COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION

o Attribution — You must give appropriate credit, provide a link to the license, and indicate if
changes were made. You may do so in any reasonable manner, but not in any way that
suggests the licensor endorses you or your use.

o NonCommercial — You may not use the material for commercial purposes.

o ShareAlike — If you remix, transform, or build upon the material, you must distribute your
contributions under the same license as the original.

How to cite this thesis

Surname, Initial(s). (2012). Title of the thesis or dissertation (Doctoral Thesis / Master’s
Dissertation). Johannesburg: University of Johannesburg. Available from:
http://hdl.handle.net/102000/0002 (Accessed: 22 August 2017).
 PHOTODYNAMIC EFFECTS OF THUJA OCCIDENTALIS
HOMEOPATHIC MOTHER TINCTURE ON LUNG CANCER CELLS

A research dissertation presented to the

Faculty of Health Sciences, University of Johannesburg, as partial fulfilment for the

Degree of Master of Technology: Homeopathy

By

Ayesha Loonat

(Student number: 201575769)

Supervisor: __________________________ 08/03/2021

____________
Dr R. Chandran Date

Co-supervisor: __________________________ ____________

Dr J. Pellow Date
DECLARATION

I, Ayesha Loonat (Student Number 201575769), declare that Photodynamic effects of

Thuja occidentalis homeopathic mother tincture on lung cancer cells is my own work.
This dissertation is being submitted to the University of Johannesburg, Faculty of Heath
Sciences for the degree of Master of Technology: Homeopathy. Permission to carry out
this study was granted by the Higher Degrees Committee of the University’s Faculty of
Health Sciences Committee (HDC-01-35-2019) as well as the Research Ethics
Committee (REC-01-57-2019). This work has never been submitted for any other
qualification to any other university.

Research candidate: ___________________________ ____________

A. Loonat Date

ii
ABSTRACT

Cancer is a broad term used to describe the unregulated growth and proliferation of
cells. It is one of the world’s most insidious diseases and a major cause of death
worldwide, with lung cancer as the leading contributor to cancer fatalities (Brito et al.,
2017). Strategies aimed at treating lung cancer include chemotherapy, radiation and
surgery. Despite treatment advances, the current five-year survival rate is estimated at
15% of all stages combined (Duruisseaux & Esteller, 2018). Homeopathy is currently
the most often utilised complementary medicine by patients for the supportive treatment
of both cancer and non-cancer related diseases (Gaertner et al., 2018). Active research
on the anti-cancer properties of plants has shown promising results. Recent studies
have revealed that herbal extracts of Thuja occidentalis Linn (T. occidentalis) shows
anti-cancer and anti-proliferative properties (Silva et al., 2017). Several studies have
also shown that photodynamic therapy (PDT), a low-intensity laser irradiation therapy
that activates a photosensitizing compound to produce cytotoxic reactive oxygen
species which induces cell death, has the ability to eradicate cancer by making use of
visible light in specific wavelengths (Heiskanen & Hamblin, 2018). Currently, there is no
research on the photodynamic effects of T. occidentalis homeopathic mother tincture
(Ø) on lung cancer cells.

The aim of this study was to investigate the photodynamic effects of T. occidentalis Ø
on lung cancer cells in vitro using various morphological and biochemical assays.

Commercially available A549 lung cancer cells were used for the study. A preliminary
experiment was carried out prior to the study with cells treated with the tincture at
different volumes to optimize doses for further experiments. Doses of 5, 10, and 15 µL
were selected for further experiments. Cell models were divided into experimental and
control groups, with experimental groups receiving either a single treatment of the
tincture or laser irradiation at 660 nm, or the combined treatment (T. occidentalis Ø
mediated PDT). Various morphological and biochemical assays were carried out post-
treatment which included inverted microscopy for cellular morphology, adenosine
triphosphate (ATP) luminescent assay for cellular proliferation, lactate dehydrogenase
(LDH) assay for cytotoxicity, trypan blue assay for cellular viability, and the hoechst
stain assay for nuclear morphology. Data was statistically analysed using the one-way

iv
ANOVA and Dunnett’s multiple comparison test; statistical significance was set at
*p<0.05 when compared to the control.

Groups treated with laser irradiation alone at a wavelength of 660 nm and a fluence rate
of 5 J/cm2 had no significant effect on its activity against A549 cells when compared to
the control. Cell groups treated with T. occidentalis Ø alone and when photoactivated in
PDT demonstrated significant morphological changes in the cell and cell nuclei which is
indicative of apoptosis and hence, cancer cell death. Furthermore, these groups
exhibited a dose dependant increase in LDH release and a decrease in ATP levels and
cell viability compared to untreated control groups indicating the cytotoxic and anti-
proliferative potential of the therapies. Furthermore, at the same doses, T. occidentalis
Ø, when employed in PDT, was able to induce enhanced cytotoxic and antiproliferative
responses thereby surpassing the effects of treatment with the tincture alone. Results
demonstrate how the direct cytotoxic effects of the tincture can act synergistically with
the photochemical reactions produced during PDT to promote cancer cell death.

This study has confirmed the antiproliferative and cytotoxic effects of T. occidentalis Ø
and has implicated its efficacy as an effective anticancer agent. T. occidentalis Ø has
also shown potential as a promising photosensitzer in PDT to enhance cancer cell
death. However, further studies providing detailed mechanisms of the therapy are
needed in order to facilitate optimal therapeutic responses.

v
DEDICATION

For my parents,

Firouwz and Faeza

Everything I am is because of you.

For my Grandfathers,

Dr Rashid Ahmed Loonat and Moosa Abdoola

May their memories be eternal!

I hope that I have made you all proud!

vi
ACKNOWLEDGEMENTS

 To my supervisor, Dr Rahul Chandran, for your guidance, support and

motivation on this project; thank you. In this short time, I have learnt so much
from you. Your knowledge, wisdom and kindness is extraordinary, as are you.
 To my co-supervisor, Dr Janice Pellow, your mentorship extends far beyond
this project. I’m always in awe of your brilliance, patience and attention to detail.
You embody and encourage everything I aspire to be as a doctor. Thank you.
 To Prof Heidi Abrahamse, thank you for allowing me to be a part of the Laser
Research Centre. It has been an honour to have worked beside such an
incredible group of people. You all are inspiring!
 To Dr Radmilla Razlog and the Department of Complementary Medicine, from
each of you, I have learned invaluable lessons. I am privileged to have been
taught by some of the most brilliant and accomplished minds. Thank you!
 To my father Firouwz, for teaching me the importance of a good education as
key to my independence, and to my mother Faeza, for helping achieve it all;
thank you. It is only through your unconditional love and guidance that I have
the courage to pursue my goals. You have constantly put my dreams before
your own, when you see this; I hope you feel that your sacrifices were worth it.
 To my sisters, best friends and biggest supporters, Razina and Zakiya, for so
graciously walking through life with me with such courage and conviction.
Thank you for being my pillars of strength, support and patience.
 To my sweet niece Aleena, thank you for being the light in all our lives. Even
though your adventure has just begun, I hope somewhere along the way I can
be someone you look up to. You are greatly loved.
 To my brother-in-law Khurrum, thank you for all the joy you bring to my life.
Without realising when, you have become an inseparable part of our family.
 To Reyna and Heleen, thank you for the memories we have created, I will
cherish them for life. To the rest of my family and friends, for your unremitting
support and encouragement; I am deeply grateful for each one of you.
 To my creator, the Almighty, for blessing me with everything that I have.
 This study was financially supported by the University of Johannesburg, Laser
Research Centre, CSIR and the National Laser Centre.
vii
TABLE OF CONTENTS
Page

DECLARATION .............................................................................................................. ii

AFFIDAVIT..................................................................................................................... iii

ABSTRACT.................................................................................................................... iv

DEDICATION ................................................................................................................. vi

ACKNOWLEDGEMENTS ............................................................................................. vii

TABLE OF CONTENTS ............................................................................................... viii

LIST OF FIGURES....................................................................................................... xiv

LIST OF TABLES ......................................................................................................... xx

LIST OF SYMBOLS, ABBREVIATIONS AND ACRONYMS ....................................... xxi

CHAPTER 1: INTRODUCTION ...................................................................................... 1

1.1 Foreward ............................................................................................................... 1

1.2 Problem statement ................................................................................................ 2

1.3 Aim ........................................................................................................................ 4

1.3.1 Objectives .................................................................................................... 5

1.4 Hypothesis ............................................................................................................ 5

1.5 Null hypothesis ..................................................................................................... 5

CHAPTER 2: LITERATURE REVIEW ............................................................................ 6

2.1 Cancer................................................................................................................... 6

2.1.1 Cancer worldwide prevalence ...................................................................... 7

2.1.2 Mechanisms for cancer cell death................................................................ 7

2.1.2.1 Apoptosis ............................................................................................ 8

viii
 2.1.2.2 Necrosis.............................................................................................. 9

2.1.2.3 Autophagic cell death ......................................................................... 9

2.1.3 Lung cancer ............................................................................................... 10

2.1.4 Lung cancer pathophysiology .................................................................... 10

2.1.4.1 Small-cell lung carcinoma ................................................................. 12

2.1.4.2 Non-small-cell lung carcinoma ......................................................... 12

2.1.4.3 Common genomic and regulatory alterations

associated with lung cancer.............................................................. 13

2.1.5 Lung cancer risk factors ............................................................................. 14

2.1.6 Lung cancer signs and symptoms.............................................................. 15

2.1.7 Lung cancer diagnosis and staging............................................................ 16

2.1.8 Current treatment strategies for lung cancer .............................................. 16

2.1.8.1 Chemotherapy .................................................................................. 17

2.1.8.2 Radiotherapy .................................................................................... 18

2.1.8.3 Surgery ............................................................................................. 19

2.1.8.4 Immunotherapy ................................................................................. 20

2.1.8.5 Targeted gene therapy ..................................................................... 20

2.1.9 Prognosis ................................................................................................... 21

2.2 Homeopathy ........................................................................................................ 21

2.2.1 The principle of similars ............................................................................. 22

2.2.2 Homeopathic mother tinctures ................................................................... 23

2.3 Thuja occidentalis .............................................................................................. 24

2.3.1 Phytochemical properties of Thuja occidentalis ......................................... 25

2.3.1.1 Essential oils..................................................................................... 26

ix
 2.3.1.2 Polysaccharides ............................................................................... 27

2.3.1.3 Flavonoids ........................................................................................ 27

2.3.2 Thuja occidentalis Ø .................................................................................. 28

2.4 Photodynamic therapy ........................................................................................ 29

2.4.1 Advantages and disadvantages of photodynamic therapy ......................... 29

2.4.2 Photosensitizers......................................................................................... 30

2.4.2.1 Characteristics of a good photosensitizer ......................................... 30

2.4.2.2 The advantages of using plant sources as natural photosensitizers . 31

2.4.3 Light ........................................................................................................... 32

2.4.3.1 Wavelength....................................................................................... 33

2.4.5 Oxygen ...................................................................................................... 33

2.4.5 Photodynamic therapy mechanism of action ............................................. 34

2.4.5.1 Anti-cancer effects of photodynamic therapy .................................... 36

CHAPTER 3: METHODOLOGY ................................................................................... 39

3.1 Research design and procedure ......................................................................... 39

3.2 Cell culture treatment ......................................................................................... 39

3.2.1 Plating of cells ............................................................................................ 39

3.3 Thuja occidentalis Ø ............................................................................................ 40

3.3.1 Thuja occidentalis Ø dose determination .................................................. 40

3.4 Laser set-up and parameters .............................................................................. 41

3.5 Groups and set-up .............................................................................................. 41

3.5.1 Cellular morphology- Inverted light microscopy ......................................... 42

3.5.2 Cytotoxicity- Lactate dehydrogenase assay............................................... 43

x
 3.5.3 Cellular proliferation- Adenosine triphosphate luminescent

assay ........................................................................................................ 43

3.5.4 Cellular viability- Trypan blue assay........................................................... 43

3.5.5 Nuclear morphology- Hoechst stain assay................................................. 44

3.6 Reliability and validity measures ......................................................................... 44

3.7 Statistical analysis ............................................................................................... 45

3.8 Ethics .................................................................................................................. 45

3.8.1 Biological safety ......................................................................................... 45

CHAPTER 4: RESULTS ............................................................................................... 47

4.1 Preliminary study ................................................................................................. 47

4.1.1 Ethanol cytotoxicity study ........................................................................... 47

4.1.1.1 Inverted light microscopy .................................................................. 47

4.1.1.2 Trypan blue assay ............................................................................ 49

4.2 Experimental study .............................................................................................. 50

4.2.1 Inverted light microscopy ........................................................................... 50

4.2.2 Lactate dehydrogenase assay ................................................................... 52

4.2.3 Adenosine triphosphate luminescent assay ............................................... 54

4.2.4 Trypan blue assay...................................................................................... 57

4.2.5 Hoechst stain assay ................................................................................... 60

CHAPTER 5: DISCUSSION ......................................................................................... 62

5.1 Contextualising the ethanol cytotoxicity study ..................................................... 62

5.2 Contextualising the experiments ........................................................................ 64

5.2.1 Inverted light microscopy ........................................................................... 64

xi
 5.2.2 Lactate dehydrogenase assay ................................................................... 65

5.2.3 Adenosine triphosphate luminescent assay ............................................... 66

5.2.4 Trypan blue assay...................................................................................... 68

5.2.5 Hoechst stain assay ................................................................................... 68

5.3 Limitations of the study ....................................................................................... 69

CHAPTER 6: CONCLUSION ........................................................................................ 71

6.1 Future directives ................................................................................................. 72

REFERENCES ............................................................................................................ 74

APPENDICES ............................................................................................................. 112

APPENDIX A .............................................................................................................. 112

Letter of permission to work in the laboratory.......................................................... 112

APPENDIX B .............................................................................................................. 113

Diagrammatic representation of project plan ........................................................... 113

APPENDIX C .............................................................................................................. 114

Higher Degrees Committee clearance form ............................................................ 114

APPENDIX D .............................................................................................................. 115

Ethics clearance form .............................................................................................. 115

APPENDIX E............................................................................................................... 116

List of consumables ................................................................................................ 116

APPENDIX F ............................................................................................................... 117

List of equipment ..................................................................................................... 117

APPENDIX G .............................................................................................................. 118

Calculations............................................................................................................. 118

xii
 G1. Calculation to determine the total amount of cells in

suspension........................................................................................................ 118

G2. Calculation to determine seeding ratios in flasks ...................................... 118

G3. Calculation to determine cell seeding density for culture

plates ......................................................................................................... 118

G4. Laser irradiation calculations...................................................................... 119

APPENDIX H .............................................................................................................. 121

Certificate of analysis for T. occidentalis Ø ............................................................. 121

APPENDIX I ................................................................................................................ 122

Certificate of analysis for A549 cells ....................................................................... 122

APPENDIX J ............................................................................................................... 124

Product sheet (A549 cells) ...................................................................................... 124

APPENDIX K .............................................................................................................. 127

Graph values, standard error and statistical significance ........................................ 127

M1. ATP assay ................................................................................................. 127

M2. LDH assay ................................................................................................. 128

M3. Trypan blue assay...................................................................................... 129

APPENDIX L ............................................................................................................... 130

Biosafety clearance from ATCC to receive cells ..................................................... 130

APPENDIX M ............................................................................................................ 1131

Plagiarism report ..................................................................................................... 131

xiii
LIST OF FIGURES

Figure 2.1: Schematic representation of the three major pathways of cell

death (Madkour, 2020): Apoptosis, Necrosis and Autophagy. (A)
Normal cell; (B) Apoptosis: damaged cell undergoes shrinkage,
chromatin condensation, and finally the formation of apoptotic
bodies and membrane blebbing; (C) Autophagy: damaged cell
contents are enveloped into autophagosomes and; (C) Regulated
necrosis (necroptosis): damaged cell undergoes swelling and
membrane breakdown. 8

Figure 2.2: The histological classification of lung cancer. (A): Histological

classification of lung cancer can be divided into two main groups,
small-cell lung cancer/ carcinoma (SCLC) and non-small-cell lung
cancer/ carcinoma (NSCLC). The latter is subdivided into lung
adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC)
and, large cell carcinoma (LCC). (B): Location of tumours and cell
origins (Filho et al., 2019). 11

Figure 2.3: (A) Thuja occidentalis tree, (B) Branch of the Thuja Occidentalis
tree (Bigstock, 2020). 24

Figure 2.4: Schematic Jablonski’s diagram demonstrating the Type 1 and

Type 2 reactions in PDT. The photosensitizer (PS) absorbs
photons from the light source and reaches an excited singlet state
(PSEs). The PSEs through intersystem crossing directs the PS to its
excited triplet sate (PSEt), which can react in two ways: The PSEt
can react with biomolecules to produce reactive oxygen species
(ROS) (Type 1 reaction); or it can directly react with molecular
oxygen (O2) to generate singlet oxygen (1O2) (Type 2 reaction)
(Calixto et al., 2016). 35

xiv
Figure 2.5: A flow diagram summarising the antineoplastic effects of
photodynamic therapy. Antitumour effects can be elicited through
three main mechanisms: direct cell death, cell death via vascular
damage, and activation of the immune system (Li et al., 2012). 36

Figure 2.6: Type of cell death induced by localization of photosensitizers

(Abrahamse et al., 2017). 37

Figure 4.1: Morphology of A549 cells after exposure to different doses of 61%
ethanol (EtOH) under inverted light microscope (200x
Magnification). Figure (A) Untreated control, (B) 5 µL EtOH, (C) 10
µL EtOH, (D) 15 µL EtOH, (E) 20 µL EtOH, (F) 25 µL EtOH, (G) 50
µL EtOH, (H) 100 µL EtOH, (I) 150 µL EtOH and (J) 200 µL EtOH.
Control cells showed high density of attached cells with normal
shape. Cells treated with 5-25 µL demonstrated no significant
changes compared to the control. At 50-100 µL there is a gradual
decrease in the density of attached cells, rounded up dead cells can
be seen floating in the media. At 150-200 µL, the majority of cells
are rounded up and can be seen floating in the media. Scale bar 50
µm. 48

Figure 4.2: The percentage (%) of viable cells using trypan blue assay in
A549 cells treated with 61% Ethanol (EtOH). The control group
demonstrates the highest viability indicating an average of 97%
living cells. Cells treated with EtOH at doses 5-50 μL shows no
significant changes in cell viability when compared to the control.
A gradual decrease in cell viability can be seen in cells treated
with 100 μL of EtOH. The maximum decrease in cell viability can
be seen in groups treated with 150 and 200 μL of EtOH. Data is
represented as the mean ± SE from ten independent experiments.
Statistically significant at **p<0.01 and ***p<0.001 when compared
to the control. 49

xv
Figure 4.3: Cell morphology of A549 cells after exposure to different doses of
T. occidentalis Ø (TO) and TO mediated photodynamic therapy
(TO-PDT) under an inverted light microscope (200x Magnification).
Figure 1(A) Untreated control, (B) irradiation only, (C) 5 µL TO, (D)
10 µL TO, (E) 15 µL TO, (F) 5 µL TO-PDT, (G) 10 µL TO-PDT, (H)
15 µL TO-PDT. Control cells showed high density of attached cells
with normal shape. Cells treated with irradiation alone
demonstrated no significant changes compared to the control. At all
doses, TO and TO-PDT treated cells decreases in the density of
attached cells can be observed and rounded up dead cells can be
seen floating in the media. White arrows indicates examples of
rounded up dead cells. Scale bar 50 µm. 51

Figure 4.4: Lactate dehydrogenase (LDH) release from A549 cells treated
with T. occidentalis Ø (TO). The control group demonstrates the
lowest LDH release. Cells treated with TO at different doses
shows a dose-dependent gradual increase in LDH release. Data is
represented as the mean ± SE from four independent
experiments. Statistically significant at *p<0.05, **p<0.01 and
***p<0.001 when compared to the control. 52

Figure 4.5: Lactate dehydrogenase (LDH) release from A549 cells treated
with T. occidentalis Ø mediated photodynamic therapy (TO-PDT).
The control group demonstrates the lowest LDH release. Cells
treated with irradiation alone demonstrate no significant difference
in LDH release (p=0.805) compared to the control. Cells treated
with TO-PDT at different doses shows a dose-dependent increase
in LDH. Data is represented as the mean ± SE from five
independent experiments. Statistically significant at ***p<0.001
when compared to the control. 53

xvi
Figure 4.6: A comparison of the amount of lactate dehydrogenase (LDH)
released from A549 cells treated with T. occidentalis Ø (TO) and
TO mediated photodynamic therapy (TO-PDT) at the same doses.
Cells treated with TO-PDT showed increased LDH levels
compared to cells treated with TO alone. Data is represented as
the mean ± SE from six independent experiments. 54

Figure 4.7: The level of adenosine triphosphate (ATP) in relative light units
(RLU) on A549 cells treated with T. occidentalis Ø (TO). The
control group demonstrates the highest ATP level. Cells treated
with TO at different doses shows a dose-dependent decrease in
ATP levels. Data is represented as the mean ± SE from four
independent experiments. Statistically significant at **p<0.01 and
***p<0.001 when compared to the control. 55

Figure 4.8: The level of adenosine triphosphate (ATP) metabolism in relative

light units (RLU) on A549 cells treated with T. occidentalis Ø
mediated photodynamic therapy (TO-PDT). The control group
demonstrates the highest ATP level. Cells treated with irradiation
only demonstrate no significant difference in ATP levels (p=0.756)
compared to the control. Cells treated with TO-PDT at different
doses show a dose-dependent decrease in ATP levels. Data is
represented as the mean ± SE from five independent experiments.
Statistically significant at ***p<0.001 when compared to the
control. 56

Figure 4.9: A comparison of the level of adenosine triphosphate (ATP) in

relative light units (RLU) on A549 cells treated with T. occidentalis
Ø (TO) and TO mediated photodynamic therapy (TO-PDT) at the
same dose. Cells treated with TO-PDT shows decreased ATP
levels compared to cells treated with TO alone. Data is
represented as the mean ± SE from six independent experiments. 57

xvii
Figure 4.10: The percentage (%) of viable cells using trypan blue assay in
A549 cells treated with T. occidentalis Ø (TO). The control group
demonstrates the highest viability indicating an average of 85%
living cells. Cells treated with TO at different doses shows a dose-
dependent decrease in cell viability thus indicating cell death. Data
is represented as the mean ± SE from four independent
experiments. Statistically significant at ***p<0.001 when compared
to the control. 58

Figure 4.11: The percentage (%) of viable cells using trypan blue assay in
A549 cells treated with Thuja occidentalis Ø mediated
photodynamic therapy (TO-PDT). The control group demonstrates
the highest viability indicating an average of 97% living cells. Cells
treated with irradiation only demonstrate no significant difference
in viability (p=0.605) compared to the control. Cells treated with
TO-PDT at different doses shows a dose-dependent decrease in
cell viability thus indicating cell death. Data is represented as the
mean ± SE from five independent experiments. Statistically
significant at ***p<0.001 when compared to the control. 59

Figure 4.12: A comparison of cell viability on A549 cells treated with T.

occidentalis Ø (TO) and TO mediated photodynamic therapy (TO-
PDT) at the same doses of the tincture. At the same doses of TO,
cells treated with TO-PDT showed decreased viability percentages
compared to cells treated with TO only. Data is represented as the
mean ± SE from six independent experiments. 60

xviii
Figure 4.13: Nuclei morphology of A549 cells after exposure to different doses
of T. occidentalis Ø (TO) and TO mediated photodynamic therapy
(TO-PDT) under a fluorescent microscope (400x Magnification).
Figure 1(A) Untreated control, (B) irradiation only, (C) 5 µL TO, (D)
10 µL TO, (E) 15 µL TO, (F) 5 µL TO-PDT, (G) 10 µL TO-PDT, (H)
15 µL TO-PDT, (I) Normal nucleus, and (J) Abnormal nucleus.
Control cells show normal, evenly stained nuclei. Cells treated with
irradiation alone show no significant changes compared to the
control. Cells treated at different doses of TO and TO-PDT
demontrated nuclei with abnormal shape, nuclei shrinkage,
chromatin condensation and nuclear fragmentation. Figure (I)
provides an enlarged example of a normal nucleus that is
homogenously stained and hence remains dense. Figure (J)
provides an enlarged example of an abnormally shaped nulcleus
demonstrating nuclear fragmentation. Scale bar 20 µm. 61

xix
LIST OF TABLES

Table 3.1: A table showing irradiation parameters. 41

Table 3.2: A table showing a summary of the groups and variables in the
experiment. 42

xx
LIST OF SYMBOLS, ABBREVIATIONS AND ACRONYMS

% Percentage

< Less than

= Equal to

® Registered

°C Degree Celsius

µL Microliter

µm Micrometre

A549 Adenocarcinoma human alveolar basal epithelial cells

ADCC Antibody-dependent cellular cytotoxicity

AIDS Acquired immunodeficiency syndrome

ALK Anaplastic lymphoma kinase

ANOVA Analysis of variance

ATCC American Type Culture Collection

ATP Adenosine triphosphate

BAX Bcl-2-associated X protein

Bcl-2 B-cell lymphoma 2

C/ cH Centesimal/ Centesimal Hahnemanni (1:100 dilution

scale)

cm Centimetre

CM Complementary medicine

cm2 Centimetre squared

CO2 Carbon dioxide

COPD Chronic obstructive pulmonary disease

CSIR Council for Scientific and Industrial Research

D Decimal (1:10 dilution scale)

DAPI 4′,6-diamidino-2-phenylindole

xxi
DNA Deoxyribonucleic acid

EGFR Epidermal growth factor receptor

EtOH Ethanol

FBS Foetal bovine serum

FDA Food and Drug Administration

GmbH Gesellschaft mit beschränkter Haftung translated as

“company with limited liability”

h Hours

HBSS Hank’s Balanced Salt Solution

HIV human immunodeficiency virus

HPD Hematoporphyrin derivative

HPT Homeopathic pathogenic trials

HPTLC High-performance thin-layer chromatography

IARC International Agency for Research on Cancer

ISO International Organization for Standardization

J/cm2 Joules per Centimetre Squared

KRAS Kirsten rat sarcoma

LDL Low-density lipoprotein

LED Light emitting diode

LILI Low intensity laser irradiation

LRC Laser Research Centre

MET Mesenchymal-epithelial transition

mL Millilitre

min Minutes

mW Milliwatt

mW/cm2 Milliwatt per centimetre squared

n Sample size

NAD+ Nicotinamide adenine dinucleotide

xxii
NADH Nicotinamide adenine dinucleotide hydrogen

NK Natural killer

NLC National Laser Centre

nm Nano meter

NSCLC Non-small cell lung carcinoma

Ø Homeopathic mother tincture

O2 Oxygen

p53 Protein 53

PBM Photobiomodulation

PBS Phosphate buffered saline

PDT Photodynamic therapy

PS Photosensitizer

PS0 Photosensitizer ground state

PSEs Photosensitizer excited singlet state

PSEt Photosensitizer excited triplet state

QoL Quality of life

RET Rearranged during transfection

RLU Relative Light Units

ROS Reactive oxygen species

ROS1 ROS proto-oncogene

RPM Revolutions per minute

RPMI Roswell Park Memorial Institute 1640 Medium

s Seconds

SABR Stereotactic ablative radiotherapy

SCLC Small cell lung carcinoma

SE Standard error

SHS Second-hand smoke

xxiii
T. occidentalis Thuja occidentalis Linn

TLC Thin-layer chromatography

TME Tumour microenvironment

TMN Tumour, Nodal, and Metastasis

TO Thuja occidentalis Ø

TO-PDT Thuja occidentalis Ø mediated photodynamic therapy

VATS Video-assisted thoracoscopic surgery

W Watt

WHO World Health Organisation

α Alpha

β Beta

xxiv
CHAPTER 1: INTRODUCTION

1.1 Forward
Cancer is an umbrella term that refers to a set of diseases that describes the
unregulated growth and proliferation of abnormal cells to form a tumour that eventually
acquires the ability to invade other tissues (Ortega & Campos, 2019). Cancer is a
rapidly emerging disease and a principal cause of death equally in both developed and
developing countries (Abbas & Rehman, 2018). Lung cancer continues to be a major
contributor of cancer mortality rates worldwide, with sufferers having a poor five-year
survival rate (Duruisseaux & Esteller, 2018). The initiation and progression of lung
cancer ascribes to certain genetic and environmental exposures and their interactions;
however, smoking accounts as a principal risk factor in the majority of cases (Casal-
Mourino et al., 2019; Cryer & Thorley, 2019). Lung cancer is initially asymptomatic;
therefore diagnosis is usually made at advanced stages (Nasim et al., 2019). Standard
lung cancer treatment relies on a combination of therapies including surgery,
chemotherapy and radiation, which all present with significant drawbacks (Xu et al.,
2019).

Homeopathy is a holistic complementary system of medicine founded by Dr Samuel

Hahnemann, which is based upon the fundamental principle of likes cures likes (Relton
et al., 2017). Homeopathy is currently one of the most commonly used complementary
therapies by cancer patients (Unlu et al., 2017). Homeopathic remedies are both
economical and effective, which makes them easily accessible to the population (Singh
et al., 2014). Homeopathic mother tinctures (Ø’s) are derived from plant, animal and
mineral raw materials. Plant based Ø’s are acquired by ethanolic extraction of the entire
plant, or parts of the plant, prepared in a very precise manner (Das et al., 2019).

Thuja occidentalis Linn (T. occidentalis) is an evergreen plant of the Cupressaceae

family. Recent pre-clinical studies have identified several active constituents of this plant
which exhibit antitumour activities (Biswas et al., 2011). One such active ingredient,
thujone, is said to have anti-carcinogenic potential, and according to an in vitro study on
glioblastoma multiforme cells, this compound was shown to exert pro-apoptotic and
anti-invasive effects (Pudełek et al., 2019). In homeopathy, T. occidentalis is indicated

1
in the treatment of pathological growths of vegetative condylomata, warty excrescences
and tumours (Vermeulen, 2015).

