOI 2, MH at 62.5 µg/ml, and their combination, and comparison with control at
*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 112
4.8.3 ATP assay

From the results shown in (Figure (4-35)), it has been noticed that ATP
concentration decreased after treatment with MH (62.5µg/ml), NDV (MOI = 2),
and their combination for 72 hrs. (Table (4-13)) shows the statistical analysis. The
virus reduced the concentration of ATP significantly when compared with control
or more than MH. MH also reduced ATP significantly in comparison with control
or less than the virus. The combination of MH and virus was a significantly better
inhibitor and enhancer of growth inhibition when compared with separate treatment
or with control of breast cancer cell lines. Whereas, the effect was insignificant in
normal REF cell line.
Table (4-13): Statistical data of inhibition of ATP production after treatment of cell
lines with NDV, MH, and their combination, in comparison with the control.
ATP ATP
Mean ± SD Mean ± SD
(mmol/L) - REF (mmol/L) – AMJ13
Control
The group
1.1 ± 0.05 Control

The group
1.2 ± 0.057
NDV 1 ± 0.11 NDV 0.58 ± 0.056
MH 1 ± 0.04 MH 0.61 ± 0.077
Com 1 ± 0.08 Com 0.39 ± 0.059
ns, P= 0.33 ns, P= 0.06 ****S, ****S,
(P <0.05) (P <0.05) (P >0.0001) (P >0.0001)
ns, P= 0.09 ns, P= 0.658 ****S, **S, P= 0.0032
(P <0.05) (P <0.05) (P >0.0001) (P > 0.01)
ns, P= 0.64 ns, P= 0.874 **S, P= 0.0036 ns, P= 0.512
(P <0.05) (P <0.05) (P >0.01) (P <0.05)
P value (T-test)
****S, ****S,
ATP (P >0.0001) (P >0.0001)
Mean ± SD
(mmol/L) – MCF7 Control Com NDV Com
Control 1.3 ± 0.074 ****S, ***S, P= 0.0002
(P >0.0001) (P > 0.001)
The group
NDV 0.65 ± 0.061

MH Com NDV MH
MH 0.69 ± 0.065
***S, P= 0.0001 ns, P= 0.397
Com 0.35 ± 0.04 (P >0.001) (P <0.05)
Page 113
The results showed insignificant differences between (NDV -control), (NDV -

Com), (NDV - MH), (MH - control), (Com - control) and (Com -MH) in normal
REF cell line, and (NDV - MH) in AMJ13 and MCF-7 cell line, whereas the
difference between them was significant in breast cancer cell lines (Figure (4-35)).
It was identified a significant reduction in the ATP concentration in all the
treatment modalities in the cancer cells but not in the normal cells. MH–NDV
combination efficiently inhibits glycolysis and inhibit ATP production in the
treated breast cancer cells but not in normal cells.
REF
AMJ13
MCF-7
Figure (4-35) Effect of NDV and MH on the ATP concentration in glycolysis inhibition
represented in (mmol/L). A) in REF, B) AMJ13 and C) MCF7 cell lines after treatment with
NDV at MOI 2, MH at 62.5 µg/ml, and their combination, and comparison with control at
*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
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4.8.4 pH measurements
Measurements of the pH scales of the treated and control cell lines are shown
B after treatment of MCF-7CC
in (Figure (4-36)). Elevation was noticed in the pH scale
and AMJ13 cell lines with 62.5µg/ml MH, 2 MOI NDV, and their combination for
72 hrs. The reduction of acidity (lactic acid) was caused by the reduced of lactate
concentration in comparison with the control. When used separately, the virus and
MH inhibited the acidity significantly when compared with the control. The
combination of MH and virus was a significantly better inhibitor and induced
proliferation inhibition when compared with control of breast cancer cell lines,
whereas the effect of NDV, MH, and their combination was less in normal REF
cell line (Figure (4-36)). The statistical analysis data of acidity is shown in (Table
(4-14)).
Normalization measurements revealed that, when used separately, the virus

and MH inhibited the acidity significantly in breast cancer cell lines when
compared with normal REF cell line. The combination of MH and virus was a
significantly better inhibitor when compared with normal REF cell line (Figure
(4-37)).
Table (4-14): Statistical data of acidity after treatment of cell lines with NDV, MH,
and their combination, and comparison with control.
pH - REF Mean ± SD pH – AMJ13 Mean ± SD

Control 7.3 ± 0.1 Control 6.3 ± 0.12
The group
The group
NDV 7.7 ± 0.15 NDV 7.3 ± 0.1

MH 7.6 ± 0.1 MH 7.3 ± 0.1
Com 8.2 ± 0.15 Com 8.2 ± 0.2
pH – MCF-7 Mean ± SD
Control 6.3 ± 0.15
The group
NDV 7.4 ± 0.1

MH 7.4 ± 0.15
Com 8.2 ± 0.15
Page 115
Results showed that normalization measurements have significant differences

between (AMJ13 and REF) and (MCF7 and REF) after treatment with NDV, MH,
and their combination. MH–NDV efficiently decrease cancer cell glycolysis
products pyruvate, ATP, and acidity (represent lactic acid) compared to
monotherapies.
REF AMJ13
MCF-7
Figure (4-36) Effect of NDV and MH on the acidity of REF, AMJ13, and MCF7 cell lines
based on pH values: pH values of A- the REF cell line, B- the AMJ13 cell line, and C- the
MCF7 cell line after treatment with MH at 62.5 µg/ml, NDV at MOI 2, and their combination,
and comparison with control. at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 116
MH–NDV induce significant reduction on the level of acidity in both breast

cancer cell lines but not in normal embryonic REF cells, see (Figure (4-36)). A
significant reduction was identified in the acidity in all the treatment modalities in
the cancer cells but not in the normal cells. MH–NDV combination efficiently
inhibits glycolysis products in the treated breast cancer cells but not in normal cells.
NDV MH
Normalization pH = [Sample] / [ Control]
Com
Figure (4-37) Normalization (Nor): effect of treatment with MH at 62.5 µg/ml, NDV at MOI
2, and their combination (Com) on the acidity of cell lines based on pH values. (A)
Normalization of NDV–cell lines, (B) Normalization of MH–cell lines, (C) Normalization of
Com–cell lines. at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Page 117
Further investigation to the mechanism by which MH–NDV inhibit breast

cancer cell proliferation was done through examination whether MH–NDV
efficiently decrease cancer cell glycolysis production of pyruvate, ATP and acidity
(represent lactic acid) compared to monotherapies as shown in comparison between
tests (Figure (4-38)).
Remarkably, MH–NDV combination induce significant reduction of the levels

of pyruvate, ATP and acidity in both breast cancer cell lines but not in normal
embryonic REF cells.
4.8.5 Comparison between tests

MH–NDV combination efficiently inhibits glycolysis products in the treated
breast cancer cells but not in normal cells in compare to monotherapies (Figure (4-
38 A)). MH–NDV combined therapy induce significant decrease in the activity of
hexokinase in AMJ13 and MCF-7 cancer cell lines, while there is no significant
reduction in normal REF cells.
(B and C) measurement of pyruvate and ATP levels in cancer cells

significantly reduced in MH-NDV combination therapy in compare to both
monotherapies and untreated control cells. However, the reduction in the normal
REF cells was not significant.
(D) Measurements of pH levels in AMJ13 and MCF-7 breast cancer cells

indicate that MH-NDV combination treated cells supernatant was more alkaline in
compare to both monotherapies significantly, while untreated control cells in breast
cancer cells showing acidity, as well as the control and treated non-cancerous REF
cells was alkaline. All data shown are means ± SD (*P < 0.05) data from three
different experiments.
Page 118
Figure (4-38). Comparison between tests (HK, Pyruvate, ATP and pH)
Page 119
CHAPTER-FIVE
Chapter Five …………………….…..…………..……………… …..………… Discussion
5. Discussion
5.1. Newcastle disease Virus Preparation and Collection

In tumor pathogenesis and metastasis, defects in apoptosis are essential. They
allow tumor cells to overcome nutrient deprivation, absence of growth-stimulating
signals and presence of growth-inhibitory signals. Newcastle disease virus Iraqi
Strain is interesting oncolytic agent with promising anti-tumor properties. One of
the major anti-tumor properties is apoptotic induction (Al-Shammari, 2018). (Al-
Shammari, 2018) revealed that NDV had powerful effect on inducing apoptosis in
AMN3 mammary adenocarcinoma in vitro, this study is similar to the current study
that revealed that NDV increase apoptosis induction in breast cancer cell lines.
The results showed that Iraqi attenuated strain of NDV has the ability to kill
most chicken embryos within 48 hrs as described by (Alexande, 2001). The isolated
virus showed Syncytia formation which is a hallmark of NDV infection in host
cells. It was reported by (Qin et al., 2008) that Syncytia are a typical cytopathic
effect caused by the virus and can lead to tissue necrosis, and that it might also be a
mechanism of virus spread. NDV strains can be categorized as velogenic (highly
virulent), mesogenic (intermediate virulence), or lentogenic (non-virulent).
Mesogenic strains in 60–90 hrs and lentogenic strains in more than 90 hrs.
(Alexander and Senne, 1998).
Newcastle disease virus was quantified by the hemagglutination assay.

Hemagglutination (adhesion of RBC) test showed a positive result as a typical
hemagglutination mesh pattern of chicken red blood cell 256 HAU, the virus is able
to agglutinate human, or animal red blood cells (Wood et al., 2014). The virus
induces syncytia by fusing infected cells with neighboring cells leading to the
formation of multinucleated enlarged cells. This event is induced by surface
expression of viral fusion protein that is fusogenic directly at the host cell
Page 120
membrane. Syncytia is an ideal cytopathic effect caused by the virus, which leads
to tissue necrosis. Usually these syncytia are the result of expression of a viral
fusion protein at the host cell membrane during viral replication, it is the mark of
the mechanism of virus spread (Freeman et al., 2014).
The family Paramyxoviridae includes many viruses such as NDV, that

significantly affect animal health. An essential step in the paramyxovirus life cycle
is viral entry into host cells, mediated by virus-cell membrane fusion. Upon viral
entry, infection results in expression of the paramyxoviral glycoproteins on the
infected cell surface. This can lead to cell-cell fusion (syncytia formation), often
linked to pathogenesis. Thus, membrane fusion is essential for both viral entry and
cell-cell fusion, and it is an attractive target for therapeutic development (Aguilar et
al., 2016).
NDV Iraqi strain is a broad-spectrum oncolytic agent that can destroy tumor
cells (Al-Shamery, 2003) demonstrated that NDV Iraqi strain exhibits oncolytic
activity to wide range of tumor cells such as mammary adenocarcinoma,
Rhabdomyosarcoma (malignant tumor involving striated muscle tissue), and
freshly cultured melanoma cell lines, where the virus showed the characterized
features of NDV cytopathic effects and syncytia formation in the tumor cells, this
were also confirmed by other researchers (Bar-Eli et al., 1996).
(Wu et al. 2016) demonstrated that NDV Anhinga strain could not only
directly kill tumor, but could also elicit immune reaction and a potent
immunological memory in vivo which could become an effectual vector for tumor
treatment.
(Al-Shammari et al., 2016) revealed that NDV is a very promising anti-
malignancy agent when combined with other antitumor agents and when the
combinations of NDV and Doxorubicin were tested on normal rat embryo
fibroblasts, there seemed to be no favorable interactions at the same low doses; but,
Page 121
at higher doses there was toxicity on the normal cells, this similar to current study
that revealed no effect on the normal cell line at low doses. NDV has been shown
to be selective in replicating in tumor cells and not replicating on normal rat
embryo fibroblast cell lines (Reichard et al., 1992), This proves the increased effect
of the virus on cancer cell lines and its non-effect on normal REF cells in the
present study.
5.2. Tissue Culture Infective Dose 50 (TCID 50) of NDV in Vero cell line
Tissue culture infective dose fifty percent (TCID 50) is an assay used to
determine the titer of virus (Eliyana and Redhuan, 2008). According to (Florence et
al., 1992), TCID50 refers to the ability of a certain virus dilution to infect 50% of
the cell cultures inoculated. Therefore, a titer is defined as a number of infectious
virus units per unit volume.
The TCID50 titer provides qualitative information on dose, or dilution that

causes cytopathic effect (CPE) in 50% of the sample, instead of giving the
quantitative amount of virus infection that is present in a sample. Newcastle disease
virus formed plaques on Vero cell line and normal tissue bird such as chick embryo
fibroblast in vitro. It did not form plaques on any normal fibroblast cell line from
other sources (Cuadrado - Castanoa et al., 2015).
The gated of TCID50 (124.6 × 106) used in the present study ensure the high
virulent of NDV. When each infected cell produces virus and eventually lyses, only
the immediately adjacent cells become infected. Each group of infected cells is
referred to as a plaque that is surrounded by uninfected cells. After several
infection cycles, the infected cells in the center of the plaques begin to lyse, and
then the virus moves to the uninfected cells gradually (Fu et al., 2011). (Ravindra et
al. 2008) showed that NDV can induce apoptosis in Vero cells after exposure and
replication for 48 hrs.
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5.3 Cytotoxicity and IC50 of NDV, MH, and DXL in cancer cell lines
The in vitro results of this study revealed that an increased concentration of
MH, DXL, and MOI of NDV increased in cytotoxicity and antiproliferation effect
against breast cancer cell lines, because MH caused inhibition of glycolysis and this
effect on glycolysis pathway reduced HK activity, [Pyruvate], [ATP], and acidity
(lactic acid). The results are similar to those related to inhibition of glycolysis for
3-bromopyruvate and 2-deoxyglucose, results in mitochondrial pathway-induced
apoptosis (Danos et al., 2008); (Kim et al., 2008).
Docetaxel, a chemotherapy drug, was used as a positive control for

comparison with the effect of D-Mannoheptulose as phytotherapy, NDV as
virotherapy, and their combination on normal and breast cancer cell lines. The
effect of MH and NDV had a significant killing level in breast cancer cells, while it
had a non-significant killing level in normal cells. This was caused by differences
between normal and breast cancer cells in receptors, cell environment, pH,
apoptosis, and the effect of morphological changes. These results support the study
(Mikirova et al., 2017) as glycolysis increased in tumor cells in comparison with
normal cells. D-Mannoheptulose has been found to inhibit glucokinase and
hexokinase of rat liver (Coore and Randle, 1964).
The half maximal inhibitory concentration (IC50) is a measure of the potency

of a virus, drug, and inhibitor in inhibiting breast cancer cell lines. IC50 is a
quantitative measure that indicates the amount of a particular inhibitory substance
(DXL, NDV, and MH) needed to inhibit proliferation of breast cancer. The results
showed that there are significant differences in IC50 between breast cancer cell
lines in comparison to REF cell line after treatment with virus, inhibitor, and drug.
This is because their effect in cells differs according to their type as virotherapy,
phytotherapy, and chemotherapy, and it is due to the difference between the nature
of normal cells and cancer cells. Insignificant difference was found between MCF-
Page 123
7 and AMJ13 cell lines. Cytotoxicity was measured between REF, AMJ13, and
MCF-7 cell lines at specific concentrations after treatment with NDV, MH, and
DXL. It was found that, the effect of NDV, MH, and DXL on breast cancer cell
lines was very clear compared to normal REF cell line where a significant
difference was found between them.
5.4 Synergistic effect of NDV and MH on cells lines

Inhibition of growth induced by high concentrations of the virus, inhibitor,
and their combination is the best. Also, treatment of NDV and MH showed
enhancement to the growth inhibition properties in breast cancer cell lines more
than normal cell line. The combination of MH and virus was a significantly better
promoter and enhancer of growth inhibition when compared with separate
treatment in breast cancer cell lines.
While in normal REF cell line, the effect of virus, inhibitor, and their
combination was insignificant and induced cytotoxicity less than in breast cancer
cell lines. From the results, a significant induction was identified in growth
inhibition in all the treatment modalities in the breast cancer cell lines but not in the
normal cells line. Dose response evaluation of treatments differed significantly
between REF and breast cancer cell lines. Combination between NDV and MH in
cell lines showed high synergism in MCF-7 at all interactions between MH and
NDV, and also in AMJ13 with the exception one value which was considered
additive.
On the other hand, all REF cell line values indicated antagonism and additive
effect which is neglected effect as there was no killing effect reached 50% at all
tested concentrations. The results implied that synergism between inhibitor and
virus had more effect in inhibition of proliferation, anticancer growth action, and
caused increase in cytotoxicity, inhibition of HK, pyruvate, ATP, and acidity which
Page 124
lead to induced morphological changes and apoptosis. Isobologram analysis, or

