FINAL YEAR PROJECT PROPOSAL 2017

TITLE
INVESTIGATING THE EFFECT OF PHARMACOLOGICAL INHIBITION OF

AUTOPHAGY ON NEWCASTLE DISEASE VIRUS (NDV) - INDUCED ONCOLYSIS

IN BREAST CANCER CELLS.

Department of Microbiology

Faculty of Biotechnology and Biomolecular Sciences

NAME : NUR ERRA IZANIE BINTI AHMAD DAUD

MATRIC NUMBER : 180077

SUPERVISOR : DR SAILA ISMAIL

DATE : 30TH OCTOBER 2017

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TABLE OF CONTENTS

CONTENT PAGE NUMBER

List of Abbreviation 4

1.0 Introduction 5

2.0 Hypothesis 6

3.0 Objectives 6

4.0 Problem Statement 6

5.0 Materials 7

6.0 Methodology

6.1 Cell culture and maintenance of the mcf-7 cell line 7

6.2 Growth of virus in embryonated eggs 7

6.3 Titration of virus stocks 8

6.4 Purification of virus from the allantoic fluid of 8

infected eggs
8
6.5 Plaque assay
9
6.6 NDV infection of mcf-7 cancer cells
9
6.7 RNA expression
10
6.8 Flow cytometry
10
6.9 Statistical analysis

7.0 Flow chart 11

8.0 Gantt chart 12

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9.0 Literature review

9.1 NDV as Biological Structure and Properties 13-14

9.2 NDV as Oncolytic Virus Application 14-15

9.3 NDV as a Vaccine Vector 15

9.4 Breast cancer 16

9.5 Autophagy 17-18

9.6 Autophagy and cancer 19

9.7 Autophagy and cancer immunology 20-21

9.8 Chemical inhibitors of autophagy 22

List of References 23-28

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LIST OF ABBREVIATION

ATCC: American Type Culture Collection

CQ: chloroquine
DAMPS: Damage-associated molecular pattern molecules
ERK: extracellular signal-regulated kinase
HCQ: hydroxyl chloroquine
IFN: Interferon
JNK : c-jun N-terminal kinase
mTOR : mammalian target of rapamycin
mTORC1: mammalian target of rapamycin complex 1
NLRs: nucleotide oligomerization domain (NOD)-like receptors
PI3K : phosphatidylinositol 3-kinase
PAMPS: Pathogen-associated molecular pattern molecules
PRR: pattern recognition receptor
TLRs: Toll-like receptors
TNF-a: tumour necrosis factor
TRAIL: TNF-related apoptosis-inducing ligand
3-M: 3-methyladenine

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1.0 INTRODUCTION

Newcastle disease virus is a family of paramyxoviridae of avulavirus which is first isolated

in 1927. It is well studied due to its pathogenic potential, oncolytic activity and vaccine

vector for both humans and animals (Ganar, Das, Sinha, & Kumar, 2014). It is well-studied

due to potential of oncolytic cancer therapy, gene therapy and immune stimulation. NDV are

categorised into three classification depending on the severity of the disease which are

lentogenic , mesogenic , or velogenic (Zamarin & Palese, 2012).

Globally, breast cancer is one of the most frequent diagnosed cancers (Zervoudis et al.,

2014). There are 1.38 million new cancer cases were reported in 2008 (23% of all cancers),

and ranks second overall with 10.9% of all cancers (Ferlay et al., 2010). There are studies

showed that the breast cancer have an association with exogenous hormones, ionizing

radiation and beverage alcohol (MacMahon, 2006).

Autophagy is a conserved process where the cell self-digests its own component. This

process can be classified into three types; macroautophagy, microautophagy and chaperone-

mediated autophagy (CMA) (Jiang et al., 2014).

Cancer occur due to lack of regulatory control over cell death mechanism. Cancer therapy

known as removal of tumour by surgery has been implemented recently. However, new

treatments like immunotherapy, radiotherapy, chemotherapy were recently discovered (Kelly

& Russell, 2007). A clear example is usage of viruses in tumour therapy to treat human

diseaser using a potential microorganism. Besides, when X-rays and radiotherapy were first

discovered in 1895 and 1898, their application in treating cancer become wide and three

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months after the report published, there was treatment of breast cancer that have been done

using X-rays (Bernier, Hall, & Giaccia, 2004).