Photodynamic therapy (PDT) is a non-invasive treatment modality that has gained

considerable attention for its anti-cancer properties (Zhen et al., 2019). PDT relies on
the transformation of light energy into cytotoxic reactive oxygen species (ROS) by a
photosensitizer (PS) that has been irradiated by a specific wavelength of light (Zhao et
al., 2020). The cytotoxic ROS can induce cell death directly through apoptotic or
necrotic pathways, or indirectly by harming tumour vasculature to stimulate hypoxia and
starvation of tumour cells (Abo-Zeid et al., 2018). PDT has the advantage of having dual
selectivity. The PS can be targeted to a cell type or tissue, and simultaneously, the
source of irradiation can be spatially directed to the target tissue (Siewert & Stuppner,
2019).

1.2 Problem statement

According to the World Health Organization (WHO), approximately 8.2 million people
die from cancer every year. This number is projected to rise significantly over the next
few years, with 17 million new cancer cases expected in 2020 alone (Bhattacharjee et
al., 2020). Lung cancer is at the forefront of cancer related deaths in both men and
women globally, accounting for an estimated 1.4 million deaths annually (Jhanwar et al.,
2020).

Despite improvements in standard lung cancer therapies and the development of new
targeted therapy in the past two decades, the overall five year survival rate remains
unsatisfactory (Xie et al., 2020). Hence, targeting the early stages of the disease for
treatment and cure should be preferred, whereby the benefits of surgery and adjuvant
therapy are optimal. However, the majority of patients with lung cancer are diagnosed
with end-stage disease when the cancer is inoperable, and patients find themselves
deteriorating physiologically and psychologically throughout the course of the disease
(Quist et al., 2020).

Even with the availability of resources employed for the detection, intervention and care
of South African citizens inflicted with cancer, many cases are misdiagnosed and
misunderstood, particularly in low economic rural and urban areas within the country
(Mendenhall et al., 2019). The marginalisation of resources is a result of a dysfunctional
2
health system that continues to live with the consequences of the political and economic
decisions made during the apartheid era (Maphumulo & Bhengu 2019).

Surgery, chemotherapy, and radiation have long been the foundation for lung cancer
treatment. However, the lack of targeting precision and the accompanying adverse
effects continues to be a challenge for modern medicine even in the 21 st century
(Mottaghitalab et al., 2019). Additionally, the acquisition of resistance to both
conventional and targeted anti-cancer drugs remains a primary cause of therapeutic
failure (Zargar et al., 2019). This has pronounced negative effects on the patients’
quality of life (QoL) and overall functioning, both in the short- and long- term (Shrestha
et al., 2019). In South Africa, these modalities are available primarily in the private
health sector, while patients with lung cancer in the public health sector have limited
access to treatment (van Eeden et al., 2020). As a result, curative-intended therapy is
rendered inadequate and the dying patient is attended to by untrained and informal
caregivers, mainly family and friends who lack the knowledge needed to provide
competent health care (O’Neil et al., 2018).

The current trends in lung cancer cases are not reciprocated by the ability of
international health care systems to successfully treat it. This mainly reflects the inability
to deliver appropriate therapeutic regimens at sufficient concentrations to the tumour
site, without subsequent collateral damage to healthy cells and with limited side-effects
(Cryer & Thorley et al., 2019). The severe complications of the disease, poor survival
rate and the continuous rise in lung cancer cases demonstrate a serious unmet need
that underpins the necessity and urgency of developing effective anti-cancer therapies
with curative intent (Zhou et al., 2020).

The application of PDT in lung cancer has a history of just over fifteen years (Shafirstein
et al., 2016). The known advantages of PDT are no drug resistance, fewer
complications, and good target selectivity that promote faster healing of normal tissue
with minimal scarring (Dong et al., 2020; da Silva et al., 2019). Effective PDT outcomes
are highly dependent on selecting an appropriate tumour-specific PS. However, current
medically approved PSs such as photophrin® are only commercially available in small
amounts. In addition, only a few of these PSs benefit patients, and none are entirely
satisfactory in clinical practice (Hu et al., 2020). Several studies have further
demonstrated how photophrin-related PSs are eliminated slowly after entering the body,
3
which can lead to skin sensitivity. This emphasizes the need to develop more effective
and safer PSs (Shi et al., 2019). Therefore, agents derived from alternate systems are
gaining potential interest to improve therapeutic PDT outcomes. The search for novel
PSs has shifted its focus towards finding natural sources of PSs for PDT applications
(Mamone et al., 2016). Much research has concentrated its efforts on isolating bioactive
phytochemicals found in plant extracts to treat various diseases due to its improved risk-
benefit ratio, low cost, easy availability, and negligible side-effects (Uttra et al., 2018).
Hence, the use of these compounds as PSs are being widely studied to increase the
efficacy of cancer therapy.

Homeopathic supportive care in cancer patients centres on the prevention and

treatment of symptoms for which there is no satisfactory regime in conventional
medicine (Bagot, 2020a). Many studies have only begun to demonstrate the effects of
homeopathy in oncology care, and its effects on cancer cells in vitro is still in its infancy
(Fuselier et al., 2019). A diverse range of natural substances, including plant sources, is
recruited for the development and employment of Ø’s and potency remedies (Bell et al.,
2014). T. occidentalis Ø is frequently prescribed in clinical practice, yet there continues
to be a massive gap in research related to the efficacy of this tincture in both in vitro and
in vivo experiments. Although there have been some substantially positive results from
studies on herbal extracts of T. occidentalis and some significant results from a few
scarce studies on the anti-cancer effects of homeopathic preparations of T. occidentalis
Ø, there is no data on its application in PDT.

Therefore, this project is focused as a starting point towards understanding the role of
the effects of T. occidentalis Ø mediated PDT to observe the potential synergetic
benefits in enhancing cancer cell death, which will allow further research in the field.
Additionally, this study was the first to introduce the role of T. occidentalis Ø as a
possible natural PS in PDT applications.

1.3 Aim
The aim of this study was to investigate the in vitro photodynamic effects of T.
occidentalis Ø on lung cancer cells using various morphological and biochemical
assays.

4
1.3.1 Objectives
 Culture and maintenance of A549 lung cancer cells to carry out further
experiments.
 To optimize the treatment dose of T. occidentalis Ø through preliminary
experiments.
 To evaluate the cytotoxic and antiproliferative effects of T. occidentalis Ø on
A549 lung cancer cells by means of morphological and biochemical analyses.
 To determine the photodynamic effects (combined laser and tincture) of T.
occidentalis Ø at 660 nm on A549 cancer cells.
 To observe and compare the cellular responses in pre- and post-irradiation
groups treated with T. occidentalis Ø.

1.4 Hypothesis:
The application of T. occidentalis Ø alone, and as a PS in PDT, has effective cytotoxic
and antiproliferative properties on A549 cancer cells compared to untreated controls.

1.5 Null hypothesis:

The application of T. occidentalis Ø alone, and as a PS in PDT, does not have effective
cytotoxic and antiproliferative properties on A549 cancer cells compared to untreated
controls.

5
CHAPTER 2: LITERATURE REVIEW

2.1 Cancer
The ancient lineage of cancer can be traced backed to the early Egyptians. The origin of
the word cancer was coined over 2500 years ago by Hippocrates who used the Greek
word karkinoma meaning “crab” to describe the evil animal-like appearance of tumours
(Davis, 2011). Cancer is a generic term used to describe over 100 diseases that result
in the unregulated growth and proliferation of cells (Brito et al., 2017). The basic
mechanisms that produce cancer are quite similar; any diversification between the
different cancers that may occur, happens during polyclonal expansion of cancer cells
(Kunimasa et al., 2019). Carcinogenesis is a dynamic process which induces changes
in the body’s tissue architecture and cell phenotype to initiate cancer. This results from
the interaction between environmental factors and genetic and epigenetic changes
ensuing in the complex modification of the host’s genome and the gradual accumulation
of errors in vital regulatory pathways (Bidram et al., 2019).

Carcinogenesis is initiated after the acquisition of a primary driver mutation in a single

cell or a group of cells. The primary mutation confers a selective growth advantage to
the cells. These cells undergo subsequent rounds of cellular divisions, obtaining further
genetic aberrations (Shenoy, 2020). The atypical cells start to spread over a limited
area of tissue where they begin to progressively lose their normal function (Abbas and
Rehman, 2018). At this point, the tumour is non-invasive and is considered benign.
These abnormalities constantly accumulate onto the genome of normal cells thereby
governing the transition of atypical cells into highly malignant derivatives (Habli et al.,
2017). When the tumour reaches an advanced stage, it is able to invade the
surrounding tissues, gaining the ability to metastasize. This stage is considered
malignant and is difficult to treat (Ortega & Campos, 2019).

The hallmarks of cancer comprise of six biological capabilities acquired during

carcinogenesis to ensure that the cells reprogram to switch to their phenotypic identity:
(i) sustaining proliferative signalling, (ii) evading growth suppressors, (iii) activating
invasion and metastases, (iv) enabling replicative immortality, (v) inducing angiogenesis
and (vi) resisting cell death (Hanahan & Weinberg, 2011).

6
2.1.1 Cancer worldwide prevalence
Cancer continues to be a major public health concern and is the second leading cause
of mortality and morbidity worldwide (Siegel et al., 2019). Global cancer statistics have
identified the five most common cancers in 2018 which were lung cancer (2.09 million
cases), breast cancer (2.09 million cases), colorectal cancer (1.80 million cases),
prostate cancer (1.28 million cases), and skin cancer (1.04 million cases) (Adjei, 2019).
According to the World Health Organization (WHO), approximately 9.6 million deaths
were caused by cancer in 2018. This number is projected to rise by 70% over the next
20 years (Chen et al., 2020). The reasons for the substantial rise in cancer cases are
complex but reflect the aging and growth of the population, as well as the changes in
the prevalence and distribution of the main risk factors for cancer, many of which are
associated with socioeconomic development (Bray et al., 2018). Cancer mortality rates
remain largely unreported in South Africa. In 2012, the International Agency for
Research on Cancer (IARC) estimated 47 350 deaths caused by cancer in South Africa
alone, but efforts to combat the disease were minimal due to resources been aimed at
the HIV/AIDS epidemic burdening the health care system concurrently. Within South
Africa, lung cancer remains the leading cause of cancer-related mortality in men, while
cervical cancer is the leading cause of cancer death in women (Made et al., 2017).

2.1.2 Mechanisms for cancer cell death

Cell death mechanisms are crucial to the regulation of many critical physiological and
pathological events to maintain tissue homeostasis (Wimmer et al., 2020). When the
haemostatic mechanism of a normal cell is disturbed beyond its limit of adaptation
response (hypertrophy or atrophy), cell injury culminates. This triggers one or several
cell death pathways to induce cell death (Madkour, 2020). These mechanisms eliminate
excess and defective cells in the body and can therefore be used as a biomarker to
monitor disease progression and treatment responses (Wimmer et al., 2020). There are
three main forms of morphologically distinct cell death mechanisms: apoptosis, necrosis
and autophagic cell death (Madkour, 2020) (Figure 2.1). Each type of cell death has a
unique molecular mechanism that is controlled independently by multiple signalling
pathways. These signalling pathways are interconnected and can be activated
simultaneously (Patil et al., 2020).

7
 A

D B

Figure 2.1: Schematic representation of the three major pathways of cell death: Apoptosis,
Necrosis and Autophagy. (A) Normal cell; (B) Apoptosis: damaged cell undergoes shrinkage,
chromatin condensation, and finally the formation of apoptotic bodies and membrane blebbing;
(C) Autophagy: damaged cell contents are enveloped into autophagosomes and; (C) Regulated
necrosis (necroptosis): damaged cell undergoes swelling and membrane breakdown (Madkour,
2020).

2.1.2.1 Apoptosis
Apoptosis is an adenosine triphosphate (ATP)-dependent, genetically programmed
death of unwanted and potentially harmful cells. It is characterised morphologically by
cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation
(Nowak et al., 2020). This process is modulated by precise signalling circuitry which is
facilitated by a group of enzymes termed caspases. Apoptosis is triggered in a cell
through either the intrinsic pathway or extrinsic pathway (Ouyang et al., 2012).

The intrinsic or mitochondrial pathway is mediated by signals that converge at the

mitochondrial level. It involves a variety of intracellular and/ or extracellular stimuli that
can promote changes in the mitochondrial membrane, resulting in mitochondrial outer
membrane permeabilization and the subsequent release of cytochrome c into the

8
cytosol (Mortezaee et al., 2019). Cytochrome c is responsible for the assembly of
apoptosomes (caspase-activating complexes), which initiates the molecular programme
in cells leading to apoptosis (Dhuriya et al., 2019). The extrinsic or death receptor
pathway is activated in response to external signalling proteins. This generates an
intracellular signal via transmembrane death receptor-mediated interactions on the cell
surface (McBride et al., 2019). The hallmark feature of apoptosis is pyknosis, which is
the irreversible condensation of the nuclear chromatin leading to karyorrhexis.
Karyorrhexis is the dissolution of the nuclear membrane, and the slicing of deoxy
ribonucleic acid (DNA) into short fragments called apoptotic bodies. The apoptotic
bodies break off from the cell and are eventually removed by macrophages for cell
recycling through a process called efferocytosis (Pistritto et al., 2016).

2.1.2.2 Necrosis
Traditionally, necrosis is defined as a rapid, uncontrolled or accidental cell death
occurring in an extreme environment or in response to severe changes in pathological
conditions, including hypoxia, ischaemia, and exposure to reactive oxygen species
(ROS) (Thornton et al., 2017). Necrosis is characterised by cell swellings, cell
membrane disruptions, and cell lysis. The breakdown of the cellular membrane releases
the intracellular contents onto neighbouring cells, eliciting a strong inflammatory
response (Handali & Rezaei, 2020). Up until recently, necrosis was thought of as an
accidental form of cell death. However, after more sophisticated research tools and data
have been made available, it has become clear that necrosis can function as an
alternate programmed cell death pathway. This type of programmed necrosis is termed
necroptosis to distinguish it from passive necrosis (Nikoletopoulou et al., 2013).
Necroptosis shares common morphological features as necrosis, but differs in that cells
undergoing necroptosis retain their integral nuclei while cells undergoing necrosis do
not. Necroptosis is triggered by the same death signals as apoptosis, but is independent
of caspase activation. Therefore, pro-apoptotic agents can induce necroptosis in the
absence of caspases when apoptosis is blocked (Wu et al., 2020).

2.1.2.3 Autophagic cell death

Autophagy is a conservative caspase-independent programmed cell death mechanism
which occurs as an adaptive response to cell stress. It is considered as a lysosome-
mediated catabolic process that plays a fundamental role in cell homeostasis
(Yonekawa & Thorburn, 2013). During autophagy, damaged organelles and proteins are
9
enveloped into double membrane vesicles called autophagosomes. Autophagosomes
delivers the damaged intracellular components to lysosomes for degradation. The
degraded products are then recycled into bioenergetic substrates (Yun & Lee, 2018).
Autophagy can play both cytoprotective and cytotoxic roles during carcinogenesis.
While malignant cells adapt autophagy as a pro-survival mechanism to maintain
metabolic demands in conditions of stress, an excessive induction of autophagy under
conditions which permanently enhance organelle damage can lead to autophagic cell
death (Ajoolabady et al., 2020).

2.1.3 Lung cancer

Lung cancer is a heterogeneous disease defined as the uncontrolled and abnormal
growth of cells that can occur in various anatomical locations in the epithelium of one or
both lungs (Sarode et al., 2019). Lung cancer is considered to be the leading contributor
to cancer deaths worldwide, surpassing the combined mortality rates of breast, prostate
and colorectal cancer (Shah & Gregory, 2019). Even with recent advancements in both
old and new cancer therapeutic agents, lung cancer continues to uphold its position as
the most lethal cancer among males in the world and has overtaken breast cancer as
the most fatal cancer among females in many developed countries (Lee & Cheah,
2019).

2.1.4 Lung cancer pathophysiology

The pathophysiology of lung cancer is complex and incompletely understood. Lung
cancer can be divided morphologically, histologically, and clinically into two types,
namely, small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC),
the latter accounting for the majority of lung cancer cases (Ryan & Burke, 2017) (Figure
2.2). Lung cancer cells arise through a multistep process involving a series of cellular
and genetic changes which result in the clonal expansion of cells that ultimately evolves
into an invasive cancer (Lemjabbar-Alaoui et al., 2015). The genetic changes result in
the activation of oncogenic pathways and/ or inhibition of tumour suppressor genes,
both of which are crucial for malignant transfer of tumour precursor cells (Salehi-Rad et
al., 2020). Once the primary cancer has developed, molecular, genetic and epigenetic
abnormalities and mutations continue to progressively accumulate. This results in the
disruption of multiple cellular pathways, which influences further invasion, metastasis
and resistance to cancer therapeutics (Lemjabbar-Alaoui et al., 2015).

10
Figure 2.2: The histological classification of lung cancer. (A): Histological classification of lung
cancer can be divided into two main groups, small-cell lung cancer/ carcinoma (SCLC) and non-
small-cell lung cancer/ carcinoma (NSCLC). The latter is subdivided into lung adenocarcinoma
(LUAD), lung squamous cell carcinoma (LUSC) and, large cell carcinoma (LCC). (B): Location of
tumours and cell origins (Barros-Filho et al., 2019).

The pathophysiology of lung cancer however, cannot be attributed to the deregulation of

the pathways alone but rather encompass both the tumour microenvironment (TME)
and host immune response. These factors are both a cause and a consequence of
carcinogenesis, and work synergistically to promote the progression and survival of lung
cancer cells (Salehi-Rad et al., 2020). The TME is comprised of the cellular and
noncellular components including immune cells, growth factors, proteases, and the
extracellular matrix of the tumoural niche that enables tumour cells to acquire the
hallmarks of cancer (Wang et al, 2017). Cancer development is highly associated with
the physiological state of the TME (Genova et al., 2017). In a healthy state, the
microenvironment can help protect against carcinogenesis; in contrast, if in an
unhealthy state, it becomes an accomplice to tumour development (Wang et al., 2017).
Tumour cells can modify the microenvironment by releasing extracellular signals,
promoting immune tolerance and neoangiogenesis. The now-adapted TME supports the
proliferation and survival of cancer cells (Genova et al., 2017). The lungs present a
11
unique niche that enables tumours to progress in complicity with the TME. Pulmonary
disorders that promote inflammation, such as chronic obstructive pulmonary disease
(COPD), can contribute to the pathologic alteration of the lung microenvironment in a
way that supports and promotes carcinogenesis (Altorki et al., 2019).

2.1.4.1 Small-cell lung carcinoma

SCLC is a highly aggressive neuroendocrine tumour of the lung and is associated with
the most unfavourable prognosis (Messaritakis et al., 2019). It occurs in approximately
15% of lung cancer cases and has a median overall survival rate of around 8-12 months
for patients with extensive-stage SCLC and around 12-20 months for those with limited-
stage SCLC (Iams et al., 2019). Patients who are at the forefront for developing SCLC
include males usually above 70 years of age, who are active smokers or have a past
history of being a heavy smoker; and those who suffer from cardiovascular and
respiratory co-morbidities (van Meerbeeck et al., 2011). SCLC is usually diagnosed
when it has already widely metastasized. Although initially responding to cytotoxic
treatment, it almost always relapses with emerging resistance to further therapeutics
(Gazdar et al., 2017). SCLC is associated with a higher prevalence of genomic
instability compared to most other cancers (Krencz et al., 2019).

2.1.4.2 Non-small-cell lung carcinoma

NSCLC represents the most common type of lung cancer, accounting for 80-85% of
cases (Cao et al., 2017). Patients at risk of developing NSCLC include tobacco
smokers, and people exposed to radon and air pollution (Gridelli et al., 2015). The
outcome of patients with NSCLC continues to remain poor, and despite combined
therapy of chemotherapy and radiation, the overall five year survival rate is only around
15% (Hubbard et al., 2012). NSCLC is further subdivided into three categories:
adenocarcinomas, squamous cell carcinomas and large cell carcinomas (Inamura,
2017).

Adenocarcinomas are the most common type of lung cancer, accounting for 40% of
lung cancer cases, and usually occur in the periphery of the lung. It arises due to the
transformation of small airway epithelial type II alveolar cells (Zappa & Mousa, 2016).
Squamous cell carcinomas represent 25-30% of cases, and occur due to the
transformation of the bronchial epithelium. They usually present in the centre of the lung
(Savini et al., 2015). Large cell carcinomas are poorly differentiated subtypes of
12
NSCLC, occurring in only 5-10% of lung cancer cases. It is a malignant epithelial
neoplasm lacking glandular or squamous differentiation and cytological features of
SCLC; therefore, it is essentially diagnosed by exclusion of all other possibilities (Rajdev
et al., 2018). NSCLC usually progresses into a metastatic disease that damages various
end organs, including the adrenal glands, brain, liver, and bone, and involves much
higher mortality rates. The prime site of NSCLC metastases, the bone (40%), is
enriched with resident bone marrow that affords a highly supported environment for the
development of metastases (Attar-Schneider et al., 2020).

2.1.4.3 Common genomic and regulatory alterations associated with lung cancer
Studies have identified genetic and regulatory aberrations in the cell signalling pathways
that promote cell division, suppress cell death, and induce lung carcinogenesis
(Bethune et al., 2010). Activation of epidermal growth factor receptor (EGFR)
represents one of the most prominent clinical biomarkers in lung cancer growth and
development. In the western population, EGFR mutations occur in approximately 15%
of lung cancer cases, and in 40% of lung cancer cases in Asians (Choi et al., 2019).
Anaplastic lymphoma kinase (ALK) is another common actionable driver gene in
NSCLC. Activation of ALK genes lead to downstream signals that promote proliferation
and differentiation of cancer cells through fusion with other genes (Song et al., 2020).
Kirsten rat sarcoma (KRAS) viral oncogene homolog mutations are common alterations
in NSCLC accounting for approximately 25% of lung cancer cases in the Caucasian
population, and occur even more frequently in patients with adenocarcinoma histology
and those with a smoking history. These KRAS-mutated tumours have been associated
with co-occurring alterations in multiple genes as well as with a higher mutatational
burden (Gibert et al., 2020).

Rearrangements in ROS proto-oncogene 1 (ROS1) are targetable driver alterations

occurring in around 1-2% of lung cancer cases. Patients with ROS1-rearranged tumours
are prone to be younger and non-smokers, with an adenocarcinoma histology
(Digumarthy et al., 2020). Mesenchymal-epithelial transition (MET) genes are NSCLC-
associated driver alterations that have the ability to induce cell proliferation and tumour
growth. The oncogenic MET exon14 skipping mutation drives 1.3-5.7% of NSCLCs
(Pruis et al., 2020). Fusions in the rearrangement during transfection (RET) proto-
oncogene occur in 1-2% of patients with NSCLC. Rearrangements in these genes

13
results in the expression of oncogenic fusion proteins leading to downstream ligand-
dependent growth and proliferation (Hida et al., 2019).

2.1.5 Lung cancer risk factors

Many environmental and lifestyle causes have been linked to the development of lung
cancer. Although the majority of lung cancer cases are attributed to active tobacco
smoking, around 25% of cases occur in lifetime non-smokers. Lung cancer in non-
smokers represents the seventh most common cause of cancer-mortality globally, and
is therefore regarded as a separate and distinct disease entity to smoking-related
cancer (Soo et al., 2017). These non-smoking types of carcinogens may often act in a
synergistic and additive manner in people who actively smoke tobacco (Field & Withers,
2012).

Tobacco smoking is known to be a powerful carcinogenic agent and accounts as a

major risk factor and a largely modifiable and preventable cause of cancer (Balata et al.,
2020). The WHO has estimated that in the year 2019, there were approximately 1.1
billion smokers worldwide, with 80% of users living in low to middle-income countries,
where the burden of tobacco-related ill effects and death is at its highest (WHO, 2019).
The interlude between tobacco smoking and the development of lung cancer is between
30 to 40 years (Martín-Sánchez et al., 2017). There are several hypotheses regarding
the biological mechanisms that link cancer to tobacco smoking. The most accepted
mechanism involves the binding of inhaled carcinogens to DNA which result in the
formation of DNA adducts. This causes DNA miscoding and permanent mutations in
proto-oncogenes or tumour suppressor genes, which leads to the loss of normal cellular
proliferation and the consequent development of lung cancer (Poirier et al., 2019). By
1986, the IARC had already found sufficient evidence to determine second-hand smoke
(SHS) to be carcinogenic to humans (Hori et al., 2016). Exposure to SHS has
demonstrated to have several adverse health effects on both children and adults, and
therefore, contributes to lung cancer vulnerability (Carreras et al., 2019).

The correlation between lung cancer and air pollution can mainly be seen in non-
smokers (Yang et al., 2020). Studies have demonstrated how air pollution can cause
respiratory inflammatory reactions and impaired lung functions (Wang et al., 2019). Next
to increasing cancer risks, air pollution can increase mortality rates in cancer patients.
Lung cancer patients suffer from immune defects from the cancer itself and/or as a
14
result of the treatment thereof. This makes them more susceptible to the toxicity of air
pollutants which may exacerbate the condition and aid in the deterioration of the patient
(Espín-Pérez et al., 2018). Between 3-14% of global lung cancer cases can be
attributed to inhalation exposure to radon. Therefore, radon represents the second
leading cause of lung cancer after smoking (Elío et al., 2018). Lung cancer attributed to
asbestos-exposure is estimated at around 5-7% (Nymark, 2008), and people subjected
to exposure are typically blue-collar workers, especially those who are most often
smokers (El Zoghbi et al., 2017). The latency period between asbestos exposure and
the onset of lung cancer is around 20 to 40 years, and contrary to other neoplasms of
the lung, asbestos malignancies occur in the lower part of the lung (Świątkowska et al.,
2015).

Studies have demonstrated that between 50-70% of lung cancer patients suffer from
COPD, which is characterised by airflow limitation. COPD represents a significant and
independent risk factor for the development of lung cancer in both screened and non-
screened lung cancer cases (Hopkins et al., 2019). The incidence of both conditions
has been increasing in recent years, and this trend is projected to continue for the next
decade. One evident explanation for the co-existence of these conditions is their shared
risk factors; where tobacco-smoking is the most important (Husebø et al., 2019).

2.1.6 Lung cancer signs and symptoms

Lung cancer encompasses a high symptom burden. Researchers have identified a
number of predominant concurrent symptom clusters occurring in lung cancer patients,
all of which are associated with poor patient performance and outcomes (Cheville et al.,
2011). Patients with lung cancer are usually asymptomatic for many months prior to the
presentation of symptoms, which include both lung-related symptoms (cough, dyspnoea
and chest pain) and systemic symptoms (weight loss, appetite loss and fatigue) (Walter
et al., 2015). Symptoms arising in patients with cancer have been hypothesized to result
from a complex interaction between the malignancy (local tumour, intrathoracic spread,
distant metastases, or paraneoplastic syndromes) and the host characteristics, which
includes the state of the host’s immune system, any pre-existing comorbidities, and the
types of anti-cancer treatments being applied (Reyes-Gibby et al., 2013).

15
Cough is a primary symptom occurring in more than half of lung cancer patients and it is
one of the most important determinants of the patients’ quality of life (QoL) (Luckett et
al., 2019). Besides being a direct symptom of lung cancer, a chronic cough itself can
lead to its own array of diverse complications including incontinence, hernias, syncope,
loss of employment and social isolation (Badri & Smith, 2019). A study by Walter et al.
(2015) found haemoptysis to be the strongest predictor of lung cancer, despite
occurring in only one fifth of patients. Pain represents one of the most feared,
distressing and debilitating symptoms lung cancer patients may experience, which may
cause or exacerbate feelings of depression and anxiety (Mercadante & Vitrano, 2010).
Digital clubbing, although rare, is a highly predictive sign of lung cancer (Latimer, 2018).
Lung cancer is regarded as being the most psychologically disabling of all cancer types.
The surveillance, epidemiology, and end results programme data indicates that lung
cancer is associated with the highest suicide mortality rates compared to all other
cancer types (Andersen et al., 2019). Additionally, when compared to patients with other
cancers, those with lung cancer also exhibit the greatest prevalence of mood and
anxiety disorders (Linden et al., 2012).

2.1.7 Lung cancer diagnosis and staging

The initial diagnostic procedure for lung cancer requires a chest x-ray. If results are
inconclusive and suspicion for lung cancer remains, a computed tomography scan with
contrast imaging must be obtained (Latimer, 2018). Immunohistochemistry is
recommended to confirm lung cancers which prove difficult to diagnose. Precise
histological diagnosis is based either on biopsy or cytological specimens and molecular
testing (Travis, 2020). Lung cancer staging provides the foundation for informed patient
care and prognosis. The Union for International Cancer Control Tumour, Metastasis,
and Nodal (TMN) classification is the internationally accepted standard for cancer
staging (Kutob & Schneider, 2020). The definition of disease progression typically used
in lung cancer includes local recurrence, regional recurrence, and distant metastasis
(Nantavithya et al., 2020).

2.1.8 Current treatment strategies for lung cancer

Despite ongoing developments in novel lung cancer treatment strategies, multiple
barriers still lie ahead of successful clinical implementation. Drug resistance, insufficient
curative effect, and tumour relapse remain huge challenges and drawbacks of current
cancer therapeutics (Mei et al., 2019). The optimal treatment regimen in patients with
16
lung cancer remains unclear. The choice of surgery versus radiotherapy as well as the
optimal adjuvant therapy is undefined; therefore, recommendations are based largely on
the findings from observational studies. Several clinical practice guidelines have
recommended surgery for stage I lung cancer patients, followed by adjuvant
chemotherapy (Putora et al., 2020). For advanced stage lung cancer with unresectable
and metastatic cancers, cytotoxic combination chemotherapy is generally the first line
treatment choice (Zappa & Mousa, 2016). Local recurrence after first-line treatment
usually presents as an endobronchial tumour. Endobronchial tumours occlude the
airways and are therefore highly symptomatic and lethal if untreated (Weinberg et al.,
2010).