Chou–Talalay equation (Combination index) showing the synergistic effect
between NDV and MH in 9 different doses used on normal and breast cancer cell
lines. Isobologram analyses in AMJ13 and MCF-7 cell line revealed synergism
between NDV and MH, indicating that the effect is synergistic, while1 point in
AMJ13 indicated that the effect is antagonism. Also, isobologram analyses in REF
cell line revealed antagonism between NDV and MH, indicating the effect is
antagonistic, or less interaction in combination between virus and inhibitor.
Combination therapies give great advantages represented by better

effectiveness, decreased cancer toxicity, and reduced development of drug
resistance. Therefore, these advantages became a standard for the treatment of
several diseases and are a promising approach in indications of unmet medical need
(Foucquier and Gued, 2015). There are many methods to determine the synergy of
combination of two, or more chemotherapy combined for each other to treat a lot of
diseases and cancers, these methods were applied by CompuSyn program (Chou,
2010; Rodea-Palomares et al., 2010; W. Humphrey et al., 2011). The selective
nature of NDV makes it an ideal virotherapy agent. Its ability to replicate in tumor
tissue allows massive amplification of the input dose at the tumor site, and at the
same time, its lack of replication in normal tissues allows efficient clearance and
reduction of toxicity (Ravindra et al., 2009).
The results of cytotoxicity activity of combination (NDV and MH) on cell

lines showed the inhibitory rates. These results reported a synergistic effect of 3
combination concentrations that were used on MCF-7 and AMJ13 cell lines. The
results of the present study reflects the great importance of combination therapy in
the treatment of breast cancer and it turned out that the high rates from the
combination of MH and NDV that were obtained in the two cell lines tumors
AMJ13 and MCF-7, may also be useful in treating other types of cancer, even if
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they have different genetic makeups (AL-Shammari et al., 2016). The synergistic
effect was a concept that NDV may increase anti-tumor activity by rising cellular
sensitivity to the therapeutic agents due to enhanced susceptibility that is partially
caused by the induction of apoptosis which was previously confirmed to be induced
by NDV (Naik et al., 2011).
The use of MH with NDV decreased the proliferation activity of cancer cell
culture, decrease signaling via the major pathways that promote cancer cell culture
progression as in the study of (Applegate and Lane, 2015). This interpretation
outcome of combining MH with NDV increased apoptosis of cancer cells.
A synergistic interaction allows the use of lower doses of the combination

constituents, a situation that may reduce adverse reactions. Most previous study
results, which relate to the effect of Newcastle disease virus and MH separately on
some cancers, showed the effects and results closely. Combination of NDV and
MH synergistically enhanced the cytotoxic effect on cancer cells than their separate
effect. Another study showed that synergistic antitumor combination of NDV and
2-Deoxy glucose had a synergistic and greater effect on cancer cells than their
separate use in breast cancer cell lines (human AMJ13 and murine AMN3) cell line
(Salim, 2016).
From the results of cytotoxicity of NDV, MH, and their combination in

AMJ13, MCF7, and REF cell lines, which have already been explained. It can be
concluded that increased MOI of NDV and concentration of MH increased
cytotoxicity, while their combination caused a better result by inducing
cytotoxicity, decreasing proliferation of cancer cells, and elevating growth
inhibition as the virus induced apoptosis and MH inhibited hexokinase which lead
to inhibition of glycolysis. A significant difference was found in cytotoxicity
between normal REF cell line and breast cancer cell lines of the virus, inhibitor,
and drug, while insignificant difference was found between them in MCF-7 and
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AMJ13 cell lines. There is a significant difference between effect the DXL at 1.25
μg/ml and combination at (2MOI-NDV and 62.5 μg/ml MH) on the treatment of
normal and breast cancer cell lines. This shows that combined therapy is better than
chemotherapy alone. The results of some studies such as (Aghi et al., 2006)
showed antagonism when they used a combination of NDV with other
chemotherapies. Also, this type of analysis determines synergy and it is an
acceptable method of assessing the efficacy of chemotherapy combination therapies
as well as combination chemotherapy and viral therapy (Mullerad et al., 2005).
The findings of the present study showed that the combination of NDV and
MH gave a high percentage of inhibition rates in three concentrations with cancer
cell lines such as MCF-7. Therefore, the present study suggests that, the result of
additive in one concentration of the AMJ13 cell line is possibly due to low
concentrations of the combination.
On the other hand, the remaining concentrations showed a synergistic activity

on AMJ13 cell line, which means that the synergism in the combined effect
observed, is significantly greater than the expected activity. Other studies have used
different methods, one by halving the administered dose of chemotherapeutic agent
(rituximab, and doxorubicin) and completing the eliminated dose with NDV in
order to reduce the toxicity of chemotherapy (Al shammari et al., 2016).
One investigator has hypothesized that combination will act synergistically
(McCart et al., 2000).
The theory supported that combination chemotherapy in assaulting tumor cells

using different mechanisms of action can prevent tumor cells from having enough
time to improve resistant to treatment (Post et al., 2003). The strategy of
combination therapy is by attacking tumor cells through different mechanisms of
action to prevent cancer cells from developing resistance to therapy (Kumar et al.,
2014).
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The mechanism of synergistic activity for the combination of MH with NDV

is unknown, but it can be proposed a few hypotheses. First, NDV may augment the
anti-tumor activity of MH by increasing the cellular sensitivity to phytotherapy
agent (inhibitor) (MH), and this enhanced sensitivity is partially caused by the
induction of apoptosis in response to virulent NDV strain. Second, a synergistic
dose of MH may augment the viral replication. Each agent may work
independently on different cell populations. In addition, virotherapy with NDV
may complement the anti-tumor activity of MH which selectively targets tumor cell
populations that are resistant to chemotherapy or other conventional treatments.
This may be of important value because most human tumors consist of a mixture of
cells that have a different genetic makeup. Heterogeneity in the tumor cell
populations may be the major reason most monotherapies fail to achieve complete
tumor remission (Henderson and Yu. 2002).
Moreover, one of the objectives of this study was to reduce the toxic side effects
of chemotherapy or other conventional treatments by using combination therapy
(virotherapy and phytotherapy) in breast cancer cell lines. Also, this was clear by
comparison with Docetaxel drug (DXL) as positive control and a chemotherapy.
This can be achieved by reducing the administered dose while maintaining the
same or stronger for anti-tumor activity when using chemotherapy with
combination therapy.
5.5 Study of morphological changes and apoptosis in cell lines

The morphological changes showed the presence of cytopathic effect, which
was detected in treated cells, a large number of cramped cells with granulation,
formation of syncytial, and shrinkage of cells were noticed when compared with
the control. Morphological changes and apoptosis showed after treatment with MH
(62.5µg/ml) and NDV (MOI = 2) depends on the combination index that has been
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measured for NDV and MH for 72 hrs. Apoptosis was seen in stained cells by
AO/PI and in treated cells as red cells, while viable cells were green.
Apoptosis is a spontaneous process of programmed cell death, which can be

induced by a variety of physical and chemical factors, and it is precisely regulated
by an organism. Although there are three predominant signaling pathways in
apoptosis (the mitochondrion, the death receptor, and the endoplasmic reticulum
signaling pathways), the integration and amplification of apoptotic signaling
usually occurs at the mitochondrial level (Guo et. al, 2016). Cancer cells continue
to divide uncontrollably as they can ignore signals that normally tell cells to stop
dividing or that begin a process known as programmed cell death, or apoptosis
(Gatenby and Gillies, 2004).
Also study of apoptosis using AO/PI staining by fluorescent microscopy was

similar with the study (Danos et al., 2008) (Kim et al., 2008) which revealed that
inhibition of glycolysis, for specific inhibitors, 3-bromopyruvate and 2-
deoxyglucose, results in mitochondrial pathway-induced apoptosis. Also, a
compatible research study confirmed that over-expression of LDH-B by
panepoxydone inhibitor in cancer cell lines leads to enhanced apoptosis and
mitochondrial damage (Arora et al., 2015).
NDV induced apoptosis in various cancerous cells, the critical relationship

between apoptosis and cancer signifies that any therapeutic strategy aimed at
specifically triggering apoptosis in cancer cells will have a potential therapeutic
effect (Ravindra et al., 2008). NDV induced apoptosis in the infected cells (Al-
Shammari et al., 2015).
NDV stimulated specific anti-tumor immune response and was able to induce
endoplasmic reticulum stress; all these mechanisms beside the direct cytolytic
effect are secondary to virus replication (Al-Shammari and Yaseen, 2012). NDV
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has been demonstrated to induce apoptosis in various cancerous cells (Ravindra et

al., 2008), and human breast carcinoma cells (Washburn et al., 2003).
The results showed that NDV induced apoptosis in MCF-7 and AMJ13 cells
by inducing mitochondrial apoptosis. (Al-Shammari and Yaseen, 2012) revealed
that NDV has powerful effects on inducing apoptosis in AMN3 mammary
adenocarcinoma in vitro. Based on current results in two different cell lines (MCF-
7 and AMJ13), NDV exhibited oncolytic activity on two tumor cell lines and to a
lower degree on normal cell line (REF). Previous studies have shown that a virulent
NDV strain is oncolytic (Al-Shammari et al., 2016). This strain showed antitumor
activity both in vitro and in vivo in a clinical trial (Csatary et al., 1999).
In current study was found alternative or supportive treatment for

chemotherapy or other conventional treatments, by using the inhibitor (MH) or
virus (NDV) or their combination (MH-NDV). Also, it was obtained an anti-tumor
efficacy and proliferation inhibition of cancer cells through glycolysis inhibition
and induction of apoptosis. This can be an alternative treatment as combination
therapy that can be used to reduce the dose of chemotherapy or other conventional
treatments with the maintaining the same or more anti-proliferative activity and
overcoming chemotherapy resistance or other treatments.
5.7 Glycolysis inhibition

Malignant cells use glycolysis as the main source for energy, meanwhile, a
mass of lactate and pyruvate is generated during the process. However, lactate, the
end product of glycolysis, is exported to the extracellular medium and contributes
to the acidification of tumor microenvironment, which favors tumor development,
invasion, chemotherapy and radiotherapy resistance, recurrence of tumor, and
suppresses the immune defense of anticancer (Goetze et al., 2011).
D-Mannoheptulose has been found to inhibit glucokinase and hexokinase of rat
Page 130
liver and other tissues (Coore and Randle, 1964), and it competitively inhibits the
phosphorylation of glucose in homogenates of isolated islets of Langerhans
(Malaise et al., 1968). Cancer cells derive most of their energy from glycolysis that
is, glucose is converted to lactate for energy followed by lactate fermentation, even
when oxygen is available. This is termed the Warburg effect. This produces far less
energy than oxidative phosphorylation and as cancer cells require a lot of energy to
grow this seems paradoxical.
The Warburg effect may be beneficial to cancer cells because it provides

precursors for many biosynthetic pathways. These precursors include amino acid
precursors and NADPH and ribose sugars for DNA and RNA synthesis. The
Warburg effect may be caused by impaired oxygen sensing in cancer cells.
Glycolytic enzymes such as GLUT1, lactate dehydrogenase, pyruvate kinase and
the lactate exporter are unregulated in cancer cells, whilst pyruvate dehydrogenase
is inhibited leading to increased glycolytic flux and impaired ability of pyruvate to
enter oxidative phosphorylation (Fadaka et al., 2017).
The results mentioned demonstrated that MH could have inhibited glycolytic

flux in MCF-7 and AMJ13 cell lines by suppressing the activity of hexokinase
(HK), resulting in ATP depletion and decreasing lactate generation, which induced
insufficiency of energy supplement to support cancer cells mitosis, proliferation,
and invasion. The acidic microenvironment, which favors cancer cells resistance to
chemotherapy, was disrupted by decreasing production of lactate. This agrees with
(Lu et al., 2011).
The effect of MH in inhibition of hexokinase and effect of NDV in induction

of apoptosis in addition to synergism between them led to the decrease in HK
activity, pyruvate, ATP concentration, and acidity in breast cancer cell lines. The
virus, MH, and their combination inhibited the effectiveness of HK and pyruvate,
ATP concentration, and acidity significantly when compared with the control.
Page 131
Therefore, it led to prevention of proliferation of cancer cells and induced growth

inhibition, this agrees with (Mathupala et al., 2006) which mentioned that HK via
its mitochondrial location suppresses the death of cancer cells. For these reasons,
targeting key enzyme by inhibitors led to inhibition of glycolysis, therefore
suppressing proliferation of cancer cells. The combination of MH and virus was a
significantly better inhibitor and enhancer of growth inhibition when compared
with separate treatment or with control of breast cancer cell lines, while the effect
was insignificant in normal REF cell line.
A significant reduction in the ATP concentration was identified in all the

treatment modalities in the cancer cells but not in the normal cells. MH–NDV
combination efficiently inhibits glycolysis products in the treated breast cancer
cells but not in normal cells. Because the concentration of pyruvate depends on the
action of hexokinase in the glycolysis pathway, the decrease in hexokinase activity
led to deficiency in pyruvate concentration. The results in this study have shown
that MH and NDV could prominently suppress the generation of intercellular ATP
by inhibition of glycolysis and induction of apoptosis. A decrease of ATP related to
another study revealed that, mitochondrial membrane potential (Δψm) is necessary
for the activity of ATP synthase in normal cells, which generates ATP. The loss of
Δψm and damage of the mitochondrial inhibited the activity of ATP synthase,
leading to ATP deficiency (Vander Heiden et al., 2009).
In another study, decreased ATP level in MCF-7 and MDA-MB-231 cells was
identified to be effective in inhibiting energy metabolism in breast cancer cells
(Cheng et al., 2013); (Ding et al., 2012). To obtain sufficient energy for growth and
proliferation, cancer cells require an increased glucose uptake. In hypoxic
conditions, glucose via glycolysis is catabolized to lactate to produce ATP. In
hypoxic conditions, the monocarboxylate transporters MCT4 are up regulated,
enabling cells to rapidly dispose the accumulated lactic acid (Ullah et al., 2006).
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(Fadaka et al., 2017) mentioned that glycolysis generates ATP more rapidly
than OXPHOS, and this offers a selective advantage to rapidly growing tumor
cells. When glycolysis is inhibited, lactate production is completely stopped and
there is an abrupt decrease of intracellular ATP concentration (Gonin-Giraud et al.,
2002), this is consistent with the present study. This study is similar to findings
(Arora et al., 2015) by using panepoxydone inhibitor significantly reduces lactate
production. The results have also supported an increased glycolysis in transformed
cells (cancer cells) in comparison with normal cells (Mikirova et al., 2017).
Also, the decrease of ATP that was proved in the current study is in agreement
with the study (Lu et al., 2011) which proved that ATP depletion induced
insufficiency of energy supplement to support cancer cells mitosis, proliferation,
and invasion. Through the results, an increase in pH measurements (less acidity)
was noticed in breast cancer cell lines in comparison to the control, and less effect
in normal REF cell line after treatment with MH and NDV for 72 hrs, which lead to
a decrease of acidity.
This decrease of acidity results from the reduction of lactate concentration that
resulted from the decrease in pyruvate concentration which lead to growth
inhibition and anti-proliferation of cancer cells. This result is compatible with the
study (Arora et al., 2015) which proved that deficiency in lactate concentration
leads to reduction in the acidity of the cell environment, which is considered
favorable in preventing proliferation of cancer cells as they grow in acidic
environments. Release of lactate from the cell is one of the main causes of
extracellular acidosis (Fadaka et al., 2017).
One important difference is that cancer cells are less specialized than normal
cells (Elstrom et al., 2004). In the presence of MH, glycolysis is inhibited because
there is no production of glucose-6-P, hence no increase in ATP concentration
which is necessary for cancer cells. Warburg originally proposed that the aerobic
Page 133
glycolysis in cancer cells was due to a permanent impairment of mitochondrial

oxidative phosphorylation. Warburg observed that cancer cells tend to convert most
glucose to lactate (Kroemer and Pouyssegur, 2008).
Warburg originally hypothesized that cancer cells develop a defect in the