2.0 HYPOTHESIS

1. Newcastle disease virus able to induce autophagy in breast cancer cells.

2. Autophagy plays a role in enhancing Newcastle disease virus- mediated oncolysis in breast

cancer cells.

3. Autophagy show the effect on NDV replication in breast cancer cells.

3.0 OBJECTIVES

1. To determine whether NDV can induce autophagy in breast cancer cells.

2. To investigate whether pharmacological inhibition of autophagy can enhance NDV-

induced oncolysis in breast cancer cells.

3. To examine the effect of autophagy pathway inhibition on NDV replication in breast

cancer cells.

4.0 PROBLEM STATEMENT

1. Does the Newcastle disease virus able to induce autophagy in breast cancer cells?

2. Does Newcastle disease virus- mediated oncolysis in breast cancer cells can be enhanced

by autophagy?

3. Does the autophagy show the effect on NDV replication in breast cancer cells?

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5.0 MATERIALS

Breast cancer MCF-7 cell line, SW620 cell line obtained from (ATCC), tryphan blue, 200 9-

day-old embryonated eggs , 70% ethanol, nail polish or wax , Phosphate Buffer Saline, Foetal

Bovine Serum (FBS), NDV; vaccine strain AF 2240 , sucrose , 0.5% red blood cell (RBCs),

dimethylsulfoxide (DMSO), SAR405, Chloroquine.

6.0 METHODOLOGY

6.1 CELL CULTURE AND MAINTENANCE OF THE MCF-7 CELL LINE

Breast cancer MCF-7 cell line is obtained from American Type Culture Collection (ATCC).

T75 cm² will be used to grow cells until they are confluent (Ahmad, et al., 2015). Cells will

be observed under the inverted microscope until the semi-confluent monolayer form

(Ahamed, et al., 2004). Cell death will be determined using tryphan blue exclusion and the

total cells will be counting using haemocytometer (Meng, et al., 2014). Next, the cells will be

incubated at 37°C in 5% CO2 incubator (Ahmad, et al., 2015).

6.2 GROWTH OF VIRUS IN EMBRYONATED EGGS

Nine-day-old embryonated eggs will be used as the source of fibroblasts for stock virus

production (Lam, 1995). Upon arrival, the eggs will be incubated immediately at 37.5°C in

an incubator. After 10 days of incubation, position of embryo, membranes and blood vessels

will be observed. Dead embryo’s eggs will be discarded. The site of injection point will be

marking which is 5 mm above the line where the membranes meets the air sac and between

blood vessels. To sterilize the outer surface, the eggs will be rinsed with 70% ethanol. Make a

small hole in the shell at the site of injection. The needle must be inserted carefully to

penetrate the embryo. Holes in the eggs will be punctured and covered either with nail polish

or wax. Eggs are then incubated at 37.5°C in an incubator.

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6.3 TITRATION OF VIRUS STOCKS

Hemagglutination test is used to determine the virus titer (Vogel, J. et al., 1957). Cells will

be plating in 96-multiwell microtiter flat bottom plate (Nunc, Denmark) at a density of

1×10^4 cells/ml in a 100 µL/well in culture medium, incubated for 24 hours to allow

confluent (Ahmad, et al., 2015). 50 μl PBS will be added to each well together with 50 μl

viral dilution.

The above steps will be repeated for the first column, then the mixing and transferring 50 μl

to the next well will be repeated, 50 μl from the last well will be discarded into the bleach

solution. Then, 50 μl of 0.5% RBCs will be added into each well and mixing gently, incubate

for 45 minutes at 37°C.

6.4 PURIFICATION OF VIRUS FROM THE ALLANTOIC FLUID OF INFECTED EGGS

Purification will be done by centrifugation process to concentrate the virus. The virus will be

harvested from the allantoic fluid and purified centrifugation at 3000 rpm for 10 min. The

allantoic fluid containing virus will be collected and stored at –80°C (Zhirnov, 2017). The

supernatant consisting virus will be collected and cryopreservation at -80°C will be

performed (Meng , et al., 2014). The virus will be purified through a 20% sucrose gradient

onto a 65% sucrose pad by centrifugation.