2.1.8.1 Chemotherapy
Chemotherapy is defined as the application of chemically synthesized medicines to kill
cancer cells (Siveen & Kuttan, 2011). Around 40% of lung cancer patients are newly
diagnosed at stage IV; therefore, the treatment goal for these patients is to improve
survival, and reduce the symptom burden of the disease (Zappa & Mousa, 2016).
Platinum based anticancer drugs in combination with taxanes, anti-metabolites, and
vinca alkaloids are the most commonly prescribed anti-cancer drugs used as first-line
agents which have been widely accepted as an evidence-based treatment for advanced
lung cancer (Huang et al., 2017).

Apoptosis plays a vital and therefore indispensable role in chemotherapy. An increase

in the expression of anti-apoptotic proteins and a decrease in the expression of pro-
apoptotic proteins are necessary for tumour development and progression. Alterations
in anti-apoptotic and pro-apoptotic protein expressions resultantly contribute to
apoptotic resistance, a factor which is associated with chemoresistance (Xue et al.,
2020). Chemoresistance is a primary cause of treatment failure. Although initially
effective, patients undergoing chemotherapy ultimately develop resistance to both
single and multiple therapeutic agents which encourages metastatic spread (Maji et al.,
2018). Although chemotherapeutic drugs are systematically distributed throughout the
body, unfortunately for lung cancer patients, very little of it actually gets absorbed in the
lungs (Zhang et al., 2020). Furthermore, the dosages of drugs employed for
chemotherapy are generally close to the drugs’ toxic dosages. Therefore, errors in
dosage calculations carry a high risk of acute and cumulative toxicity, thereby

17
increasing the likelihood of mortality in patients. Medication errors represent the second
most common cause of death in patients undergoing treatment (Goldspiel et al., 2015).

The desired goal of chemotherapy is to eliminate tumour cells; however, a diverse

range of normal cells are affected concurrently during treatment. This causes
debilitating effects which can dramatically reduce the functional ability and QoL in
cancer survivors, even years after the discontinuation of treatment (Sak, 2012). Despite
being prescribed regularly, the use of these anticancer drugs is restricted by severe
dose-limiting side-effects, and by either primary or acquired resistance (Oun et al.,
2018). On average, a patient can experience a combination of around forty side-effects.
Nausea and vomiting are two of the most feared side-effects for patients undergoing
chemotherapy (Nurgali et al., 2018). This requires reductions in the dosage of the drug,
extensive monitoring of the patient’s biochemistry, and the additional co-prescription of
non-chemotherapy-based drugs to combat the side-effects (Oun et al., 2018).

2.1.8.2 Radiotherapy
Radiation is a physical agent used to destroy cancer cells. Ionising radiation forms ions
(electrically charged particles) which deposit energy in the cells of the tissues it is
passing through. This energy can kill cancer cells or cause genetic changes that result
in cancer cell death (Baskar et al., 2012). Radiotherapy is indicated in a lung cancer
treatment algorithm, under any circumstance and at any stage of the disease with
different therapeutic goals. For patients with unresectable stage III cancer,
chemoradiotherapy is the treatment of choice either in combination or sequentially
(Vojtíšek, 2019).

Stereotactic ablative body radiotherapy (SABR) was pioneered during the mid-1990s for
the treatment of tumours within the thorax (Rulach et al., 2020). SABR has become the
international standard of care for medically inoperable early stage NSCLC. It delivers a
high biological equivalent dose, in fewer highly focused doses, and within a shorter
treatment time compared to other forms of radiation (Phillips et al., 2019). Even though
patients undergoing SABR treatment have promising outcomes in terms of very high
loco regional control rates, local failure rates of 5-15% have been reported (Nantavithya
et al., 2020).

18
The radical doses of radiotherapy are delivered in close proximity to organs at risk
highlighting its limitation in clinical practice. This leads to unacceptable toxicities
reaching collateral organs, which can cause a wide array of complications including
radiation pneumonitis, oesophagitis, cardiac injury and neuropathy (Kapoor et al.,
2020). Lymphocytopaenia has been recognised as a black box warning for thoracic
radiotherapy which is associated with poor treatment outcomes. It is likely due to the
irradiation of a large volume of circulatory blood compared to other parts of the body,
since the pulmonary circulation receives around half of the cardiac output (Joseph et al.,
2019). One third of patients undergoing radiotherapy will suffer treatment failure due to
local recurrence, radioresistance and distant metastasis (Sun et al., 2019).

2.1.8.3 Surgery
To date, surgery remains one of the mainstay treatments for localised solid cancer
treatment. The standard care for patients with early stage lung cancer is surgical
resection (lobectomy) with curative intent, which has a five-year survival rate of around
70%. However, many patients find themselves unfit for a major operation due to
inoperable lesions at the time of diagnosis, fragility, or cardiorespiratory co-morbidities
(Rulach et al., 2020).

Standard resection includes the removal of the cancer infiltrated lobe, and the systemic
evaluation of ipislateral hilar and mediastinal lymph nodes (Lackey & Donington, 2013).
Video-assisted thoracoscopic surgery (VATS) has marked a new beginning for thoracic
surgery. It is associated with smaller incisions, shorter hospital stays, and less pain and
bleeding after surgery (Kim & Cho, 2020). The completeness of the resection, cancer
stage, and lymph node involvement are three primary predictors of survival after
surgery, with 37% of patients experiencing some form of postoperative complication
(Lackey & Donington, 2013). Patient prognosis is usually determined by the precision of
the surgical resection, and since surgery depends highly upon visual inspection,
resection of tumours may be imprecise and suboptimal (James et al., 2016). Poor
function, bad cosmetic results, severe complications, and early post-operative morbidity
may occur as a result of surgery, and in many cases adjuvant therapy is still required
(Pedro et al., 2018).

Surgery can affect the patient’s health-related QoL. Persistent postsurgical pain is
described as the development of chronic pain after thoracic surgery persisting for more
19
than two months without other causes. Furthermore, it is described as being
characteristically different from preoperative pain (Peng et al., 2014). Persistent
postsurgical pain is a common complication after thoracic surgery that can interfere with
daily activities, sleep and mood. If poorly managed, it results in longer hospital
admissions and delays in the patient’s ability to participate in rehabilitation programmes
(Gjeilo et al., 2020).

Surgery has also been studied for its role as a trigger in metastasis. Surgery can induce
shedding of cancer cells into the circulation, suppress antitumour immunity allowing
circulating cells to survive, upregulate adhesion molecules in target organs, and recruit
immune cells capable of entrapping tumour cells. The trauma induced by surgery can
further cause local and systemic inflammatory responses that can contribute to the
accelerated growth of residual and micrometastatic disease (Tohme et al., 2017).

2.1.8.4 Immunotherapy
Cancer immunotherapy is a type of cancer treatment that helps the immune system to
fight cancer through the use of immune checkpoint inhibitors (Liu & Guo, 2018).
Immunotherapy is a first-line treatment choice for patients with advanced NSCLC.
Preoperative immunotherapy is superior to chemotherapy in terms of reduced toxicity
and pathological effectiveness. It can be performed prior to surgery as part of
neoadjuvant treatment combined with chemotherapy in order to obtain better results (Lu
& Su, 2019). Despite the initial encouraging promises of immune checkpoint inhibition,
most patients do not respond and/or subsequently develop resistance to
immunotherapy (Zhang et al., 2020). Additionally, patients with relatively weaker
immune systems do not produce adequate levels of immune cells to destroy the tumour.
The blocking of certain receptors by immunotherapy causes a susceptibility to over-
express immune cells. Such a reaction may lead to severe disorders which can be fatal
(Cho, 2017).

2.1.8.5 Targeted Gene therapy

Gene therapy, consisting of a few modalities including oncolytic viro therapy and gene
transfer, holds great potential for the future but is at present not very reliable (Bidram et
al., 2019). Although these modalities are associated with better survival prognosis,
patients with specific driver genes will inevitably be faced with drug resistance and
disease progression (Zhang et al., 2020).
20
2.1.9 Prognosis
Despite treatment advances in recent years, lung cancer survival rates continue to
remain low with 15-18% of sufferers having a disappointing five-year survival rate; a
rate that has only slightly increased over the last 2.5 decades (Duruisseaux & Esteller,
2018). The poor prognosis is greatly related to the stage of the disease at diagnosis,
with the majority of patients being diagnosed with locally advanced or metastatic
disease (Kourlaba et al., 2019). This is due to the fact that the symptoms experienced
by patients have a low predictive value for cancer diagnosis (Ellis & Vandermeer, 2011).
The resultant delay in the initiation of treatment is associated with poor health outcomes
(Kourlaba et al., 2019). Owing to the poor prognosis of lung cancer; early detection,
diagnosis, primary prevention, and therapeutic advancements are of vital importance to
patient outcomes and well-being (Casal-Mourino et al., 2019).

2.2 Homeopathy
Homeopathy is a holistic complementary medicine (CM) modality, founded by Dr
Samuel Hahnemann (1755-1843) just over 200 years ago (Unlu et al., 2017). According
to the WHO, CM refers to a broad set of health care practices that are not part of that
country’s own tradition or conventional medicine and are not fully integrated into the
dominant health-care system (WHO, 2019). CM in parallel with conventional therapies
is used to aid in the treatment of various conditions, including cancer and to improve
general well-being (Dehghan et al., 2020). It has been gaining global traction over the
last few decades especially in circumstances where treatment regimens in conventional
medicine are unsatisfactory, patient’s individual needs are disregarded, and focus is
placed rather on reducing risks and simply adding years to patients’ lives (Rodrigues
dos Santos & Mendes, 2020). Patients therefore, turn towards CM in efforts to address
their own unmet needs outside of conventional medicine (Foley et al., 2020).

Integrative oncology is defined as a diverse approach to the treatment of cancer

patients that encompasses both conventional and CM therapies (Witt et al., 2017). In an
oncology setting, CM is used as supportive to conventional care before, during and/or
after treatment to alleviate cancer-induced symptoms, treat common side-effects
resulting from standard cancer treatments, improve QoL, and increase patient
satisfaction and compliance to treatment (Doğan et al., 2020). Homeopathy is the most
often used CM method by patient’s worldwide (Gaertner et al., 2018). The application of
homeopathy in oncology care has been recognised especially for improving patient’s

21
QoL and prolonging survival. Furthermore, in vitro studies demonstrating the effects of
homeopathic remedies on cancer cell lines has only just begun (Fuselier et al., 2019).

2.2.1 The principle of similars

Homeopathy is derived from the Greek words "homoios", meaning similar, and "pathos",
meaning suffering (Şenel, 2019). This terminology reflects the core and most vital
principle of homeopathy; “Similia similibus curantur” translated as “like is cured by like”
(Ahmad et al., 2018). This principle states that a substance capable of causing
symptoms in a healthy patient can be used in minute doses to cure similar symptoms in
a diseased patient (Schmidt, 2020). Hahnemann further strengthened this key concept
with the addition of drug provings and drug potentisation as crucial to the principle of the
similimum (Das et al., 2019).

The foundation of homeopathic doctrine learning stems from homeopathic pathogenic

trials (HPT) (also known as a proving). Homeopathic pathogenic trials are aimed at
investigating the effects of undiluted homeopathic remedies on healthy individuals under
a rigorous methodology (Renoux, 2018). After the administration of the homeopathic
remedy, healthy subjects will produce a set of symptoms within a specified timeframe.
These symptoms are analysed and synthesised to produce a remedy picture that is
characteristic of the particular homeopathic remedy. The remedy is then applied in
cases of illnesses which produce similar symptoms (Unlu et al., 2017). The principle of
similars dictates that the efficacy of the homeopathic remedy increases when the
similarity between the patients’ symptom picture and the remedy picture increases
(Almirantis, 2013).

Homeopathic remedies are prepared from either plant, animal, chemical or mineral
based crude substances (Bell et al., 2014). Homeopathic remedy production begins with
a homeopathic mother tincture (Ø) for soluble substances or a triturate (successive
grinding of a starting substance with lactose) for insoluble substances. The substance is
triturated until it is able to be suspended in a liquid medium (Culbert & Olness, 2010). In
either case, the resulting solutions undergo a series of dilutions using either the
centesimal (C) scale (1:100 dilution) or the decimal (D) scale (1:10 dilution), with at
every step, vigorous shakings termed 'succussions' (Basu et al., 2017). The combined
application of dilutions and succussions is termed potentisation. During this process, the
biological activity of the source material is activated through physiochemical changes.
22
This is generated by the serial application of a certain quantum of force. The
succussions after each dilution are required to retain the activity of the original
substance (Shah, 2016). Potentisation is implemented in order to reduce drug toxicity
and strengthen, enhance and extend the pharmacodynamic effects of the crude
substance through each additional ascending potency (Das et al., 2019).

2.2.2 Homeopathic mother tinctures

Homeopathic mother tinctures are liquid preparations arising from the extraction of a
raw material that is macerated in an alcohol or water solution within a specific ratio in
accordance with the concise guidelines laid out in the homeopathic pharmacopoeia
(Scheepmaker & Gower, 2011). These Ø’s are used worldwide and represent a
substantial proportion of the global pharmaceutical market (Singh et al., 2014). The Ø’s
act as a precursor for the preparation of other potencies and represent the starting point
for the production of homeopathic remedies (Maiti et al., 2013).

Like herbal tinctures, Ø’s have therapeutic indications which require the same
registration procedures to prove their safety and efficacy (Csupor et al., 2013).
According to the WHO, to ensure the quality of Ø’s, the following data must be made
available: the method of the preparation, its appearance, description, identity tests,
purity tests, stability testing procedures, and the determination of content and active
constituents (WHO, 2009). Since Ø’s contain detectable amounts of active ingredients,
they can therefore be subjected to chemical profiling and quantification using analytical
techniques including Thin-Layer Chromatography (TLC) and High-Performance Thin-
Layer Chromatography (HPTLC) fingerprint profiles to ensure standardization (Jadhav
et al., 2016). The distinction between a Ø and a herbal tincture is based on the clinical
context, the rationale behind its prescription, and the method of production of the
tincture (Jütte & Riley, 2005). A Ø is typically prepared in a hydroalcoholic solvent in a
1:10 dilution (Hedayat & Lapraz, 2019), whereas a herbal tincture is in a 1:5 dilution,
and herbal extracts are far more concentrated, in a 1:2 or 1:1 dilution (Romm et al.,
2010).

23
2.3 Thuja occidentalis
Thuja occidentalis Linn (T. occidentalis), is a monoecious coniferous plant belonging to
the Cupressaceae family (Figure 2.3). It is commonly known as the white cedar or arbor
vitae and its name is Latin for tree of life (Silva et al., 2017). T. occidentalis was first
identified in the 16th century by the French explorer Jacques Cartier during his second
voyage of discovery to Canada (Sunila et al., 2011). T. occidentalis is a slow growing,
shade-tolerant tree with a typical lifespan of 80-400 years, reaching heights on average
of 12-15 m and diameters ranging between 30-60 cm. Although it is able to tolerate a
range of substrates, its growth is maximised on moist, well-drained soils derived from
calcareous bedrock (Kincaid, 2016).

Figure 2.3: (A) Thuja occidentalis tree, (B) Branch of the Thuja Occidentalis tree (Bigstock, 2020).

T. occidentalis is indigenous to the north-eastern mixed and boreal forests of North

America, and is grown as an ornamental tree in Europe (Danneyrolles et al., 2017). It is
an important Native American ceremonial plant which has been widely utilised in
ethnomedicine. The plant is greatly honoured by the Ojibwa culture by the name

24
Nookomis Giizhik, meaning Grandmother Cedar. These people used the soft twigs to
make soups and teas for the treatment of various ailments (Pudełek et al., 2019). In
traditional medicine, T. occidentalis has been used to treat diseases of the respiratory
system (bronchial catarrh), urinary and reproductive systems (enuresis, cystitis,
amenorrhoea), as well as rheumatic and autoimmune diseases (Stan et al., 2019).

Several pre-clinical studies have demonstrated the efficacy of T. occidentalis in tumour

suppression. A study by Saha et al. (2014) investigated the antitumourogenic potential
of a bioactive extract of T. occidentalis on human mammary epithelial carcinoma cell
lines, MCF-7 and MDA-MB-231. T. occidentalis was shown to induce apoptosis in the
functional p53 expressing mammary epithelial carcinoma cell lines while sparing normal
cells. Mukherjee et al. (2012) showed that ethanolic extracts of T. occidentalis blocks
proliferation of A549 NSCLC cells and induces apoptosis in vitro. The study
demonstrated significant DNA-nick generation, chromatin condensation, increase in
caspase 3 activity, Bax up-regulation, and Bcl-2 down-regulation; all in favour of
apoptosis.

Sunila & Kuttan (2006) demonstrated the in vivo effects of T. occidentalis extract on
lung metastasis induced by B161F-10 melanoma cells on C57BL16 mice. The study
showed a remarkable reduction in tumour-nodule formation in conjunction with a
significant increase in the lifespan of the treated mice. Sunila & Kuttan (2005) also
demonstrated the protective effect of T. occidentalis extract against radiation-induced
toxicity in Swiss albino mice. T. occidentalis significantly reduced leucopoenia induced
by a sub-lethal dose of radiotherapy. Normal levels of white blood cell count were
present at the end of treatment, whereas regenerative capacity in control group animals
was low and did not regain a normal level. In addition, T. occidentalis treated groups
showed a significant increase in bone marrow cellularity.

2.3.1 Phytochemical properties of Thuja occidentalis

According to phytochemical studies, T. occidentalis based preparations contain various
constituents, namely essential oils (camphene, fenchone, limonene, terpinolene and
thujone), flavonoids (umbelliferone, kaempferol, myricetine, quercetin and tannic acid),
coumarins, proteins and polysaccharides. Recent studies have demonstrated several
pharmacological properties of T. occidentalis including anti-proliferative, radio-
protective, antioxidant and anticarcinogenic activities (Silva et al., 2017). Much of the
25
phytochemistry research of T. occidentalis has concentrated its efforts on the essential
oil thujone for which most of the plant’s pharmacological and therapeutic effects is
ascribed to. However, preclinical studies have identified the presence of
polysaccharides and flavonoids in the ethanol fraction of the plant which also exhibit
anti-tumour activity (Alves et al., 2014).

2.3.1.1 Essential oils

Essential oils are secondary metabolites of plants that play important roles in the
protection of the plant. They have been shown to possess cancer cell targeting activity,
pro-immune functions, and the ability to increase the efficacy of commonly used
chemotherapy drugs (Blowman et al., 2018). Cedar oil is an essential oil present in the
leaves of T. occidentalis. It is extracted by steam distillation or hydro distillation of the
fresh leaves of the plant (Akkol et al., 2015). Thujone is a monoterpene ketone, a
natural principal constituent and active ingredient of T. occidentalis, which occurs as a
variable mixture of diastereoisomers α-thujone and β-thujone (Pelkonen et al., 2013).
Thujone is extracted from the dry mass of the leaf branches, and represents 60% of the
compounds found in the essential oil (Stan et al., 2019).

Immunostimulants are a heterogeneous group of compounds that act in a non-specific

manner on the immune system (Puggioni et al., 2019). These compounds are able to
enhance host defence responses by eliciting and activating both the humoral and cell-
mediated immune systems (Bascones-Martinez et al., 2014). The search for natural
products of plant origins as a new basis for the development of powerful and safe
immunostimulant agents has gained inspiring research interest (Jantan et al., 2015).
Naturally occurring immunostimulants can be used in combination with common
anticancer modalities to improve immune responses against tumours, as well as to
reduce the suppression effect produced by standard cancer treatment (Mohamed et al.,
2017). A study by Siveen & Kuttan (2011) demonstrated the immunostimulatory activity
of thujone on the humoral and cell-mediated immune system during solid tumour
development. The augmentation of the immune system by thujone increased natural
killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC), and cytotoxic T
lymphocyte generation; thereby causing a reduction in Dalton's Lymphoma Ascites
induced solid tumour development in Balb/c mice.

26
According to an in vitro study by Pudełek et al. (2019) done on glioblastoma multiforme
cells, α-thujone was shown to exert pro-apoptotic and anti-invasive effects. The
attenuating effect of α-thujone was further supported with the accumulation of ROS. In
another study, the phytochemical thujone isolated from T. occidentalis extracts was
tested on a melanoma A-375 skin cancer cell line, and demonstrated the activation of
pro-apoptotic signalling (Ijaz et al., 2018).

2.3.1.2 Polysaccharides
Polysaccharides have emerged as potential chemical entities exhibiting good anticancer
activity. Polysaccharides act on malignant cells mainly through induction of apoptosis.
They are able to induce DNA damage, cell cycle arrest, and mitochondrial membrane
disruptions. Furthermore, polysaccharides can produce nitric oxide, which is able to kill
cancer cells and prevent metastasis (Khan et al., 2019). An in vivo study by Sunila et al.
(2011) showed the effects of T. occidentalis and its polysaccharide on tumour-bearing
mice. T. occidentalis and its polysaccharide effectively stimulated cell-mediated
immunity through the enhancement of NK activity, ADCC, and antibody-dependent
complement-mediated cytotoxicity much earlier than the tumour-bearing control
animals. Furthermore, it also showed a decrease in pro-inflammatory cytokines, as a
result inhibiting metastasis of tumour cells.

2.3.1.3 Flavonoids
Flavonoids are a group of secondary plant metabolites that have various biochemical
and antioxidant effects (Panche et al., 2016). During carcinogenesis, flavonoids
interfere with multiple signal transduction pathways and thus have the capability to limit
proliferation, angiogenesis and metastasis, or increase apoptosis (Abotaleb et al.,
2019). A study by Mukherjee et al. (2014), demonstrated the in vitro and in vivo effects
of flavanol isolated from an ethanolic leaf extract of T. occidentalis in A549 NSCLC cells
and Swiss albino mice. In in vitro, the study demonstrated how flavonol isolated from T.
occidentalis reduced A549 cell viability. The cytotoxic effect was mediated through cell
cycle arrest and ROS-independent apoptosis that was found to be target-specific and
chemo-preventative. In in vivo, the flavonol demonstrated signs of anti-cancer potential
through the inhibition of cell proliferation and growth of lung tumours in mice. Moreover,
the flavonol at its specific dose was neither cytotoxic to normal L-132 lung cells in vitro,
nor was able to raise any toxicity in mouse bodies, in vivo. This makes the drug more
potent and potentially suitable for therapeutic use against lung cancer.
27
2.3.2 Thuja occidentalis Ø
T. occidentalis Ø has been widely prescribed in homeopathy for the treatment of various
conditions and is most commonly indicated in the treatment of pathological growths of
vegetative condylomata, warty excrescences and tumours (Vermeulen, 2015). T.
occidentalis Ø is mainly used in the treatment of acute and chronic infections of the
upper respiratory tract, and as an adjuvant to antibiotics for severe bacterial infections
(Alves et al., 2014). T. occidentalis Ø is prepared according to the methods outlined in
the Homeopathic Pharmacopeia from fresh, young, non-woody branches with leaves,
using a high percentage of ethanol as the solvent (Stan et al., 2019).

Currently, there is a paucity of research related to the homeopathically prepared T.

occidentalis Ø. An in vitro study by Biswas et al. (2011) on A375 NSCLC cells
demonstrated how T. occidentalis Ø caused a decrease in cell-viability. Additionally, it
induced mitochondrial transmembrane collapse, inter-nucleosomal DNA fragmentation,
increased ROS generation, and the activation of caspase-3; all of which are related to
the apoptotic induction of A375 cancer cells. Results of the study also indicated that T.
occidentalis Ø has four chromatographically separated fractions, of which the thujone-
rich fraction was found to be the most bioactive (anti-cancer, pro-apoptotic) component.
Furthermore, TO demonstrated no significant cytotoxic effects on normal peripheral
blood mononuclear cells.

T. occidentalis 1M potency (M = 1000 homeopathic succussions and dilutions of a

remedy), used in combination with two other homeopathic remedies, namely Hydrastis
canadensis 1M and Lycopodium clavatum 1M, showed anti-metastatic effects in B16F-
10 melanoma-bearing animals (Ahmad et al., 2018). Another in vitro study on T.
occidentalis 30cH demonstrated convincing evidence of anti-carcinogenic effects in lung
cancer cells through its possible action at the molecular level of gene regulation.
(Mukherjee et al., 2013). In a reading by Bagot (2020b) on the in vitro and in vivo
effects of T. occidentalis, seven research studies demonstrated a positive activity of the
remedy in tincture or in high dilution on cancer cells without harmful action on healthy
cells.

28
2.4 Photodynamic therapy
The earliest accounts of interventions using photodynamic therapy (PDT) can be traced
back to the ancient Egyptian and Indian healers who used sunlight in combination with
herbal extracts to treat certain diseases (Hönigsmann, 2013). PDT is an alternate
therapy that uses coherent monochromatic low intensity laser irradiation (LILI) to induce
photobiological processes at a cellular level (Heiskanen & Hamblin, 2018), and it has
emerged as a promising tool for the diagnosis and treatment of cancer (Chen et al.,
2020). The primary principal of PDT involves a photochemical reaction whereby a
photosensitizer (PS) is excited with a light of a specific wavelength in the presence of
molecular oxygen (O2) to form cytotoxic ROS, that then destroys hyperproliferating
cancer cells (Hosokawa et al., 2020). In order to achieve this, PDT requires three basic
yet fundamental components: A PS, light of a specific wavelength, and molecular O 2
(Agostinis et al., 2011).

2.4.1 Advantages and disadvantages of photodynamic therapy

PDT has several known advantages over traditional anticancer therapies. PDT can
significantly reduce the side-effects caused by standard cancer therapies, and improve
the target specificity of drug delivery. Compared to standard cancer therapeutics, PDT
has low systemic cytotoxicity, negligible drug resistance, high spatio-temporal precision,
and localised photo-irradiation (da Silva et al., 2019; Fan et al., 2019). Furthermore,
PDT has shown effectiveness against multi-drug-resistant tumours (Yi et al., 2018).

The disadvantages of PDT can mainly be observed when there is a lack of available O 2.
When there is an insufficient amount of oxygen in tumour environment, photodamage is
reduced or no PDT reaction occurs at all (Song et al., 2020). In addition, despite the
initial response to the therapy, the rapid consumption of oxygen during the PDT reaction
and microvascular damage further exacerbates the tumour hypoxia, leading to
compromised treatment efficacy, and poor prognosis (Zhang et al., 2020).Besides
tumour hypoxia, the limited tissue penetration depth and delivery efficiency of light has
emerged as the “Achilles heel” of PDT. Cancers that are deep seated or have internally
metastasized are harder to treat with PDT, since it is almost impossible to deliver
adequate laser light irradiation to these tissues (Yu et al., 2018).

29
2.4.2 Photosensitizers
Photosensitizers (PSs) are defined as molecules that are capable of absorbing
ultraviolet radiation and visible light through photochemical reactions (Vázquez-Ortega.,
2020). PSs are categorized into three broad chemical families: (i) Porphyrins, (ii)
chlorophyll derivatives, and (iii) dye substances (Iyer et al., 2018). There are currently
three generations of PSs developed for the improved efficacy of PDT. First-generation
PSs was synthesised as early as the 1970s, which consisted of complex mixtures of
porphyrins (Fan et al., 2019). A water‐soluble mixture of porphyrins called
hematoporphyrin derivative (HPD), was the first ever PS to be clinically trialled for
cancer therapy. HPDs are used today as Porfimer Sodium, commonly known under its
trade name Photophrin®- a commercially available PS (Agostinis et al., 2011). Many
PSs including Photophrin® faced the shortcomings of poor chemical purity, poor tissue
penetration, and prolonged half-life. In addition, the accumulation of Photophrin® in the
skin can lead to skin phototoxicity which may persist for two or even three months after
administration (Zhang et al., 2018).

In an attempt to overcome these disadvantages, second-generation PSs began

developing in the 1980s in an effort to develop PSs with improved drug delivery.
Second-generation PSs are chemically more effective primarily due to its higher
chemical purity, good absorption in the visible and/or near infrared light spectrum, and
higher production yield of singlet O2 (Senge et al., 2012). In addition, second-generation
PSs are associated with fewer adverse effects, since they have a higher selectivity for
malignant tissues and are eliminated faster from the body (Dobson et al., 2018).
Second-generation PSs are modified into third-generation PSs in order to meet specific
demands (Darwish et al., 2020). Additional biological criteria are included in the design
principle of third-generation PSs, which consists of finding special drug delivery and
formulation techniques in order to improve selectivity and specificity of PDT (Senge et
al., 2012).

2.4.2.1 Characteristics of a good photosensitizer

The successful application of PDT relies on the appropriate selection of a PS. In order
to gain full therapeutic benefit, it is desirable that PSs should ideally embody certain
physiochemical and biological characteristics which include: (i) chemically pure and of a
specific composition, (ii) stable at room temperature, (iii) minimal cytotoxicity in the dark,
(iv) photosensitive effects only in the presence of a specific wavelength of light (600-800
30
nm), (v) selective retention by target tissues, (vi) high photochemical reactivity, (vii) are
inexpensive and commercially available, (viii) easy solubility in body tissues, and (IX)
high capacity for quantum generation of O2 with the objective of generating singlet O2
(Kou et al., 2017).

2.4.2.2 The advantages of using plant sources as natural photosensitizers

Nature is an inexhaustible source of natural substances with preventative and curative
properties. Ancestral knowledge of plant properties has led to the extraction of active
compounds, as well as the purification and chemical modification of natural scaffolds in
order to enhance therapeutic benefit and increase cytotoxic and anti-proliferative
potential (Diederich & Cerella, 2016). The pharmaceutical industry has long been driven
by the diverse reservoir of chemical compounds found in natural products. Natural
products generally contain a higher number of novel chemicals than that obtained
synthetically. To date, among all therapeutic agents approved by the Food and Drug
Administration (FDA), an estimated 50% of medicines, and around 48.6% of anticancer
drugs are directly or indirectly derived from natural resources (Jiang et al., 2019).
Recent years has shown a steady growth towards the use of natural substances and
herbal drugs that are commercially available and environmentally sustainable (Mansoori
et al., 2019). Natural products are therefore resourceful tools for the development of
new drugs (Shi et al., 2019). Selected natural compounds can target multiple
deregulated cellular pathways to induce apoptosis in an efficacious and selective
manner with minimal toxicity on normal cells, and increase the sensitivity of the cancer
cells to anticancer agents (Millimouno et al., 2014).