mitochondria that leads to impaired aerobic respiration and a subsequent reliance
on glycolytic metabolism (Vander Heiden et al., 2009). Each reaction in the
glycolytic pathway is catalyzed by a specific enzyme or enzyme complex (Pelicano
et al., 2006). This effect means higher glucose uptake of cancer cells compared to
normal cells, which can be used as a target for selective therapy by targeting
glucose metabolism (Aft et al., 2002).
Through the current results, a decrease in the ATP concentration was observed
by the effect of the inhibitor and the virus or their combination, and this led to
treatment of breast cancer, based on the study (Xu et al., 2005), which showed that
cellular ATP depletion is the main event for cancer treatment. It has also been
reported that the Warburg effect is generally attributed to a "breathing injury" or a
defect in the mitochondrial oxidative phosphorylation process, forcing cells to use
the glycolic pathway to generate ATP, which affects ATP concentration later. It
should be noted that previous studies have suggested that apoptosis is an ATP-
dependent process, and depletion of ATP tends to cause cell death by necrosis
(McConkey, 1998).
(Khalifa et al., 2013) mentioned that the methanolic extracts of Avocado fruit
and leaves are highly promising to be used in cancer therapy and antioxidant
treatments, this corresponds with the current study in which the inhibitor (MH) was
used, which is originally extracted from the avocado plant. D-mannoheptulose is a
seven-carbon sugar commonly found in avocado fruits that inhibits HK, thus
reducing glycolysis. According to (Granchi & Minutolo, 2012) study, D-
mannoheptulose is considered safe to use as a hexokinase inhibitor to treat breast
Page 134
cancer cell lines and thus does not cause side effects on human health. The
phenomenon of aerobic glycolysis increase in cancer cells was first described by
Otto Warburg.
Otto Warburg showed that compared to normal cells, malignant cells exhibit
significantly elevated glycolytic activity even in the presence of sufficient oxygen,
and considered this phenomenon as the most fundamental metabolic alteration in
malignant transformation, or ‘the origin of cancer cells’ (Warburg, 1956), This is
agree with the current study, where an increase in the concentration of pyruvate,
ATP and acidity was observed in cancer cells compared to normal cells as control.
also, it was observed by increasing the effectiveness of treatment by NDV and MH
and their combination in breast cancer cell lines compared to normal cells.
Cancer cells consume far more glucose than normal cells to maintain sufficient ATP
supply for their active metabolism and proliferation. As such, maintaining a high
level of glycolytic activity is essential for cancer cells to survive and growth.
This metabolic feature has led to the hypothesis that inhibition of glycolysis
may severely abolish ATP generation in cancer cells and thus may preferentially kill
the malignant cells (Munoz-Pinedo et al., 2003; Izyumov et al., 2004; Xu et al.,
2005), and this is agree with the present study, where an increase in the
concentration of ATP was observed in cancer cells compared to normal cells as
control. Also, A decrease in ATP concentration was also observed after treatment
with NDV, MH and their combination, and thus agree with the hypothesis.
Inhibition of glycolysis alone may not be sufficient to effectively kill the

malignant cells. It has been suggested that ATP depletion should reach certain
thresholds in order to trigger cell death by apoptosis or necrosis processes, with a
depletion of 25–70% ATP leading to apoptosis ( shrinkage, active process), and an
over 85% ATP depletion causing necrosis (swelling , passive process, inflammation
and cell lysis) (Lieberthal et al., 1998).
Page 135
In current study was used the combination therapy by synergism between NDV
and MH to get ATP depletion. Where it was observed a significant decrease in ATP
concentration after combination therapy in AMJ13 and MCF-7 cell lines. One way
to achieve a high level of ATP depletion and improve therapeutic activity is to
combine multiple ATP-depleting agents with different mechanisms of action
(Martin et al., 2001). This is what happened in present study through the use of two
agents (NDV and MH) to depletion of ATP. Where a significant decrease in ATP
concentration was also observed after treated with combination therapy in
comparison with virus and inhibitor separately.
Other studies have provided compelling evidence showing that cancer cells
with mitochondrial defects or under hypoxia are highly sensitive to glycolysis
inhibition (Pelicano and Martin, 2006). It should be noted that previous studies have
suggested that apoptosis is an ATP-dependent process and that depletion of ATP
tends to cause cell death (Xu et al., 2005).
Through this clarification, it was concluded that there is a relationship between

ATP depletion and apoptosis and in this study, it has been proven that ATP
depletion led to an increase in cell death, its mean that it promoted programmed cell
death. In the current study was suggested that inhibition of glycolysis is an effective
strategy to kill cancer cells. This novel approach may have broad applications in
cancer treatment, considering the prevalent Warburg effect observed in a wide
spectrum of human cancers.
(Wang et al., 2016) described HK2- targeting modulate Warburg effect to

stimulate cancer cell apoptosis. HK inhibition can cause ATP depletion, thus
resulting in insufficient energy supply for cancer cell mitosis, proliferation, and
invasion as previously described (Coore and Randle, 1964). HK activity in breast
cancer cell lines was lower than that in normal cells, and a non-significant reduction
in HK activity was observed in the REF normal cell line.
Page 136
(Patra et al., 2013) discovered that deletion of HK2 gene in adult mice does not
considerably disturb normal tissues. Moreover, MH is a specific inhibitor for HK II
(Dakubo, 2010) and normal cells rely more on HK I (Xu et al., 2018) this is
explained why REF cells showed mild non-significant reduction in HK activity.
There are several studies described the role of HK2 as essential in tumor initiation
and development in breast cancer; therefore, HK2 deletion as cancer treatment have
therapeutic value with no adverse physiological side effects (Patra et al., 2013).
It was noticed a significant reduction in pyruvate concentration, after treatment

with combination in breast cancer cell lines, and a non-significant difference in the
REF normal cell line after 72 h of treatment. Given that pyruvate concentration
depends on the action of HK in the glycolysis pathway (Ding et al., 2012), the
decrease in HK resulted in a deficiency in pyruvate concentration which confirmed
by the study. In correlation with previous experiments results, it was noticed that
MH-NDV combination suppress the generation of intercellular ATP. It was found
that the ATP concentrations in AMJ13 and MCF-7 cell lines treated with MH and
NDV were significantly lower than to those of the control. Reducing ATP levels
effectively inhibits energy metabolism in MCF-7 and AMJ13 cells.
The pH values of treated breast cancer cell lines secretions were higher than
those of the control. A non-significant effect was observed in the normal REF cell
line after treatment with MH and NDV. Acidity decreased because lactate
concentration decreased as a result of reduced pyruvate concentration and inhibited
the growth and proliferation of cancer cells. It is proved that lactate deficiency
reduces the acidity of the cell environment. This effect is favorable for preventing
cancer cell proliferation because tumor or cancer cells grow in acidic environments.
Elevated glucose metabolism decreases the pH in the microenvironment due to
lactate secretion (Estrella et al., 2013).
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Conclusion …………………….……………………………………………………
CONCLUSION
The following conclusions were made from the present study:
1- Combination index analysis (CI) showed presence of synergistic inhibitory

effect between NDV and MH against breast cancer cells. While, it showed
presence of antagonistic and additive effect for NDV and MH combination in
normal REF cell line.
2- Increase concentration of MH and MOI of NDV induce cytotoxicity,

morphological changes, and apoptosis. The combined MH and NDV treatment
had potent cytotoxic effects against breast cancer cell lines but not against the
normal REF cell line that proved safety.
3- The combination of MH and NDV was a significantly better inhibitor and

enhancer of growth inhibition and glycolysis inhibition.
4- A significant reduction was identified in the HK activity, [Pyruvate], [ATP], and

acidity in all the treatment modalities in the breast cancer cells but not in the
normal cells, especially with the combination therapy.
5- Synergism of NDV and MH can be considered as a future treatment of cancer

cells and a new strategic way to be used as an effective treatment. This strategy
has potential applications as an effective cancer treatment.
Page 138
Recommendation………………………………………………………………………
RECOMMENDATIONS
❖ Study the combination treatment of MH and NDV in vivo using mouse tumor
models.
❖ Study the internal organs histology to confirm the safety of the combination
therapy.
❖ Testing the combination of NDV with MH in vitro against more cancer cell
lines.
❖ Study more glycolysis parameters such as Glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) enzyme in the treated cells.
❖ Increase the efficacy of the treatment by combining more anti-glycolysis agent

such as pyruvate kinase inhibitor with NDV and MH.
Page 139
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Appendix ………………………………………………..…………………………………………………..
100 ml TDW 10 ml (FBS) (10%)
Sterile
Acidic solution (PH> 7)
1.6 gm media RPMI 1 ml antibiotic (Pen-Strep)

1640 (powder)
KOH (1N) drops
0.22 μm
Filtration Store in cooling (4Co)

Reddish orange (neutral solution) Completed culture Media
Appendix (1): Preparation of completed culture media (with FBS) by using powder
Sterile
89 ml Media RPMI
1640 (liquid) 10 ml (FBS) (10%)
KOH (1N) drops
1 ml antibiotic (Pen-Strep)
0.22 μm
Reddish orange
Filtration
(neutral solution)
Store in cooling
(4Co)
Appendix (2): Preparation of completed culture media
(with FBS) by using solution Completed culture Media
Page i
Appendix ………………………………………………..…………………………………………………..
100 ml TDW
Sterile
1.6 gm media RPMI 1 ml antibiotic (Pen-Strep)

1640 (powder)
KOH (1N) drops
0.22 μm
Filtration
Store in cooling (4Co)
Reddish orange (neutral solution)
SFM Solution
Appendix (3): Preparation of Serum Free Media (SFM) (without FBS) by using powder
99 ml Media RPMI Neutral solution (PH=7) 1 ml antibiotic

1640 (liquid) (Pen-Strep)
100ml SFM Solution
Store in cooling (4Co)
Appendix (4): Preparation of SFM (without FBS) by using solution
Page ii
Appendix ………………………………………………..…………………………………………………..
Known titer Cooled centrifuge known titer
Collected Titer (7-12) Collect Titer (5-7)
Filtration 0.45 µm
Filtration 0.45 µm
Distribution Distribution
High titer Low titer
Calculate TCID50 Used for injection

to use for cell lines Virus in ECE as stock
Appendix (5): Propagation of NDV in Chicken Embryo eggs: Check eggs, Injection,
collection, purification, filtration, and distribution of NDV
Page iii
Appendix ………………………………………………..…………………………………………………..
discard
7ml PBS/ mix washing separation
3 times
2ml blood
50 µl RBC RBC solution 25ml PBS
250 µl RBC
(1:100)
Appendix (6): Preparation of RBC Solution
30min
8 lines
PBS 50 µl Virus 50 µl was added Serially diluted

8 lines
A-G lines linesli
Virus 50 µl nes
RBS 50 µl
H control Calculation of titer of NDV
Appendix (7) Determination of Titer of NDV by Hemagglutination Test (HA)
Page iv
Appendix ………………………………………………..…………………………………………………..
Inverted Microscope
TCF (Contain CM and Vero cells)

Incubator Discard CM
CM
Monolayer cells
Or more cells
Fewer cells
CM Remove Cells Vero Cells
Trypsin Trypsinization
0.25% (3 ml) Mix
TCF (Vero cells +

Trypsin) Suspension
Mix 3min
vigorously
Neutralization TCF (Vero cells +

Trypsin) Suspension
Incubator 37Co, 5min
light Microscope
Trypsin
CM 40 cells
Hemocytometer
10 µl Solution Calculate Cells
CM
Remain cells
3 ml
100 µl
7 ml Suspension
All wells
10 ml
TCF (Vero cells + CM) Suspension, TCM Petri Dish TCM 96MP
Page v
Appendix ………………………………………………..…………………………………………………..
24 - 48 hr
96MP contain (Vero cells +CM) 96MP contain (Cells +CM)

100 µl for each well Aspirate CM by Cells adhesive on surface
Multipipette
Treatment of NDV
Vero cells must be monolayer
Vero cells only
Appendix (8): cell culture of Vero cell line
Appendix (9) Preparation of the diluted virus solution from stock virus solution
Page vi
Appendix ………………………………………………..…………………………………………………..
50 µl 50 µl
50 µl
100 µl SFM 100 µl SFM

50 µl virus solution
Control Control
50 µl SFM
Column
Row
Covered by Parafilm Save for 5 days
Calculate TCID50
Incubator 37Co
Appendix (10) Exposure NDV to Vero cell line to measure TCID50
Page vii
Appendix ………………………………………………..…………………………………………………..
Preparation of diluted MOI of NDV solution from stock virus and exposure NDV to cells
line
NDV stock solution
that have TCID50
Volume of NDV MOI Appendix (11) Preparation of different MOI

solution/ 5 well for NDV depend on TCID50 and exposure
of NDV to cell line
0.04 µl 0.08 µl 0.16 µl 0.32µl 0.64 µl 1.28 µl 2.56 µl 5.12 µl
Diluted
MOI of
NDV
solution
1 2 3 4 5 6 7 8
MOI 0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8
100 µL 500 µl SFM

100 µL
SFM (25ml) with

250 µl Antibiotic
Control
Cell line
treated
with
NDV
Wrap plate
by parafilm
Read OD by
MTT assay
Save in incubator 37Co for 72 hour
Page viii
Appendix ………………………………………………..…………………………………………………..
Preparation of MH stock solution and duplicated dilution solution
500 µl
Filtration
0.22 µm
PBS (14.87ml), MH solution (14.87ml)

MH (50mg) Each one eppendorf (500µl)
9.86 g/L (0.986 16 mM, mmol/L) or 3.360
g/100ml), 1% mg/ ml, 3360µg/ml No. (1 - 30), 3360 µg/ml CONC.
(w/v) Stock solution
1 2 ……………………………………………………………………………….…………………. 30
Each one eppendorf (500µl) MH solution, No. (1 - 30), 3360 µg/ml

Concentration
Experiment 1
500µl (3360 µg/ml, 16mM)

MH stock solution
300 µl
Concentration of solutions (8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625) mM
300 µl 300 µl 300 µl 300 µl 300 µl 300 µl 300 µl 300 µl
300 µl
Discard
Mixed
300 µl 300 µl 300 µl 300 µl 300 µl 300 µl 300 µl 300µl
SFM (25ml) with

250 µl Antibiotic
Appendix (12) Preparation of MH stock solution and diluted MH solution
Page ix
Appendix ………………………………………………..…………………………………………………..
Preparation of DXL stock solution and duplicated dilution solution
500 µl
Filtration
0.22 µm
Docetaxel (20 mg) Docetaxel Stoke

DMSO Each one eppendorf (500µl)
Solution 2000 µg/ml
(10ml) (1%) No. (1 - 20), 2000 µg/ml CONC.
1 2 ……………………………………………………………………………….…………………. 20
Each one eppendorf contain (500µl) Docetaxel Stock Solution (DXLSS), No. (1 - 20), 2000 µg/ml
Concentration
500µl (2000 µg/ml) DXLSS

5 ml 50 µl
DMSO 1%
Solution 5ml, Diluted DXLSS (20 µg/ml)
µl
Concentration of solutions (10, 5, 2.5, 1.25, 0.625 and 0.313) µg/ml
600 µl
DDXLSS 600 µl 600 µl 600 µl 600 µl 600 µl
600 µl
Discard
Mixed
600 µl 600 µl 600 µl 600 µl 600 µl 600 µl
SFM (25ml) with

250 µl Antibiotic
Appendix (13) Preparation of stock and diluted DXL drug solution
Page x
Appendix ………………………………………………..…………………………………………………..
Diluted MH concentrations Exposure MH to cell lines
1680 840 420 210 105 52.5 26.25

µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml
13.125
µg/ml
100 µl 100 µl 100 µl 100 µl 100 µl
Covered by parafilm
100 µl Control SFM
Sample (Cells + MH + SFM) (Control) (Cells +SFM)

FSM
Result
Incubator 37Co
Appendix (14) Exposure of MH to cell lines
Page xi
Appendix ………………………………………………..…………………………………………………..
Diluted DXLSS concentrations Exposure Docetaxel to cell lines
10 5 2.5 1.25 0.625 0.313

µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml
Each one eppendorf contain 600 µl
100 µl 100 µl 100 µl 100 µl 100 µl 100 µl
AMJ13
MCF7
Covered by parafilm 100 µl Control SFM
Sample (Cells + DXL+ FSM) (Control) (Cells +SFM)