6.5 PLAQUE ASSAY

SW620 cell is grown to ~90% confluency in 96-well plate. Cells will be infecting with NDV

at a MOI of 1 PFU, 2.067 × 10^5 for 1 hour at 37°C. After the incubation with NDV for 1

hour, the virus will be discarded and 200 μl of maintenance media with 1% of FBS will be

adding to each well. Cells will be incubated at 37°C in 5% CO2 incubator for the required

time points which are 24, 48, 72 hours.

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6.6 NDV INFECTION OF MCF-7 CANCER CELLS

After 24 hours of subculturing, semi-confluent of the cells will be forming, and at this stage,

cells will be infecting with the wild NDV (Ahamed, et al., 2004). The cells will be treated

with IC50 concentration (25.99 HAU/mL) value of the virus and incubated in a humidified

incubator at 37°C in an atmosphere of 5% CO2 for 24, 48, and 72 hours (Ahmad, et al.,

2015). Cells will be rinsed using PBS and will be infecting with the virus in empty DMEM at

a 10 HAU/10^6 cells for about 3 hours, and then each well will be added with the completed

medium (Meng , et al., 2014).Cells will be pre-treated with dimethylsulfoxide (DMSO),

Chloroquine or SAR405 for 1hour and then infected at MOI of 1 with virus, followed by

incubation in standard environment (Kang, et. al., 2017).

6.7 RNA EXPRESSION

Total cellular RNA will be extracting using TRIZOL (Invitrogen, 15596-026) and reverse-

transcription of RNA will be performed (Meng , et al., 2014). RNA interference will be used

to study ATG5, a gene for formation of autophage. The RT-PCR procedure will be started

with an initial denaturation at 94˚C for 5 minutes, followed by 30 cycles at 94˚C for 30 sec.

Then, annealing process at 53˚C (RVG) or 55˚C (NDV and GAPDH) will be done for 30

seconds, followed by extension at 72˚C for 30 sec, with a final extension at 72˚C for 10

minutes. PCR product will be loaded for electrophoresis and visualization will be done using

ethidium bromide. The resulting bands will be analyzed using Quantity One software (Bio-

Rad) (Bu et.al .,2016).

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6.8 FLOW CYTOMETRY

Tumour cells will be grown in 1×10^5 cells/dish in 60-mm dishes and treated with

NDV/FMW at an MOI of 10 (Jiang , et al., 2014). The cells will be harvested and rinsed with

PBS.. Apoptotic cell death will be identified by Annexin V/propidium iodide (PI) staining

assay (Meng , et al., 2014). Total amount of apoptosis will be indicated by the numbers of

early and apoptotic cells. All experiments shall be performed for three times (Bu et.al .,2016).

6.9 STATISTICAL ANALYSIS

Data will be analyzed by (ANOVA) using SPSS software. Differences with p <0.05 or p

<0.01 will be considering as statistically significant. All experiments will be repeated at least

three times (Bu et.al ., 2016). 2-tailed Student’s t test will be using for all analyses, P < 0.05

will be considered as significant difference (Meng, et. al., 2014).

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7.0 FLOW CHART

NDV propagation and quantification of viral stock titre by plaque assay

Cancer cell tissue culturing

Measurement of autophagy gene induction in cancer cells following NDV infection with or

without pharmacological autophagy inhibitor, SAR405 and Chloroquine

Determination of the effect of pharmacological inhibition of autophagy pathway on NDV-

induced oncolysis in cancer cells by flow cytometer

Quantification of viral replication following pharmacological inhibition of autophagy by

plaque assay

Thesis writing

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8.0 GANTT CHART

YEAR 2017 2018

PROJECT ACTIVITIES NOVEMBER DECEMBER JANUARY FEBRUARY MAC APRIL MAY

NDV propagation and

quantification of viral
stock titre by plaque assay

Cancer cell tissue

culturing

Measurement of
autophagy gene
induction in cancer cells
following NDV
infection with or
without
pharmacological
autophagy inhibitor,
SAR405 and
Chloroquine
Determination of the
effect of
pharmacological
inhibition of autophagy
pathway on NDV-
induced oncolysis in
cancer cells by flow
cytometer

Quantification of viral
replication following
pharmacological
inhibition of
autophagy by plaque
assay

Thesis writing

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9.0 LITERATURE REVIEW

9.1 NDV AS BIOLOGICAL STRUCTURE AND PROPERTIES

Newcastle disease virus is a family of paramyxoviridae under the genus of avulavirus which

is first isolated in 1927. It is well studied due to its pathogenic potential, oncolytic activity

and vaccine vector for both humans and animals (Ganar, Das, Sinha, & Kumar, 2014).