Chlorophyll exhibits desirable photosensitive activity as observed during photosynthesis

- the natural method of harvesting sunlight (Gao et al., 2020). Chlorophyll derivatives
are second-generation PSs that have excellent singlet O2 production and
photosensitivity. Furthermore, these PSs have a high extinction co-efficient in the range
of 400-670 nm; a range that is characterised by good tissue penetration, high affinity to
biomolecules, and long-lasting excited states (Gomaa et al., 2017; Li et al., 2012).
Therefore, plant extracts hold great potential as PSs for the successful employment of
PDT. An additional advantage of using plant extracts as a PS lies in their selective
action against malignant cells, while still maintaining a low systemic cytotoxicity against
normal cells (Mansoori et al., 2019).

31
Previous studies have shown exciting possibilities of using plant extracts as natural PSs
in PDT. An in vitro study by Lopes et al. (2019) demonstrated the effectiveness of the
plant alkaloid berberine in PDT. Berberine was able to generate ROS to induce
autophagy and apoptosis through the activation of caspase-3 in renal carcinoma cells.
Furthermore, it triggered metabolite changes related to the inhibition of cell proliferation,
migration and angiogenesis. A study by Villacorta et al. (2017) showed that the plant
extracts Lumnitzera racemosa and Albizia procera, when irradiated, induced apoptosis
in mammary cell adenocarcinoma cell lines. The crude ethanolic extracts were shown to
be non-toxic to non-cancer cells. Another study showed that a plant extract of Brassica
napus induced light-dependant cell death against leukaemia U937 and human liver
cancer SK-HEP-1 cells (Choi et al., 2017).

In an in vivo study by Laszló et al. (2020), Cornus mas extract was shown to enhance
the responsiveness of PDT in terms of apoptosis, inflammation, DNA damage and
increased antioxidant effects in 35 Wistar albino rats. The findings in an in vitro and in
vivo study by Kim et al. (2018) suggested that hypericin-mediated PDT induced cell
death via the associated oxidative stress mechanism. In an in vitro study, hypericin-
mediated PDT produced a significant generation of ROS and mitochondrial damage on
anaplastic thyroid cancer FRO cells. In an in vivo study, hypericin and laser therapy was
shown to prevent proliferation of anaplastic thyroid cancer in mice. A study on the
combined effects of Curcumin and photodynamic therapy in breast adenocarcinoma cell
lines (MCF-7 cells) showed a decrease in the proliferation of the MCF-7 cells (Machado
et al., 2019). Furthermore, in a study by Shi et al. (2019), thirteen traditional plant
extracts tested on human laryngeal epithelial cancer cells demonstrated strong
fluorescence intensities comparable to those of commercial hematoporphyrin PSs.

2.4.3 Light
Light is an external, non-invasive stimulus that can be modulated in a temporal and
spatial manner to achieve precise control of stimulus-responsive platforms (Lin et al.,
2018). Illumination parameters including irradiation source, wavelength and fluency are
fundamental not only for the appropriate PS excitation but also for tumour ablation and
sparing of normal tissue (Ferraz et al., 2011). Diverse sources of light are used for PDT
protocols including white light lamps, lasers and light emitting diodes (LEDs)
(Schaberle, 2018). The source of irradiation is generally unique to each PS, since each
PS has a determined optimal wavelength and fluency of light required to activate it
32
(Gallardo-Villagrán et al., 2019). Lasers have unique properties that make it a
favourable light source in PDT. These properties include its excellent coherency, high
intensity, good penetration range, and monochromatic beam (Navaeipour et al., 2016).
LEDs were introduced in the year 2000; it has since become the most commonly used
form of irradiation. It is defined as sources of optical semiconductor devices that are
able to convert electrical energy into light energy (Oh & Jeong, 2019). Advantages in
the developments in LED technology have provided higher power, narrower spectral
characteristics, lower energy consumption, lower costs, and longer life spans making
them a desirable alternative to standard lasers (Quirk et al., 2015).

2.4.3.1 Wavelength
The wavelength of light employed for the optimal use of PDT ranges between 600-800
nm, known as the therapeutic window. Within this range, the energy of each photon
from the laser is high enough to excite the PS, and still low enough so that the light
sufficiently penetrates into the tissue (Donohoe et al., 2019). The number of photons
absorbed by the PS will determine the efficacy of the PDT effect (Schaberle, 2018).
Since the tissues surrounding the tumour does not completely absorb the photon
energy from light, the choice of the wavelength would therefore depend on the targeted
depth of penetration and the wavelength absorption range of the particular PS being
administered (Oh & Jeong, 2019).

2.4.4 Oxygen
Molecular O2 is indispensable to the PDT reaction. PDT outcomes are optimal when the
target region is well-oxygenated (Kamanli & Çetinel, 2020). A possible advantage of
employing PDT for lung cancers may be due to the locally sufficient O 2 supply in the
lungs. When PSs are irradiated with a specific wavelength of light, energy is transferred
to the surrounding O2 to create cytotoxic ROS (Zhang et al., 2020). ROS are by-products
of aerobic metabolism, which poses inherent chemical properties that confers reactivity
to various biological and chemical targets (Schieber & Chandel, 2014). It plays vital and
diverse roles in regulating components of cellular behaviour. It is therefore associated
with many aspects of health and disease (Cheung et al., 2020). ROS includes peroxide,
free radicals, ions and singlet O2 all of which play key pathological roles in cell injury
and death (Liu et al., 2020).

33
The pathological and physiological effects are determined by the concentration and
compartmentation of the ROS (Tefani et al., 2016). Cancer cells produce a high rate of
ROS which is counterbalanced by an equally high rate of antioxidant activity in order to
maintain redox balance (Schieber & Chandel, 2014). The continuous rise of ROS
production above the toxic threshold, and defects in antioxidant systems, leads to
oxidative stress which exerts detrimental effects during the process of carcinogenesis
(Tefani et al., 2016). This imbalance provides an advantageous opportunity for ROS to
elicit genotoxic damage (Cheung et al., 2020).

2.4.5 Photodynamic therapy mechanism of action

The PS is administered systematically, and is distributed throughout the body via the
circulation where it hyper accumulates in the malignant tissue. The PS is prone to
selectively accumulate in the malignant tissue rather than in the healthy tissue since the
tumour endothelium is highly fenestrated and its interstitium lacks lymphatic drainage
(Weijer et al., 2015). Additionally, PSs have a tendency to combine with low density
lipoproteins (LDL). LDLs are essential for the formation of cell membranes during cell
division (Cruz et al., 2013). Since cancer cells constantly and rapidly undergo mitosis,
they exhibit an increased uptake of LDLs and LDL receptor expression on the cell
surface as compared to healthy cells. The LDL receptors act as a transporter for the PS
into the cancer tissue (Kwiatkowski et al., 2018).

The PS enters the cell which is irradiated with a specific wavelength of light in the
presence of molecular O2. The excitation of the PS initiates the PDT reaction (Bacellar
et al., 2015). The photons from the light source are absorbed by cellular components
converting the singlet ground energy state (PS0) of the PS into a short-lived excited
singlet state (PSEs). Part of the energy is radiated in the form of quantum florescence or
by internal conversion to heat. The remaining energy through intersystem crossing
directs the PS molecules to its excited triplet state (PSEt) - the therapeutic form of the
compound (Yi et al., 2018). The unabiding PSEt can undergo one of two alternate
mechanisms thereafter (Figure 2.4).

34
Figure 2.4: Schematic Jablonski’s diagram demonstrating the Type 1 and Type 2 reactions in PDT.
The photosensitizer (PS) absorbs photons from the light source and reaches an excited singlet
state (PSEs). The PSEs through intersystem crossing directs the PS to its excited triplet sate (PS Et),
which can react in two ways: The PSEt can react with biomolecules to produce reactive oxygen
species (ROS) (Type 1 reaction); or it can directly react with molecular oxygen (O2) to generate
1
singlet oxygen ( O2) (Type 2 reaction) (Calixto et al., 2016).

During the type 1 mechanism of the PDT reaction, energy passes from the PS Et state of
the PS to biomolecules (cancerous tissue) through electron transfer to produce radicals
(dos Santos et al., 2019). The radical species can further interact with O 2 molecules to
form cytotoxic ROS. This stimulus initiates a cascade of reactions leading to oxidative
stress and the subsequent destruction of cancer cells (Dobson et al., 2018). The type 1
mechanism typically generates superoxide anions, hydroxyl radicals, and hydrogen
peroxide type ROS (Agazzi et al., 2019). The type 2 mechanism of the PDT reaction
represents the most dominant type of ROS produced by PDT (Weijer et al., 2015). This
mechanism relies on the energy or electrons released from the excited PS (Abo-Zeid et
al., 2018). Energy or electrons from the PSEt state of the PS are directly transferred to
the molecular O2 to form excited state singlet O2 molecules which can interact with cells
within the target tissue to induce cell death (Dobson et al., 2018).

35
2.4.5.1 Anti-cancer effects of PDT
The anti-cancer effects of PDT can be attributed to three main mechanisms resulting in
cellular, local and systemic destruction of cancer cells: i) the generation of ROS directly
induces cell death, ii) death of tumour cells secondarily to killing tumour associated
vascular endothelial cells; and iii) activation of the immune system to fight against the
tumour (Hamblin, 2018) (Figure 2.5).

Figure 1.5: A flow diagram summarising the antineoplastic effects of photodynamic therapy.
Antitumour effects can be elicited through three main mechanisms: direct cell death, cell death
via vascular damage, and activation of the immune system (Li et al., 2012).

Cytotoxic ROS can cause direct cell death either through apoptosis, necrosis and
autophagic cell death depending on the location and concentration of the PS and the
fluency of light (Figure 2.6). Low to mild doses of PDT (low concentrations of the PS or
a low fluency of light) can damage the mitochondria, thereby triggering apoptosis
(Zheng et al., 2020). In an attempt to repair low-dose PDT-induced cell damage, cells
will adapt autophagy. However, when the protective capacity of autophagy is
overwhelmed or malfunctioned, it will induce cell death directly or indirectly through the
activation of other cell death mechanisms (Sun et al., 2020). At high doses of PDT
(high concentrations of the PS or a high fluency of light) a necrotic reaction usually

36
dominates. As a result, there is a release of calcium and metabolic by-products which
are incompatible with cell functions. This in turn overwhelms the repair function of the
cell therefore, leading to the rapid destruction of the cellular and subcellular membranes
and the subsequent ablation of tumour cells (Zheng et al., 2020). Additionally, the
release of cytokines and toxic chemicals from this reaction causes lethal damage in
cells nearby creating both regional and systematic reactions (Allison & Moghissi, 2013).

Figure 2.6: Type of cell death induced by localization of photosensitizers (Abrahamse et al., 2017).

When irradiated with light, the tumour vasculature just like the tumour itself will respond
to the photodynamic reaction (Allison & Moghissi, 2013). The activated PS concentrates
in the endothelial cells of the vasculature, destroying vascular membranes, resulting in
the deprivation of blood flow to the tumour (Zheng et al., 2020). This leads to the
induction of the vascular shutdown effect, which enhances the haemostatic and
anticancer effects as a result of the ischaemic environment (Ikeda et al., 2018).

Research has demonstrated how PDT can evoke an acute inflammatory response
following treatment, which can trigger both the adaptive and innate immune system.
This plays a crucial role in inhibiting tumour growth and recurrence in the long-term
(Shen et al., 2020). PDT has the ability to promote wider antitumor immune stimulation
37
and responses by inducing different cellular pathways through the activation of NK cells,
neutrophils, tumour infiltration by leukocytes, presentation of tumour antigens to T cells,
and the appearance of heat shock proteins (Kaleta-Richter et al., 2019). These all
converge onto the treatment region to initiate a tumour-specific immunity (Shen et al.,
2020).

38
CHAPTER 3: METHODOLOGY

3.1 Research design and procedure

This is a quantitative and qualitative experimental in vitro study that was conducted at
the Laser Research Centre (LRC), Faculty of Health Sciences at the University of
Johannesburg with permission granted (Appendix A). This research study investigated
the role of T. occidentalis Ø (TO) and laser irradiation at 660 nm at fluency of 5 J/cm2
alone or in combination on A549 human lung cancer cells. A diagram representing the
project plan can be found under appendix B. A preliminary experiment was carried out
prior to the study with cells treated with TO at different volumes to optimize doses for
further experiments. This research project was approved by the Higher Degrees
Committee of the Faculty of Health Sciences of the University of Johannesburg
(Appendix C), and by the Research Ethics Committee of the Faculty of Health Sciences
of the University of Johannesburg (Appendix D). A list of consumables, equipment and
calculations used in the study can be found under appendices E, F, and G.

3.2 Cell culture and treatment

A commercially purchased A549 human lung cancer cell line (ATCC CCL-185) was
used for the anti-cancer study. Cells were cultured in Roswell Park Memorial Institute
1640 Medium (RPMI, Sigma-Aldrich, R8758) supplemented with 10% foetal bovine
serum (FBS, Gibco, 306.00301), 1% antibacterial (Penicillin-streptomycin) and 1%
antifungal (Amphotericin-B) agents. The culture was maintained at 37°C with 5% carbon
dioxide (CO2) and 85% humidity. Every 2-3 days, old media was discarded and
replaced with fresh growth medium, after rinsing with Hank’s Balanced Salt Solution
(HBSS, Invitrogen, 10-543F). Once cells reached a confluency of around 90%, they
were rinsed with HBSS, and dissociated using TrypLE Express (Invitrogen, 12605-028)
for subculturing.

3.2.1 Plating of cells

The confluent flasks were rinsed with HBSS, and dissociated using TrypLE Express.
Trypan blue exclusion test was performed to quantify the number of viable cells per mL.
Approximately 5 × 105 cells were seeded in 3.4 cm diameter culture plates, containing 2
mL of RPMI with growth supplements and antibiotics. Culture dishes were incubated at

39
37°C with 5% CO2 and 85% humidity overnight to allow the cells to attach. Culture
plates with more than 90% confluence were used for further experiments.

3.3 Thuja occidentalis Ø

T. occidentalis Ø (TO) was bought from a reputable and registered homeopathic
company (Fusion Homeopathics, South Africa). This company imports their mother
tinctures from Gehrlicher Pharmaceutical Extracts (GmbH). The tincture was stored at
room temperature in a 100 mL amber glass bottle, away from direct sunlight. A
certificate of analysis from the manufacturer of the tincture is provided which shows that
the tincture was manufactured according to the appropriate monograph rule 3a of the
German and European Homeopathic Pharmacopoeia (Appendix H). According to the
certificate of analysis, TO has a pH of 5.7 therefore it is essentially a weak acid. Drugs
with a lower pH have the ability to exploit the pH gradient. Since the acidic tumour
extracellular environment can lower the overall charge it is able to facilitate diffusion of
the drug across the cell membrane. Furthermore, according to the pH partition theory,
once inside the cell, weak acids will mobilize into alkaline compartments such as the
mitochondria, which are favourable sites to initiate cancer cell death (Adams & Morgan,
2011).

3.3.1 Thuja occidentalis Ø dose determination

After overnight incubation, the media from culture plates were discarded and replaced
with 2 mL of fresh medium. Different doses of TO (5, 10, 15, 20, 25, 50, 100, 150 and
200 μL) were added to their respective culture plates. The use of 9 different groups was
used to asses for optimal dosing parameters and to identify relevant treatment related
toxicities. An ethanol control group was included as a Ø solvent control. The ethanol
control group received predetermined doses of 61% ethanol (identical to the
homeopathic tincture but without the active ingredients). The untreated control group
neither received any tincture nor ethanol. Cellular morphology and trypan blue assays
were carried out after 24 hours (h) of incubation at 37°C, with 5% CO2 and 85%
humidity. TO at 5, 10, 15 μL were selected as the final doses for further experiments. As
the ethanol control had no significant change with respect to the untreated control at
these doses, the group was not included in further experiments.

40
3.4 Laser set-up and parameters
For treatment regimens with light, cells were irradiated using a diode laser supplied by
the Council for Scientific and Industrial Research (CSIR), National Laser Centre (NLC)
of South Africa, at a wavelength of 660 nm at a 5 J/cm2 fluence rate. The fluence rate
was based on research studies that demonstrate how low fluencies employed in PDT
can enhance the cytotoxic effects of treatment (Hartl et al., 2015). Before the irradiation,
the laser was switched on and allowed to stand for 10 minutes (min) to stabilize and
reach maximum power output. A power meter (FieldMate) was used to determine the
power output of the laser at bench level in the dark. To obtain a fluency of 5 J/cm 2, the
duration of laser irradiation was calculated using the following formulae:

𝑭𝒍𝒖𝒆𝒏𝒄𝒚 (𝑱⁄𝒄𝒎𝟐 ) = 𝒕𝒊𝒎𝒆(𝒔) × [𝒑𝒐𝒘𝒆𝒓 𝒐𝒖𝒕𝒑𝒖𝒕 (𝑾)⁄𝑺𝒖𝒓𝒇𝒂𝒄𝒆 𝒂𝒓𝒆𝒂 (𝒄𝒎𝟐 )]

Culture plates were protected from extraneous light sources, and one culture dish was
illuminated at a time. The culture dishes requiring irradiation were placed directly under
the laser beam with their lids removed. All control and experimental culture plates were
covered in foil and placed back in the incubator for 24 h prior to carrying out post-
irradiation assays. Irradiation parameters can be found in table 3.1.

Table 3.1: A table showing irradiation parameters

Name and type Diode laser

Spectrum 660 nm
Wave emission Continuous wave
Spot size 9.1cm2
Power output 87.6 mW
Power density 9.3 mW/cm2
Fluency 5 J/cm2
Irradiation time 9 min, 3 seconds (s)

3.5 Groups and set-up

Cell models were divided into experimental and control groups, with experimental
groups receiving either a single treatment of TO or laser irradiation at 660 nm, or the
combined treatment (Table 3.2). After overnight incubation, the medium from culture
41
plates were removed and discarded. Control groups received 2 mL of fresh medium
with neither the homeopathic tincture nor any laser irradiation. The tincture treated
groups received 2 mL of fresh medium with pre-determined doses of TO (5, 10, 15 μL)
alone. Irradiation controls received 2 mL of fresh medium followed by irradiation only.
The photodynamic therapy (PDT) treated groups received 2 mL of fresh medium with
pre-determined doses of TO (5, 10, 15 μL). Cells were incubated at 37oC with 5% CO2
and 85% humidity for an additional 4 h prior to irradiation.

Table 3.2: A table showing a summary of the groups and variables in the experiment

Groups Variables
I Untreated control
II Tincture alone (TO)
III Irradiation alone
IV Tincture and Irradiation in
combination- TO mediated
PDT (TO-PDT)

The cells and media of all the experimental and control groups were collected after 24 h
of incubation to perform various morphological and biochemical assays. Cellular
morphology using inverted microscopy, adenosine triphosphate (ATP) luminescent
assay for cellular proliferation, lactate dehydrogenase (LDH) assay for cytotoxicity,
trypan blue assay for viability, and hoechst stain for nuclear morphology were the post-
treatment parameters analysed for anticancer property.

3.5.1 Cellular morphology- Inverted light microscopy

This qualitative assay was carried out to analyse cellular density and morphology of
cells in all control and experimental groups using an inverted light microscope (Wirsam,
Olympus CKX41). The microscope is connected to a camera to allow for live
visualisation of cells. Culture plates were placed individually under the microscope with
their lids removed in a darkened room. Images were captured on a computer using the
GetIT analysis programme.

42
3.5.2 Cytotoxicity-Lactate dehydrogenase assay
The membrane integrity of cells was assessed by estimating the amount of LDH present
in the culture media. The cytosolic enzyme LDH is released into the media due to
membrane damage. The CytoTox 96® Non-Radioactive Cytotoxicity Assay (Anatech
Promega G400) is a quantitative method used to measure the LDH released in all
control and experimental groups. An equal volume (50 μL) of reconstituted LDH reagent
and cell culture medium was micropipetted in a clear bottom 96 well plate. The entire 96
well plate was covered with tin foil, mixed, and incubated in the dark at room
temperature for 15 min. The colorimetric mixture was measured spectrophotometrically
at 490 nm (Perkin–Elmer, Separation Scientific, VICTOR3™).

3.5.3 Cellular proliferation- Adenosine triphosphate luminescent assay

The ATP Cell Titer-Glo® luminescent assay (Promega, G7571) is a homogeneous
method for the determination of cellular proliferation and quantification of ATP present in
metabolically active cells. Cells from all control and experimental groups were detached
using 1 mL/3.4 cm diameter plate of TrypLE Express and centrifuged at 2500 rpm for 5
min. The cell pellets were then re-suspended in HBSS. An equal volume (50 μL) of
reconstituted ATP reagent and the cell suspension was micropipetted into an opaque-
walled luminescent 96 well plate. The entire plate was covered with foil and the cells
and reagent mixture were mixed on a shaker for 5 min to induce cell lysis. The cells
were incubated in the dark at room temperature for 30 min to stabilize the luminescent
signal. The luminescent signal was read using the 1420 multilabel counter victor3
(Perkin-Elmer, Separation Scientific, VICTOR3™) in relative light units (RLU).

3.5.4 Cellular viability- Trypan blue assay

Cell viability was quantified using Trypan Blue dye exclusion assay. Trypan Blue
(Sigma-Aldrich, T8154) was used to stain the cells by diluting 10 μL of the cell
suspension into 10 μL of 0.4% trypan blue dye. A volume of 10 μL of stained cells was
loaded into each side of a plastic, disposable CountessTM cell counting chamber slide.
Cells with damaged membrane (non-viable) will take in the blue chromophore dye,
whereas the cells with intact cell membrane (viable) will not absorb the dye and hence,
remain clear. An automated cell counter (CountessTM Automated Cell Counter,
Invitrogen) was used for the cell count and viability estimation.

43
3.5.5 Nuclear morphology- Hoechst stain assay
The hoechst stain is a qualitative assay used to assess alterations in the nuclear
morphology of cells. Approximately 5 x 105 cells were seeded in 3.4 cm diameter culture
dishes over sterile cover slips and allowed to reach above 80% confluence. Cells were
then treated with TO only, laser irradiation only, or the combined treatment. After 24 h of
incubation at 37oC with 5% CO2 and 85% humidity, old media in all control and
experimental groups were discarded and cells were rinsed 2-3 times with phosphate
buffered saline (PBS). A Hoechst working stock solution stain was prepared by diluting
5 μL of Hoechst fluorescent dye stock solution (Hoechst 33258) into 25 mL of deionized
water. Each coverslip was stained with 200 μL of the working stock solution. Culture
plates were covered with tin foil and incubated at room temperature for 30 min. The
solution was removed from each cover slip, and cells were rinsed once with PBS to
remove any remaining stain. Each coverslip was carefully removed from culture plates
using sterilized forceps and placed cell side down onto a glass microscope slide,
containing a single drop of FluoromountTM Aqueous Mounting Medium (Sigma-Aldrich,
F4680). Fluorescence was observed in a dark room with a fluorescent microscope (Axio
observer Z1, Carl Zeiss) using a broadband 4',6-Diamidino-2-Phenylindole (DAPI) filter
set, measured at an excitation wavelength of 352 nm and an emission wavelength of
455 nm. The nuclei of various cell groups were then analysed for any morphological
changes.

3.6 Reliability and validity measures

Control groups were included in all the experiments, and all assays were repeated in
triplicate to ensure quality of results. Assay results were based on replicate measures
where possible (n= 6). Experimental protocols were followed as per the LRC guidelines.
TO was bought from a reputable homeopathic company (Fusion Homeopathics, South
Africa). A certificate of analysis from the manufacturer (Gehrlicher Pharmaceutical
Extracts) of the tincture is provided as a means of quality assurance to confirm that the
tincture meets its product specification. A commercially purchased A549 lung cancer
cell line obtained from ATCC (CCL-185) was used for the anti-cancer study. This
company is international standards organization (ISO) compliant that ensures the
manufacture of ATCC products are accurate, have repeatability and are of good quality.
A certificate of analysis from the ATCC is provided as a means of quality assurance to
confirm that the A549 cells meet its product specification (Appendix I). A product sheet

44
describing the basic procedures for handling A549 cells from ATCC has been provided
(Appendix J).

3.7 Statistical analysis

Data was obtained from representative independent experiments and expressed as the
mean ± standard error (SE) (Appendix K). Since the preliminary experiments with
ethanol alone was performed to justify the non-toxicity, it was important to determine the
toxicity of exact dose ranges used for the tincture. Hence rather than using focusing on
replicates 9 dose ranges were used and assays were run in duplicate (n=2). Final
experiments were repeated in triplicate, and assays run in duplicate (n= 6). The SPSS
Data Analysis Software was used to collect and process the results. Data were
statistically analysed using the one-way ANOVA and Dunnett’s multiple comparison
test. Statistically significant at *p<0.05 when compared to the control.

3.8 Ethics
A commercially purchased A549 cell line purchased from ATCC was used to conduct
the experimental in vitro study. Cells are readily available and kept in liquid nitrogen
stocks in the LRC laboratory. Guidelines on the ethical standards for obtaining human
materials from ATCC can be viewed on their website which is available for public view
(https://www.lgcstandardsatcc.org/About/AboutATCC/Ethical_Standards for Obtaining
Human Materials.aspx), and all ethical standards have been met by ATCC when
isolating these cells. Donation of the tissue is anonymous, and the cell lines cannot be
traced back to the donors. The LRC has also received the necessary biosafety
clearance required from ATCC when ordering such cells (Appendix L). This study was
approved by the Research Ethics Committee of the Faculty of Health Sciences of the
University of Johannesburg (REC-01-57-2019). A plagiarism report is provided under
the appendices (Appendix M).

3.8.1 Biological safety

The student conducting the research was adequately trained, supervised and assisted
throughout the laboratory work by the LRC staff and supervisor within the functional and
ethical guidelines of the laboratory. Rules, regulations, protocols and biosafety practices
of the laboratory were followed. The experiment was carried out in a biosafety level-2
laboratory. Standard precautions and risk assessment procedures were followed.
Required personal protective equipment was worn at all times within the laboratory. All
45
waste was disposed of in the applicable biological waste containers. The live untreated
cells were stored in liquid nitrogen, following standard protocol, and the treated and
dead cells were firmly sealed in flasks or cans and discarded into biohazard safety bins
supplied by the 'Universal Waste Solutions'. The bins are cleaned and replaced by the
said company twice a week to ensure biosafety in the lab.

46
CHAPTER 4: RESULTS

To explore the anticancer and photodynamic effects of T. occidentalis Ø (TO), A549

lung cancer cells were cultured with different doses of the tincture alone and in
combination with 660 nm laser irradiation at a fluence of 5 J/cm2. A preliminary study
was carried out to examine the effects of TO on A549 cells, and to optimize doses of TO
for further experiments. All experimental groups were compared to an untreated control
group to determine statistical significance. A value of *p<0.05, **p<0.01 and ***p<0.001
is regarded as statistically significant.

4.1 Preliminary Study

A preliminary experiment was carried out to determine the dose response of TO and
61% ethanol on A549 cells. Cells were exposed to TO at different doses to optimize
doses for further experiments. An inverted light microscope was used to assess cellular
morphology and trypan blue assay was used to assess cell viability. As the dose of TO
increased, cell viability gradually decreased. Optimized doses of 5, 10 and 15 μL of TO
were selected based on the 50% inhibition value of the tincture.

4.1.1 Ethanol cytotoxicity study

Since the tincture is prepared in a 61% ethanol solvent, it was important to establish the
cytotoxic effects of the ethanol on A549 cells. To investigate the cytotoxic effects of
ethanol, an ethanol control group treated with 61% ethanol in identical doses as the
tincture was assessed in comparison to an untreated control group. The cytotoxic
effects were observed using an inverted light microscope for cellular morphology, and
trypan blue assay for cell viability after 24 h of incubation.

4.1.1.1 Inverted light microscopy

The morphology of A549 cells after 24 h exposure to different doses of 61% ethanol
were observed under inverted light microscope and compared to the untreated control
(Figure 4.1). Ethanol alone, in doses up to 25 μL, had no significant effect on the
morphology of A549 cells compared to the control cells. Cells at these doses and in the
control retained their normal shape and remained attached to their respective culture
plates. Morphological changes became apparent in cells treated with ethanol above 50
μL which began to show signs of cell death, with increasing evidence of detachment,
rounding of cells and debris in cells treated with 150 and 200 μL of 61% ethanol.
47
 A B

C D

E F

G H

I J

Figure 4.1: Morphology of A549 cells after exposure to different doses of 61% ethanol (EtOH)
under inverted light microscope (200x Magnification). Figure (A) Untreated control, (B) 5 µL EtOH,
(C) 10 µL EtOH, (D) 15 µL EtOH, (E) 20 µL EtOH, (F) 25 µL EtOH, (G) 50 µL EtOH, (H) 100 µL EtOH,
(I) 150 µL EtOH and (J) 200 µL EtOH. Control cells showed high density of attached cells with
normal shape. Cells treated with 5-25 µL demonstrated no significant changes compared to the
control. At 50-100 µL there is a gradual decrease in the density of attached cells, rounded up dead
cells can be seen floating in the media. At 150-200 µL, the majority of cells are rounded up and
can be seen floating in the media. Scale bar 50 µm.