SFM
Media
Incubator 37Co, 72hr Discard
Appendix (15) Exposure of different

OD Results by MTT
concentrations for DXL to breast cancer cell line Microplate reader
Page xii
Appendix ………………………………………………..…………………………………………………..
Discard 50 µL SFM
Media to all plate
50 µL MTT dye
to all plate
Plate after exposure and incubation 72hr
100 µl DMSO Save in incubator

(1%) to all plate 37Co for 3 hours
Discard MTT dye

Media 2mg/ml
Wrap plate by
parafilm
Shake plate and saved
in incubator for 15 min
OD results for ELISA Plate

exposure Reader
Read OD at 492 nm
Appendix (16) MTT Cytotoxicity Assay
Page xiii
Appendix ………………………………………………..…………………………………………………..
Appendix (17) HK Kit components and preparation of reagents
Appendix (18) Transillumination of eggs by candling lamp to ensure the viability of the embryo.
Page xiv
Appendix ………………………………………………..…………………………………………………..
Appendix (19) ATP Kit components and preparation of reagent
Page xv
Appendix ………………………………………………..…………………………………………………..
Appendix (20) Save Eppendorf tubes in ice Appendix (21) Embryonated chicken eggs
(9-days-old) were incubated in a
conventional incubator at 37oC
Appendix (22) Allantoic fluid of infected embryo was harvested in Eppendorf tubes and
stored at -86oC into deep freeze after calculating TCID50.
Page xvi
Appendix ………………………………………………..…………………………………………………..
Appendix (24) Inverted and fluorescent Microscope
Page xvii
Appendix ………………………………………………..…………………………………………………..
To calculate titer for all allantoic fluid in plate culture: (2X) ( 21, 22, 23, 24….212)
Titer: HAU 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096
Results:
Control
A= 0 Titer / not contain virus 0.0%

Contain virus
agglutination
B= 12 Titer / 4096 HAU contain virus 100%
(+) Positive result
C= 9 Titer / 512 HAU contain virus 75%

Not contain virus
NO Agglutination
D= 5 Titer / 32 HAU contain virus 41.7%
(clot) (-)
(-) Negative result
E= 5 Titer / contain virus 41.7%
F= 8 Titer / 256 HAU contain virus 66.7 % Contain virus

Agglutination
G= 9 Titer / contain virus 75% (+) Positive result
H= Control / not contain virus
Page xviii
Appendix ………………………………………………..…………………………………………………..
Control
Contain virus Contain virus Not contain virus

Agglutination agglutination NO Agglutination
(+) Positive result (+) Positive result (clot) (-)
(-) Negative result
A= 0 Titer
B=0
C= 0
D= 0
E= 7
F= 9
G= 9
H= control
Appendix (25) Calculation of titer of NDV by HA test in 96-well microplate
Page xix
Appendix ………………………………………………..…………………………………………………..
Excel Microsoft V. 13
GraphPad Prism V.7 Biostatics/ figures
Photoshop V. 19 Biostatics/ figures
Google Scholar EndNote 7 Diagrams
References
SPSS V.16 & GraphPad
Biostatics/ Values
CompuSyn program V.1
Isobologram & Chou–Talalay
Synergism & CI
Soft Max Program V.6
Absorbance/Kits
ELISA plate reader
Word Microsoft V.19

Word processor
Research Programs
Word processor
Turnitin Program
Thesis
Appendix (26): Outline of the programs were used in the study
Appendix (27): Hemagglutination (the clumping together of red blood cells) by NDV (Karakus, 2018)
Page xx
La Prensa Médica Argentina®
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Al Muthanna International Trauma Conference
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Acceptance letter
LPMA-Conference Special Issue
Received: May 9-11, 2020
Accepted: June 20, 2020
Dear Authors;
1 2 3
Ahmed Ghdhban Al-Ziaydi , Ahmed Majeed Al-Shammari , Mohammed I. Hamzah , Haider Sabah
4 5
kadhim , Majid Sakhi Jabir
1
Department of Medical Chemistry, College of Medicine, University of Al-Qadisiyah, Iraq, E-mail:
ahmed.alziaydi@qu.edu.iq
2
Iraqi Center of Cancer and Medical Genetics Research, Mustansiriyah University, Iraq, E-mail:
Ahmed.Alshammari@iccmgr.org
3
College of Medicine, University of Al-Nahrain, Iraq, E mail: moh_alsafi75@colmed-alnahrain.edu.iq
4
Department of Microbiology, College of Medicine, Al-Nahrain University, Iraq, Email:
haider_kadhim@yahoo.com
5
Department of Applied Sciences, Technology University, Iraq, E mail: msj_iraq@yahoo.com
*Corresponding author, E-mail: Ahmed.Alshammari@iccmgr.org
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Acceptance Letter
TMREES Conference Series

Technologies and Materials for Renewable Energy, Environment and Sustainability
TMREES20-Greece Int'l Conf. Athens - Greece, June 25-27, 2020.
Sunday, May 10, 2020
Speaker: …Ahmed Majeed Al-Shammari…. / Oral-Visio /ID: #.58../SN: 859…

Authors: …Ahmed Ghdhban Al-Ziaydi, Mohammed I. Hamzah, Ahmed Majeed Al-Shammari, Haider Sabah Kadhim and
Majid Jabir……..
Paper Title: …The Anti-proliferative Activity of D- Mannoheptulose against Breast Cancer Cell line through Glycolysis
Inhibition……………….
Country: …Iraq……….// Affiliation: …Iraqi Center for Cancer and Medical Genetic Research, Mustansiriyah
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1st International Virtual Conference on Pure Science
College of Science, University of Al-Qadisiyah
10th-11th June 2020
Date: 8/06/2020
Acceptance Letter
We are pleased to inform you that your manuscript entitled
Propagation of Newcastle Disease Virus in Embryonated Chicken Eggs and its Research
Applications in Cell lines
has been accepted for online publication in the 1st International Virtual Conference on Pure Science that
will held in College of Science, University of Al-Qadisiyah, Iraq, at the IOP publishing, Journal of
Physics: Conference Series, June, 2020.
Authors and Affiliation Details:
1
Ahmed Ghdhban AL-Ziaydi, 1Department of Medical Chemistry, College of Medicine, University of
Al-Qadisiyah, Iraq.
2
Ahmed M. Al-Shammari, 2Iraqi Center of Cancer and Medical Genetics Research (ICCMGR)
Al-Mustansiria University, Iraq
3
Mohammed I. Hamzah, 3College of Medicine, University of Al-Nahrain, Iraq.
Thank you for your contribution to 1st International Virtual Conference on Pure Science
Sincerely
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Conference Chairman
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VirusDis.
https://doi.org/10.1007/s13337-020-00612-z
ORIGINAL ARTICLE
Newcastle disease virus suppress glycolysis pathway and induce

breast cancer cells death
Ahmed Ghdhban Al-Ziaydi1 • Ahmed Majeed Al-Shammari2 • Mohammed I. Hamzah3 •
Haider Sabah Kadhim4 • Majid Sakhi Jabir5
Received: 17 January 2020 / Accepted: 18 June 2020

Ó Indian Virological Society 2020
Abstract Newcastle disease virus (NDV) can modulate replicates efficiently in cancer cells and spare normal cells
cancer cell signaling pathway and induce apoptosis in and induce morphological changes and apoptosis in breast
cancer cells. Cancer cells increase their glycolysis rates to cancer cells but not in normal cells. NDV infected cancer
meet the energy demands for their survival and generate cells showed decreased in the HK activity, pyruvate and
ATP as the primary energy source for cell growth and ATP concentrations, and acidity, which reflect a significant
proliferation. Interfering the glycolysis pathway may be a decrease in the glycolysis activity of the NDV infected
valuable antitumor strategy. This study aimed to assess the tumor cells. No effects on the normal cells were observed.
effect of NDV on the glycolysis pathway in infected breast In conclusion, oncolytic NDV ability to reduce glycolysis
cancer cells. Oncolytic NDV attenuated AMHA1 strain pathway activity in cancer cells can be an exciting module
was used in this study. AMJ13 and MCF7 breast cancer to improve antitumor therapeutics.
cell lines and a normal embryonic REF cell line were
infected with NDV with different multiplicity of infections Keywords Warburg effect Pyruvate Virotherapy
(moi) to determine the IC50 of NDV through MTT assay. Cancer metabolism Oncolytics
Crystal violet staining was done to study the morphological
changes. NDV apoptosis induction was assessed using AO/
PI assay. NDV interference with the glycolysis pathway Introduction
was examined through measuring hexokinase (HK) activ-
ity, pyruvate, and ATP concentrations, and pH levels in Newcastle disease virus (NDV) is belonging to the genus
NDV infected and non-infected breast cancer cells and in Avulavirus of the family Paramyxoviridae. Viruses from
normal embryonic cells. The results showed that NDV this family are enveloped, nonsegmented, negative-sense
RNA viruses that cause the inflammation of the respiratory
and gastrointestinal tracts in a wide variety of poultry
& Ahmed Majeed Al-Shammari species [28]. The virulent NDV strain still inducing out-
ahmed.alshammari@iccmgr.org breaks in different countries, including Iraq, leading to
1
Department of Medical Chemistry, College of Medicine,
severe economic damages in the poultry industry [11]; yet,
University of Al-Qadisiyah, Al Diwaniyah, Iraq attenuated and lentogenic NDV has promising antitumor
2
Department of Experimental Therapy, Iraqi Center of Cancer
activity and excellent safety in laboratory animals [26].
and Medical Genetics Research, Mustansiriyah University, The Iraqi NDV strain AMHA1 is an oncolytic virus that is
Baghdad, Iraq attenuated strain isolated originally from an outbreak [7].
3
College of Medicine, University of Al-Nahrain, Baghdad, The NDV induces apoptosis in cancer cells through both
Iraq intrinsic and extrinsic pathways [8], causing DNA frag-
4
Department of Microbiology, College of Medicine, Al- mentation and FAS ligand expression [13]. Furthermore,
Nahrain University, Baghdad, Iraq AMHA1 NDV strain found to induce apoptosis in cancer
5
Department of Applied Sciences, Technology University, cells in caspase-dependent and independent pathways [29].
Baghdad, Iraq Oncolytic NDV strain found to interfere with biological
123
A. G. Al-Ziaydi et al.
processes such as angiogenesis in cancer cells and reduce MTT cytotoxicity assay
its expression [5, 6].
Breast cancer cells rely on fermentative glycolysis as Human breast cancer AMJ13, MCF7, and normal REF cell
the main source of energy [18]. Increased glycolysis is lines were used. Cells were seeded at a density of 1 9 104
the primary source of energy in cancer cells for ATP cells in a 96-well microplate and incubated at 37 °C for
generation through the Warburg effect [22]. Cancer cells 72 h until monolayer confluence was achieved. Cytotoxi-
mainly produce energy by increasing the rate of glycol- city was investigated through 3-(4,5-dimethylthiazol-2-yl)-
ysis by 200 times that in their normal cells of origin; the 2,5-diphenyltetrazolium bromide (MTT) assay. The cells
increment in glycolysis is followed by lactate fermenta- were exposed to a range of multiplicity of infections (MOI)
tion in the cytosol of the cells even if oxygen is plentiful of NDV (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8). After
[2]. Cancer cells are highly proliferative relative to nor- 72 h of infection, MTT dye solution (2 mg/ml) was added
mal cells and thus require increased ATP to meet their to each well. The incubation was continued for three hours.
metabolic demands [16]. Therefore, high energy demands A total of 100 ll of DMSO was added to each well and
characterize malignant cells [31]. It is found that Influ- incubated for 15 min. The optical density was measured at
enza virus-infected non-tumor permissive cells have 492 nm using a microplate reader. Cytotoxicity % was
substantial changes in glycolysis and general cell meta- calculated using the following equation:
bolism [17]. Thus, it is interesting to assess the effect of
Cytotoxicity % ¼ ODControl ODsample =ODControl 100;
NDV infection on the cancer cell glycolysis pathway and
its interference with the proliferation of breast cancer were ODcontrol is the mean optical density of untreated
cells. wells, and ODSample is the optical density of treated wells
[12].
Materials and methods Morphological changes
NDV propagation To visualize cell morphology under inverted microscopy,

200 ll of the AMJ13, MCF7, and REF cell suspensions
The Experimental Therapy Department provided NDV were seeded in 96-well microtitration plates at the density
AMHA1 attenuated strain, Iraqi Center of Cancer and of 1 9 104 cells/ml. The cells were exposed to NDV MOI
Medical Genetics Research (ICCMGR), Mustansiriyah (2) and compared with the control (untreated cells). The
University. A stock of attenuated NDV was propagated in medium was removed, and NDV was added. Then, the
embryonated chicken eggs (Al-Kindi Company, Baghdad, plates were stained with 50 ll of crystal violet and incu-
Iraq), harvested from allantoic fluid, and then purified from bated at 37 °C for 15 min. The stain was removed through
debris by centrifugation (at 3000 rpm and 4 °C for gentle washing with tap water. The cells were observed by
30 min). NDV was quantified through the hemagglutina- using an inverted microscope (Leica Microsystems, Ger-
tion test, aliquoted, and stored at - 86 °C. Viral titers were many) under 9 200 magnification and photographed by
determined through a 50% tissue culture infective dose using a digital camera at four haphazardly selected cultured
(TCID50) titration on Vero cells following the standard fields. The images were analyzed using the ImageJ soft-
procedure [7]. ware (http://rsb.info.nih.gov/ij/). Percentage (%) of cells
stained by crystal violet were calculated, and statistical
Cell lines and cell culture analysis was performed [3].
The Cell Bank Unit provided AMJ13 human breast Apoptosis assessment (propidium iodide/acridine
cancer cell line, MCF7 human breast cancer cell line and orange staining)
normal REF cell line, Experimental Therapy Department,
ICCMGR, Mustansiriyah University, Baghdad, Iraq. The The propidium iodide/acridine orange (PI/AO) dual stain-
cell lines were cultured in RPMI-1640 medium (US ing method was used to quantify the apoptotic death rates
Biological, USA) supplemented with 10% (v/v) fetal of the MCF7 and AMJ13 cell lines. The cells were seeded
bovine serum (FBS) (Capricorn-Scientific, Germany) and in a 96-well plate at the density of 7000 cells/well on the
1% (v/v) penicillin–streptomycin (Capricorn-Scientific, night prior to treatment. The cells were treated with NDV
Germany) under a humidified atmosphere of 5% CO2 at MOI (2) at 37 °C in an incubator at 72 h prior to PI/AO
37 °C. Exponentially growing cells were used for staining. Then, 1 ml of the cell suspension was used for
experiments [9]. conventional PI/AO staining, and 100 ll of the cell
123
Newcastle disease virus suppress glycolysis pathway and induce breast cancer cells death
Fig. 1 NDV induced significant cytotoxicity in breast cancer cells but not in normal cells. a NDV cytotoxicity (CT %) in REF normal cells;
b NDV CT % in AMJ13 cell line; c NDV CT % in MCF-7 cell-line
suspension was transferred to a 96-well plate for modified incubated at 37 °C for 72 h. The cell suspension was col-
PI/AO staining. AO was used (10 ll AO ? 1 ml PBS), and lected by thawing. The pH values of AMJ13, MCF7, and
PI (10 ll PI ? 1 ml PBS) was then added at a 1:1 ratio. A REF cell lines were measured by litmus papers and pH
total of 50 ll of the AO/PI stain mixture was added to all of meter and compared with those of the control.
the wells of the 96-well plate. The plates were incubated
for 30 s at room temperature. Then, the dye was discarded. Statistical analysis
PBS (100 ll) was added to all of the wells of the 96-well
plate for washing and then discarded. Photographs were All results were presented as SD and mean ± SEM. A two-
taken directly by using a fluorescence microscope (Leica tailed t test was performed, and statistical analysis was
Microsystems, Germany). Fluorescence intensity was conducted with Excel version 10, GraphPad Prism version
measured using Image J version 1.47 software to count 7 (USA), and Isobologram version 1. CompuSyn software
apoptotic cells [1]. was used to compare the difference between groups under
different conditions. The level of significance was set at
Glycolysis pathway and products measurement p \ 0.05.
Treated and untreated cell samples were prepared. Gly-