Further investigation showed that NDV can be classified into three classification depending

on the disease severity in birds which are lentogenic, mesogenic, or velogenic (Zamarin &

Palese, 2012). Moura (2016) stated that velogenic and mesogenic NDV strains produce

similar distribution mode of lesions in the brain; however, lesions caused by mesogenic

strains are delayed in time.

Fusion protein (F), haemagglutinin-neuraminidase protein (HN), nucleocapsid protein (NP),

matrix protein (M), phosphoprotein (P), and large polymerase protein (L) are structural

proteins encoded NDV genome and V and W protein as 2 non- structural proteins.(Steward,

1993). The nucleocapsid (N) protein is the most sufficient protein present in the virus

particle. The genomic RNA is associated with the N, P and L thus forming the

ribonucleoprotein complex (RNP) which serves as a template for RNA synthesis. The

herringbone-like structure formed when the interaction of amino-terminus N protein with

viral RNA (Errington and Emmerson, 1997). The phosphoprotein (P) protein consists of 395

amino acids, play a different function when interacting with N protein during virus

replication. Its carboxy-terminal residue (247–291) participates in P–P and P–N interaction

(Jahanshiri et al., 2005). In another study, P protein acts as virulence depending on the cell

type and the NDV strain (Dortmans et al., 2010). Mebatsion et al., 1999 suggested that M

protein function is on maintaining the spherical shape of nucleocapsid. F protein is said play

the major role in the virulence of NDV strain (Panda et al., 2004; Romer-

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Oberdorfer et al., 2003). Presence of arginine at position at 113, 115 and 116 in the cleavage

site is observed to cause variation cleavage of virulent NDV F protein (Samal et al., 2011).

Several studies demonstrated that HN is a multifunctional protein which responsible in

receptor recognition in the host cell, receptor removal, preventing self-assembly and interact

with F protein to promote fusion. Lamb and Parks, 2007; Sergel et al., 1993a stated that HN

protein responsible for virus specific membrane fusion. Other finding claimed that HN

protein is responsible for the infection and the pathogenesis of NDV (Huang et al., 2004c;

Khattar et al., 2009; Kimet al., 2009; Yan et al., 2009). Large polymerase protein (L) consists

of 2204 amino acids and it is believed as the largest NDV genome (Lamb and Parks, 2007).

The role is synthesizing viral mRNA and support genomic RNA replication. Further, L

protein also carry out 5’ capping, methylation, and poly A polymerase activity on the newly

formed mRNA (Dortmans et al., 2010; Lamb and Parks, 2007). The V protein is another

virulence factor of NDV (Park et al., 2003). Viral RNA replication occur as a result of

interaction between N protein and V protein (Horikami et al., 1996).

9.2 NDV AS ONCOLYTIC VIRUS APPLICATION

Nowadays, Newcastle disease virus (NDV) is under on-going research for gene therapy, and

immune stimulation and oncolytic cancer therapy. Most of the oncolytic virus is genetically

engineered to be selectively replicate in tumour cells and can cause cancer cell death,

however NDV is said naturally oncolytic RNA virus (Schirrmacher, 2015). Multiple studies

have been conducted to show the mechanism underlying the NDV-mediated cytotoxicity.

Oncolytic NDV could trigger apoptosis pathways in tumour-infected cells (Phuangsab, 2001).

Other study on NDV strains related to cancer cell lines of ectodermal, endodermal, and

mesodermal origin showed that NDV exert oncolysis by extrinsic and intrinsic pathways of

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cell death (Elankumaran, 2006). In this infection activity, tumour cells led to yielding of

TNF-ɑ and soluble TRAIL where resulted caspase 8 activation though some of the cell lines

can be induced without that activation. However, in all tumour cell lines, NDV reduced

potential of mitochondrial membrane and activation of caspase 9, prove that the intrinsic

pathway is crucial. Meng, 2012 state that NDV successfully induce autophagy in U251

human ganglioma cells to produce virus (Meng, 2012). The engulfment of cytoplasmic

macromolecules and damaged organelles by autophagosome were the indicator of the

autophagy induction for virus replication ( Kraft, 2012).