48
4.1.1.2 Trypan blue assay
Trypan blue exclusion assay was used to compare the viability of ethanol treated cells
at its different doses to the untreated control (Figure 4.2). Trypan blue assay showed no
significant differences in cell viability in optimized doses of 5, 10 and 15 μL of ethanol
when compared to the control group. Cellular viability in the control group was 97%.
Cells treated with 5, 10, 15, 20, 25, and 50 μL demonstrated an average cell viability of
91% (p=0.425), 94% (p=0.701), 91.5% (p=0.470), 93.5% (p=0.657), 94% (p=0.701),
and 86.5% (p=0.131) respectively. Statistical significance is noted at doses of 100, 150,
and 200 μL with a decrease in the viability of cells at 74% (p<0.01), 19% (p<0.001), and
20.5% (p<0.001).

n= 2
100
90
80 **
70
Cell viablity (%)

60
50
40
30 *** ***
20
10
0
Control EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH
5 μL 10 μL 15 μL 20 μL 25 μL 50 μL 100 μL 150 μL 200 μL

Figure 4.2: The percentage (%) of viable cells using trypan blue assay in A549 cells treated with
61% Ethanol (EtOH). The control group demonstrates the highest viability indicating an average of
97% living cells. Cells treated with EtOH at doses 5-50 μL shows no significant changes in cell
viability when compared to the control. A gradual decrease in cell viability can be seen in cells
treated with 100 μL of EtOH. The maximum decrease in cell viability can be seen in groups treated
with 150 and 200 μL of EtOH. Data is represented as the mean ± SE from ten independent
experiments. Statistically significant at **p<0.01 and ***p<0.001 when compared to the control.

49
4.2 Experimental study
The groups were divided into an untreated control group and experimental groups. The
first round of experiments was subdivided into a tincture only treated group (TO) and an
untreated control group. The second round of experiments was subdivided into an
untreated control group, an irradiation only treated group, and a TO mediated
photodynamic therapy (TO-PDT) treated group. Cells were pre-treated with TO in doses
of 5, 10, and 15 μL for further experiments in both the TO and TO-PDT treated groups.
To obtain a fluency of 5 J/cm2, the duration of laser irradiation was calculated using the
power output from the 660 nm laser. The power output for the 660 nm laser was 87.6
mW. Therefore, the time calculated to deliver a fluence of 5 J/cm 2 to a culture plate
requiring irradiation was 9 min and 3 s. As the ethanol control had no significant change
with respect to the untreated control at the optimized doses, the group was not included
in further experiments.

To investigate the antiproliferative, cytotoxic and photodynamic effects of TO, all control
and experimental groups were subjected to various morphological and biochemical
assays. Qualitative assays including light microscopy and hoechst stain were used to
investigate the morphological changes in the cell and the cell nuclei respectively. The
lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and trypan blue assays
were used to explore the cytotoxic and antiproliferative responses of the various groups.

4.2.1 Inverted light microscopy

An inverted light microscope was used to assess morphological changes after 24 h
incubation in all control and experimental groups, which may be indicative of cell death
(Figure 4.3). Untreated cells in the control group retained their characteristic shape and
remained attached to their respective culture plates. Cells treated with TO at all doses
demonstrated morphological changes attributed to cell death mechanisms including an
acquired rounded up morphology, cell detachment, and cell debris. Cells treated with
660 nm laser irradiation demonstrated no significant morphological changes when
compared to control cells. Cells treated with TO-PDT demonstrated significant
morphological changes when compared to control group. Cells showed an excessive
amount of cell rounding, detachment and debris in the culture media. Morphological
changes were more severe with increasing dosages of TO when combined with laser
irradiation.

50
 A B

C D

E F

G H

Figure 4.3: Cell morphology of A549 cells after exposure to different doses of T. occidentalis Ø
(TO) and TO mediated photodynamic therapy (TO-PDT) under an inverted light microscope (200x
Magnification). Figure 1(A) Untreated control, (B) irradiation only, (C) 5 µL TO, (D) 10 µL TO, (E) 15
µL TO, (F) 5 µL TO-PDT, (G) 10 µL TO-PDT, (H) 15 µL TO-PDT. Control cells showed high density of
attached cells with normal shape. Cells treated with irradiation alone demonstrated no significant
changes compared to the control. At all doses, TO and TO-PDT treated cells decreases in the
density of attached cells can be observed and rounded up dead cells can be seen floating in the
media. White arrows indicates examples of rounded up dead cells. Scale bar 50 µm.

51
4.2.2 Lactate dehydrogenase assay
The LDH colorimetric assay was used to determine in vitro cytotoxicity, and hence cell
death in all control and experimental groups. When the cell membrane integrity is
compromised, intracellular LDH is able to leak out of the cell. The amount of LDH
released from cells into the media was quantified 24 h after treatment using the
CytoTox96®Assay. TO treated cells demonstrated a gradual dose-dependent increase
in LDH levels. Cells treated with TO showed statistically significant increases in LDH
levels at 5 (p<0.05), 10 (p<0.01) and 15 (p<0.001) μL when compared to cells in control
groups (Figure 4.4).

n= 6
0.7
***
LDH Membrane Integrity (A490 nm)

0.6 **
*

0.5

0.4

0.3

0.2

0.1

0
Control TO 5 μL TO 10 μL TO 15 μL

Figure 4.4: Lactate dehydrogenase (LDH) release from A549 cells treated with T. occidentalis Ø
(TO). The control group demonstrates the lowest LDH release. Cells treated with TO at different
doses shows a dose-dependent gradual increase in LDH release. Data is represented as the mean
± SE from four independent experiments. Statistically significant at *p<0.05, **p<0.01 and
***p<0.001 when compared to the control.

The level of LDH release from irradiated cells demonstrated statistically insignificant
results showing a 0.78% (p=0.805) decrease in LDH when compared to control cells.
Cells treated with TO-PDT demonstrated a significant (p<0.001) dose-dependent
increase in LDH levels compared to the control group (Figure 4.5). Among all the TO-

52
PDT groups, cells treated with 15 μL showed highest increase in LDH release when
compared to cells in the control group.

n= 6
1.4
*** ***
LDH Membrane Integrity (A490 nm)

***
1.2

0.8

0.6

0.4

0.2

0
Control Irradiation TO-PDT TO-PDT TO-PDT
5 μL 10 μL 15 μL

Figure 4.5: Lactate dehydrogenase (LDH) release from A549 cells treated with T. occidentalis Ø
mediated photodynamic therapy (TO-PDT). The control group demonstrates the lowest LDH
release. Cells treated with irradiation alone demonstrate no significant difference in LDH release
(p=0.805) compared to the control. Cells treated with TO-PDT at different doses shows a dose-
dependent increase in LDH. Data is represented as the mean ± SE from five independent
experiments. Statistically significant at ***p<0.001 when compared to the control.

Figure 4.6 compares the LDH levels in the TO and TO-PDT treated groups. At the same
dosage of TO, cells treated with TO-PDT demonstrated higher LDH release compared
to cells treated with the tincture alone.

53
 n= 6
1.4

LDH Membrane Integrity (A490 nm)

1.2

0.8

0.6

0.4

0.2

0
5 μL 10 μL 15 μL
TO TO-PDT

Figure 4.6: A comparison of the amount of lactate dehydrogenase (LDH) released from A549 cells
treated with T. occidentalis Ø (TO) and TO mediated photodynamic therapy (TO-PDT) at the same
doses. Cells treated with TO-PDT showed increased LDH levels compared to cells treated with TO
alone. Data is represented as the mean ± SE from six independent experiments.

4.2.3 Adenosine triphosphate luminescent assay

The ATP Cell Titer-Glo® luminescent assay was used to measure cell proliferation in all
experimental and control groups after 24 h of incubation. Metabolically active cells
proliferate at a higher rate than cells that are metabolically inactive and vice versa. The
proliferation rate of cells in all experimental groups was compared to cells in the control
group. Cells treated with TO demonstrated a dose-dependent decrease in ATP levels at
5 (p<0.01), 10 (p<0.001) and 15 (p<0.001) μL when compared to cells in the control
group (Figure 4.7). The effects were more pronounced in cells treated with the highest
dose.

54
 n= 6
2.0E+6
ATP Luminescence (RLU) 1.8E+6
1.6E+6
1.4E+6
1.2E+6
**
1.0E+6
8.0E+5
***
6.0E+5
4.0E+5
***
2.0E+5
0.0E+0
Control TO 5 μL TO 10 μL TO 15 μL

Figure 4.7: The level of adenosine triphosphate (ATP) in relative light units (RLU) on A549 cells
treated with T. occidentalis Ø (TO). The control group demonstrates the highest ATP level. Cells
treated with TO at different doses shows a dose-dependent decrease in ATP levels. Data is
represented as the mean ± SE from four independent experiments. Statistically significant at
**p<0.01 and ***p<0.001 when compared to the control.

The level of ATP from irradiated cells demonstrated statistically insignificant results with
only a 1% (p=0.756) decrease in ATP when compared to control cells. Cells treated with
TO-PDT demonstrated a significant (p<0.001) dose-dependent decrease in ATP levels
compared to the control group (Figure 4.8). The effects were more pronounced in cells
treated with the highest dose.

55
 n= 6
2.5E+6
ATP Luminescence (RLU)

2.0E+6

1.5E+6

1.0E+6
***

5.0E+5
***
***
0.0E+0
Control Irradiation TO-PDT TO-PDT TO-PDT
5 μL 10 μL 15 μL

Figure 4.8: The level of adenosine triphosphate (ATP) metabolism in relative light units (RLU) on
A549 cells treated with T. occidentalis Ø mediated photodynamic therapy (TO-PDT). The control
group demonstrates the highest ATP level. Cells treated with irradiation only demonstrate no
significant difference in ATP levels (p=0.756) compared to the control. Cells treated with TO-PDT
at different doses show a dose-dependent decrease in ATP levels. Data is represented as the
mean ± SE from five independent experiments. Statistically significant at ***p<0.001 when
compared to the control.

Figure 4.9 compares the ATP levels in the TO and TO-PDT treated groups. At the same
dosage of TO, cells treated with TO-PDT demonstrated lower ATP levels compared to
cells treated with the tincture alone.

56
 n= 6
1.2E+6

1.0E+6
ATP Luminescence (RLU)

8.0E+5

6.0E+5

4.0E+5

2.0E+5

0.0E+0
5 μL 10 μL 15 μL
TO TO-PDT

Figure 4.9: A comparison of the level of adenosine triphosphate (ATP) in relative light units (RLU)
on A549 cells treated with T. occidentalis Ø (TO) and TO mediated photodynamic therapy (TO-
PDT) at the same dose. Cells treated with TO-PDT shows decreased ATP levels compared to cells
treated with TO alone. Data is represented as the mean ± SE from six independent experiments.

4.2.4 Trypan blue assay

Trypan blue exclusion assay was used to investigate the viability of cells in all control
and experimental cell groups after 24 h of incubation. Cells with damaged membrane
(non-viable/dead) took the blue chromophore dye in whereas the cells with intact-cell
membranes (viable) did not absorb the dye, and hence remained clear. Cells treated
with TO demonstrate a dose-dependent decrease in cell viability. After treatment with
TO at doses of 5, 10, and 15 μL, the average viability percentage gradually decreased
from 85% in the control to 52% (p<0.001), 33% (p<0.001), and 20% (p<0.001) in the
respective doses of the tincture (Figure 4.10). TO revealed just under 50% of cell death
at 5 µL. The effects were more pronounced in cells treated with the highest dose.
Exposure to 15 µL of TO for 24 h caused the majority of A549 cells to die.

57
 n= 6
100
90
80
70
Cell Viability (%)

60
***
50
40 ***
30
***
20
10
0
Control TO 5 μL TO 10 μL TO 15 μL

Figure 4.10: The percentage (%) of viable cells using trypan blue assay in A549 cells treated with
T. occidentalis Ø (TO). The control group demonstrates the highest viability indicating an average
of 85% living cells. Cells treated with TO at different doses shows a dose-dependent decrease in
cell viability thus indicating cell death. Data is represented as the mean ± SE from four
independent experiments. Statistically significant at ***p<0.001 when compared to the control.

Cells treated with irradiation alone demonstrated a viability of 96% (p=0.605) when
compared to the control group that showed a viability of 97%. Cells treated with TO-
PDT demonstrated a significant dose-dependent decrease in cell viability. After
treatment with TO-PDT at doses of 5, 10, and 15 μL, the average viability percentage
gradually decreased from 97% in controls to 48% (p<0.001), 22% (p<0.001), and 12%
(p<0.001) in the respective doses of the treatment (Figure 4.11). The effects were more
pronounced in cells treated with the highest dose. Exposure to 15 µL of TO-PDT for 24
h caused the majority of A549 cells to die.

58
 n= 6

100
90
80
70
Cell Viability (%)

60
***
50
40
30
***
20
***
10
0
Control Irradiation TO-PDT TO-PDT TO-PDT
5 μL 10 μL 15 μL

Figure 4.11: The percentage (%) of viable cells using trypan blue assay in A549 cells treated with
Thuja occidentalis Ø mediated photodynamic therapy (TO-PDT). The control group demonstrates
the highest viability indicating an average of 97% living cells. Cells treated with irradiation only
demonstrate no significant difference in viability (p=0.605) compared to the control. Cells treated
with TO-PDT at different doses shows a dose-dependent decrease in cell viability thus indicating
cell death. Data is represented as the mean ± SE from five independent experiments. Statistically
significant at ***p<0.001 when compared to the control.

Figure 4.12 compares the viability percentages in the TO and TO-PDT treated groups.
At the same dosage of TO, cells treated with TO-PDT demonstrated lower viability
percentages compared to cells treated with the tincture alone.

59
 n= 6
100
90
80
70
Cell viablity (%)

60
50
40
30
20
10
0
5 μL 10 μL 15 μL
TO PDT

Figure 4.12: A comparison of cell viability on A549 cells treated with T. occidentalis Ø (TO) and TO
mediated photodynamic therapy (TO-PDT) at the same doses of the tincture. At the same doses of
TO, cells treated with TO-PDT showed decreased viability percentages compared to cells treated
with TO only. Data is represented as the mean ± SE from six independent experiments.

4.2.5 Hoechst stain assay

The apoptotic morphological changes of the cell nucleus induced by TO and TO-PDT
treatment was detected by hoechst fluorescent staining. The nuclei of cells in
experimental groups were compared to the nuclei of cells in the control group to assess
the nuclear damage (Figure 4.13). The control group displayed characteristic dense
ellipsoidal shaped, homogeneously stained nuclei. Cells treated with TO demonstrated
changes in nuclei morphology including irregular shaped nuclei, nuclear shrinkage,
chromatin condensation and nuclear fragmentation. The nuclei of irradiated cells
demonstrated nuclei with intact chromatin and no significant morphological changes
when compared to the control group. Cells treated with TO-PDT demonstrated changes
in nuclei morphology including irregular shaped nuclei, nuclear shrinkage and nuclear
fragmentation.

60
 A B

C D
I

E F J

G H

Figure 4.13: Nuclei morphology of A549 cells after exposure to different doses of T. occidentalis Ø
(TO) and TO mediated photodynamic therapy (TO-PDT) under a fluorescent microscope (400x
Magnification). Figure 1(A) Untreated control, (B) irradiation only, (C) 5 µL TO, (D) 10 µL TO, (E) 15
µL TO, (F) 5 µL TO-PDT, (G) 10 µL TO-PDT, (H) 15 µL TO-PDT, (I) Normal nucleus, and (J) Abnormal
nucleus. Control cells show normal, evenly stained nuclei. Cells treated with irradiation alone
show no significant changes compared to the control. Cells treated at different doses of TO and
TO-PDT demontrated nuclei with abnormal shape, nuclei shrinkage, chromatin condensation and
nuclear fragmentation. Figure (I) provides an enlarged example of a normal nucleus that is
homogenously stained and hence remains dense. Figure (J) provides an enlarged example of an
abnormally shaped nulcleus demonstrating nuclear fragmentation. Scale bar 20 µm.

61
CHAPTER 5: DISCUSSION

Lung cancer has afflicted humanity for centuries. The continuous rise of lung cancer
cases has overwhelmed health care systems and their ability to effectively treat the
disease (Faguet, 2015). Although significant progress has been made in an attempt to
combat lung cancer, it has not always translated into successful clinical outcomes. This
encourages the necessity to investigate alternate treatment options aimed at providing
new perspectives in the search for optimal lung cancer therapies. Therefore, this study
proposed a synergistic approach based on photodynamic therapy (PDT) combined with
homeopathy in order to advance the development of safer and more effective
anticancer therapies.

PDT and homeopathy offer considerable opportunities as alternative lung cancer

therapeutic modalities. Research has already confirmed the anticancer effects of Thuja
occidentalis Linn (T. occidentalis) extracts in both in vitro and in vivo studies, with a
particular focus on its active ingredient thujone (Blowman et al., 2018). Thujone is
associated with the activation of pro-apoptotic signalling via the activation of BAX,
caspase 3 and cytochrome c (Ijaz et al., 2018). Although there have only been a few
studies on the anticancer effects of the homeopathically prepared T. occidentalis Ø (TO)
in vitro, it has shown evidence of inducing pro-apoptotic induction, and hence cancer
cell death. To date there is no research on the effects of TO mediated photodynamic
therapy (TO-PDT). This study therefore aimed to investigate the photodynamic effects
of TO on A549 lung cancer cells in vitro.

5.1 Contextualising the ethanol cytotoxicity study

The introduction of extraneous solvents such as ethanol to the growth medium of cell
cultures has the potential to alter the cellular environment, which can consequently
affect the outcomes of the experiment and treatment (Timm et al., 2013). Therefore, in
order to obtain accurate results, it is imperative to ensure that the solvent at its specific
dose is biocompatible and non-toxic to cells (Nguyen et al., 2020).

TO is prepared using a 61% ethanol solvent. The high ethanol percentage raises some
concern about ethanol toxicology on cell cultures and organisms in experimental
research studies (Chirumbolo & Bjørklund, 2018). In order to keep the volume of the

62
tincture solvent at the most suitable doses for biological experimentation, an ethanol
control group was included in the preliminary study as a mother tincture solvent control.
The ethanol control group consisted of 61% ethanol (identical to the ethanol vehicle in
TO but without any of the active ingredients), delivered to cells in identical doses as the
homeopathic tincture. Cell morphology was assessed using inverted microscopy, while
viability was assessed by trypan blue staining.

Morphological studies demonstrated no significant differences between the ethanol

control group at doses of 5-25 μL compared to the untreated control group, which is
associated with the absence of cell death and proliferation. At doses up to 25 μL, the
viability of A549 cells was not significantly different from the control as reflected in the
morphological study, and is hence associated with the absence of cytotoxicity. This
would suggest that 61% ethanol in optimized doses of TO (5-15μL) does not have a
significant cytotoxic effect on A549 lung cancer cells, and any signs of cell death can be
attributed to TO. Morphological changes including cell shrinkage, rounding up of cells,
and cell detachment were apparent at 50 and 100 μL, and became increasingly
noticeable at 150 and 200 μL, which is suggestive of the anti-proliferative and cytotoxic
effects of ethanol at these doses. A gradual decrease in cell viability can be seen at a
dosage of 100 μL. At dosages of 150-200 μL, the viability decreased significantly thus
indicating the cytotoxic effects of 61% ethanol on A549 cells. Results did demonstrate a
slight increase in viable cells at 200 μL compared to cells treated with 150 μL. This
could be due to a number of reasons including human error and small sample size. This
trend can be observed between the other ethanol doses as well but results reveal
statistically insignificant changes when compared to the control which may be the case
between the above two doses as well. Ethanol above its toxic threshold can interfere
with the membrane of cells and cause disruptions in the physical activities of the cell
which can promote cell death (Nguyen et al., 2020).

This result can be further substantiated using a study by Ghosh et al. (2013). In this
study, Phytolacca decandra Ø prepared in a 65% ethanol vehicle induced apoptosis in
skin melanoma cells. The tincture-treated cells showed apoptotic DNA fragmentation,
while its absence was noted in the ethanol control group. After treatment with the
remedy, the apoptotic percentages increased from 10.11% in the ethanol controls to
54.93%, 65.20%, and 66.73% in the respective doses of the tincture. This study and
other similar studies may suggest that ethanol, if optimized for an experiment (if the
63
doses are below the toxic threshold), should have minimal or no chemical significance
on the therapeutic outcomes of homeopathic tinctures.

5.2 Contextualising the experiments

A549 cells were pre-treated with TO, irradiation only or TO-PDT at 5, 10, and 15 μL for
24 hours to observe the anticancer effects. Cells were irradiated using a 660 nm laser at
a fluence rate of 5 J/cm2. PDT was shown to be most effective at low fluence rates.
When PDT is employed using high fluence rates, O2 consumption and depletion
exceeds the rate of O2 replenishing which leads to photobleaching of the available PSs.
Furthermore, PDT at low fluence rates demonstrates an increased induction of selective
apoptosis in cancer cells (Hartl et al., 2015).

Cells were investigated for cytotoxicity, viability and proliferation by means of various
morphological and biochemical assays. An inverted light microscope was used to
assess morphological alterations in cells growing in the different groups that could be
attributed to cell death mechanisms. After initial validation of cell morphology,
extracellular lactate dehydrogenase (LDH) was used to assess the membrane integrity
of cells and thus acted as a measurement for cytotoxicity. Measurement of intracellular
adenosine triphosphate (ATP) was used to assess cell proliferation. Trypan blue
staining was used to asses cell viability and cell death. Hoechst stain was used to
observe alterations in cell nuclear morphology. All cell groups were compared to an
untreated control group.

5.2.1 Inverted light microscopy

Direct observation of cell morphology provides a preliminary judgement to assess cell
death (Wu et al., 2020). Cells treated with TO and TO-PDT at all doses were able to
induce morphological changes with signs of apoptosis, and hence cell death, as
observed under inverted microscope. This included cell shrinkage, rounding up of cells,
reduced cell density, and cell detachment from the culture plate, which is associated
with decreased proliferation and viability of cells. Apoptosis after PDT is associated with
mitochondrial photodamage and hence irreversible cell death (Villacorta et al., 2017).

Cells in the control group and those treated with irradiation alone were unable to induce
morphological changes in the cells which were suggestive of healthy and proliferating
cells. When laser is applied to the cells as a stand-alone treatment without the addition
64
of a PS, it is referred to as photobiomodulation (PBM). Literature data provides
contradictory evidence that demonstrates how PBM can have multidirectional effects.
PBM has both stimulating and inhibiting effects on intact tumour cells (Cherkasova et
al., 2020). The effects are dependent on the type of cell line that is irradiated, as well as
the specific irradiation parameters used. Core dose parameters for irradiation include
treatment frequency and time, as well as the intensity and power density of the laser
(Watson & Goh, 2015). Therefore, insufficient power density or too short a treatment
time has no effect on the pathology, while too much power density or time may have
inhibitory effects. When the power density and treatment time are in optimal balance,
the maximal biostimulatory effect will ensue for a particular disease (Huang, 2011).

5.2.2 Lactate dehydrogenase assay

Lactate dehydrogenase is a cytoplasmic enzyme that catalyses the conversion of
pyruvate to lactate, with the concomitant interconversion of its cofactor nicotinamide
adenine dinucleotide hydrogen (NADH) to nicotinamide adenine dinucleotide (NAD +).
These NAD+ molecules are essential for the continuous generation of ATP to maintain
glycolysis (Klein et al., 2020). During cell death, ATP levels begin to decline. The initial
decline in ATP disrupts the action of plasma membrane transporters, resulting in the
influx of sodium, chloride and calcium ions along with water. The increase in
intracellular volume causes the cell membrane to collapse and release its cell contents
including LDH into the extracellular space (Thornton et al., 2017). Therefore, beyond its
role in metabolism, LDH is a well-known marker of cytotoxicity and cell death. The
cytotoxicity is determined by measuring the amount of LDH leakage from cells into the
cell culture medium following cell lysis (Chan et al., 2013).

Cells treated with TO demonstrated a dose-dependent increase in LDH levels when

compared to the untreated control group. Results are indicative of the independent
ability of TO in inducing cytotoxic effects on A549 cells. An elevated level of LDH serves
as an indicator suggestive of disturbances in the cellular integrity and hence irreversible
cell death due to cell membrane damage (Klein et al., 2020). The TO-PDT treated
group showed a significant increase in LDH levels when compared to the untreated
control group and cells treated with TO alone, which resulted from the cytotoxic effects
of the treatment. This indicates how the direct cytotoxic effects of TO can act
synergistically with the photochemical reactions produced during PDT to promote cell
death.
65
Since TO when used as a PS in PDT induced enhanced cytotoxic effects than that
produced independently, we can assume that TO has favourable photosensitizing
properties. That means TO has a positive absorption wavelength at the red region
specifically at 660 nm, which falls within therapeutic window of biological tissue where
increased transmission and high singlet oxygen production is at its maximum. This
suggests the presence of photosensitising molecules in TO that would most likely be
found in the phytochemicals of the plant. The first law of photochemistry dictates that
light must be absorbed by a compound to initiate a photochemical reaction. Serving this
law are chromophores; structures responsible for absorbing light/photons. While the
phytochemicals responsible for the photosensitizing properties of TO need to be further
investigated; research data has provided evidence that demonstrate the presence of
chromophores in various phytochemicals of plants including furanocoumarins,
polyacetylenic molecules and thiophenes, curcumins, xanthanoids, alkaloids, and
antraquininones. These phytochemicals showed maximum absorption of light at various
wavelengths and hence are associated with positive photosensitizing properties and
PDT effects (Siewert & Stuppner, 2019).

Cells treated with irradiation alone demonstrated no significant changes in the levels of
LDH when compared to the control group, and is thus associated with the absence of
cytotoxicity. Since PBM is not considered an ablative mechanism but rather a
photochemical effect which can be compared to photosynthesis where light is absorbed
and synthesised in order to exert chemical change (Huang, 2011), the absence of
cytotoxicity would be an expected result.

5.2.3 Adenosine Triphosphate luminescent assay

ATP plays an essential role as a basis for generating cellular energy, and is commonly
referred to as the “energy currency” of the cell (Greiner & Glonek, 2020). The
uncontrolled proliferation of cancer cells requires metabolic adaptation to meet the high
demand for its unlimited growth and survival. Cell death mechanisms, including
apoptosis, necrosis and autophagy, are executed through unique molecular signalling
cascades. However, ATP deprivation is a common feature occurring in all three
mechanisms (Zhou et al., 2012). To elucidate the alteration of ATP levels after
treatment, the ATP luminescence assay was performed which represents one of the
most sensitive endpoints in measuring cell viability and proliferation.

66
Cells treated with TO alone demonstrated a dose-dependent decrease in ATP levels
after treatment, which indicates the anti-proliferative effects of the tincture. The anti-
proliferative and cytotoxic effects of the tincture is in accordance with the results
obtained by Biswas et al. (2011) that revealed how TO was able to induce decreased
proliferation and cell death in A375 human malignant melanoma cells. The proliferation
of A549 cells was strongly inhibited by TO-PDT, as exhibited by the significant dose-
dependent decrease in ATP levels after irradiation. Decreased ATP levels is directly
associated with a decrease in the metabolic activity of the cells. As damaged cells lose
their membrane integrity, they consequently lose their ability to synthesize ATP. Hence,
ATP levels begin to decline (Aslantürk, 2017). When there is an inadequate amount of
ATP, cancer cells cannot maintain growth signalling, thereby resulting in the down
regulation of cell proliferation (Kim, 2018). ATP levels rapidly decline during late-stage
apoptosis. As the intracellular ATP levels become depleted, there is a switch between
the energy-requiring apoptotic cell death to necrotic cell death (Nikoletopoulou et al.,
2013). ATP insufficiency prior to cell death is also associated with autophagy (Kim,
2018).

The decrease in ATP levels were consistent with increasing LDH levels, which
corroborates the cytotoxic and antiproliferative effects of TO and TO-PDT. Furthermore,
decreased ATP levels in cells treated with TO-PDT again shows how TO, when
photoactivated, is able to produce enhanced and more effective anticancer responses.

Cells treated with irradiation alone demonstrated no significant change in ATP levels
when compared to the control, which may suggest metabolically active and proliferating
cells. Observations from research studies have shown how PBM in the red wavelength
(380-750 nm) region can have a variance between results, with studies demonstrating
either a decrease, increase or no significant differences in ATP levels from irradiated
cell lines (da Silva et al., 2019). It is noted in this study that laser irradiation at 660 nm at
a fluence rate of 5 J/cm2 was unable to elicit any significant changes in ATP levels. This
could result from any variations in the dosimetric parameters employed in this study,
including the wavelength used at its specific fluency, as well as in the differences in the
treatment time and cell line when compared to other studies using the same
wavelength.

67
5.2.4 Trypan blue assay
Trypan blue is a negatively charged dye that quantifies the presence of viable and
nonviable cells. The trypan blue exclusion assay is based on the principle that non-
viable cells have comprised membranes that rapidly take up the dye, indicating cell
death. In contrast, viable cells have an intact membrane that is impermeable to trypan
blue dye, since both the membrane and the dye are negatively charged (Aslantürk,
2017).

Cells treated with TO and TO-PDT demonstrated a dose-dependent decrease in cell

viability. The decrease in cell viability is directly associated with cell death. The LDH and
ATP results are complemented by the trypan blue results, which confirms the cytotoxic
and anti-proliferative potential of TO when used alone and as a PS in PDT. Cells treated
with irradiation alone were unable to induce any significant change in cell viability
compared to the control. Therefore, cells treated with laser irradiation alone at a
wavelength of 660 nm and a fluence rate of 5 J/cm2 has no significant effect on its
activity against A549 cells and is hence, associated with the absence of any positive
anticancer effects.

5.2.5 Hoechst stain

The nucleus of a cell is of vital importance since it contains and preserves genetic
information that is encoded into chromatin fibres. The structural and mechanical
features of the nucleus ensure that its protective role is conserved for normal
functioning (Lionetti et al., 2020). Therefore, the nuclear structure of normal cells has a
distinctive size and shape that is generally maintained within a defined range. Healthy
cells usually have ellipsoidal shaped nuclei, with an even distribution of deoxy
ribonucleic acid (DNA) (Crowley et al., 2016). Therefore, the Hoechst stain assay can
be used to observe morphological alterations in the cell nucleus that may be indicative
of nuclear damage and hence, cell cycle progression and cell death.