colysis pathway efficiency was examined through mea- Results
suring hexokinase (HK) activity using the colorimetric
method. It was performed by using an HK activity assay kit NDV induces significant killing rate in cancer cells
(ElabScience, USA). Glycolysis products, pyruvate, and
ATP were measured. Pyruvate measured using a pyruvate Cytotoxicity percentage was measured at different NDV
colorimetric method. It was conducted by using a pyruvate treatment MOI (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8
assay kit (ElabScience, USA). ATP was measured through MOI) through the MTT cytotoxicity assay. Figure 1
a colorimetric method by using an ATP assay kit (ElabS- showed that NDV induces significant cytotoxicity %
cience, USA). in both cancer cell lines AMJ13 and MCF-7, while
there was no significant killing in the normal embryonic
pH measurements cells.
The cell lines were cultured in RPMI-1640 medium sup- NDV induces morphological changes and apoptosis
plemented with 10% (v/v) FBS and 1% (v/v) penicillin– in the infected cancer cells
streptomycin and incubated in a humidified atmosphere of
5% CO2 at 37 °C overnight prior to treatment. Exponen- The morphological changes in the infected cancer cells
tially growing cells were used for experiments. Cancer within 72 h of treatment were attributed to the NDV
cells were seeded at a density of 1 9 104 cells in a 96-well induced cytopathic effect. Cell detachment and rounding
microplate and incubated at 37 °C for 72 h until monolayer were observed in the NDV infected cells only (Fig. 2).
confluence was achieved as observed under inverted Quantitative image analysis showed that NDV infected
microscopy. Cells were exposed to NDV (MOI 2) and then cells have lower cell density, as shown in Fig. 2. AO/PI
123
Fig. 2 Morphological changes

in REF, AMJ13, and MCF7 cell
lines treated with NDV for 72 h
and stained with crystal violet,
photographed under 940
magnification (a–f).
Morphological changes in REF,
AMJ13, and MCF7 cell lines
after treatment with NDV:
untreated cells (control), treated
cells with NDV. The
quantitative image analysis
revealed a significant decrease
in treated cell density, which
reflects the destructive effect
induced by NDV infection, in
which there was no decrease in
the infected normal cells REF
staining and imaging under fluorescence inverted micro- NDV interfere with glycolysis pathway and reduce
scope, showed untreated control cells appeared green, glycolysis products
which indicate viable cells, whereas the NDV-treated cells
appeared yellow or orange as apoptotic cells, (Fig. 3). In NDV treated cancer cells showed a reduction in HK
contrast to breast cancer cell lines, normal REF cell lines activity. An insignificant reduction was found in the NDV
showed minimal morphological changes and apoptosis infected normal REF cell line (Fig. 4). The pyruvate and
after NDV treatment. Quantitative image analysis of the ATP levels were decreased in cancer treated cells signifi-
fluorescence intensity revealed significant apoptosis cantly when compared to the control untreated cancer cells.
induction by NDV treatment in cancer cells but not in Normal embryonic REF cells pyruvate and ATP levels did
normal cells (Fig. 3). not decrease significantly when infected with NDV (Figs. 5
123
killing effect in our experiment, which proves safety that

reported by others [4, 30]. The efficient replication and
killing inside cancer cells and crippled replication with no
killing effect in normal cells explained as selectivity.
The AO/PI apoptosis assay findings of the present work
also confirmed that NDV induces apoptosis in cancer cell
lines but not in normal cells. NDV triggers both apoptosis
pathways in cancer cells [21], in recent work about
AMHA1 NDV strain, it is found to induce apoptosis in
caspase-dependent and independent pathways [29]. The
HK activity in the NDV infection AMJ13 and MCF-7
breast cancer cell lines decreased relative to that in the
uninfected control cells, while no changes in infected and
non-infected REF cell lines, which again confirm the NDV
oncolytics selectivity.
In the present study, we discovered that hexokinase
activity reduced significantly after NDV infection. In a
previous study by our group, we discovered another gly-
colytic enzyme is downregulated, which is glyceraldehy-
de3-phosphate (GAPDH) after cancer cell infection by
NDV [10]. Deng et al. [20] conducted a proteomic analysis
of chicken peripheral blood mononuclear cells after NDV
infection; they found expression reduction of phospho-
glycerate kinase (PGK), which suggests that NDV may
decrease glycolytic pathway activity in the host. These
findings together confirm that NDV negatively interferes
with the glycolysis pathway.
To further confirm this negative regulation, we tested
Fig. 3 All cells were stained with AO/PI and viewed under the glycolytic pathway products level in infected and non-
fluorescence microscopy at 940 magnification to determine the infected cancer cells. The findings showed that NDV could
NDV induction of apoptosis. Yellow-orange cells are representing
apoptosis, and green cells are viable cells. Quantitative image analysis cause ATP depletion, while insignificant relation was
for fluorescence intensity revealed significant apoptosis induction in observed between NDV infection and normal REF cell
the NDV infected cells while there was no induction in normal REF lines. When glycolysis is inhibited, lactate production is
cells completely terminated, and intracellular ATP concentration
abruptly decreases [23]. Reduced pyruvate concentration
was recorded as well, a significant difference between the
and 6). The pH values of the AMJ13 and MCF7 cell lines effect of NDV and the control in breast cancer cell lines,
increased after 72 h of treatment with NDV (MOI 2) and an insignificant difference in the REF cell line after
because the reduction in lactate concentration attenuated treatment with NDV for 72 h was also observed. Pyruvate
the acidity. A significant difference was observed between concentration depends on HK action in the glycolysis
NDV and control (Fig. 7). The pH value of the REF cell pathway [24]. Therefore, the ATP concentration of AMJ13
line non-significantly increased after treatment with NDV. and MCF7 cell lines became deficient after NDV treatment
relative to that of the control.
We found that the pH of breast cancer cell lines was
Discussion higher than that of the control, and normal REF cell lines
showed limited effects after 72 h of treatment with NDV.
The in vitro results of this study showed that increasing the The reduction in acidity may be attributed to the decreased
MOI of NDV intensified the cytotoxicity and enhanced lactate concentration caused by the depleted pyruvate
cytopathic and killing effect against breast cancer cell concentration [27]. Lactate deficiency reduces the acidity
lines. Our findings are similar to several previously pub- of the cell environment. This phenomenon is favorable for
lished works confirming that NDV oncolytic to the human preventing the proliferation of cancer cells because tumors
breast cancer cells [10, 14, 25]. Exposing normal embry- or cancer cells grow in acidic environments [19]. This
onic REF cells to NDV had no significant cytopathic and phenomenon is consistent with the results of our study.
123
Fig. 4 The effect of NDV on glycolysis inhibition. The activity of HK (U/ml) in REF, AMJ13, and MCF7 cell lines after NDV treatment with
MOI 2 was compared with that of the control. a REF, b AMJ13, and c MCF7, at values represent the (mean ± SD) at *P \ 0.05, **P \ 0.01
Fig. 5 Effect of NDV on pyruvate. REF, AMJ13 and MCF7 cell lines under NDV treatment (MOI 2) in comparison with that under the control
at *P \ 0.05, **P \ 0.01 and ***P \ 0.001. a REF, b AMJ13, and c MCF7. Pyruvate lmol/ml
This provided evidence confirm the decreased glycolysis in In Conclusion, NDV exerted cytotoxicity, morphologi-
the NDV infected cancer cells compared with that in nor- cal changes, and apoptosis in cancer but not normal cells,
mal cells. Therefore, inhibition of the glycolysis process which proves a selective oncolytic effect. NDV reduced
leads to a decrease in the growth and proliferation of glycolytic pathway activity by reducing HK activity,
affected cells as described by others [15]. pyruvate, ATP, and tumor micro-environmental acidity in
123
Fig. 6 Effect of NDV on ATP production in REF, AMJ13, and MCF7 cell lines under treatment with NDV (MOI 2) compared with that in cell
lines under the control treatment at *P \ 0.05, **P \ 0.01, ***P \ 0.001 and ****P \ 0.0001. a REF, b AMJ13, and c MCF7. (ATP mmol/L)
Fig. 7 Levels of acidity of NDV infected and non-infected cells, levels. a pH values of the REF cell line, b pH values of the AMJ13
normal cells have neutral pH value in both infected and non-infected cell line and c pH values of the MCF7 cell line). Values represent the
cells. Non-infected cancer cells showing a high level of acidity, but (mean ± SD) at t *P \ 0.05, **P \ 0.01 and ***P \ 0.001
this situation reversed by NDV infection to show a rise to alkaloid
cancer but not normal cells, which again confirm the Acknowledgements The authors thank the Experimental Therapy
selective oncolytic effect that can be considered as one of Department, Iraqi Center for Cancer and Medical genetic research,
Mustansiriyah University, for their support.
therapeutic strategies for breast cancer and other types of
cancer cells in the near future.
123
Compliance with ethical standards disease virus expressing human IL12 against human breast can-
cer. Sci Rep. 2019;9(1):13999. https://doi.org/10.1038/s41598-
Conflict of interest The authors disclose no potential conflict of 019-50222-z.
interest. 15. Arora R, Schmitt D, Karanam B, Tan M, Yates C, Dean-Colomb
W. Inhibition of the Warburg effect with a natural compound
reveals a novel measurement for determining the metastatic
potential of breast cancers. Oncotarget. 2015;6(2):662–78. https://
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123
Al‑Ziaydi et al. Cancer Cell Int (2020) 20:420
https://doi.org/10.1186/s12935-020-01514-2 Cancer Cell International
PRIMARY RESEARCH Open Access
Hexokinase inhibition using

D‑Mannoheptulose enhances oncolytic
newcastle disease virus‑mediated killing
of breast cancer cells
Ahmed Ghdhban Al‑Ziaydi1, Ahmed Majeed Al‑Shammari2* , Mohammed I. Hamzah3, Haider Sabah kadhim4
and Majid Sakhi Jabir5
Abstract
Background: Most cancer cells exhibit increased glycolysis and use this metabolic pathway cell growth and pro‑
liferation. Targeting cancer cells’ metabolism is a promising strategy in inhibiting cancer cell progression. We used
D-Mannoheptulose, a specific hexokinase inhibitor, to inhibit glycolysis to enhance the Newcastle disease virus anti-
tumor effect.
Methods: Human breast cancer cells were treated by NDV and/or hexokinase inhibitor. The study included cell
viability, apoptosis, and study levels of hexokinase enzyme, pyruvate, ATP, and acidity. The combination index was
measured to determine the synergism of NDV and hexokinase inhibitor.
Results: The results showed synergistic cytotoxicity against breast cancer cells by combination therapy but no cyto‑
toxic effect against normal cells. The effect was accompanied by apoptotic cell death and hexokinase downregulation
and inhibition to glycolysis products, pyruvate, ATP, and acidity.
Conclusions: The combination treatment showed safe significant tumor cell proliferation inhibition compared to
monotherapies suggesting a novel strategy for anti-breast cancer therapy through glycolysis inhibition by hexokinase
downregulation.
Keywords: Cytotoxicity, Anticancer therapy, Warburg effect, Hexokinase inhibitor, Pyruvate
Background energy by increasing the rate of glycolysis by 200 times