9.3 NDV AS A VACCINE VECTOR

Samal, 2011 stated that NDV as a potential vaccine vector candidate in both human and

animal use. The nature of the transcription, minimum recombination frequency and DNA

phase absence during virus replication, make NDV as a favourable designed live attenuated

vaccine and vaccine vectors. There are several properties of NDV contributing to the ability

of the virus as a viral vectors. The replication of NDV in host cytoplasm cause the integration

with the host genome does not occur. Besides, recombinant NDV posses a high and stable

expression of foreign protein after many passages both in vivo and in vitro (Huang et al.,

2003a).

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9.4 BREAST CANCER

Breast cancer is the highest diagnosed cancer in women worldwide. Even though there are

several treatments available, it remains rank number one as the most common causes of

women’s death (Zhang, Cao, Sun et al., 2016). There are several factors of breast cancer

identified such as population growth, aging, lifestyle changes, and migration to urban

communities (Porter, 2008). The early menarche, late menopause, and obesity in

postmenopausal women increase the risk of developing breast cancer (Key et al., 2001).

Diagnosed breast cancer found that the largest portion for the positive breast cancer are

hormones receptor, estrogen and progesterone. These female hormones are sufficient for the

cells’s growth and survival (Stanford, 1986). A study has revealed that women who have

family history of the disease are at increased risk especially those who are older than 50

years (Veronesi et al., 2005).

A study conducted by a group of researchers found that the etiology of breast cancer are

associated with childbearing, ovary and age-specific risks (Hankinson and Hunter, 2002). It is

well-known that bearing-children could reduced the chances of getting breast cancer among

women. In western country where the breastfeeding was decreasing, the breast cancer risk

being high (Claypon, 1926). Increase of breast cancer’s risk is related to the ovary’s function

where women in an early onset and late natural menopause have high chances to this

exposure. In 2013, there were 232,340 new cases reported and specifically 39, 6200 deaths

are among US women. In 2013 , 79% of new cases have been reported and from the amount,

and 88% of breast cancer deaths occur among women aged 50 years and older (DeSantis, Ma,

Bryan et al., 2014).

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9.5 AUTOPHAGY

Autophagy is a homeostasis preservation especially under metabolic stress and nutrient-lack

condition (Michigami, Hiraga, Williams, et al., 2002). Levine and Kroeme, 2008 in

Autophagy in the Pathogenesis of Disease state that the autophagy is a pathway of lysosome

degradation which is crucial for survival, differentiation, development and homeostasis.

Pathological and physiological conditions are the factors contributing to autophagy. There are

three forms of autophagy studied: microautophagy, chaperone mediated autophagy and

macroautophagy (Ravanan, Srikumar, Talwar et al., 2017).

Among those three, macroautophagy is said as a major machinery to clear away died cellular

organelles and other debris. Macroautophagy is a process in which the substrates are

accumulated within cytosolic double-membrane vesicles, so called autophagosomes

(Yorimitsu and Klionsky, 2005). As shown in Figure 1, autophagy is a complex process

involving multiple phases (reviewed in Yang et al., 2015). The first step in autophagy is the

formation of phagophore that can be induced by various stimuli such as intracellular or

extracellular signals and stress such as growth factors depletion, nutrient starvation,

Endoplasmic Reticulum (ER) stress, and pathogen infection (He & Klionsky, 2009).

Phagophore formation is regulated by ULK1, Atg13, FIP200 and Atg101 complex,

downstream of mTORC1. Next step is nucleation/expansion/elongation. Beclin 1 and

hVPS34/Class III phosphatidylinositol 3-kinases (PI3K) complex plays an important role in

this process as Beclin 1 act as mediator in process of nucleation (Huang et al., 2012). End of

autophagy process is the production of autolysosome where acidic lysosomal hydrolases

digested the unused organelles (Saftig, 2009). To analyse the autophagy, light chain 3 (LC3)

and p62/ SQSTM1 were analyzed since they serve different function in distinct stages of

autophagosome formation (Klionsky, 2016).