On the hoechst stain, cells treated with TO and TO-PDT displayed irregularly shaped
nuclei, chromatin condensation, nuclear fragmentation and shrinkage, all of which is
associated with the typical features of apoptosis and negative consequences for cell
survival. When cells undergo apoptosis, nuclear changes as described above can be
observed (Abel & Baird, 2018). Any deformities in the morphology of the nucleus can
affect nuclear contents by causing local density changes in the chromatin, and rupture
68
of the nuclear lamina and membrane. These changes are then able to alter gene
expression and cell fate. Additionally, this is accompanied by excess DNA damage
which is associated with downstream consequences. As a result, there is a delay or a
block in the cell cycle, with the subsequent delay in cell proliferation and differentiation
of cancer cells, both of which are essential for cancer cell survival and progression
(Pfeifer et al., 2019).

Cells treated with irradiation only were unable to induce morphological changes in the
cell nuclei, and retained their normal morphological characteristics, which are
associated with healthy cells undergoing normal cell cycle progression and proliferation.
The result is in agreement with those observed in morphology, LDH, ATP and trypan
blue experiments.

5.3 Limitations of the study

It is necessary to overcome the limitations and realize the standardization of PDT and
homeopathic approaches through continuous research developments, which could be
used to improve therapeutic outcomes and enable more effective treatment protocols
and combinatory strategies in order to further enhance long-lasting responses and
patient survival. Limitations of the study include:

 Detailed studies on the in vitro mechanisms of TO-PDT were not carried out,
which could have provided a better understanding regarding the cooperation of
TO-PDT on a molecular basis.
 The study only considered the effects of TO-PDT at a wavelength of 660 nm and
a fluence of 5 J/cm2; the effects of other dosimetric parameters (fluencies and
wavelengths) that could be more optimal were not taken into account.
 Studies on the effects of TO and TO-PDT at its specific doses on healthy cells
were not included in the research design, which could have provided important
insight regarding the safety of the therapy.
 The experiment was carried out on A549 lung cancer cells, therefore, the efficacy
of TO and TO-PDT on other types of cancer was not determined.
 T. occidentalis in potency is frequently prescribed in clinical practice; however
this study did not include the effects of the remedy in different potencies on A549
cells nor on its efficacy in PDT.

69
 This study was carried out in vitro, therefore, the effects of the therapy in vivo
was not ascertained.

70
CHAPTER 6: CONCLUSION

The incidence of lung cancer is on the rise. Surgery, radiotherapy and chemotherapy
have been established as the three pillars for fighting lung cancer. However, its
implementation as front-line therapies has not been entirely successful, and its efficacy
has reached a therapeutic plateau (Lee & Cheah, 2019). The gap in the medical
advancement for effective and curative lung cancer therapies has led to the
identification of alternate platforms in an attempt to overcome critical obstacles facing
current conventional treatments.

Photodynamic therapy (PDT) has the ability to act independently, simultaneously, or in

combination with other therapeutics to elicit wider anticancer responses. The ability of
PDT to evoke both local and systemic immunity contributes to its long-term control. This
constitutes as a major advantage over traditional therapies which are usually
immunologically silent and immunosuppressive (Donohoe et al., 2019). The expenditure
of complementary medicine (CM) is growing rapidly and significantly, with homeopathy
employed as the most common CM method used by cancer patients. Although
homeopathy has shown potential as a supportive therapy in an oncological setting, a
great deal of research is needed to meet mainstream medical standards before it can
be introduced into routine treatment regimens (Pramanick et al., 2020). Research
studies on T. occidentalis have demonstrated how herbal and homeopathic
preparations of this plant can act as a natural obstacle to limit cancer cell development
and progression, as well as induce cell death. To date, no research has been carried
out to explore the effects of T. occidentalis Ø (TO) in PDT.

Therefore, this study aimed to investigate the photodynamic effects of T. occidentalis Ø

on A549 lung cancer cells in vitro. Findings from the study revealed the anticancer
potential of T. occidentalis Ø alone and when used as a photosensitizer (PS) in PDT.
Cells treated with TO exhibited a dose dependant increase in LDH release, and a
decrease in ATP levels and cell viability compared to untreated control groups, thereby
demonstrating its cytotoxic and antiproliferative efficacy. This study was the first to
report the use of TO as a PS in PDT. TO, when used as a PS in PDT, had the ability to
remarkably enhance the cytotoxic and anti-proliferative potential in A549 lung cancer
cells by favouring higher LDH release and lower ATP production and cell viability,

71
thereby surpassing the effects of treatment with TO alone. This indicates a positive
photodynamic effect of the tincture on A549 cells when photoactivated at a wavelength
of 660 nm and fluence rate of 5 J/cm2. Furthermore, cells treated with TO and TO
mediated PDT (TO-PDT) were able to induce morphological changes in the cell and cell
nuclei that was indicative of apoptosis, and hence cell death. Groups treated with laser
irradiation alone at a wavelength of 660 nm and a fluence rate of 5 J/cm 2 had no
significant effect on its activity against A549 cells compared to the control

This study proposed the combination of homeopathy and PDT as an alternative

multidisciplinary cancer treatment option. The implementation of TO in PDT presents a
promising strategy for the treatment of lung cancer. Results from the study represent
what the future might hold for personalised oncological care, which should encourage
continuous research efforts to ensure a substantial role of PDT-homeopathy based
synergistic approaches.

6.1 Future directives

This study was the first to introduce the combined application of TO and laser therapy;
therefore, extending the potential of TO in PDT applications require extensive and
unremitting research efforts which may include:

 More sophisticated in vitro models that will provide a detailed understanding of

the molecular and biological mechanisms behind the photodynamic effects of TO
needs to be carried out.
 The effects of TO-PDT using variant dosimetric parameters (fluencies and
wavelengths) can be carried out to investigate optimal irradiation parameters.
 TO in different homeopathic potencies can be investigated for its activity on A549
cells and its application in PDT.
 Further research can investigate the effects of TO and TO-PDT on normal cells
under the same experimental conditions which will provide more insight
regarding the safety of the therapy.
 The experiment can be replicated on different types of cell lines to determine the
efficacy of TO and TO-PDT on other types of cancer.
 Further research should aim to compare the photodynamic effects of TO to other
medically approved PSs, to determine if TO-PDT has any additional and

72
 synergistic benefits aimed at overcoming the limitations of current conventional
PSs.
 TO-PDT can be applied before and after commonly prescribed anticancer
therapies to investigate if it can improve the efficacy of these treatments.
 Data from the in vitro studies can then be extrapolated and implemented in in
vivo research in order to confirm and investigate local and systemic findings of
the treatment.

73
References:

Abbas, Z. & Rehman, S. (2018). An overview of cancer treatment modalities.

IntechOpen, DOI:10.5772/intechopen.76558.

Abel, S.D.A. & Baird, S.K. (2018). Honey is cytotoxic towards prostate cancer cells but
interacts with the MTT reagent: Considerations for the choice of cell viability
assay. Food Chemistry, 241:70-78. DOI:10.1016/j.foodchem.2017.08.083.

Abotaleb, M., Mathews, S.S., Varghese, E., Varghese, S., Kubatka, P., Liskova, A. &
Büssel-berg D. (2019). Flavonoids in cancer and apoptosis. Cancers, 11(1):28.
DOI:10.3390/cancers11010028.

Abo-Zeid, M.A.M., Abo-Elfadl, M.T. & Mostafa, S.M. (2018). Photodynamic therapy
using 5-aminolevulinic acid triggered DNA damage of adenocarcinoma breast cancer
and hepatocellular carcinoma cell lines. Photodiagnosis and Phodynamic Therapy,
21:351-356. DOI:10.1016/j.pdpdt.2018.01.011.

Abrahamse, H., Kruger, C.A., Kadanyo, S. & Mishra, A. (2017). Nanoparticles for
advanced photodynamic therapy of cancer. Photomedicine and Laser Surgery,
35(11):581-588. DOI:10.1089/pho.2017.4308.

Adams, D.J. & Morgan, L.R. (2011). Tumor physiology and charge dynamics of
anticancer drugs: implications for camptothecin-based drug development. Current
medicinal chemistry, 18(9):1367–1372. DOI:10.2174/092986711795029609.

Adjei, A.A. (2019). Lung cancer worldwide. Journal of thoracic oncology, 14(6):956.
DOI:10.1016/j.jtho.2019.04.001.

Agazzi, M.L., Ballatore, M.B., Durantini, A.M., Durantini, E.N. & Tomé, A.C. (2019).
BODIPYs in antitumoral and antimicrobial photodynamic therapy: An integrating
review. Journal of photochemistry and photobiology C: Photochemistry reviews, 40:21-
48. DOI:10.1016/j.jphotochemrev.2019.04.001.

74
Agostinis, P., Berg, K., Cengel, K.A., Foster, T.H., Girotti, A.W., Gollnick, S.O., Hahn,
S.M., Hamblin, M.R., Juzeniene, A., Kessel, D., Korbelik, M., Moan, J., Mroz, P., Nowis,
D., Piette, J., Wilson, B.C. & Golab, J. (2011). Photodynamic therapy of cancer: An
update. A Cancer Journal for Clinicians, 61(4):250-281. DOI:10.3322/caac.20114.

Ahmad, S., Rehman, T. & Abbasi, W.M. (2018). Homeopathic approach for the
treatment of cancer. Indian Journal of Research in Homoeopathy, 12(3):157-163.
DOI:10.4103/ijrh.ijrh_61_17.

Ajoolabady, A., Aghanejad, A., Bi, Y., Zhang, Y., Aslkhodapasandhukmabad, H.,
Abhari, A. & Ren, J. (2020). Enzyme-based autophagy in anti-neoplastic management:
From molecular mechanisms to clinical therapeutics. Biochimica et biophysica acta-
Reviews on Cancer, 1874(1):188366. DOI:10.1016/j.bbcan.2020.188366.

Akkol, E.K., İlhan, M., Demirel, M.A., Keleş, H., Tümen, I. & Süntar, İ. (2015). Thuja
occidentalis L. and its active compound, α-thujone: Promising effects in the treatment of
polycystic ovary syndrome without inducing osteoporosis. Journal of
Ethnopharmacology, 168:25-30. DOI:10.1016/j.jep.2015.03.029.

Allison, R.R. & Moghissi, K. (2013). Photodynamic therapy (PDT): Mechanisms. Clinical
Endoscopy, 46(1): 24-29. DOI:10.5946/ce.2013.46.1.24.

Almirantis, Y. (2013). Homeopathy – between tradition and modern science: Remedies

as carriers of significance. Homeopathy, 102(2):114-122.
DOI:10.1016/j.homp.2013.01.003.

Altorki, N.K., Markowitz, G.J., Gao, D., Port, J.L., Saxena, A., Stiles, B., McGraw, T., &
Mittal, V. (2019). The lung microenvironment: An important regulator of tumour growth
and metastasis. Nature reviews. Cancer, 19(1):9-31. DOI:1038/s41568-018-0081-9.

Alves, L.D.S., Figueirêdo, C.B.M., Silva, C.C.A.R., Marques, G.S., Ferreira, P.A.,
Soares, M.F.R., Silva, R.M.F. & Rolim-Neto, P.J. (2014). Thuja occidentalis L.
(Cupressaceae): review of botanical, phytochemical, pharmacological and toxicological
aspects. International Journal of Pharmaceutical Sciences and Research, 5:1163-1177.
DOI:10.13040/IJPSR.0975-8232.5(4).1163-77.
75
Andersen, B.L., Valentine, T.R., Lo, S.B., Carbone, D.P., Presley, C.J. & Shields, P.G.
(2019). Newly diagnosed patients with advanced non-small cell lung cancer: A clinical
description of those with moderate to severe depressive symptoms. Lung Cancer,
145:195-204. DOI:10.1016/j.lungcan.2019.11.015.

Aslantürk, Ö. S. (2017). In vitro cytotoxicity and cell viability assays: Principles,

advantages, and disadvantages. Intechopen. DOI: 10.5772/intechopen.71923.

Attar-Schneider, O., Dabbah, M., Drucker, L. & Gottfried, M. (2020). Niche origin of
mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC:
Primary versus metastatic. Cellular Signalling, 65:109456.
DOI:10.1016/j.cellsig.2019.109456.

Bacellar, I.O.L., Tsubone, T.M., Pavani, C. & Baptista, M.S. (2015). Photodynamic
efficiency: From molecular photochemistry to cell death. International Journal of
Molecular Science, 16(9):20523-20559. DOI:10.3390/ijms160920523.

Badri, H. & Smith, J.A. (2019). Emerging targets for cough therapies; NK1 receptor
antagonists. Pulmonary Pharmacology & Therapeutics, 59:101853.
doi.org/10.1016/j.pupt.2019.101853.

Bagot, J-L. (2020a). Homeopathy, a new tool for the prevention and treatment of
chemobrain (cerebral chemotoxicity). La Revue d'Homéopathie, 11(1): 1-9.
DOI:10.1016/j.revhom.2020.01.004.

Bagot, J-L. (2020b). How to prescribe Thuja occidentalis in oncology? Analysis of the
literature, study of practices and personal experience. La Revue
d'Homéopathie, 11(3):e26-e32. DOI:10.1016/j.revhom.2020.07.001.

Balata, H., Traverse-Healy, L., Blandin-Knight, S., Armitage, C., Barber, P., Colligan, D.,
Elton, P., Kirwan, M., Lyons, J., McWilliams, L., Novasio, J., Sharman, A., Slevin, K.,
Taylor, S., Tonge, J., Waplington, S., Yorke, J., Evison, M., Booton, R. & Crosbie, P.A.J.
(2020). Attending community-based lung cancer screening influences smoking
behaviour in deprived populations. Lung Cancer, 139:41-46.
DOI:10.1016/j.lungcan.2019.10.025.
76
Barros-Filho, M.C., Guisier, F., Rock, L.D., Becker-Santos, D.D., Sage, A.P., Marshall,
E.A. & Lam, W.L (2019). Tumour suppressor genes with oncogenic roles in lung cancer.
IntechOpen. DOI:10.5772/intechopen.85017.

Bascones-Martinez, A., Matilla, R., Gomez-Font, R. & Meurman, J.H (2014).

Immunomodulatory drugs: Oral and systemic adverse effects. Medicina Oral, Patología
Oral Y Cirugía Bucal, 19(1):e24-31. DOI:10.4317/medoral.19087.

Baskar, R., Lee, K.A., Yeo, R., & Yeoh, K.W. (2012). Cancer and radiation therapy:
Current advances and future directions. International journal of medical
sciences, 9(3):193-199. DOI:10.7150/ijms.3635.

Basu, A., Suresh, A.K., Kane, S.G. & Bellare, J.R. (2017). A review of machines and
devices to potentize homeopathic medicines. Homeopathy, 106(4):240-249.
DOI:10.1016/j.homp.2017.09.002.

Bell, I.R., Sarter, B., Koithan, M., Banerji, P., Banerji, P., Jain, S., & Ives, J. (2014).
Integrative nanomedicine: treating cancer with nanoscale natural products. Global
Advances in Health and Medicine, 3(1):36-53. DOI: 10.7453/gahmj.2013.009.

Bethune, G., Bethune, D., Ridgway, N., & Xu, Z. (2010). Epidermal growth factor
receptor (EGFR) in lung cancer: an overview and update. Journal of Thoracic Disease,
2(1):48-51.

Bhattacharjee, D.A., Patil, D.S., Talole, M.S., Singh, D.A., Chaturvedi, D.P., & Dikshit,
D.R. (2020). An impact of reduction in point prevalence of tobacco use on cancer
incidence- A challenge for global policy makers. Clinical Epidemiology and Global
Health, 8(4):1287-1296. DOI:ujlink.uj.ac.za/10.1016/j.cegh.2020.04.029.

Bidram, E., Esmaeili, Y., Ranji-Burachaloo, H., Al-Zaubai, N., Zarrabi, A., Stewart, A. &
Dunstan D.E. (2019). A concise review on cancer treatment methods and delivery
systems. Journal of Drug Delivery Science and Technology, 54:101350.
DOI:10.1016/j.jddst.2019.101350.

77
Bigstock. (2020). Thuja occidentalis 'Smaragd' Isolated On White Background. Available
from: https://www.bigstockphoto.com/image-6963349/stock-photo-thuja-occidentalis-
smaragd-isolated-on-white. (Accessed 18 October 2020).

Biswas, R., Mandal, S.K., Dutta, S., Bhattacharyya, S.S., Boujedaini, L & Khuda-
Bukhsh, A.R. (2011). Thujone-rich fraction of Thuja occidentalis demonstrates major
anti-cancer potentials: Evidences from in-vitro studies on A357 cells. Evidence-based
Complementary and Alternative Medicine, 2011:568148. DOI:10.1093/ecam/neq042.

Blowman, K., Magalhães, M., Lemos, M.F.L., Cabral, C. & Pires, I.M. (2018). Anticancer
properties of essential oils and other natural products. Evidence-Based Complementary
and Alternative Medicine, 2018:3149362. DOI:10.1155/2018/3149362.

Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R.L.,Torre, L.A. & Jemal, A. (2018).
Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality
worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians,
68(6):394-424. DOI:10.3322/caac.21492.

Brito, L.D.C., Berenger, A.L.R. & Figueiredo, M.R. (2017). An overview of anticancer
activity of Garcinia and Hypericum. Food and chemical toxicology, 109(2):847-862. DOI:
10.1016/j.fct.2017.03.053.

Calixto, G., Bernegossi, J., de Freitas, L.M., Fonatana, C.R. & Chorilli, M. (2016).
Nanotechnology-based drug delivery systems for photodynamic therapy of cancer: A
review. Molecules, 21(3):342. DOI:10.3390/molecules21030342.

Cao, L., Chen, J., Ou, B., Liu, C., Zou, Y. & Chen, Q. (2017). GAS5 knockdown reduces
the chemo-sensitivity of non-small cell lung cancer (NSCLC) cell to cisplatin (DDP)
through regulating miR-21/PTEN axis. Biomedicine & Pharmacotherapy, 93:570-579.
DOI:10.1016/j.biopha.2017.06.089.

78
Carreras, G., Lugo, A., Gallus, S., Cortini, B., Fernández, E., López, M.J., Soriano, J.B.,
López-Nicolás, A., Semple, S., Gorini, G., Castellano, Y., Fu, M., Ballbè, M., Amalia, B.,
Tigova, O., Continente, X., Arechavala, T., Henderson, E., Lugo, A., Liu, X., Bosetti, C.,
Davoli, E., Colombo, P., O'Donnell, R., Dobson, R., Clancy, L., Keogan, S., Byrne, H.,
Behrakis, P., Tzortzi, A., Vardavas, C., Vyzikidou, V.K., Bakellas, G., Mattiampa, G.,
Boffi, R., Ruprecht, A., De Marco, C., Borgini, A., Veronese, C., Bertoldi, M., Tittarelli,
A., Verdi, S., Chellini, E., Trapero-Bertran, M., Guerrero, D.C., Radu-Loghin, C.,
Nguyen, D., Starchenko, P., Ancochea, J., Alonso, T., Pastor, M.T., Erro, M., Roca, A. &
Pérez, P. (2019). Burden of disease attributable to second-hand smoke exposure: A
systematic review. Preventative Medicine, 129:105833.
DOI:10.1016/j.ypmed.2019.105833.

Casal-Mourino, A., Valdes, L., Barros-Dios, J.M. & Ruano-Ravina, A. (2019). Lung
cancer survival among never smokers. Cancer Letters, 451:142-149.
DOI:10.1016/j.canlet.2019.02.047.

Chan, F.K-M., Moriwaki, K. & De Rosa, M.J. (2013). Detection of necrosis by release of
lactate dehydrogenase activity. Methods in Molecular Biology, 979:65-70.
DOI:10.1007/978-1-62703-290-2_7.

Chen, J., Fan, T., Xie, Z., Zeng, Q., Xue, P., Zheng, T., Chen, Y., Luo, X. & Zhang, H.
(2020). Advances in nanomaterials for photodynamic therapy applications: Status and
challenges. Biomaterials, 237:119827. DOI:10.1016/j.biomaterials.2020.119827.

Cherkasova, E., Babak, K., Belotelov, A., Labutina, J., Yusupov, V., Vorobieva, N.,
Nerush, A. & Maslennikova, A. (2020). Effects of photobiomodulation in relation to HeLa
kyoto tumor cells exposed to ionizing radiation. Journal of Photochemistry and
Photobiology B: Biology, 209:111936. DOI:10.1016/j.jphotobiol.2020.111936.

Cheung, E.C., DeNicola, G.M., Nixon, C., Blyth, K., Labuschagne, C.F., Tuveson, D.A.
& Vousden, K.H. (2020). Dynamic ROS control by TIGAR regulates the initiation and
progression of pancreatic cancer. Cancer Cell, 37(2):168-182.
DOI:10.1016/j.ccell.2019.12.012.

79
Cheville, A.L., Novotny, P.J., Sloan, J.A., Basford, J.R., Wampfler, J.A., Garces, Y.I.,
Jatoi, A. & Yang, P. (2011). Fatigue, dyspnea, and cough comprise a persistent
symptom cluster up to five years after diagnosis with lung cancer. Journal of Pain and
Symptom Management, 42(2):202-212. DOI:10.1016/j.jpainsymman.2010.10.257.

Chirumbolo, S. & Bjørklund, G. (2018). Homeopathic dilutions, Hahnemann principles,

and the solvent issue: Must we address ethanol as a "homeopathic" or a "chemical"
issue? Homeopathy, 107(1):40-44. DOI:10.1055/s-0037-1608898.

Cho, J.H. (2017). Immunotherapy for non-small-cell lung cancer: Current status and
future obstacles. Immune network, 17(6):378-391. DOI:10.4110/in.2017.17.6.378.

Choi, B.E., Lee, M.W, Park, J.E., Lee, J.Y., Hong, C.O., Lee, S.M., Kim, Y.G. & Kim,
K.K. (2017). Photodynamic apoptosis and antioxidant activities of Brassica napus
extracts in U937 and SK-HEP-1 cells. Applied Biological Chemistry, 60:427-435.
DOI:10.1007/s13765-017-0295-7.

Choi, W., Jeong, J. & Lee, C.W. (2019). Association between EGFR mutation and
ageing, history of pneumonia and gastroesophageal reflux disease among patients with
advanced lung cancer. European Journal of Cancer, 122:101-108.
DOI:10.1016/j.ejca.2019.09.010.

Crowley, L.C., Marfell, B.J. & Waterhous, N.J. (2016). Analyzing cell death by nuclear
staining with Hoechst 33342. Cold Spring Harbor Protocols, 2016(9):27587774. DOI:
10.1101/pdb.prot087205.

Cruz, P.M.R., Mo, H., McConathy, W.J., Sabnis, N. & Lacko, A.G. (2013) The role of
cholesterol metabolism and cholesterol transport in carcinogenesis: A review of
scientific findings, relevant to future cancer therapeutics. Frontiers in Pharmacology,
4:119. DOI:10.3389/fphar.2013.00119.

Cryer, A.M. & Thorley, A.J. (2019). Nanotechnology in the diagnosis and treatment of
lung cancer. Pharmacology & Therapeutics, 198:189-205.
DOI:10.1016/j.pharmthera.2019.02.010.

80
Csupor, D., Boros, K. & Hohmann, J. (2013). Low potency homeopathic remedies and
allopathic herbal medicines: Is there an overlap? PLoS One, 8(9):e74181.
DOI:10.1371/journal.pone.0074181.

Culbert, T.B. & Olness, K. (2010). Integrative Paediatrics. New York: Oxford University
Press.

da Silva, L.P., Núnez-Montenegro, A., Magalhães, C.M., Ferreira, P.J.O., Duarte, D.,
González-Berdullas, P., Rodríguez-Borges, J.E., Vale, N. & Esteves da Silva, J.C.G.
(2019). Single-molecule chemiluminescent photosensitizer for a self-activating and
tumor-selective photodynamic therapy of cancer. European Journal of Medicinal
Chemistry, 183:111683. DOI:10.1016/j.ejmech.2019.111683.

Danneyrolles, V., Dupuis, S., Arseneault, D., Terrail, R., Leroyer, M., de Römer, A.,
Fortin, G., Boucher, Y. & Ruel, J. (2017). Eastern white cedar long-term dynamics in
eastern Canada: Implications for restoration in the context of ecosystem-based
management. Forest Ecology and Management, 400:502-510.
DOI:10.1016/j.foreco.2017.06.024.

Darwish, W.M., Bayoumi, N.A., El-Shershaby, H.M. & Allahloubi, N.M. (2020). Targeted
photoimmunotherapy based on photosensitizer-antibody conjugates for multiple
myeloma treatment. Journal of Photochemistry and Photobiology B: Biology,
203:111777. DOI:10.1016/j.jphotobiol.2020.111777.

Das, E. (2019). Understanding the principles of homeopathy on a research perspective.

Journal of Complementary Medicine and Alternative Healthcare, 9(2):555756.
DOI:10.19080/JCMAH.2019.09.555756.

Davis, D.L. (2011). Occupational cancer: Modern history. Encyclopaedia of

Environmental Health, 722-727. DOI:10.1016/B978-0-444-63951-6.00031-0.

81
Dehghan, M., Namjoo, Z., Bahrami, A., Tajedini, H., Shamsaddini-lori, Z., Zarei, A.,
Dehghani, M., Ranjbar, M. & Rafiee, S.N.F. (2020). The use of complementary and
alternative medicines, and quality of life in patients under hemodialysis: A survey in
southeast Iran. Complementary Therapies in Medicine, 51:102431.
DOI:10.1016/j.ctim.2020.102431.

Dhuriya, Y.K., Sharma, D. & Naik, A.A. (2019). Cellular demolition: Proteins as
molecular players of programmed cell death. International Journal of Biological
Macromolecules, 138:492-503. DOI:10.1016/j.ijbiomac.2019.07.113.

Diederich, M. & Cerella, C. (2016). Non-canonical programmed cell death mechanisms

triggered by natural compounds: Bioactive natural products in cancer prevention and
therapy. Seminars in Cancer Biology, 40-41:4-34.
DOI:10.1016/j.semcancer.2016.06.001.

Digumarthy, S.R., Mendoza, D.P., Lin, J.J., Chen, T., Rooney, M.M., Chin, E., Sequist,
L.V., Lennerz, J.K., Gainor, J.F. & Shaw, A.T. (2020). Computed tomography imaging
features and distribution of metastases in ROS1-rearranged non–small-cell lung cancer
with RET rearrangements. Cancers, 12(3):693. DOI:10.3390/cancers12030693.

Dobson, J., de Queiroz, G.F. & Golding, J.P. (2018). Photodynamic therapy and
diagnosis: Principles and comparative aspects. Veterinary Journal, 233:8-18.
DOI:10.1016/j.tvjl.2017.11.012.

Doğan, M.D., Savuci, Y. & Sayılan, A.A. (2020). The effect of complementary and
integrative medicine on symptom management and quality of life in oncology patients.
Advances in Integrative Medicine. DOI:10.1016/j.aimed.2020.05.004.

Dong, L., Li, X., Shen, X., Zhang, W., Zhang, J., Wang, Y. & Lu, Y. (2020). Efficacy and
safety of 5-aminolevulinic acid photodynamic therapy for the treatment of ulcerative
squamous cell carcinoma. Photodiagnosis and Photodynamic Therapy, 30:101710.
DOI:10.1016/j.pdpdt.2020.101710.

82
Donohoe, C., Senge, M.O., Arnaut, L.G. & Gomes-da-Silva, L.C. (2019). Cell death in
photodynamic therapy: From oxidative stress to anti-tumor immunity. Biochimica et
biophysica acta- Reviews on Cancer, 1872(2):188308.
DOI:10.1016/j.bbcan.2019.07.003.

dos Santos, A.F., de Almeida, D.R.Q., Terra, L.F., Baptista, M.S. & Labriola, L. (2019).
Photodynamic therapy in cancer treatment: An update review. Journal of Cancer
Metastasis and Treatment, 5(25). DOI:10.20517/2394-4722.2018.83.

Duruisseaux, M. & Esteller, M. (2018). Lung cancer epigenetics: From knowledge to

application. Seminars in Cancer Biology, 51:116-128.
DOI:10.1016/j.semcancer.2017.09.005.

El Zoghbi, M., Salameh, P., Stücker, I., Paris, C., Pairon, J.C., Gislard, A., Siemiatycki,
J., Bonneterre, V., Clin, B., Brochard, P., Delva, F. & Lacourt, A. (2017). Phenotypes of
lung cancer and statistical interactions between tobacco smoking and occupational
exposure to asbestos and crystalline silica from a large case-only study: The CaProMat
study. Lung Cancer, 112:140-155. DOI:10.1016/j.lungcan.2017.08.007.

Elío, J., Crowley, Q., Scanlon, R., Hodgson, J. & Zgaga, L. (2018). Estimation of
residential radon exposure and definition of radon priority areas based on expected lung
cancer incidence. Environmental International, 114:69-76.
DOI:10.1016/j.envint.2018.02.025.

Ellis, P.M. & Vandermeer, R. (2011). Delays in the diagnosis of lung cancer. Journal of
thoracic disease, 3(3):183-188. DOI:10.3978/j.issn.2072-1439.2011.01.01.

Espín-Pérez, A., Krauskopf, J., Chadeau-Hyam, M., van Veldhoven, K., Chung, F.,
Cullinan, P., Piepers, J., van Herwijnen, M., Kubesch, N., Carrasco-Turigas, G.,
Nieuwenhuijsen, M., Vineis, P., Kleinjans, J. C.S. & de Kok, T.M.C.M. (2018). Short-
term transcriptome and microRNAs responses to exposure to different air pollutants in
two population studies. Environmental Pollution, 242:182-190.
DOI:10.1016/j.envpol.2018.06.051.

83
Faguet, G.B. A brief history of cancer: Age‐old milestones underlying our current
knowledge database. (2015). International Journal of Cancer, 136(9):2022-2036.
DOI:10.1002/ijc.29134.

Fan, H., Yu, X., Wang, K., Yin, Y., Tang, Y., Tang, Y. & Liang, X. (2019). Graphene
quantum dots (GQDs)-based nanomaterials for improving photodynamic therapy in
cancer treatment. European Journal of Medicinal Chemistry, 182:111620.
DOI:10.1016/j.ejmech.2019.111620.