Cancer cell generally relies on aerobic glycolysis due to higher than that in the normal cells of origin; this incre-
hypoxia, mitochondrial dysfunction, and the malignant ment is succeeded by lactate fermentation in the cytosol
transformation that lead to glycolysis pathway-dependent of the cell regardless of the abundant oxygen supply [3].
for ATP generation [1]. This phenomenon is described The uptake of glucose in normal tissue is less than that
as the Warburg effect where cancer cells require high in cancer cells. This difference can be used as a target for
amounts of glucose to support the metabolic function cancer therapy [4]. Breast-cancer stem cells are fermenta-
and produce energy [2]. Cancer cells mainly produce tive glycolysis dependent, and that makes it sensitive to
glycolysis inhibitors [5].
The first and rate-limiting step of glycolysis con-
*Correspondence: ahmed.alshammari@iccmgr.org
2
Experimental Therapy, Iraqi Center for Cancer and Medical Genetics
trolled by hexokinase (HK) enzyme is glucose phos-
Research, Mustansiriyah University, Baghdad, Iraq phorylation to glucose-6-phosphate (Glu-6-P), which
Full list of author information is available at the end of the article is later employed to generate two ATP molecules [6]. In
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Al‑Ziaydi et al. Cancer Cell Int (2020) 20:420 Page 2 of 10
mammalian tissues, there are four major hexokinases are activity in permissive and poorly permissive cancer cells
expressed, designated as HK1, HK2, HK3, and HK4 [7]. [28]. Furthermore, pyruvate dehydrogenase kinase inhi-
HK1 and HK2 are the main hexokinases that are impor- bition showed to improve reovirus oncolytic anti-tumor
tant in cell survival. Mammalian adult tissues mostly efficacy in several cancer types [29]. We reported previ-
expressed HK1 isoform, while HK2 is expressed abun- ously that 2DG would enhance oncolytic NDV against
dantly only in dew adult tissues such as cardiac muscles, breast cancer cells through glycolysis inhibition by down-
skeletal, and adipose [6]. It is found that HK2 is expressed regulation of glyceraldehyde3-phosphate (GAPDH) [30].
in many types of cancers to promotes its growth through Here, we investigate using D-Mannoheptulose, a hexoki-
an increased glycolytic flux [8]. Brown et al. [9] found nase inhibitor to increase breast cancer cells sensitivity
that breast cancers were HK2-positive in 79% of stud- to oncolytic NDV, and the proposed mechanisms of this
ied tumors. HKII status in breast cancer tissue sections combination using glycolysis products analysis in breast
was significantly related to poor prognosis and relapse of cancer cells NDV-mediated oncolysis correlates with the
breast cancers [10]. Moreover, tamoxifen-resistant breast disturbance within the metabolism.
cancer cells MCF-7 showed upregulation of HK2 and
mTOR that was accompanied by an enhanced glycolysis Materials and methods
process. These findings make HK2 as a possible target to NDV propagation
be blocked to overcome resistance to tamoxifen [11]. And NDV (Iraqi AMHA1 strain) was provided by the Experi-
for paclitaxel resistance [12]. Furthermore, HK2 overex- mental Therapy Department/Iraqi Center of Cancer and
pression in ovarian cancer cells induces cisplatin resist- Medical Genetics Research (ICCMGR), Mustansiriyah
ance [13]. Therefore, targeting hexokinase-2 will block University, Baghdad, Iraq. A stock of attenuated NDV
glucose metabolism in cancer cells, which may inhibit was propagated in embryonated chicken eggs (Al-Kindi
its proliferation with minimum side effects reported Company, Baghdad, Iraq), harvested from allantoic fluid,
[14]. The non-metabolizable glucose analog D-Manno- and then purified from debris through centrifugation
heptulose (MH) inhibits hexokinase, the first enzyme in (3000 rpm, 30 min at 4 °C). NDV was quantified through
glycolysis, with anticancer effect [15, 16], which lead to a hemagglutination test, aliquoted, and stored at − 80 °C.
block cellular energy production [17]. D-Mannoheptu- Viral titers were determined based on 50% tissue culture
lose (MH) accumulates in avocado leaves and occasion- infective dose titration on Vero cells following standard
ally in avocado fruit [18]. Mannoheptulose described by procedure [31].
Dakubo [19] as an inhibitor for HK II with many other
agents including lonidamine, 3-BrPA, 2-deoxyglucose, Cell lines and cell culture
and 5-thioglucose. The Cell Bank Unit provided the estrogen, progester-
On the other hand, cancer is still a very difficult disease one receptors negative AMJ13 human breast cancer cell
to treat. Therefore, we need to find an effective and selec- line [32], The estrogen, progesterone receptors positive
tive treatment approach that can destroy cancer cells, MCF-7 human breast cancer cell line, and normal rat
such as oncolytic virotherapy [20]. One of the first onco- embryo fibroblast cell line (REF), Experimental Therapy
lytic Viruses discovered since the late 20th century is the Department, ICCMGR, Baghdad, Iraq. AMJ13 and REF
Newcastle disease virus (NDV) [21]. The AMHA1 NDV cell lines were cultured in RPMI-1640 medium. In con-
is an avirulent attenuated strain of avian paramyxovirus, trast, MCF-7 cell was cultured in MEM (US Biological,
which is enveloped, non-segmented, negative-sense RNA USA) supplemented with 10% (v/v) fetal bovine serum
viruses [22]. AMHA1 possesses onco-tropism character- (FBS) (Capricorn-Scientific, Germany) and 1% (v/v)
istics [23]. NDV induced apoptosis via caspase-dependent penicillin–streptomycin (Capricorn-Scientific, Ger-
and caspase-independent pathways [24, 25]. Deng et al. many) and incubated in a humidified atmosphere of 5%
[26] found in a proteomic study that NDV downregulate CO2 at 37 °C. Exponentially growing cells were used for
phosphoglycerate kinase (PGK) expression in the NDV- experiments.
infected chicken peripheral blood mononuclear cells
(PBMCs), PGK is a glycolytic enzyme that participates Cytotoxicity assay
in the glycolytic pathway, which suggests that NDV may Cells were seeded at a density of 1 × 104 cells/well in a
restrict the infected cell glycolytic pathway. Its reported 96-well microplate and incubated at 37 °C for 72 h until
that interfering with cancer cells metabolism through monolayer confluence was achieved as observed under
glycolysis inhibition may enhance oncolytic virotherapy an inverted microscope. Cytotoxicity was investigated
activity [27]. A recent study showed that using 2-deoxy- by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-
glucose (2DG) to block glycolysis or through restrict- zolium bromide (MTT) assay. The cells were exposed to
ing glucose amount will enhance oncolytic adenoviruses a range of diluted concentrations of MH (13.125, 26.25,
52.5, 105, 210, 840, and 1680 μg/ml) (Santacruz Biotech- and control breast cancer cells. The cells were seeded at
nology, USA) and NDV over a range of diluted multi- the density of 7000 cells/well in a 96-well plate the night
plicities of infection (MOI, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, before treatment and then treated with MH of 62.5 µg/
and 12.8) for IC50 determination. After 72 h, each well ml and NDV MOI of 2 in a 37 °C incubator for 72 h
received 50 μl of MTT dye solution (2 mg/ml), incubated before PI/AO staining. Then, 1 ml of cell suspension was
for 3 h, and solubilized with 100 μl of dimethyl sulfox- used for conventional PI/AO staining (10 μl AO + 10 μl
ide (DMSO). The plates were incubated for 15 min. The PI + 1 ml PBS). Exactly 50 μl of stain mixture (AO/PI)
optical density values of treated and untreated cells were was added to the tested wells, which were allowed to
measured at 492 nm with an ELISA plate reader [32]. stand for 30 s at room temperature (RT). The dye was
Exactly 200 µL of cell suspension was seeded in 96-well then discarded. Photographs were taken directly under a
microtitration plates at the density of 1 × 104 cells/ml Leica fluorescent microscope [34].
and incubated for 72 h at 37 °C to visualize cell morphol-
ogy under an inverted microscope. The medium was Cell treatment for glycolysis pathway evaluation
removed, and NDV and MH were added. The plates were The AMJ13, MCF7, and REF cell lines were seeded in
stained with 50 µl of crystal violet and incubated at 37 °C 96-well cell culture plates at a density of 1 × 104 cells/
for 15 min. Finally, the stain was removed through gen- ml and incubated overnight. After confluence, the cells
tle washing with tap water. The cells were observed under were exposed to 62.5 µg/ml MH and NDV at MOI 2
an inverted microscope at 40 × magnification and photo- alone or in combination and compared with the control
graphed by using a digital camera. The endpoint param- (untreated cell) [35] after 72 h cell lysate were collected,
eter for each cell line included the inhibition rate of cell normalized, and stored at − 86 °C until used.
growth (cytotoxicity %), which was calculated as follows:
HK activity assay
Cytotoxicity % = ODControl − ODsample / ODControl × 100,
HK activity levels were measured in treated and
where OD control is the mean optical density of untreated untreated cell samples through a colorimetric method
wells, and OD Sample is the optical density of treated wells by using a hexokinase Assay Kit (ElabScience, USA) per
[23]. the manufacturer’s recommendation. The kit detection
principle, glucose is converted to glucose-6-phosphate
Combined cytotoxicity assays and Chou–Talalay analysis by hexokinase; the glucose-6-phosphate is oxidized by
The doses for this experiment were selected from the glucose-6-phosphate dehydrogenase (G6PD) to produce
previous cytotoxicity assay results of IC50 determina- NADH, which reduces the colorless substrate to the
tion. We took concentrations around the IC50 value for colored solution, HK activity can be calculated by deter-
NDV and MH in cancer cells. The AMJ13, MCF7, and mining the absorbance at 340 nm.
REF cell lines were seeded at the density of 1 × 104 cells/
well into 96-well plates and incubated overnight. NDV Pyruvate assay
was added first at MOI of 0.3, 1, and 2, followed by MH Pyruvate levels were measured using a colorimetric assay
at the indicated concentrations (62.5, 125, and 250 μg/ kit according to the manufacturer’s instructions (Elab-
ml) through serial dilution for the growth inhibition test. Science, USA). The detection principle is that pyruvic
Growth inhibition was measured after 72 h of infection acid reacts with chromogenic reagent to form a reddish-
through MTT assay, as described earlier. The assay was brown solution. The color intensity is directly related to
performed in triplicate. NDV and MH were studied at the pyruvate content. The OD values were measured of
nonconstant ratios to determine synergism. The Chou– each sample at 505 nm using a spectrophotometer.
Talalay combination index (CI) was calculated using
CompuSyn software (CombuSyn Inc., Paramus, NJ, USA) ATP assay
to analyze the combination of NDV and MH. The unfixed ATP was measured through a colorimetric method by
ratios of NDV and MH and mutually exclusive equations using an ATP assay kit (ElabScience, USA). Cell culture
were used to determine the combination index (CI). CI samples were prepared as follows: treated and untreated
values of 0.9–1.1, < 0.9, and > 1.1 indicate additive, syner- cell samples were collected and centrifuged at 1000–1500
gism, and antagonism, respectively [33]. r/min for 10 min. The supernatant was removed, and the
cell sediment was retained (approximately 106/ml cells).
Assessment of apoptosis (propidium iodide/acridine The cell suspension was prepared with 0.3–0.5 ml of
orange assay) boiled double-distilled water, allowed to stand in a boil-
Propidium iodide/acridine orange (PI/AO) dual staining water bath for 10 min, blended, and finally extracted
ing was used to measure the apoptotic rates in infected for 1 min. The sample was centrifuged at 3500 r/min for
10 min, and the supernatant was obtained for detection. Results

The OD values of each tube were measured at 636 nm, Cytotoxicity of NDV and MH against breast cancer
following the manufacturer’s recommendation. and normal cell lines
MTT cytotoxicity assay was used to evaluate the effect
against cancer and normal cells of different concentra-
PH measurements tions of MH and over a range of MOI of NDV (Fig. 1).
The cancer cells were seeded at a density of 1 × 104 cells/ There were no noticeable percentages of cytotoxicity
plate in a 96-well microplate and incubated at 37 °C (CT%) for MH against normal REF cells, as the CT%
for 72 h until monolayer confluence was achieved as ranged from 1.67 to 24.72% at higher concentrations.
observed under an inverted microscope. The cells were While there was higher cytotoxicity against breast can-
exposed to MH (62.5 μg/ml) and NDV (MOI 2) and incu- cer cells ranged from 27.29 to 58.64% for AMJ13 cells;
bated at 37 °C for 72 h. The media supernatant was col- and 26.26% to 60.49% for MCF-7 after MH treatment
lected. The pH values were measured by using PH meter (Fig. 1a–c). NDV virotherapy did not induce a cytotoxic
and litmus papers and compared with those of the con- effect against normal embryonic REF cells (Fig. 1f ).
trol [36]. Breast cancer cells were more sensitive to NDV viro-
therapy as the CT% ranged from 24.69% to 64.26% for
AMJ13; and 23.95% to 62.02% for MCF-7 cell line after
Statistical analysis NDV treatment (Fig. 1d–f ). The cytotoxicity assay
All results were presented as SD and mean ± SEM. analysis showed that IC50 values of MH (486.9 µg REF,
unpaired t test and statistical analysis were performed 124.7 µg AMJ13, and 122.6 µg MCF7) and IC50 values
with statistical software Excel version 10, GraphPad for NDV MOI (57.5 REF, 1.648 AMJ13, and 1.561 MCF-
Prism version 7 (USA). CompuSyn software was used to 7). Therefore, we chose IC50 related doses of the MH and
compare the difference between groups under different NDV for the combination study, (0.3, 1, 2 MOI) for NDV
conditions. The level of significance was set at P < 0.05. and (62.5, 125, and 250 μg/ml) for MH.
Fig. 1 MH and oncolytic AMHA1 NDV are cytotoxic against human AMJ13 and MCF-7 breast cancer cells, but not cytotoxic to normal embryonic
REF cells. The cells were treated with (a–c) D-Mannoheptulose (MH) (13.125, 26.25, 52.5, 105, 210, 840, and 1680 μg/ml) or (d–f) NDV (MOI 0.1, 0.2,
0.4, 0.8, 1.6, 3.2, 6.4, and 12.8) for 72 h. cytotoxicity was investigated using MTT assay and showed that IC50 values of MH (486.9 µg REF, 124.7 µg
AMJ13, and 122.6 µg MCF7) and IC50 values for NDV MOI (57.5 REF, 1.648 AMJ13 and 1.561 MCF-7). All data shown are mean ± SEM from three
independent experiments
(See figure on next page.)

Fig. 2 A combination of NDV and MH showed superior anticancer activity in comparison to monotherapies in both AMJ13 and MCF-7 breast
cancer cells. However, there was no enhanced toxicity against non-cancerous REF cells. (a, c and e) AMJ13, MCF-7, and REF cells were treated with
NDV (0.3, 1, and 2 MOI) and with MH (62.5, 125, and 250 μg/ml), then cell viability was measured by MTT assay. d–f Illustrations of normalized
isobologram of nonconstant combination ratios were measured by the Chou-Talalay method, where CI value quantitatively defines synergism.
(CI < 0.9), additive effect (CI = 0.9–1.1) and antagonism (CI > 1.1). All data shown are mean ± SEM (*P < 0.05 compared to mono-treatments) data
from three different experiments
Combination cytotoxicity assays and Chou–Talalay analysis Hexokinase activity assay analysis indicates strong HK
of cell lines enzyme activity reduction in both breast cancer cell
In order to investigate the effects of oncolytic NDV and lines treated by MH‑NDV combination therapy but not in
MH combination therapy, we examined the cytotoxic- normal embryonic cells
ity ratio of the NDV (0.3, 1, and 2 MOI) and for the MH In the current experiment, we quantified and analyzed
(62.5, 125, and 250 μg/ml). Synergism was observed in all the HK enzyme activity in both breast cancer cell lines
combined doses against both breast cancer (AMJ13 and and the normal embryonic cells. The HK enzyme was
MCF7) cell lines (Fig. 2a, b). Whereas no synergism rela- evaluated for the comparison of treated and untreated
tionships were detected among treatments against the cells at 72 h (Fig. 4a). We identified a significant reduction
non-cancerous REF cell line (Fig. 2e). in the HK enzyme activity in all the treatment modali-
The CI was estimated from the dose–effect data of sin- ties in the cancer cells but not in the normal cells. The
gle and combined treatments by using CompuSyn Isobol- results showed that the HK enzyme activity was signifi-
ogram. CI < 1 indicates synergism; CI = 1 to 1.1 indicates cantly reduced in the combination of MH-NDV treated
an additive effect; and CI > 1.1 indicates antagonism. The breast cancer cells in comparison to the single treatment
AMJ13 cell line had CI < 1 in eight combination points, modalities.
indicating a synergistic effect or interaction between
NDV and MH. The additive effect was seen in one com- MH–NDV combination efficiently inhibits glycolysis
bination point (Table 1A) and (Fig. 2a, b). The combina- products in the treated breast cancer cells but not in
tion points for the combined treatment to MCF-7 cell normal cells
line indicated a synergistic effect or interaction between Further investigation of the mechanism by which MH–
NDV and MH at all points (Table 1B) and (Fig. 2c, d). NDV inhibits breast cancer cell proliferation was done
In the normal REF cell line, all combination points were through examination whether MH–NDV efficiently
CI > 1, indicating antagonism and additive effect, which is decreases cancer cell glycolysis products pyruvate, ATP,
a neglected effect as there was no killing effect reached and acidity (represent lactic acid) compared to mono-
50% at all tested concentrations (Table 1C) and (Fig. 2e, therapies. Remarkably, MH–NDV induces a significant
f ). reduction on the levels of pyruvate, ATP, and acidity in
both breast cancer cell lines but not in normal embryonic
Apoptosis and morphological changes of breast cancer REF cells (Fig. 4b–d).
and normal cell lines
The morphological changes exhibited by treated cells Discussion
after 72 h of treatment were attributed to an intense cyto- Malignant cells use glycolysis as their main source of
pathic effect for the combination therapy. AO/PI assay, energy. The in vitro results of this study showed that
as seen under the fluorescent microscope, showed the by increasing the concentration of MH and the MOI of
untreated apoptotic cells appeared green (viable cells), NDV, there is an increase in cytotoxicity and enhanced
whereas the apoptotic treated cells with MH and NDV antiproliferation effect against breast cancer cell lines
appeared yellow or orange (dead cells) (Fig. 3). Morpho- but not in normal cells. MH-NDV combination, in
logical changes and apoptosis were more intense in the turn, found to reduce HK activity, pyruvate, ATP, and
cells treated by combined NDV and MH than in those acidity levels. Our findings are similar to previous
treated with NDV or MH alone. This result indicated that results showing that NDV in combination with 2-Deox-
the synergism between the inhibitor and virus enhanced yglucos, may inhibit glycolysis as the main pathway
the morphological changes and the percentage of apop- to induce breast cancer-killing effect [30]. Moreover,
tosis in cancer cells (Fig. 3j, k). We noticed weaker or less inhibiting glycolysis with 3-bromopyruvate and 2-deox-
intense morphological changes and apoptosis in the nor- yglucose results in mitochondrial pathway-induced
mal REF cell line (Fig. 3m) than in the breast cancer cell apoptosis in cancer cells [37]. We found that MH and
lines treated with MH, NDV, and their combination. NDV had a significant effect on breast cancer cells but
Table 1 Cytotoxicity of NDV and MH combination or alone the prevention of tumor formation and cancer growth
against AMJ13, MCF-7, and REF cells [39]. Our results support the aim of our study.
Points NDV (MOI) MH (µg/ml) CI Effect In addition, our results demonstrated that MH-NDV
inhibits the glycolytic pathway in MCF-7 and AMJ13
A-AMJ13 cell lines by suppressing the activity of HK more than
1 2 250 0.10595 Synergism the specific inhibitor alone and more than NDV treat-
2 2 125 0.24635 Synergism ment alone. Wang et al. [40] described HK2- targeting
3 2 62.5 0.49989 Synergism modulate Warburg effect to stimulate cancer cell apopto-
4 1 250 0.20755 Synergism sis. HK inhibition can cause ATP depletion, thus result-
5 1 125 0.41359 Synergism ing in insufficient energy supply for cancer cell mitosis,
6 1 62.5 0.49220 Synergism proliferation, and invasion, as previously described [39].
7 0.3 250 0.35256 Synergism HK activity in breast cancer cell lines was lower than that
8 0.3 125 0.34603 Synergism in control cells, and a non-significant reduction in HK
9 0.3 62.5 1.07844 Additive activity was noticed in the REF normal cell line.
B-MCF-7 Patra et al. [14] discovered that the deletion of the HK2
1 2 250 0.06841 Synergism gene in adult mice does not considerably disturb normal
2 2 125 0.20042 Synergism tissues. Moreover, MH is a specific inhibitor for HK II
3 2 62.5 0.51245 Synergism [19], and normal cells rely more on HK I [41] this explains
4 1 250 0.10016 Synergism why REF cells showed a mild non-significant reduction in
5 1 125 0.15299 Synergism HK activity. There are several studies described the role
6 1 62.5 0.34948 Synergism of HK2 as essential in tumor initiation and development
7 0.3 250 0.09957 Synergism in breast cancer; therefore, HK2 deletion as a cancer
8 0.3 125 0.13597 Synergism treatment have therapeutic value with no adverse physi-
9 0.3 62.5 0.22648 Synergism ological side effects [14, 42]. This effect prevented cancer
C-REF cell proliferation. Thus, the targeting of this key enzyme
1 2 250 1.01291 Additive by inhibitors inhibits glycolysis and therefore suppresses
2 2 125 1.07549 Additive cancer cell proliferation [43]. We noticed a significant
3 2 62.5 1.03328 Additive reduction in pyruvate concentration, after treatment with
4 1 250 1.10258 Antagonism the combination in breast cancer cell lines, and a non-
5 1 125 1.04811 Additive significant relation in the REF normal cell line after 72 h
6 1 62.5 1.38593 Antagonism of treatment. Given that pyruvate concentration depends
7 0.3 250 1.03584 Additive on the action of HK in the glycolysis pathway [44], the
8 0.3 125 1.56003 Antagonism decrease in HK resulted in a deficiency in pyruvate con-
9 0.3 62.5 1.51414 Antagonism centration, which confirmed by our experiment.
CI was measured by CompuSyn software. (A) CI values from the treatment of In correlation with previous experiment results, we
AMJ13 cancer cells; (B) CI values from treatment in MCF-7 cancer cells; (C) CI noticed that the MH-NDV combination suppresses the
values from the treatment of non-cancer cells (REF)
generation of intercellular ATP. We found that the ATP
concentrations in AMJ13 and MCF-7 cell lines treated
with MH and NDV were significantly lower than those
a non-significant effect on normal cells. The combina-
of the control. Reducing ATP levels effectively inhibits
tion of NDV and MH had the strongest effect on cancer
energy metabolism in MCF-7 and MDA-MB-231 cells.
cell lines in comparison to monotherapies. CI values
When glycolysis is inhibited, lactate production is com-
revealed high synergism between MH and NDV (CI < 1)
pletely terminated, and intracellular ATP concentration
in both MCF-7 and AMJ13 cell lines. All values for
abruptly decreases [45]. These phenomena are consistent
the REF cell line showed no synergism and they have
with the results of our study. Our results also provided
neglected cytotoxicity due to the fact of absence any
further evidence that glycolysis is increased in trans-
death percentage above 50%.
formed cells (cancer cells) in comparison with that in
AO/PI assay observations revealed that combination
normal cells, which reported before [46].
therapy was the best inducer for apoptosis, and that
The pH values of treated breast cancer cell lines secre-
was compatible with our previous results. NDV induces
tions were higher than those of the control. A non-signif-
apoptosis in virus-treated cells [38]. Furthermore, it’s
icant effect was detected in the normal REF cell line after
found that glycolysis inhibitors enhance apoptosis and
treatment with MH and NDV. Acidity decreased because
mitochondrial damage [38]. Apoptosis is essential for
lactate concentration decreased as a result of reduced
Fig. 3 Investigation the MH-NDV combined therapy to induce apoptosis in treated cells using acridine orange and propidium iodide: (a–d)
AMJ13 cancer cells indicated that MH-NDV induces apoptosis as evidenced red-stained cells, Untreated control cells emitting green fluorescence.
e–h MCF-7 cells were showing that the number of apoptotic cells is higher in the combination treatment. i–l There was no effect against REF
cells by combination therapy using MH-NDV. j, k MH-NDV treated AMJ13, and MCF-7 breast cancer cells expressed significant apoptotic cell
death in comparison to monotherapies and the untreated control cells. m no significant changes in all treatment modalities. Values represent the
(mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. The magnification of all images was 400×
pyruvate concentration and inhibited the growth and reductions in HK activity by the MH-NDV combina-
proliferation of cancer cells. It is proved that lactate defi- tions leading to a decrease in pyruvate, ATP, and micro-
ciency reduces the acidity of the cell environment [38]. environmental acidity. In addition, our results showed
This effect is favorable for preventing cancer cell pro- that the combined MH and NDV treatment had potent
liferation because tumors or cancer cells grow in acidic cytotoxic effects against breast cancer cell lines but not
environments. against the normal REF cell line that proved safety. This
treatment affected and enhanced cancer cell apoptosis
Conclusion as a result of glycolysis inhibition. Our investigation
Our results revealed that MH, NDV, and their com- proved the effect of MH and NDV and the strong effect
bination could inhibit the growth and proliferation of synergism between these compounds as the best
of breast cancer cell lines by increasing cytotoxicity therapy for cancerous cells in vitro. This strategy has
through inhibiting the glycolysis pathway and induc- potential applications as an effective cancer treatment.
ing apoptosis. These effects were attributed to specific Our observations will provide new insight into the
Fig. 4 MH–NDV combination efficiently inhibits glycolysis products in the treated breast cancer cells but not in normal cells in comparison to
monotherapies. a MH–NDV combined therapy induces a significant decrease in the activity of hexokinase in AMJ13 and MCF-7 cancer cell lines.
At the same time, there is no significant reduction in normal REF cells (b, c) measurement of pyruvate and ATP levels in cancer cells significantly
reduced in MH-NDV combination therapy in comparison to both monotherapies and untreated control cells. However, the reduction in the normal
REF cells was not significant. d Measurements of pH levels in AMJ13 and MCF-7 breast cancer cells indicate that MH-NDV treated cells supernatant
was more alkaline in comparison to both monotherapies significantly, while untreated control cells showing acidity as well as the control and
treated non-cancerous REF cells. All data shown are mean ± SEM (*P < 0.05) data from three different experiments using unpaired t-test, stars on MH
and NDV column means it is significant in compare to each other and star on the com means it is significant in compare to monotherapies (MH and
NDV). Star on the control column means it is significant when compared to MH, NDV and Com
development of therapeutic strategies for breast cancer Competing interests