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Figure 1: Autophagy involving stages of initiation, elongation and formation of

autophagosome ,fusion and autolysosome. Reproduced from (Yang et al., 2015).

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9.6 AUTOPHAGY AND CANCER

Accumulating evidence shows a role for autophagy in cancer, although it remains

controversial as it can be tumour suppressive during cancer development, but can lead to

tumour cell survival during cancer progression (Rouschop & Wouters, 2009). Last decade

research showed several genetics links were related to defects of autophagy and cancer, led to

sufficient proof that autophagy is a bona fide tumour suppressor pathway (Levine, 2007;

Mathew et al., 2007a). The discovery of the specific relation of autophagy mechanism and

human cancer is due to the report that ATG Beclin 1 as tumour suppressor (Liang et al.,

1999) yet the molecular mechanisms on how autophagy is functioning is not well studied.

Levine (2007) stated that oncogenes and tumour suppressors are the main regulators for

autophagy thus led to the finding of unknown role of autophagy in cancer. There is specific

mechanism required by the cells to become cancerous. Cancer cells suppress several anti-

autophagic genes where they resist the normal cells from transformed into a cancerous one.

The example of the genes are Bcl-2, AKT and PI3KC1. Inhibition of autophagy has been

shown to increase therapeutics benefits in cancer therapies (Chen et al., 2010). In contrast,

Gozuacik and colleagues reported that induction of autophagy was able to kill cancer cells

through apoptosis (Gozuacik & Kimchi, 2004).

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9.7 AUTOPHAGY AND CANCER IMMUNOLOGY

In recent past, role of autophagy is associated with immune systems where innate and

adaptive immune responses were administered. A great development of study claimed that

stimulation of autophagy is related to cancer immune responses (Pan et al., 2015). Crotzer &

Blum, (2010) stated that autophagy and genes associate with autophagy can effect the innate

and adaptive immunity by regulating antigen processing and presentation. Figure 2 shows the

regulation of autophagy by innate immune receptors. The machinery requires PAMPs and

DAMPs with PRR. PRR incorporate TLRs, NLRs and receptors for cytokines such as

interferon and TNF-α.

With the aid of TLRs, the interference of phagocytosis and autophagy occurred, thus increase

the host innate immune response. This process is done by activation of JNK and ERK

signalling pathways (Fang et al., 2014). NLRs are said to be responsible in intracellular

bacteria sensing and consists of important element of host innate immune system. NLRs

which constitutes of Nod1 and Nod2 are interacting with ATG16L1, a component for

autophagosome formation in order to trigger autophagy (Travassos, 2010). Maturation of

autophagy is due to the presence of interferon regulatory factor 8 (IRF8) by stimulating

autophagosome formation and lysosomal fusion (Gupta, 2015).

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Figure 2: Autophagy-associated immune signals and cancer immune responses. Different

signalling pathway can activate innate and adaptive receptors thus recruit autophagy proteins

to phagosomal membrane which derived to anti-tumorigenic effects or pro-tumorigenic

effects (Pan et al., 2015).

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9.8 CHEMICAL INHIBITORS OF AUTOPHAGY

Boya et al. claimed that inhibition of macroautophagy cause apoptosis either by chemical

inhibitors or RNA interference. In common, autophagy is regulated by the mTOR and PI3K

pathways. Target of rapamycin, TOR kinase, is the major inhibitory signal that block

autophagy in the presence of growth factors and abundant nutrients. Autophagy inhibition

could decreased the cancer cell’s resistance to Bcl2-inhibitors, Tyrosine kinase inhibitor,

Bortezomib and conventional radiotherapy (Han, 2014). In recent years, there are great

findings of autophagy inhibitors studied. Some of the inhibitors are said to inhibit autophagy

at the early stage while some will do so at late stage.

Autophagy at the early stage can be inhibited by 3-MA, Wortmannin, and LY294002 , while

the late stage can be inhibited by CQ/HCQ and Bafilomycin A1. Uses of CQ in pretreatment

could block autophagy which resulted in an increased of anti-tumour activity of NDV

(Cuadrado-Castano et al., 2016). SAR405 has a significant role in autophagy as it is able to

inhibit PI3K catalytic activity. Besides, this chemical also prevent autophagy stimulated by

starvation or MTOR (Pasquier, 2015).

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