Ferraz, R.C.M.C., Fontana, C.R., Ribeiro, A.P.D., Trindade, F.Z., Bartoloni, F.H.,
Baader, J.W., Lins, E.C., Bagnato, V.S. & Kurachi, C. (2011). Chemiluminescence as a
PDT light source for microbial control. Journal of Photochemistry and Photobiology B:
Biology, 103(2):87-92. DOI:10.1016/j.jphotobiol.2011.01.018.

Field, R.W. & Withers, B.L. (2012). Occupational and environmental causes of lung
cancer. Clinics in Chest Medicine, 33(4):681-703. DOI:10.1016/j.ccm.2012.07.001.

Foley, H., Steel, A., McIntyre, E., Harnett, J., Sibbritt, D., Wardle, J. & Adams, J. (2020).
Complementary medicine practitioner consultations amongst 1,314 individuals with
chronic conditions: Characteristics of users, reasons for and predictors of use.
Complementary Therapies in Clinical Practice, 40:101194.
DOI:10.1016/j.ctcp.2020.101194.

Fuselier, C., Terryn, C., Berquand, A., Crowet, J-M., Bonnomet, A., Molinari, M.,
Dauchez, M., Martiny, L. & Schneider, C. (2019). Low-diluted Phenacetinum disrupted
the melanoma cancer cell migration. Scientific Reports, 9(9109). DOI:10.1038/s41598-
019-45578-1.

Gaertner, K., Luer, S., Frei-Erb, M. & Ammon, K. (2018). Complementary individual
homeopathy in paediatric cancer care: A case series from a University Hospital,
Switzerland. Complementary Therapies in Medicine, 41:267-270.
DOI:10.1016/j.ctim.2018.10.010.C.

84
Gallardo-Villagrán, M., Leger, D.Y., Liagre, B. & Therrien, B. (2019). Photosensitizers
used in the photodynamic therapy of rheumatoid arthritis. International Journal of
Molecular Sciences, 20(13):3339. DOI:10.3390/ijms20133339.

Gao, R., Fu, R., Jiao, W., Fan, G., Liang, C., Chen, J., Ren, H., Wang, Y., Liu, W., Ren,
S., Ren, X., Wei, Q. & Sun, M. (2020). Opto-acoustic effect of Au nanoparticles in water
under irradiation of pulse laser. Optik., 202:163512. DOI:10.1016/j.ijleo.2019.163512.

Gazdar, A.F., Bunn, P.A. & Minna, J.D. (2017). Small-cell lung cancer: What we know,
what we need to know and the path forward. Nature Reviews. Cancer, 17(12):725-737.
DOI:10.1038/nrc.2017.87.

Genova, C., Rijavec, E. & Grossi, F. (2017). Tumor microenvironment as a potential

source of clinical biomarkers in non-small cell lung cancer: Can we use enemy territory
at our advantage? Journal of Thoracic Disease, 9(11):4300–4304.
DOI:10.21037/jtd.2017.10.66.

Ghosh, S., Bishayee, K., Paul, A., Mukherjee, A., Sikdar, S., Chakraborty, D.,
Boujedaini, N. & Khuda-Bukhsh, A.R. (2013). Homeopathic mother tincture of
phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase–
mediated signaling via reactive oxygen species elevation. Journal of Integrative
Medicine, 11(2):116-124. DOI:10.3736/jintegrmed2013014.

Gibert, J., Clavé, S., Hardy-Werbin, M., Taus, Á., Rocha, P., Longarón, R., Piquer, G.,
Chaib, I., Carcereny, E., Morán, T., Salido, M., Dalmases, A., Bellosillo, B. & Arriola, E.
(2020). Concomitant genomic alterations in KRAS mutant advanced lung
adenocarcinoma. Lung Cancer, 140: 42-45. DOI:10.1016/j.lungcan.2019.12.003.

Gjeilo, K.H., Oksholm, T., Follestad, T., Wahba, A. & Rustøen, T. (2020). Trajectories of
pain in patients undergoing lung cancer surgery: A longitudinal prospective study.
Journal of Pain and Symptom Management, 59(4):818-828.
DOI:10.1016/j.jpainsymman.2019.11.004.

85
Goldspiel, B., Hoffman, J.M., Griffith, N.L., Goodin, S., DeChristoforo, R., Montello,
C.M., Chase, J.L., Bartel, S. & Patel, J.T. (2015). ASHP guidelines on preventing
medication errors with chemotherapy and biotherapy. American Journal of Health-
System Pharmacy, 72(8):e6-35. DOI:10.2146/sp150001.

Gomaa, I., Sebak, A., Afifi, N. & Abdel-Kader, M. (2017). Liposomal delivery of ferrous
chlorophyllin: A novel third generation photosensitizer for in vitro PDT of melanoma.
Photodiagnosis and Photodynamic Therapy, 18:162-170.
DOI:10.1016/j.pdpdt.2017.01.186.

Greiner, J.V. & Glonek, T. (2020). Hydrotropic function of ATP in the crystalline lens.
Experimental Eye Research, 190:107862. DOI:10.1016/j.exer.2019.107862.

Gridelli, C., Rossi, A., Carbone, D.P., Guarize, J., Karachaliou, N., Mok, T., Petrella, F.,
Spaggiari, L. & Rosell, R. (2015). Non-small-cell lung cancer. Nature Reviews. Disease
Primers, 1:15009. DOI:10.1038/nrdp.2015.9.

Habli, Z., Toumieh, G., Fatfat, M., Rahal, O.N. & Gali-Muhtasib, H. (2017). Emerging
cytotoxic alkaloids in the battle against cancer: Overview of molecular mechanisms.
Molecules, 22(2):250. DOI:10.3390/molecules22020250.

Hamblin, M.R. Photodynamic therapy and photobiomodulation: Can all diseases be

treated with light? (2018). Encyclopaedia of Modern Optics. Academic Press.
DOI:10.1016/B978-0-12-803581-8.09688-0.

Hanahan, D. & Weinberg, R.A. (2011). Hallmarks of cancer: The next generation. Cell,
144(5):646-674. DOI:10.1016/j.cell.2011.02.013.

Handali, S. & Rezaei, M. (2020). Interlink between improved formulations, inhibitory

concentrations and cell death mechanism investigations of cytotoxic drugs: What really
matters? Journal of controlled release, 320:404-411.
DOI:10.1016/j.jconrel.2020.02.007.

86
Hartl, B.A., Hirschberg, H., Marcu, L. & Cherry, S.R. (2015). Characterizing low fluence
thresholds for in vitro photodynamic therapy. Biomedical optics express, 6(3):770–779.
DOI:10.1364/BOE.6.000770.

Hedayat, K.M. & Lapraz, J-C. (2019). Introduction to the usage of medicinal plants.
Chapter 16 in The Theory of Endobiogeny. Academic Press. DOI: 10.1016/B978-0-12-
816903-2.00016-1.

Heiskanen, V. & Hamblin, M.R. (2018). Photobiomodulation: Lasers vs. light emitting
diodes? Photochemical and Photobiological Sciences, 17(8):1003-1017.
DOI:10.1039/c8pp90049c.

Hida, T., Velcheti, V., Reckamp, K.L., Nokihara, H., Sachdev, P., Kubota, T., Nakada,
T., Dutcus, C.E., Ren, M. & Tamura, T. (2019). A phase 2 study of lenvatinib in patients
with RET fusion-positive lung adenocarcinoma. Lung cancer, 138124-130.
DOI:10.1016/j.lungcan.2019.09.011.

Hönigsmann, H. (2013). History of phototherapy in dermatology. Photochemical and

Photobiological Sciences, 12(1):16-21. DOI:10.1039/c2pp25120e.

Hopkins, R.J., Ko, J., Gamble, G.D. & Young, R.P. (2019). Airflow limitation and survival
after surgery for non-small cell lung cancer: Results from a systematic review and lung
cancer screening trial (NLST-ACRIN sub-study). 135:80-87.
DOI:10.1016/j.lungcan.2019.07.015.

Hori, M., Tanaka, H., Wakai, K., Sasazuki, S. & Katanoda, K. (2016). Secondhand
smoke exposure and risk of lung cancer in Japan: A systematic review and meta-
analysis of epidemiologic studies. Japanese Journal of Clinical Oncology, 46(10):942-
951. DOI:10.1093/jjco/hyw091.

Hosokawa, S., Takahashi, G., Sugiyama, K., Takebayashi, S., Okamura, J., Takizawa,
Y. & Mineta, H. (2020). Porfimer sodium-mediated photodynamic therapy in patients
with head and neck squamous cell carcinoma. Photodiagnosis and Photodynamic
Therapy, 29:101627. DOI:10.1016/j.pdpdt.2019.101627.

87
Hu, J., Lei, Q. & Zhang, X. (2020). Recent advances in photonanomedicines for
enhanced cancer photodynamic therapy. Progress in Materials Science, 114:100685.
DOI:10.1016/j.pmatsci.2020.100685.

Huang, C.Y., Ju, D.T., Chang, C.F., Muralidhar Reddy, P., & Velmurugan, B.K. (2017).
A review on the effects of current chemotherapy drugs and natural agents in treating
non-small cell lung cancer. BioMedicine, 7(4):23. DOI:10.1051/bmdcn/2017070423.

Huang, Y.Y., Sharma, S.K., Carroll, J., & Hamblin, M.R. (2011). Biphasic dose response
in low level light therapy - an update. Dose-Response, 9(4):602-618. DOI:10.2203/dose-
response.11-009.Hamblin.

Hubbard, M.O., Fu, P., Margevicius, S., Dowlati, A. & Linden, P.A. (2012). Five-year
survival does not equal cure in non–small cell lung cancer: A Surveillance,
Epidemiology, and End Results–based analysis of variables affecting 10- to 18-year
survival. The Journal of Thoracic and Cardiovascular Surgery, 143(6):1307-1313.
DOI:10.1016/j.jtcvs.2012.01.078.

Husebø, G.R., Nielsen, R., Hardie, J., Bakke, P.S., Lerner, L., D'Alessandro-Gabazza,
C., Gyuris, J., Gabazza, E., Aukrust, P. & Eagan, T. (2019). Risk factors for lung cancer
in COPD – results from the Bergen COPD cohort study. 152:81-88.
DOI:10.1016/j.rmed.2019.04.019.

Iams ,W.T., Shiuan, E., Meador, C.B., Roth, M., Bordeaux, J., Vaupel, C., Boyd, K.L.,
Summitt, I.B., Wang, L.L., Schneider, J.T., Warner, J.L., Zhao, Z. & Lovly, C.M. (2019).
Improved prognosis and increased tumor-infiltrating lymphocytes in patients who have
SCLC with neurologic paraneoplastic syndromes. Journal of Thoracic Oncology,
14(11):1970-1981. DOI:10.1016/j.jtho.2019.05.042.

Ijaz, S., Akhtar, N., Khan, M.S., Hameed, A., Irfan, M., Arshad, M.A., Ali, S. & Asrar, M.
(2018). Plant derived anticancer agents: A green approach towards skin
cancers. Biomedicine & pharmacotherapy, 103:1643-1651.
DOI:10.1016/j.biopha.2018.04.113.

88
Ikeda, H., Ohba, S., Egashira, K. & Asahina, I. (2018). The effect of photodynamic
therapy with talaporfin sodium, a second-generation photosensitizer, on oral squamous
cell carcinoma: A series of eight cases. Photodiagnosis and Photodynamic Therapy,
21:176-180. DOI:10.1016/j.pdpdt.2017.11.016.

Inamura, K. (2017). Lung Cancer: Understanding its molecular pathology and the 2015
WHO classification. Frontiers in oncology, 7:193. DOI:10.3389/fonc.2017.00193.

Iyer, R., Wolf, J., Zhukova, D., Padanilam, D. & Nguyen, K.T. (2018). Nanomaterial
Based Photo-Triggered Drug Delivery Strategies for Cancer Theranostics. Handbook of
Nanomaterials for Cancer Theranostics, 2018:351-39. DOI:10.1016/B978-0-12-813339-
2.00012-8.

Jadhav, H.P., Chaudhari, G.G., Patil, D.D., Jadhav, R.B., Reddy, N.M., Shirkhedkar,
A.A., Goyal, S.N. & Patil, C R. (2016). Standardization of homeopathic mother tincture
of Toxicodendron pubescens and correlation of its flavonoid markers with the biological
activity. Homeopathy, 105(1):48-54. DOI:10.1016/j.homp.2015.08.003.

James, N.S., Joshi, P., Ohulchanskyy, T.Y., Chen, Y., Tabaczynski, W., Durrani, F.,
Shibata, M. & Pandey, R.K. (2016). Photosensitizer (PS)-cyanine dye (CD) conjugates:
Impact of the linkers joining the PS and CD moieties and their orientation in tumor-
uptake and photodynamic therapy (PDT). European Journal of Medicinal Chemistry,
122:770-785. DOI:10.1016/j.ejmech.2016.06.045.

Jantan, I., Ahmad, W. & Bukhari, S.N. (2015). Plant-derived immunomodulators: An

insight on their preclinical evaluation and clinical trials. Frontiers in Plant Science, 6:655.
DOI:10.3389/fpls.2015.00655.

Jhanwar, S.C., Xu, X.L., Elahi, A.H. & Abramson, D.H. (2020). Cancer genomics of lung
cancer including malignant mesothelioma: A brief overview of current status and future
prospects. Advances in Biological Regulation, 78:100723.
DOI:10.1016/j.jbior.2020.100723.

89
Jiang, D., Rasul, A., Batool, R., Sarfraz, I., Hussain, G., Mateen Tahir, M., Qin, T.,
Selamoglu, Z., Ali, M., Li, J., & Li, X. (2019). Potential anticancer properties and
mechanisms of action of formononetin. BioMed Research International, 2019:5854315.
DOI:10.1155/2019/5854315.

Joseph, N., McWilliam, A., Kennedy, J., Haslett, K., Mahil, J., Gavarraju, A., Mistry, H.,
Van Herk, M., Faivre-Finn, C. & Choudhury, A. (2019). Post-treatment
lymphocytopaenia, integral body dose and overall survival in lung cancer patients
treated with radical radiotherapy. Radiotherapy and Oncology, 135:115-119.
DOI:10.1016/j.radonc.2019.03.008.

Jütte, R. & Riley, D. (2005). A review of the use and role of low potencies in
homeopathy. Complementary Therapies in Medicine, 13(4):291-296.
DOI:10.1016/j.ctim.2005.10.003.

Kaleta-Richter, M., Kawczyk-Krupka, A., Aebisher, D., Bartusik-Aebisher, D., Czuba, Z.

& Cieślar, G. (2019). The capability and potential of new forms of personalized colon
cancer treatment: Immunotherapy and photodynamic therapy. Photodiagnosis and
Photodynamic Therapy, 25:253-258. DOI:10.1016/j.pdpdt.2019.01.004.

Kamanli, A.F. & Çetinel, G. (2020). Comparison of pulse and super pulse radiation
modes’ singlet oxygen production effect in antimicrobial photodynamic therapy.
Photodiagnosis and Photodynamic Therapy, 30:101706.
DOI:10.1016/j.pdpdt.2020.101706.

Kapoor, R., Das, N., Miriyala, R., Sood, A., Oinam, A. & Singh, N. (2020). Challenges of
radical chemoradiation planning in stage III non-small-cell lung cancer: Can volumetric
modulated arc radiotherapy overcome an unfavourable location? Physics and Imaging
in Radiation Oncology, 13:50-54. DOI:10.1016/j.phro.2020.03.005.

Khan, T., Date, A., Chawda, H. & Patel, K. (2019). Polysaccharides as potential
anticancer agents- A review of their progress. Carbohydrate Polymers, 210:412-428.
DOI:10.1016/j.carbpol.2019.01.064.

90
Kim, H., Kim, S.W., Soek, K.H., Hwang, C.W., Ahn, J.C., Jin, J.O. & Kang, H.W. (2018).
Hypericin-assisted photodynamic therapy against anaplastic thyroid cancer.
Photodiagnosis and Photodynamic Therapy, 24:15-21.
DOI:10.1016/j.pdpdt.2018.08.008.

Kim, S.Y. (2018). Cancer energy metabolism: Shutting power off cancer factory.
Biomolecules & therapeutics, 26(1):39-44. DOI:10.4062/biomolther.2017.184.

Kim, T.H. & Cho, J.H. (2020). Nonintubated video-assisted thoracoscopic surgery lung
biopsy for interstitial lung disease: Thoracic Surgery Clinics, 30(1):41-48.
DOI:10.1016/j.thorsurg.2019.08.005.

Kincaid, J.A. (2016). Structure and dendroecology of Thuja occidentalis in disjunct

stands south of its contiguous range in the central Appalachian Mountains, USA. Forest
Ecosystems, 3(25). DOI:10.1186/s40663-016-0085-4.

Klein, R., Nagy, O., Tóthová, C., & Chovanová, F. (2020). Clinical and diagnostic
significance of lactate dehydrogenase and its isoenzymes in animals. Veterinary
Medicine International, 5346483. DOI:10.1155/2020/5346483.

Kou, J., Dou, D. & Yang, L. (2017). Porphyrin photosensitizers in photodynamic therapy
and its applications. Oncotarget, 8(46):81591-81603. DOI:10.18632/oncotarget.20189.

Kourlaba, G., Gkiozos, I., Kokkotou, E., Stefanou, G., Papaspiliou, A. & Syrigos, K.
(2019). Lung cancer patients' journey from first symptom to treatment: Results from a
Greek registry. Cancer Epidemiology, 60:193-200. DOI:10.1016/j.canep.2019.04.014.

Krencz, I., Sebestyen, A., Papay, J., Lou, Y., Lutz, G.F., Majewicz, T.L. & Khoor, A.
(2019). Correlation between immunohistochemistry and RICTOR fluorescence in situ
hybridization amplification in small cell lung carcinoma. Human Pathology, 93:74-80.
DOI:10.1016/j.humpath.2019.08.018.

91
Kunimasa, K., Hirotsu, Y., Nakamura, H., Tamiya, M., Iijima, Y., Ishida, H., Hamamoto,
Y., Maniwa, T., Kimura, T., Nishino, K., Goto, T., Amemiya, K., Mochizuki, H., Oyama,
T., Nakatsuka, S., Kumagai, T., Okami, J., Higashiyama, M., Imamura, F. & Omata, M.
(2019). Rapid progressive lung cancers harbouring multiple clonal driver mutations with
big bang evolution mode. Cancer Genetics, 241:51-56.
DOI:10.1016/j.cancergen.2019.12.006.

Kutob, L. & Schneider, F. (2020). Lung cancer staging. Surgical Pathology Clinics,
13(1):57-71. DOI:10.1016/j.path.2019.10.003.

Kwiatkowski, S., Knap, B., Przystupski, D., Saczko, J., Kędzierska, E., Knap-Czop, K.,
Kotlińska, J., Michel, O., Kotowski, K. & Kulbacka, J. (2018). Photodynamic therapy –
mechanisms, photosensitizers and combinations. Biomedicine and Pharmacotherapy,
106:1098-1107. DOI:10.1016/j.biopha.2018.07.049.

Lackey, A. & Donington, J.S. (2013). Surgical management of lung cancer. Seminars in
Interventional Radiology, 30(2):133–140. DOI:10.1055/s-0033-1342954.

Laszló, I., Laszló, M.R., Toma, V., Baldea, I., Olteanu, D., David, L., Moldovan, B., Ion,
R.M., Moldovan, R., Filip, G.A., Kacso, G., Cainap, C., Clichici, S. & Muresan, A.
(2020). The in vivo modulatory effects of Cornus mas extract on photodynamic therapy
in experimental tumors. Photodiagnosis and Photodynamic Therapy, 30:101656.
DOI:10.1016/j.pdpdt.2020.101656.

Latimer, K.M. (2018). Lung cancer: Clinical presentation and diagnosis. FP Essentials,
464: 23-26.

Lee, S.S. & Cheah, Y.K. (2019). The interplay between microRNAs and cellular
components of tumour microenvironment (TME) on non-small-cell lung cancer (NSCLC)
progression. Journal of immunology research. DOI:10.1155/2019/3046379.

Lemjabbar-Alaoui, H., Hassan, O.U., Yang, Y. & Buchannan, P. (2015). Lung cancer:
Biology and treatment option. Biochimica et BiophysicaActa- Reviews on Cancer,
1856(2):189-210. DOI:10.1016/j.bbcan.2015.08.002.

92
Li, W., Ma, Q. & Wu, E. (2012). Perspectives on the role of photodynamic therapy in the
treatment of pancreatic cancer. International Journal of Photoenergy.
DOI:10.1155/2012/637429.

Lin, X., Wu, M., Li, M., Cai, Z., Sun, H., Tan, X., Li, J., Zeng, Y., Liu, X. & Liu, J. (2018).
Photo-responsive hollow silica nanoparticles for light-triggered genetic and
photodynamic synergistic therapy. Acta Biomaterialia, 76:178-192.
DOI:10.1016/j.actbio.2018.07.007.

Linden, W., Vodermaier, A., MacKenzie, R. & Greig, D. (2012). Anxiety and depression
after cancer diagnosis: Prevalence rates by cancer type, gender, and age. Journal of
Affective Disorders, 141(3):343-351. DOI:10.1016/j.jad.2012.03.025.

Lionetti, M.C., Bonfanti, S., Fumagalli, M.R., Budrikis, Z., Font-Clos, F., Costantini, G.,
Chepizhko, O., Zapperi, S. & La Porta, C.A.M. (2020). Chromatin and cytoskeletal
tethering determine nuclear morphology in progerin-expressing cells. Biophysical
Journal, 118(9):2319-2332. DOI:10.1016/j.bpj.2020.04.001.

Liu, M. & Guo, F. (2018). Recent updates on cancer immunotherapy. Precision Clinical
Medicine, 1(2):65-74. DOI:10.1093/pcmedi/pby011.

Liu, Z., Li, J., Chen, W., Liu, L. & Yu, F. (2020). Light and sound to trigger the Pandora's
box against breast cancer: A combination strategy of sonodynamic, photodynamic and
photothermal therapies. Biomaterials, 232:119685.
DOI:10.1016/j.biomaterials.2019.119685.

Lopes, T.Z., de Moraes, F.R., Tedesco, A.C., Arni, R.K., Rahal, P. & Calmon, M.F.
(2019). Berberine associated photodynamic therapy promotes autophagy and apoptosis
via ROS generation in renal carcinoma cells. Biomedicine and Pharmacotherapy,
123:109794. DOI:10.1016/j.biopha.2019.109794.

Lu, M. & Su, Y. (2019). Immunotherapy in non-small cell lung cancer: The past, the
present, and the future. Thoracic cancer, 10(4):585-586. DOI:10.1111/1759-
7714.13012.

93
Luckett, T., Phillips, J., Currow, D.C., Agar, M. & Molassiotis, A. (2019). Cough in lung
cancer: A survey of current practice among Australian health professionals: Palliative
care. Collegian, 26(6):629-633. DOI:10.1016/j.colegn.2019.09.002.

Machado, F.C., Adum de Matos, R.P., Primo, F.L., Tedesco, C., Rahal, P. & Calmon,
M.F. (2019). Effect of curcumin-nanoemulsion associated with photodynamic therapy in
breast adenocarcinoma cell line. Bioorganic & Medicinal Chemistry, 27(9):1882-1890.
DOI:10.1016/j.bmc.2019.03.044.

Made, F., Wilson, K., Jina, R., Tlotleng, N., Jack, S., Ntlebi, V & Kootbodien, T. (2017).
Distribution of cancer mortality rates by province in South Africa. Cancer Epidemiology,
51:56-61. DOI:10.1016/j.canep.2017.10.007.

Madkour, L.H. (2020). Programmed cell death mechanisms and nanoparticle toxicity.
Chapter 10 in Reactive Oxygen Species (ROS), Nanoparticles, and Endoplasmic
Reticulum (ER) Stress-Induced Cell Death Mechanisms. Saudi Arabia: Academic Press.
DOI:10.1016/B978-0-12-822481-6.00010-4.

Madkour, L.H.( 2020). Cell death mechanisms—Apoptosis pathways and their

implications in toxicology. Chapter 9 in Reactive Oxygen Species (ROS), Nanoparticles,
and Endoplasmic Reticulum (ER) Stress-Induced Cell Death Mechanisms. Saudi
Arabia: Academic Press. DOI: 10.1016/B978-0-12-822481-6.00009-8.

Maiti, S., Ali, K.M., Jana, K., Chatterjee, K., De, D. & Ghosh, D. (2013). Ameliorating
effect of mother tincture of Syzygium jambolanum on carbohydrate and lipid metabolic
disorders in streptozotocin-induced diabetic rat: Homeopathic remedy. Journal of
Natural Science, Biology, and Medicine, 4(1):68-73. DOI:10.4103/0976-9668.107263.

Maji, S., Panda, S., Samal, S.K., Shriwas, O., Rath, R., Pellecchia, M., Emdad, L., Das,
S.K., Fisher, P.B. & Dash, R. (2018). Bcl-2 antiapoptotic family proteins and
chemoresistance in cancer. Advances in Cancer Research. 137:37-75. DOI:
10.1016/bs.acr.2017.11.001.

94
Mamone, L., Di Venosa, G., Sáenz, D., Batlle, A. & Casas, A. (2016). Methods for the
detection of reactive oxygen species employed in the identification of plant
photosensitizers. Methods, 109:73-80. DOI:10.1016/j.ymeth.2016.05.020.

Mansoori, B., Mohammadi, A., Doustvandi, M.A., Mohammadnejad, F., Kamari, F., G-
jerstorff, M.F., Baradaran, B. & Hamblin, M.R. (2019). Photodynamic therapy for cancer:
Role of natural products. Photodiagnosis and Photodynamic Therapy, 26:395-404.
DOI:10.1016/j.pdpdt.2019.04.033.

Maphumulo, W.T. & Bhengu, B.R. (2019). Challenges of quality improvement in the
healthcare of South Africa post-apartheid: A critical review. Curationis, 42(1):1901.
DOI:10.4102/curationis.v42i1.1901.

Martín-Sánchez, J.C., Bilal, U., Clèries, R., Lidón-Moyano, C., Fu, M., González-de Paz,
L., Franco, M., Fernandez, E. & Martínez-Sánchez, J.M. (2017). Modelling lung cancer
mortality rates from smoking prevalence: Fill in the gap. Cancer Epidemiology, 49:19-
23. DOI:10.1016/j.canep.2017.04.012.

McBride, A., Houtmann, S., Wilde, L., Vigil, C., Eischen, C.M., Kasner, M., &
Palmisiano, N. (2019). The role of inhibition of apoptosis in acute leukemias and
myelodysplastic syndrome. Frontiers in Oncology, 9:192.
DOI:10.3389/fonc.2019.00192.

Mei, D., Chen, B., He, B., Liu, H., Lin, Z., Lin, J., Zhang, X., Sun, N., Zhao, L., Wang, Z.
& Zhang, Q. (2019). Actively priming autophagic cell death with novel transferrin
receptor-targeted nanomedicine for synergistic chemotherapy against breast cancer.
Acta Pharmaceutica Sinica B, 9(5):1061-1077. DOI:10.1016/j.apsb.2019.03.006.

Mendenhall, E., Bosire, E.N., Kim, A.W & Norris, S.A. (2019). Cancer, chemotherapy,
and HIV: Living with cancer amidst comorbidity in a South African township. Social
Science & Medicine, 237:112461. DOI:10.1016/j.socscimed.2019.112461.

Mercadante, S. & Vitrano, V. (2010). Pain in patients with lung cancer: Pathophysiology
and treatment. Lung Cancer, 68(1):10-15. DOI:10.1016/j.lungcan.2009.11.004.

95
Messaritakis, I., Nikolaou, M., Koinis, F., Politaki, E., Koutsopoulos, A., Lagoudaki, E.,
Vetsika, E.K., Georgoulias, V. & Kotsakis, A. (2019). Characterization of DLL3-positive
circulating tumor cells (CTCs) in patients with small cell lung cancer (SCLC) and
evaluation of their clinical relevance during front-line treatment. Lung Cancer, 135:33-
39. DOI:10.1016/j.lungcan.2019.06.025.

Millimouno, F.M., Dong, J., Yang, L., Li, J. & Li, X. (2014). Targeting apoptosis
pathways in cancer and perspectives with natural compounds from mother nature.
Cancer Prevention Research, 7(11):1081-1107. DOI: 10.1158/1940-6207.CAPR-14-
0136.

Mohamed, S.I.A., Jantan, I. & Haque, M.A. (2017). Naturally occurring

immunomodulators with antitumor activity: An insight on their mechanisms of action.
International Immunopharmacology, 50:291-304. DOI:10.1016/j.intimp.2017.07.010.

Mortezaee, K., Najafi, M., Farhood, B., Ahmadi, A., Potes, Y., Shabeeb, D. & Musa,
A.E. (2019). Modulation of apoptosis by melatonin for improving cancer treatment
efficiency: An updated review. Life Sciences, 228:228-241.
DOI:10.1016/j.lfs.2019.05.009.

Mottaghitalab, F., Farokhi, M., Fatahi, Y., Atyabi, F. & Dinarvind, R. (2019). New
insights into designing hybrid nanoparticles for lung cancer: Diagnosis and treatment.
Journal of Controlled Release, 295:250-267. DOI:10.1016/j.jconrel.2019.01.009.

Mukherjee, A., Boujedaini, N. & Khuda-Bukhsh, A.R. (2013). Homeopathic Thuja 30C
ameliorates benzo(a)pyrene–induced DNA damage, stress and viability of perfused lung
cells of mice in vitro. Journal of Integrative Medicine, 11(6):397-404.
DOI:10.3736/jintegrmed2013054.

Mukherjee, A., Sikdar, S., Bishayee, K., Boujedaini, N. & Khuda-Bukhsh. (2014).
Flavonol isolated from ethanolic leaf extract of Thuja occidentalis arrests the cell cycle
at G2-M and induces ROS-independent apoptosis in A549 cells, targeting nuclear DNA.
Cell Proliferation, 47(1):56-71. DOI:10.1111/cpr.12079.