The authors declare that there is no competence of financial interests.
and other types of cancer in the near future.
Author details
Acknowledgements 1
Department of Medical Chemistry, College of Medicine, University of Al-
The authors would like to thank the Department of Experimental Therapy,
Qadisiyah, Al Diwaniyah, Iraq. 2 Experimental Therapy, Iraqi Center for Cancer
Iraqi Center for Cancer and Medical Genetic Research, Mustansiriyah University
and Medical Genetics Research, Mustansiriyah University, Baghdad, Iraq.
for their support during the work. 3
College of Medicine, University of Al-Nahrain, Baghdad, Iraq. 4 Depart‑
ment of Microbiology, College of Medicine, Al-Nahrain University, Baghdad,
Authors’ contributions
Iraq. 5 Division of Biotechnology, Department of Applied Science, University
AMA, HSK and MIH designed the experiments. AGA, AMA, and MSJ conducted
of Technology, Baghdad, Iraq.
experiments. AGA, AMA, MIH wrote the manuscript. AGA, AMA, MIH and HSK
approved the drafts. All authors read and approved the final manuscript.
Received: 2 January 2020 Accepted: 24 August 2020
Funding
The authors declare that no specific funding was provided for the work.
Availability of data and materials

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‫(د‪ .‬احمد غضبان ثعبان الزيادي) ينشر بحثا علميا مشتركا في مجلة علمية عالمية‬
‫مصنفة ضمن مستودعات سكوبس وكالريفيت‪.‬‬
‫(د‪ .‬احمد غضبان ثعبان الزيادي) ينشر بحثا علميا مشتركا في مجلة علمية عالمية مصنفة ضمن‬
‫مستودعات سكوبس وكالريفيت وذلك بنشره بحثه في المجلة االنكليزية ‪Cancer Cell‬‬
‫‪ International‬والمدرجة ضمن المستوعبين (‪ Scopus‬بـ ‪ Cite Score‬مقداره ‪ 4.1‬و‬
‫‪ Clarivate‬بـ ‪ Impact Factor‬مقداره ‪ )3.4‬والتي تصدر عن الـ ‪ BMC‬وهي احدى مجالت‬
‫دار النشر ‪Springer Nature‬‬
‫‪https://cancerci.biomedcentral.com/articles/10.1186/s12935-020-01514-2‬‬
‫نشر (د ‪ .‬احمد غضبان ثعبان) ‪ /‬التدريسي في فرع الكيمياء الطبية ‪ /‬كلية الطب ‪ /‬جامعة القادسية‬
‫باالشتراك مع (أ‪.‬م‪ .‬د‪ .‬احمد مجيد الشمري) ‪ /‬رئيس القسم التجريبي ‪ /‬المركز العراقي للسرطان‬
‫والوراث ة الطبية ‪ /‬الجامعة المستنصرية و (أ‪.‬م‪ .‬د‪ .‬محمد عمران حمزة) التدريسي في قسم‬
‫الكيمياء والكيمياء الحياتية ‪ /‬كلية طب‪ /‬جامعة النهرين بحثا بعنوان‪:‬‬
‫‪((Hexokinase inhibition using D‑Mannoheptulose enhances oncolytic Newcastle‬‬

‫‪disease virus‑mediated killing of breast cancer cells)).‬‬
‫في مجلة )‪ (Cancer Cell International‬وهي مجلة عالمية مصنفة ضمن مستودعات سكوبس وكالريفيت‬
‫وتم نشرها من قبل )‪(Publisher: Springer Nature‬‬
‫تضمن البحث تنمية فيروس‬
‫كـ‬ ‫((‪NDV‬‬ ‫نيوكاسل‬ ‫مرض‬
‫(‪ )Virotherapy‬في بيض الدجاج‬
‫المخصب ومن ثم عزلة وتنقيته‬
‫وقياس ضراوة الفايروس الـ(‪)Titer‬‬
‫من خالل قياس التراص الدموي‬
‫(‪ )HA‬وتعريضه الى خاليا فيرو‬
‫لقياس قيمة الـ(‪ )TCID50‬ثم دراسة‬
‫تأثيره على خطوط خاليا سرطان‬
‫مثبط‬ ‫تأثير‬ ‫دراسة‬ ‫مع‬ ‫الثدي‬
‫)‪Hexokinase‬‬ ‫‪(D-‬‬ ‫الـ(‬
‫‪ )Mannoheptulose‬كـ (‪ )Phytotherapy‬ومن ثم دراسة تأثيرهما معا على خطوط الخاليا ومقارنة تأثيرهما‬
‫مع تأثير دواء الـ(‪ )Docetaxel‬كـ (‪ )Chemotherapy‬ويتم حساب تأثير هذه العوامل من خالل قياس السمية‬
‫الخلية بواسطة اختبار الـ (‪ )MTT‬وقياس الـ(‪ )IC50‬وتقدير التغيرات المورفولوجية باستخدام صبغة الـ( ‪Crystal‬‬
‫‪ )Violate‬كذلك توضيح تأثير االعتالل الخلوي للخاليا من خالل برنامج موت الخلية (‪ )Apoptosis‬باستخدام‬
‫صبغة ( ‪ ) Acridine Orange/ Propidium Iodide‬لتحديد نسبة قتل الخاليا السرطانية ومقارنة تأثيرها على‬
‫الخاليا الطبيعية (‪ .)Rat Embryo Fibroblast‬كذلك قياس تأثير هذه العوامل (الفايروس والمثبط ومزجهما)‬
‫على تثبيط مسار التحلل السكري (‪ )Glycolysis‬من خالل قياس تركيز الـ(‪)Hexokinase, Pyruvate, ATP‬‬
‫وقياس الحامضية الـ(‪ )pH‬المعبرة عن حامض الـ(‪ .)Lactate‬توصلت هذه الدراسة الى ان هذه العوامل ممكن ان‬
‫تثبط مسار التحلل السكري ونواتجه وتخفض من مستوى الطاقة المطلوبة لنمو الخاليا السرطانية وتحفز على موت‬
‫الخاليا المبرمج وبالتالي تمنع انتشار الخاليا السرطانية‪ .‬ولهذا ممكن اعتبار هذه العوامل كعالج واعد ومناسب‬
‫كبديل عن العالجات التقليدية للسرطان او مساند لها مما يقلل من التأثيرات الجانبية الضارة‪.‬‬
‫‪TMREES Conference Series‬‬
‫‪Technologies and Materials for Renewable Energy,‬‬
‫‪Environment and Sustainability‬‬
‫‪TMREES20-Greece Int'l Conf. Athens - Greece, June 25-27, 2020‬‬
‫تمت مشاركة المدرس المساعد احمد غضبان ثعبان الزيادي في مؤتمر اليونان الدولي لتقنيات‬
‫الطاقة المستدامة برعاية األكاديمية األوروبية للتنمية المستدامة والذي تم انعقاده في أثينا بتاريخ‬
‫‪ .2002/6 /27-25‬عن بحثه الموسوم‪:‬‬
‫‪((The Anti-Proliferative Activity of D- Mannoheptulose against Breast‬‬
‫))‪Cancer Cell line through Glycolysis Inhibition‬‬
‫)‪Ahmed Ghdhban Al-Ziaydi (1), Mohammed I. Hamzah (2), Ahmed M. Al-Shammari )3‬‬
‫تضمن البحث‪:‬‬
‫‪ -1‬تقدير تأثير الـ (‪ )D-Mannoheptulose‬كمثبط (‪ )Inhibitor‬للـ (‪ (Hexokinase‬على ايض تحلل السكر‬
‫(‪ ) Glycolysis Metabolism‬وموت الخاليا المبرمج ( ‪.) Apoptosis‬‬
‫‪ -2‬منع انتشار الخاليا السرطانية وتثبيط نمو خاليا سرطان الثدي في خطوط الخاليا نوع (‪AMJ13 Cell Lines‬‬
‫‪ )and MCF-7‬في المختبر (‪ )In Vitro‬من خالل تأثير المثبط‪.‬‬
‫‪ -3‬تقدير السمية الخلوية (‪ )Cytotoxicity‬وقيمة الـ (‪ )IC50‬لتراكيز مختلفة من المثبط‬
‫(‪ )D-Mannoheptulose‬عن طريق فحص السمية الخلوية باستخدام اختبار الـ(‪.)MTT cytotoxicity assay‬‬
‫‪ -4‬تقييم التغيرات المورفولوجية (‪ )Morphological Changes‬في الخاليا بواسطة صبغة البنفسج البلوري‬
‫(‪ )Crystal Violet‬من خالل تأثير المثبط‪.‬‬
‫‪ -5‬تقييم موت الخاليا المبرمج )‪ (Programmed Cell Death‬بواسطة تلوين األكريدين البرتقالي ‪ /‬يوديد‬
‫البروبيديوم (‪ (Acridine Orange/Propidium Iodide staining) )AO / PI‬حسب تأثير المثبط‪.‬‬
‫‪ -5‬قياس نشاط انزيم الهيكسوكيناز)‪ (Hexokinase activity‬والبيروفات )‪ )pyruvate‬وتركيز الـ ‪، ATP‬‬
‫والحموضة (‪ (pH( )Acidity‬في خطوط خاليا سرطان الثدي (‪ )Breast Cancer Cell Lines‬والخاليا الطبيعية‬
‫لجنين الفار)‪.)REF ( )Rat Embryo Fibroblast‬‬
‫كما اظهرت نتائج الدراسة أن المثبط يمكن أن يمنع انتشار خاليا سرطان الثدي عن طريق تثبيط مسار تحلل السكر‬
‫وتحريض موت الخاليا المبرمج‪ .‬كذلك لديه سمية خلوية قوية ضد خطوط خاليا سرطان الثدي وليس له تأثير على‬
‫خط الخاليا الطبيعي‪ .‬وبالتالي ستضيف هذه الدراسة ضو ًءا جديدًا في مجال تطوير االستراتيجيات العالجية لسرطان‬
‫الثدي في المستقبل القريب ويمكن اعتبار المثبط كعالج مستقبلي للخاليا السرطانية وطريقة استراتيجية مناسبة‬
‫الستخدامه كعالج فعال‪.‬‬
‫الخالصة……………………‪………………………………………………..‬‬
‫الخالصة‬
‫حديثا‪ ،‬كانت هناك عدة طرق جديدة تهدف إلى عالج السرطان‪ ،‬وسرطان الثدي على وجه الخصوص‪،‬‬
‫من خالل الحد من اآلثار الجانبية للعالج التقليدي‪ ،‬وغيرها من األساليب الجديدة مثل العالجات المستهدفة ‪،‬‬
‫العالج الفيروسي ومجموعة من هذه العالجات الجديدة‪ .‬يقلل عالج التآزر من اآلثار الجانبية والسمية‪ ،‬ويتغلب‬
‫على مقاومة الخاليا السرطانية للعالج‪.‬‬
‫تظهر معظم الخاليا السرطانية زيادة انحالل السكر حيث تستخدم هذا المسار األيضي لتوليد األدينوسين‬
‫ثالثي الفوسفات لنمو الخاليا واالنتشار‪ .‬يوضح تأثير واربورغ أن تثبيط انحالل السكر قد يكون ذا قيمة‬
‫عالجية في العالج المضاد لألورام‪ .‬يمكن لفيروس مرض نيوكاسل (الساللة العراقية) ومثبطات اإلنزيم أن‬
‫تعدل التمايز وتحفز موت الخاليا المبرمج في خطوط سرطان الثدي‪ .‬يعمل الـ د‪ -‬مانوهيبتولوز بايلوجيا عن‬
‫طريق تثبيط انحالل السكر‪ ،‬وبالتالي يثبط نمو الورم‪.‬‬
‫الهدف‪ :‬لتقييم اآلثار التآزرية لفايروس مرض نيوكاسل كعالج فيروسي ود‪ -‬مانوهيبتولوز كمثبط‬
‫للهيكسوكاينيز في تثبيط مسار انحالل السكر وبالتالي التمثيل الغذائي‪ ،‬كذلك لمنع التكاثر وتحفيز موت الخاليا‬
‫المبرمج في خطوط خاليا سرطان الثدي بالمقارنة مع خطوط الخاليا اللمفية في جنين الفأر الطبيعي‪.‬‬
‫الطريقة‪ :‬يتم تكاثر فايروس مرض نيوكاسل في بيض الدجاج الجنيني ويقدر كميا باستخدام اختبار‬
‫التراص الدموي ومتوسط الجرعة المصابة بالزراعة النسيجية على خط خاليا فيرو‪ .‬تم استخدام خط سرطان‬
‫الثدي البشري أحمد مرتضى جابرية ‪ ،2013‬ومؤسسة ميشيغان للسرطان ‪ ،7-‬وخط الخاليا الليفية الجنينية‬
‫الطبيعية للفئران‪ .‬ثم تم قياس نسبة السمية الخلوية للفيروس والمثبط ضد خطوط أحمد مرتضى جابريا ‪2013‬‬
‫‪ ،‬ومؤسسة ميشيغان للسرطان ‪ ، 7‬والخاليا الليفية الجنينية للفئران لمدة ‪ 72‬ساعة باستخدام فحص ميثيل‬
‫ثيازوليل تترازوليوم‪ ،‬لتحديد نصف اقصى تركيز للمثبط عند إصابات متعددة مختلفة تتراوح بين (‪-0.1‬‬
‫‪ )12.8‬وتراكيز مخففة تتراوح بين (‪ 1680-13.125‬ميكروغرام ‪ /‬مل) للفيروس والمثبط ‪ ،‬على التوالي ‪،‬‬
‫تم استخدام نصف اقصى تركيز للمثبط لدراسة الجمع او التأزر بينهما‪ ،‬باستخدام برنامج كومبوساين ‪ ،‬تم‬
‫قياس مؤشر االقتران لتحديد الجرعات الفعالة للفيروس والمثبط‪ .‬بعدها تم قياس التغيرات الشكلية ‪ /‬باستخدام‬
‫صبغة الكريستال البنفسجية‪ ،‬وموت الخاليا المبرمج باستخدام مزيج من صبغة األكريدين البرتقالية ‪ /‬يوديد‬
‫البروبيديوم‪ ،‬وفعالية الهيكسوكاينيز‪ ،‬وتركيز البيروفات‪ ،‬وتركيز األدينوسين ثالثي الفوسفات‪ ،‬والرقم‬
‫‪Page I‬‬
‫الخالصة……………………‪………………………………………………..‬‬
‫الهيدروجيني (الذي يعبر عن الحموضة) في سرطان الثدي المعالج وغير المعالج وخطوط الخاليا الطبيعية‪،‬‬
‫لفحص التأثير على مسار التحلل السكري‪.‬‬
‫النتائج‪ :‬كشفت هذه الدراسة أن الفايروس والمثبط بشكل فردي يثبط االنتشار والنمو‪ ،‬ويخفض قابلية‬
‫الحياة والنمو للخلية‪ ،‬ويحث على التغيرات الشكلية وموت الخاليا المبرمج في خطوط الخاليا السرطانية‬
‫للثدي‪ .‬من خالل تثبيط تحلل السكر وتحريض موت الخاليا المبرمج‪ ،‬لم تكن هناك نسب ملحوظة من السمية‬
‫الخلوية للـ د‪ -‬مانوهيبتولوز ضد الخاليا الليفية الجنينية للفئران حيث تراوحت نسبة السمية الخلوية من‬
‫(‪ ٪1.67‬إلى ‪ )٪24.72‬عند التراكيز العالية‪ .‬بينما كان هناك ارتفاع في السمية الخلوية ضد خاليا سرطان‬
‫الثدي تراوحت بين (‪ ٪27.29‬إلى ‪ )٪58.64‬لخط خاليا أحمد مرتضى جابرية ‪ .2013‬و (‪ ٪26.26‬إلى‬
‫‪ )٪60.49‬لخط خاليا مؤسسة ميشيغان للسرطان ‪ 7-‬بعد عالج المثبط‪ .‬لم يسبب الفيروس تأثيرا ساما للخاليا‬
‫على خط الخاليا الطبيعي حيث تراوحت نسبة السمية الخلوية من (‪ ٪4.31‬إلى ‪ )٪28.75‬للخاليا الليفية‬
‫الجنينية للفئران‪ .‬كانت خاليا سرطان الثدي أكثر حساسية للفيروس حيث تراوحت نسبة السمية الخلوية من‬
‫(‪ ٪24.69‬إلى ‪ )٪64.26‬لخط خاليا أحمد مرتضى جابرية ‪ .2013‬و (‪ ٪23.95‬إلى ‪ )٪62.02‬لخط خاليا‬
‫مؤسسة ميشيغان للسرطان ‪ 7-‬بعد عالج الفيروس‪.‬‬
‫لوحظ تثبيط تحلل السكر من خالل انخفاض فعالية الهيكسوكاينيز من (‪ 3.2‬وحدة ‪ /‬مل كسيطرة إلى‬
‫‪ 2.0‬وحدة ‪ /‬مل مع فيروس و ‪ 1.4‬وحدة ‪ /‬مل مع مثبط و ‪ 1.1‬وحدة ‪ /‬مل مع الجمع بينهما في خط خاليا‬
‫أحمد مرتضى جابرية ‪ 2013‬ومن ‪ 3.0‬وحدة ‪ /‬مل كسيطرة إلى ‪ 1.9‬وحدة ‪ /‬مل مع فيروس ‪ 1.4 ،‬وحدة ‪/‬‬
‫مل مع مثبط و ‪ 1.2‬وحدة ‪ /‬مل مع الجمع بينهما في خط خاليا مؤسسة ميشيغان للسرطان ‪ ، )7-‬وتركيز‬
‫البيروفات من (‪ 0.48‬ميكرو مول ‪ /‬مل كسيطرة إلى ‪ 0.28‬ميكرو مول ‪ /‬مل بالفيروس ‪ 0.25 ،‬ميكرو مول‬
‫‪ /‬مل مع مثبط و ‪ 0.14‬ميكرومول ‪ /‬مل مع الجمع بينهما في خط خاليا أحمد مرتضى جابرية ‪ 2013‬ومن‬
‫‪ 0.5‬ميكرومول ‪ /‬مل كسيطرة إلى ‪ 0.23‬ميكرومول ‪ /‬مل مع فيروس ‪ 0.23 ،‬ميكرومول ‪ /‬مل مع مثبط و‬
‫‪ 0.15‬ميكرومول ‪ /‬مل مع الجمع بينهما في خط خاليا مؤسسة ميشيغان للسرطان ‪ )7-‬وتركيز األدينوسين‬
‫ثالثي الفوسفات بتراكيز تتراوح من (‪ 1.2‬مليمول ‪ /‬لتر كسيطرة إلى ‪ 0.58‬مليمول ‪ /‬لتر مع فيروس ‪،‬‬
‫‪ 0.61‬مليمول ‪ /‬لتر مع مثبط و ‪ 0.39‬مليمول ‪ /‬لتر مع الجمع بينهما في خط خاليا أحمد مرتضى جابرية‬
‫‪ 2013‬ومن ‪ 1.3‬مليمول ‪ /‬لتر كسيطرة إلى ‪ 0.65‬مليمول ‪ /‬لتر مع فيروس ‪ 0.69 ،‬مليمول ‪ /‬لتر مع مثبط‬
‫و ‪ 0.35‬مليمول ‪ /‬لتر مع الجمع بينهما في خط خاليا مؤسسة ميشيغان للسرطان ‪ .7-‬لوحظ انخفاض‬
‫الحموضة من خالل زيادة الرقم الهيدروجيني من (‪ 6.3‬كسيطرة إلى ‪ 7.3‬مع فيروس و‪ 7.3‬مع مثبط و‪8.2‬‬
‫‪Page II‬‬
‫الخالصة……………………‪………………………………………………..‬‬
‫مع الجمع بينهما في خط خاليا أحمد مرتضى جابرية ‪ 2013‬ومن ‪ 6.3‬كسيطرة إلى ‪ 7.4‬مع فيروس و‪7.4‬‬
‫مع مثبط و‪ 8.2‬مع الجمع بينهما في خط خاليا مؤسسة ميشيغان للسرطان ‪.7-‬‬
‫كشفت نتائج العالج المركب أن التآزر بين الفايروس والمثبط أنتج تأثير أقوى مضاد للورم من تأثير‬
‫العالج األحادي مع أي منهما‪ .‬نتج عن الجمع بينهما زيادة تثبيط الخاليا الحية‪ ،‬وزيادة التغيرات الشكلية‬
‫الخلوية‪ ،‬وارتفاع تحريض موت الخاليا المبرمج في خطوط الخاليا السرطانية المعالجة عن طريق تثبيط‬
‫مسار تحلل السكر‪ ،‬كما يتضح من خالل انخفاض فعالية الهيكسوكاينيز‪ ،‬وتركيز البيروفات‪ ،‬واألدينوسين‬
‫ثالثي الفوسفات والحموضة البيئية التي تعزى الى انخفاض تركيز الالكتات (حامض الالكتيك)‪ ،‬مقارنة مع‬
‫خطوط الخاليا الطبيعية‪ .‬عالوة على ذلك‪ ،‬كان هذا العالج آمنا نسبيا في الخاليا الطبيعية‪.‬‬
‫االستنتاج‪ :‬التآزر بين الفيروس والمثبط يزيد من السمية الخلوية ويسبب تثبيط نمو كبير للخاليا عن‬
‫طريق تثبيط تحلل السكر وتحفيز موت الخاليا المبرمج في خاليا سرطان الثدي ولكن ليس في الخاليا‬
‫الطبيعية في المختبر‪ .‬كما أنه أفضل من العالج الفردي‪ .‬تم تحديد انخفاض كبير في فعالية الهيكسوكاينيز‪،‬‬
‫وتركيز البيروفات‪ ،‬واألدينوسين ثالثي الفوسفات‪ ،‬والحموضة في جميع طرق العالج في خاليا سرطان الثدي‬
‫ولكن ليس في الخاليا الطبيعية‪ .‬يمكن اعتبار التآزر بين الفيروس والمثبط عالجا مستقبليا للخاليا السرطانية‬
‫وطريقة استراتيجية جديدة الستخدامها كعالج فعال‪.‬‬
‫‪Page III‬‬
‫جمهورية العراق‬
‫وزارة التعليم العالي والبحث العلمي‬
‫جامعة النهرين‬
‫كليـة الطـب‬
‫الفعالية األحادية والمختلطة المضادة للتكاثر للمانوهيبتولوز‬

‫وفايروس مرض نيوكاسل ضد خطوط خاليا سرطان الثدي‬
‫التي تستهدف تثبيط تحلل الكلوكوز‬
‫أطروحة‬
‫مقدمة إلى مجلس كلية الطب‪/‬جامعة النهرين‬
‫وهي جزء من متطلبات نيل درجة الدكتوراة في فلسفة الكيمياء الطبية‬
‫من قبل‬
‫امحد غضبان ثعبان الزايدي‬
‫بكالوريوس علوم كيمياء ‪( /‬جامعة القادسية‪) 1993 /‬‬
‫دبلوم عالي علوم كيمياء‪ /‬تحليالت كيميائية ودوائية ‪( /‬جامعة كربالء‪)2008 /‬‬
‫ماجستير علوم كيمياء‪/‬كيمياء حياتية‪( /‬الجامعة العثمانية‪ /‬الهند ‪)2015/‬‬
‫بأشراف‬
‫ا‪.‬م‪.‬د‪ .‬احمد مجيد حمزة‬ ‫أ‪.‬م‪.‬د‪ .‬محمد عمران حمزة‬

‫دكتوراة علم االمراض‬ ‫دكتوراة كيمياء حياتية سريرية‬
‫‪ 2020‬م‬ ‫‪ 1441‬هـ‬

The Mono and Combined Anti-Proliferative Activity of D - Mannoheptulose and Newcastle Disease Virus Against Breast Cancer Cell Lines Targeting Glycolysis Inhibition

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The Mono and Combined Anti-Proliferative Activity of D - Mannoheptulose and Newcastle Disease Virus Against Breast Cancer Cell Lines Targeting Glycolysis Inhibition

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Ministry of Higher Education

& Scientific Research

The Mono and Combined Anti-Proliferative Activity of

Assistant Professor Assistant Professor

2020 A.D 1441 A.H

I dedicate this humble work…..To

My dear and beloved wife ……

My dear son and daughters…

My dear brothers and sisters….

Ahmed Ghdhban Al-Ziaydi

I would like to express my sincere thanks and gratitude and appreciation to my

Ahmed Ghdhban Al-Ziaydi

Aim: To assess the synergistic effects of Newcastle disease virus as viral

Method: Newcastle disease virus is propagated in embryonated chicken eggs

combination in Michigan cancer foundation-7) and Adenosine tri phosphate

Conclusion: Synergism between Newcastle disease virus and

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4-14 Comparison of IC50 between cell lines 84

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A-24 Inverted and fluorescent Microscope xvii

Cancer is a major public health problem worldwide and it is the second

disease virus (NDV) is a member of the genus avulavirus, in the family of

Aim of the study

1- To determine the anti-proliferative activity of NDV, D-Mannoheptulose (MH) and

2- Determination of TCID50 of NDV and IC50 values of NDV and MH.

3- Determination of synergism, or antagonism between NDV and MH.

5- Investigate the apoptosis induction by mono- and combination therapies in cancer

2.1 Tumor (Neoplasia)

Cancer is a group of diseases characterized by uncontrolled growth and spread

2.3 Cancer and cell death

2.4 Breast cancer

2.4.1 Etiology of breast cancer

Evidence indicates that breastfeeding reduces the risk of developing breast

2.4.2 Signs and symptoms of breast cancer

2.4.3 Genetics and breast cancer

Genetic changes can occur at different levels and by different mechanisms.

2.4.4 Breast cancer Spread and metastasis

2.4.5 Types of breast cancer

B. Lobular Carcinoma in Situ (LCIS):

2.4.5.2 Invasive Carcinoma

A. Invasive Ductal Carcinomas (IDC)

B. Invasive Lobular Carcinoma (ILC)

C. Less Common Types of Invasive Breast cancer

2.5 Glycolysis Pathway

Under certain conditions, especially under oxygen deprivation, OXPHOS

2.5.1 Glycolysis in cancer cells

Lactate dehydrogenase (LDH) is a tetrameric enzyme with 2 subtypes: LDH-A

2.5.2 Metabolic pathways in cancer

Cancer is a disease condition arising from uncontrolled division of cells in the

Aerobic breakdown of glucose consists of four stages: glycolysis, pyruvate

Figure (2-5): Glycolysis in tumor cell (Fadaka et al., 2017).

2.5.4 Warburg effect and the glycolytic inhibitors

Figure (2-6): Schematic representation of the differences between oxidative phosphorylation,

Oxygen is essential for this process as it is the final electron acceptor to

In proliferating cells, ~10% of the glucose is diverted into biosynthetic

metabolism during nutrient limitation and non-oxidative metabolism during cell

Hexokinase is a tissue-specific isoenzyme that catalyzes the phosphorylation of

phosphorylation (Chen et al., 2017). As anaerobic glycolysis allows more rapid