96
Mukherjee, A., Sikdar, S., Bishayee, K., Paul, A., Ghosh, S., Boujedaini, N. & Khuda-
Bukhsh, A.R. (2012). Ethanolic extract of Thuja occidentalis blocks proliferation of A549
cells and induces apoptosis in vitro. Journal of Chinese Integrative Medicine,
10(12):1451-1459. DOI:10.3736/jcim20121218.

Nantavithya, C., Gomez, D.R., Chang, J.Y., Mohamed, A.S.R., Fuller, C.D., Li, H.,
Brooks, E.D. & Gandhi, S.J. (2020). An improved method for analyzing and reporting
patterns of in-field recurrence after stereotactic ablative radiotherapy in early-stage non-
small cell lung cancer. Journal of the European Society for Therapeutic Radiology and
Oncology, 145:209-214. DOI:10.1016/j.radonc.2020.01.002.

Nasim, F., Sabath, B.F., & Eapen, G.A. (2019). Lung cancer. The Medical Clinics of
North America, 103(3):463-473. DOI:10.1016/j.mcna.2018.12.006.

Navaeipour, F., Afsharan, H., Tajalli, H., Mollabashi, M., Ranjbari, F., Montaseri, A. &
Rashidi, M. (2016). Effects of continuous wave and fractionated diode laser on human
fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic
therapy: A comparative study. Journal of Photochemistry and Photobiology B: Biology,
161:456-462. DOI:10.1016/j.jphotobiol.2016.06.017.

Nguyen, S.T., Nguyen, H.T-L. & Truong, K.D. (2020). Comparative cytotoxic effects of
methanol, ethanol and DMSO on human cancer cell lines. Biomedical Research and
Technology, 7(7):3855-3859. DOI :10.15419/bmrat.v7i7.614.

Nikoletopoulou, V., Markaki, M., Palikaras, K. & Tavernarakis, N. (2013). Crosstalk

between apoptosis, necrosis and autophagy. Biochimica et Biophysica Acta,
1833(12):3448-3459. DOI:10.1016/j.bbamcr.2013.06.001.

Nowak, K.L. & Edelstein, C.L. (2020). Apoptosis and autophagy in polycystic kidney
disease (PKD). Cellular Signalling, 68:109518. DOI:10.1016/j.cellsig.2019.109518.

Nurgali, K., Jagoe, R.T. & Abalo, R. (2018). Editorial: Adverse effects of cancer
chemotherapy: Anything new to improve tolerance and reduce sequelae? Frontiers in
Pharmacology, 9:245. DOI:10.3389/fphar.2018.00245.

97
Nymark, P., Wikman, H., Hienonen-Kempas, T. & Anttila, S. (2008). Molecular and
genetic changes in asbestos-related lung cancer. Cancer Letters, 265(1):1-15.
DOI:10.1016/j.canlet.2008.02.043.

Oh, P. & Jeong, H. (2019). Therapeutic application of light emitting diode: Photo-
oncomic approach. Journal of Photochemistry and Photobiology B: Biology, 192:1-7.
DOI:10.1016/j.jphotobiol.2019.01.003.

O'Neil, D.S., Prigerson, H.G., Mmoledi, K., Sobekwa, M., Rratsgikana-Moloko, M.,
Tsitsi, J.M., Cubasch, H., Wong, M.L., Omoshoro-Jones, J.A.O., Sackstein, P.E.,
Blinderman, C.D., Jacobson, J.S., Joffe, M., Ruff, P., Neugut, A.I. & Blanchard, C.L.
(2018). Informal caregiver challenges for advanced cancer patients during end-of-life
care in Johannesburg, South Africa and distinctions based on place of death. Journal of
Pain and Symptom Management, 56(1):98-106.
DOI:10.1016/j.jpainsymman.2018.03.017.

Ortega, M.A.M. & Campos, S.M.R. (2019). Medicinal plants and their bioactive
metabolites in cancer prevention and treatment. Chapter 5 in Bioactive Compounds.
Woodhead Publishing. DOI: 10.1016/B978-0-12-814774-0.00005-0.

Oun, R., Moussa, Y.E. & Wheate, N.J. (2018). The side effects of platinum-based
chemotherapy drugs: A review for chemists. Dalton Transactions, 47(19):6645-6653.
DOI:10.1039/c8dt00838h.

Ouyang, L., Shi, Z., Zhao, S., Wang, F.T., Zhou, T.T., Liu, B. & Bao, J.K. (2012).
Programmed cell death pathways in cancer: A review of apoptosis, autophagy and
programmed necrosis. Cell Proliferation, 45(6):487-498. DOI:10.1111/j.1365-
2184.2012.00845.x.

Panche, A.N., Diwan, A.D., & Chandra, S.R. (2016). Flavonoids: An overview. Journal
of Nutritional Science, 5:e47. DOI:/10.1017/jns.2016.41.

Patil, A.A., Bhor, S.A. & Rhee, W.J. (2020). Cell death in culture: Molecular
mechanisms, detections, and inhibition strategies. Journal of Industrial and Engineering
Chemistry, 91:37-53. DOI:10.1016/j.jiec.2020.08.009.
98
Pedro, C., Mira, B., Silva, P., Netto, E., Pocinho, R., Mota, A., Labareda, M.,
Magalhães, M., Esteves, S. & Santos, F. (2018). Surgery vs. primary radiotherapy in
early-stage oropharyngeal cancer. Clinical and Transitional Radiation Oncology, 9:18-
22. DOI:10.1016/j.ctro.2017.12.002.

Pelkonen, O., Abass, K. & Wiesner, J. (2013). Thujone and thujone-containing herbal
medicinal and botanical products: Toxicological assessment. Regulatory Toxicology and
Pharmacology, 65(1):100-107. DOI:10.1016/j.yrtph.2012.11.002.

Peng, Z., Li, H., Zhang, C., Qian, X., Feng, Z., & Zhu, S. (2014). A retrospective study of
chronic post-surgical pain following thoracic surgery: Prevalence, risk factors, incidence
of neuropathic component, and impact on quality of life. PloS One, 9(2):e90014.
DOI:10.1371/journal.pone.0090014.

Pfeifer, C.R., Vashisth, M., Xia, Y. & Discher D.E. (2019), Nuclear failure, DNA damage,
and cell cycle disruption after migration through small pores: a brief review. Essays in
Biochemistry, 63(5):569-577. DOI:10.1042/EBC20190007.

Phillips, I., Sandhu, S., Lüchtenborg, M. & Harden, S. (2019). Stereotactic ablative body
radiotherapy versus radical radiotherapy: Comparing real-world outcomes in stage I
lung cancer. Clinical Oncology, 31(10):681-687. DOI:10.1016/j.clon.2019.07.013. Epub
2019 Jul 31.

Pistritto, G., Trisciuoglio, D., Ceci, C., Garufi, A., & D'Orazi, G. (2016). Apoptosis as
anticancer mechanism: Function and dysfunction of its modulators and targeted
therapeutic strategies. Aging, 8(4):603–619. DOI:10.18632/aging.100934.

Poirier, A.E., Ruan, Y., Grevers, X., Walter, S.D., Villeneuve, P.J., Friedenreich, C.M. &
Brenner, D.R. (2019). Estimates of the current and future burden of cancer attributable
to active and passive tobacco smoking in Canada. Preventative Medicine, 122:9-19.
DOI:10.1016/j.ypmed.2019.03.015.

99
Pramanick, J., Uchat, U., Chattopadhyay, A., Mir, A.A., Koley, M. & Saha, S. (2020). An
open-label randomized pragmatic non-inferiority pilot trial comparing the effectiveness
of Curare 30CH against individualized homeopathic medicines in post-stroke
hemiparesis. Advances in Integrative Medicine, 7(2):79-88.
DOI:10.1016/j.aimed.2019.06.002.

Pruis, M.A., Geurts-Giele, W.R.R., von der, T.J.H., Meijssen, I.C., Dinjens, W.N.M.,
Aerts, J.G.J.V., Dingemans, A.M.C., Lolkema, M.P., Paats, M.S. & Dubbink, H.J.
(2020). Highly accurate DNA-based detection and treatment results of MET exon 14
skipping mutations in lung cancer. Lung Cancer, 140:46-54.
DOI:10.1016/j.lungcan.2019.11.010.

Pudełek, M., Catapano, J., Kochanowski, P., Mrowiec, K., Janik-Olchawa, N., Czyż, J. &
Ryszawy, D. (2019). Therapeutic potential of monoterpene α-thujone, the main
compound of Thuja occidentalis L. essential oil, against malignant glioblastoma
multiforme cells in vitro. Fitoterapia, 134:172-181. DOI:10.1016/j.fitote.2019.02.020.

Puggioni, F., Alves-Correia, M., Mohamed, M-F., Stomeo, N., Mager, R., Marinoni, M.,
Racca, F., Paoletti, G., Varricchi, G., Giorgis, V., Melioli, G., Canonica, G.W. & Heffler,
E. (2019). Immunostimulants in respiratory diseases: Focus on Pidotimod.
Multidisciplinary Respiratory Medicine, 14:31. DOI:10.1186/s40248-019-0195-2.

Putora, P.M., De Ruysscher, D., Glatzer, M., Widder, J., Van Houtte, P., Troost, E.G.C.,
Slotman, B.J., Ramella, S., Pöttgen, C., Peeters, S., Nestle, U., McDonald, F., Le
Pechoux, C., Dziadziuszko, R., Belderbos, J. & Faivre-Finn, C. (2020). The role of
postoperative thoracic radiotherapy and prophylactic cranial irradiation in early stage
small cell lung cancer: Patient selection among ESTRO experts. Radiotherapy and
Oncology, 145:45-48. DOI:10.1016/j.radonc.2019.11.022.

Quirk, B.J., Brandal, G., Donlon, S., Vera, J.C., Mang, T.S., Foy, A.B., Lew, S.M.,
Girotti, A.W., Jogal, S., LaViolette, P.S., Connelly, J.M. & Whelan, H.T. (2015).
Photodynamic therapy (PDT) for malignant brain tumors – Where do we stand?
Photodiagnosis and Photodynamic Therapy, 12(3):530-544.
DOI:10.1016/j.pdpdt.2015.04.009.

100
Quist, M., Langer, S.W., Lillelund, C., Winther, L., Laursen, J.H., Christensen, K.B.,
Rørth, M. & Adamsen, L. (2020). Effects of an exercise intervention for patients with
advanced inoperable lung cancer undergoing chemotherapy: A randomized clinical trial.
Lung Cancer, 145:76-82. DOI:10.1016/j.lungcan.2020.05.003.

Rajdev, K., Siddiqui, A.H., Ibrahim, U., Patibandla, P., Khan, T. & El-Sayegh, D. (2018).
An unusually aggressive large cell carcinoma of the lung: Undiagnosed until autopsy.
Cureus, 10(2):e2202. DOI:10.7759/cureus.2202.

Relton, C., Cooper, K., Viksveen, P., Fibert, P. & Thomas, K. (2017). Prevalence of
homeopathy use by the general population worldwide: A systematic review.
Homeopathy, 106(2):69-78. DOI:10.1016/j.homp.2017.03.002.

Renoux, H. (2018). Methodology, importance, aims and objectives of provings carried

out by homeopathic students. La Revue d'Homéopathie, 9(1):e1-3.
DOI:10.1016/j.revhom.2018.01.022.

Reyes-Gibby, C.C., Wang, J., Spitz, M., Wu, X., Yennurajalingam, S. & Shete S. (2013).
Genetic variations in interleukin-8 and interleukin-10 are associated with pain,
depressed mood, and fatigue in lung cancer patients. Journal of Pain and Symptom
Management, 46(2):161-172. DOI:10.1016/j.jpainsymman.2012.07.019.

Rodrigues dos Santos, M. & Mendes, C.S.N. (2020). Traditional & complementary
medicine legislation in Portugal- Progress, challenges and flaws. European Journal of
Integrative Medicine, 35:101104. DOI:10.1016/j.eujim.2020.101104.

Romm, A., Ganora, L., Hoffman, D., Yarnell, E., Abascal, K. & Coven, M. (2010).
Fundamental Principles of Herbal Medicine. Chapter 3 in Botanical Medicine for
Women's Health. Saint Louis: Churchill Livingston. DOI:10.1016/B978-0-443-07277-
2.00003-9.

101
Rulach, R., McLoone, P., Lumsden, G., McKay, S., MacLaren, V., Macphee, J., Moore,
K., Omand, M., Sproule, M., Currie, S., Aitken, A., Ferguson, R., Valentine, R., Houston,
P., Harrow, S. & Hicks, J. (2020). Toxicity and efficacy of stereotactic ablative body
radiotherapy for moderately central non-small cell lung cancers using 50 Gy in five
fractions. Clinical Oncology, 32(4)250-258. DOI:10.1016/j.clon.2019.09.055.

Ryan, C. & Burke, L. (2017). Pathology of lung tumours. Surgery, 35(5):234-242.

DOI:10.1016/j.mpsur.2017.02.002.

Saha, S., Bhattacharjee, P., Mukherjee, S., Mazumdar, M., Chakraborty, S., Khurana,
A., Nayak, D., Manchanda, R., Chakrabarty, R., Das, T. & Sa, G. (2014). Contribution of
the ROS-p53 feedback loop in thuja-induced apoptosis of mammary epithelial
carcinoma cells. Oncology Reports, 31(4):1589-1598. DOI:10.3892/or.2014.2993.

Sak, K. (2012). Chemotherapy and dietary phytochemical agents. Chemotherapy

Research and Practice, 282570. DOI:10.1155/2012/282570.

Salehi-Rad, R., Li, R., Paul, M.K., Dubinett, S.M. & Liu, B. (2020). The Biology of Lung
Cancer: Development of more effective methods for prevention, diagnosis, and
treatment. Clinics in Chest Medicine, 41(1):25-38. DOI:10.1016/j.ccm.2019.10.003.

Sarode, P., Mansouri, S., Karger, A., Schaefer, M.B., Gimminger, F., Seeger, W. &
Savai, R. (2019). Epithelial cell plasticity defines heterogeneity in lung cancer. Cellular
Signalling, 65:109463. DOI:10.1016/j.cellsig.2019.109463.

Savini, A., Berardi, R., Mazzanti, P., Caramanti, M., Santoni, M., Lisa, M.D., Morgese,
F., Rinaldi, S., Torniai, M., Fiordoliva, I., Onofri, A. & Cascinu, S. (2015). Squamous cell
carcinoma of the lung: Clinical criteria for treatment strategy. Journal of Cancer
Metastasis and Treatment, 1:90-93. DOI:10.4103/2394-4722.157974.

Schaberle, F.A. (2018). Assessment of the actual light dose in photodynamic therapy.
Photodiagnosis and Photodynamic Therapy, 23:75-77.
DOI:10.1016/j.pdpdt.2018.06.009.

102
Scheepmaker, M.M. & Gower, N.T. (2011).The quality of selected South African and
international homeopathic mother tinctures. African Journal of Traditional,
Complementary, and Alternative Medicines, 8(5):46-52. DOI:10.4314/ajtcam.v8i5S.14.

Schieber, M. & Chandel, N.S. (2014). ROS function in redox signaling and oxidative
stress. Current Biology, 24(10):453-462. DOI:10.1016/j.cub.2014.03.034.

Schmidt, J.M. (2020). Sustainability as a challenge to therapeutics – The

Hahnemannian and Gandhian approach. Explore, 16(4):237-241.
DOI:10.1016/j.explore.2019.11.009.

Şenel, E. (2019). Evolution of homeopathy: A scientometric analysis of global

homeopathy literature between 1975 and 2017. Complementary Therapies in Clinical
Practice, 34:165-173. DOI:10.1016/j.ctcp.2018.11.018.

Senge, M.O. (2012). mTHPC – A drug on its way from second to third generation
photosensitizer? Photodiagnosis and Photodynamic Therapy, 9(2):170-179.
DOI:10.1016/j.pdpdt.2011.10.001.

Shafirstein, G., Battoo, A., Harris, K., Baumann, H., Gollnick, S.O., Lindenmann, J. &
Nwogu, C.E. (2016). Photodynamic therapy of non-small cell lung cancer. Narrative
review and future directions. Annals of the American Thoracic Society, 13(2):265-275.
DOI:10.1513/AnnalsATS.201509-650FR.

Shah, D.R. & Gregory, A. (2019). Precision medicine in lung cancer treatment. Surgical
Oncology Clinics of North America, 29(1):15-21. DOI:10.1016/j.soc.2019.08.002.

Shah, R. (2016). Standardization of the potentizing machine and quantification of impact

of potentization. Indian Journal of Research in Homeopathy, 10(2):126-132.
DOI:10.4103/0974-7168.183879.

Shen, L., Zhou, T., Fan, Y., Chang, X., Wang, Y., Sun, J., Xing, L. & Jiang, H. (2020).
Recent progress in tumor photodynamic immunotherapy. Chinese Chemical Letters,
31(7):1709-1716. DOI:10.1016/j.cclet.2020.02.007.

103
Shenoy, S. (2020). Cell plasticity in cancer: A complex interplay of genetic, epigenetic
mechanisms and tumor micro-environment. Surgical Oncology, 34:154-162.
DOI:10.1016/j.suronc.2020.04.017.

Shi, S., Cho, H., Sun, Q., He, Y., Ma, G., Kim, Y., Kim, B. & Kim, O. (2019).
Acanthopanacis cortex extract: A novel photosensitizer for head and neck squamous
cell carcinoma therapy. Photodiagnosis and Photodynamic Therapy, 26:142-149.
DOI:10.1016/j.pdpdt.2019.02.020.

Shrestha, A., Martin, C., Burton, M., Walters, S., Collins, K. & Wyld, L. (2019). Quality
of life versus length of life considerations in cancer patients: A systematic literature
review. Psychooncology, 28(7):1367-1380. DOI:10.1002/pon.5054.

Siegel, R.L., Miller, K.D. & Jemal, A. (2019). Cancer statistics, 2019. A Cancer Journal
for Clinicians, 69(1):7-34. DOI:10.3322/caac.21551.

Siewert, B. & Stuppner, H. (2019). The photoactivity of natural products – An

overlooked potential of phytomedicines? Phytomedicine, 60:152985.
DOI:10.1016/j.phymed.2019.152985.

Silva, I.S., Nicolau, L.A.D., Sousa, F.B.M., Araújo, S.D., Oliveira, A.P., Araújo, T.S.L.,
Souza, L.K.M., Martins, C.S., Aquino, P.E.A., Carvalho, L.L., Silva, R.O., Rolim-Neto,
P.J. & Medeiros, J.V.R. (2017). Evaluation of anti-inflammatory potential of aqueous
extract and polysaccharide fraction of Thuja occidentalis Linn. in mice. International
Journal of Biological Macromolecules, 105(1):1105-1116.
DOI:10.1016/j.ijbiomac.2017.07.142.

Singh, S., Kumar, R., Karwasra, R., Kalra, P., Rani, S., Nayak, D. & Gupta, Y.K. (2014).
Evaluation of safety profile of homeopathic mother tinctures. Indian Journal of Research
in Homeopathy, 8(2):81-86. DOI:10.4103/0974-7168.135640.

Siveen, K.S. & Kuttan, G. (2011). Augmentation of humoral and cell mediated immune
responses by Thujone. International Immunopharmacology, 11(12):1967-1975.
DOI:10.1016/j.intimp.2011.08.006.

104
Song, L., Wang, G., Hou, X., Kala, S., Qiu, Z., Wong, K.F., Cao, F. & Sun, L. (2020).
Biogenic nanobubbles for effective oxygen delivery and enhanced photodynamic
therapy of cancer. Acta Biomaterialia, 108:313-325. DOI:10.1016/j.actbio.2020.03.034.

Soo, R.A., Kubo, A., Ando, M., Kawaguchi, T., Ahn, M. & Ou, S.I. (2017). Association
between environmental tobacco smoke exposure and the occurrence of EGFR
mutations and ALK rearrangements in never-smokers with non–small-cell lung cancer:
Analyses from a prospective multinational ETS registry. Clinical Lung Cancer,
18(5):535-542. DOI:10.1016/j.cllc.2017.01.005.

Stan, M.S., Voicu, S.N., Caruntu, S., Nica, I.C., Olah, N-K., Burtescu, R., Balta, C.,
Rosu, M., Herman, H., Hermenean, A. & Dinischiotu, A. (2019). Antioxidant and anti-
Inflammatory properties of a Thuja occidentalis mother tincture for the treatment of
ulcerative colitis. Antioxidants, 8(9):416. DOI:10.3390/antiox8090416.

Sun, C., Li, Y., Tan, Y., Zhang, H., Liang, Y., Zeng, J., Yu, J. & Zou, H. (2019). A novel
role for NFIA in restoring radiosensitivity in radioresistant NSCLC cells by
downregulating the AKT and ERK pathways. Biochemical and Biophysical Research
Communications, 515(4):558-564. DOI:10.1016/j.bbrc.2019.06.011.

Sun, Y., Zhao, D., Wang, G., Wang, Y., Cao, L., Sun, J., Jiang, Q. & He, Z. (2020).
Recent progress of hypoxia-modulated multifunctional nanomedicines to enhance
photodynamic therapy: Opportunities, challenges, and future development. Acta
pharmaceutica sinica B, 10(8):1382-1396. DOI:10.1016/j.apsb.2020.01.004.

Sunila, E.S. & Kuttan, G. (2005). Protective effect of Thuja occidentalis against
radiation-induced toxicity in mice. Integrative Cancer Therapies, 4(4):322-328.
DOI:10.1177/1534735405282251.

Sunila, E.S. & Kuttan, G. (2006). A preliminary study on antimetastatic activity of Thuja
occidentalis L. in mice model. Immunophramacology and Immunotoxicology, 28(2):269-
280. DOI:10.1080/08923970600809017.

105
Sunila, E.S., Hamsa, T.P. & Kuttan, G. (2011). Effect of Thuja occidentalis and its
polysaccharide on cell-mediated immune responses and cytokine levels of metastatic
tumor-bearing animals. Pharmaceutical Biology, 49(10):1065-1073.
DOI:10.3109/13880209.2011.565351.

Świątkowska, B., Szubert, Z., Sobala, W. & Szeszenia-Dąbrowska, N. (2015).

Predictors of lung cancer among former asbestos-exposed workers. Lung Cancer,
89(3):243-248. DOI:10.1016/j.lungcan.2015.06.013.

Tefani, M., Sansone, L., Limana, F., Arcangeli, T., De Santis, E., Polese, M., Fini, M. &
Russo, M.A. (2016). The interplay of reactive oxygen species, hypoxia, inflammation,
and sirtuins in cancer initiation and progression. Oxidative Medicine and Cellular
Longevity, 2016:3907147. DOI:10.1155/2016/3907147.

Thornton, C., Leaw, B., Mallard, C., Nair, S., Jinnai, M., & Hagberg, H. (2017). Cell
death in the developing brain after hypoxia-ischemia. Frontiers in Cellular
Neuroscience, 11:248. DOI:10.3389/fncel.2017.00248.

Timm, M., Saaby, L., Moesby, L., & Hansen, E. W. (2013). Considerations regarding
use of solvents in in vitro cell based assays. Cytotechnology, 65(5):887-894.
DOI:10.1007/s10616-012-9530-6.

Tohme, S., Simmons, R.L., & Tsung, A. (2017). Surgery for cancer: A trigger for
metastases. Cancer research, 77(7):1548–1552. DOI:10.1158/0008-5472.CAN-16-
1536.

Travis, W.D. (2020). Lung cancer pathology: Current concepts. Clinics in Chest
Medicine, 41(1):67-85. DOI:10.1016/j.ccm.2019.11.001.

Unlu, A., Kirca, O. & Ozdogan, M. (2017). Homeopathy and cancer. Journal of
Oncological Sciences, 3(2):77-80. DOI:10.1016/j.jons.2017.05.006.

106
Uttra, A.M., Ahsan, H., Hasan, U.H. & Chaudhary, M.A. (2018). Traditional medicines of
plant origin used for the treatment of inflammatory disorders in Pakistan: A review.
Journal of Traditional Chinese Medicine, 38(4):636-656. DOI:10.1016/S0254-
6272(18)30897-5.

van Eeden, R., Tunmer, M., Geldenhuys, A., Nayler, S. & Rapoport, B.L. (2020). Lung
cancer in South Africa. Journal of Thoracic Oncology, 15(1):22-28.
DOI:10.1016/j.jtho.2019.06.032.

van Meerbeeck, J.P., Fennell, D.A. & De Ruysscher, D.K. (2011). Small-cell lung
cancer. Lancet, 378(9804):1741-1755. DOI:10.1016/S0140-6736(11)60165-7.

Vázquez-Ortega, F., Lagunes, I. & Trigos, Á. (2020). Cosmetic dyes as potential

photosensitizers of singlet oxygen generation. Dyes and Pigments, 176:108248.
DOI:10.1016/j.dyepig.2020.108248.

Vermeulen, F. (2015). Concordant Reference. Saltire Books Ltd, Glascow, Scotland.

2068-2079.

Villacorta, R.B., Roque, K.F.J., Tapang, G.A. & Jacinto, S.D. (2017). Plant extracts as
natural photosensitizers in photodynamic therapy: In vitro activity against human
mammary adenocarcinoma MCF-7 cells. Asian Pacific Journal of Tropical Biomedicine,
7(4):358-366. DOI:10.1016/j.apjtb.2017.01.025.

Vojtíšek, R. (2019). Cardiac toxicity of lung cancer radiotherapy. Reports of Practical

Oncology & Radiotherapy, 25(1):13-19. DOI:10.1016/j.rpor.2019.10.007.

Walter, F., Rubin, G., Bankhead, C., Morris, H.C., Hall, N., Mills, K., Dobson, C.,
Rintoul, R.C., Hamilton, W. & Emery, J. (2015). Symptoms and other factors associated
with time to diagnosis and stage of lung cancer: A prospective cohort study. British
Journal of Cancer, 112(1):6-13. DOI:10.1038/bjc.2015.30.

107
Wang, M., Zhao, J., Zhang, L., Wei, F., Lian, Y., Wu, Y., Gong, Z., Zhang, S., Zhou, J.,
Cao, K., Li, X., Xiong, W., Li, G., Zeng, Z. & Guo, C. (2017). Role of tumor
microenvironment in tumorigenesis. Journal of Cancer, 8(5):761-773.
DOI:10.7150/jca.17648.

Wang, N., Mengersen, K., Tong, S., Kimlin, M., Zhou, M., Wang, L., Yin, P., Xu, Z.,
Cheng, J., Zhang, Y. & Hu, W. (2019). Short-term association between ambient air
pollution and lung cancer mortality. Environmental Research, 179:108748.
DOI:10.1016/j.envres.2019.108748.

Watson, T. & Goh, A. (2015). Dose dependency with electro physical agents: Both the
arndt schulz law and the goldilocks principle provide an explanatory
model. Physiotherapy, 101:e1615-1616. DOI:10.1016/j.physio.2015.03.1630.

Weijer, R., Broekgaarden, M., Kos, M., van Vught, R., Rauws, E.A.J., Breukink, E., van
Gulik, T.M., Storm, G. & Heger, M. (2015). Enhancing photodynamic therapy of
refractory solid cancers: Combining second-generation photosensitizers with multi-
targeted liposomal delivery. Journal of Photochemistry and Photobiology C:
Photochemistry Reviews, 23:103-131. DOI:10.1016/j.jphotochemrev.2015.05.002.

Weinberg, B.D., Allison, R.R., Sibata, C., Parent, T. & Downie, G. (2010). Results of
combined photodynamic therapy (PDT) and high dose rate brachytherapy (HDR) in
treatment of obstructive endobronchial non-small cell lung cancer (NSCLC). (2010).
Photodiagnosis and Photodynamic Therapy, 7(1):50-58.
DOI:10.1016/j.pdpdt.2009.12.002.

Wimmer, K., Sachet, M. & Oehler, R. (2020). Circulating biomarkers of cell death.
Clinica Chimica Acta, 500:87-97. DOI:10.1016/j.cca.2019.10.003.

Witt, C.M., Balneaves, L.G., Cardoso, M.J., Cohen, L., Greenlee, H., Johnstone, P.,
Kücük, O., Mailman, J. & Mao, J.J. (2017). A comprehensive definition for integrative
oncology. JNCI Monographs, 2017(52). November 2017, DOI:
10.1093/jncimonographs/lgx012.

108
World health organization. (2019). Tobacco. Available from: https://www.who.int/news-
room/fact-sheets/detail/tobacco. (Accessed 17 January 2020).

World Health Organization. (2009). Safety issues in the preparation of homeopathic

medicines. Available from:
https://apps.who.int/iris/bitstream/handle/10665/44238/9789241598842_eng.pdf;jsessio
nid=7241E7326513B7B82DA8BCA96C0B91B0?sequence=1. (Accessed 3 November
2019).

World Health Organization. (2019). Global report on traditional and complementary

medicine. Available from:
https://apps.who.int/iris/bitstream/handle/10665/312342/9789241515436-eng.pdf?ua=1.
(Accessed 3 November 2019).

Wu, Y., Dong, G. & Sheng, C. (2020). Targeting necroptosis in anticancer therapy:
Mechanisms and modulators. Acta Pharmaceutica Sinica B, 10(9):1601-1618.
DOI:10.1016/j.apsb.2020.01.007.

Xie, J., Zhang, W., Liang, X., Shuai, C., Zhou, Y., Pan, H., Yang, Y. & Han, W. (2020).
RPL32 promotes lung cancer progression by facilitating p53 degradation. Molecular
Therapy. Nucleic Acids, 21:75-85. DOI:10.1016/j.omtn.2020.05.019.

Xu, M., Yang, G., Bi, H., Xu, J., Feng, L., Yang, D., Sun, Q., Gai, S., He, F., Dai, Y.,
Zhong, C. & Yang, P. (2019). Combination of CuS and g-C3N4 QDs on upconversion
nanoparticles for targeted photothermal and photodynamic cancer therapy. Chemical
Engineering Journal, 360:866-878. DOI:10.1016/j.cej.2018.12.052.

Xue, D., Pan, S., Huang, G. & Qiu, J. (2020). ROS enhances the cytotoxicity of cisplatin
by inducing apoptosis and autophagy in tongue squamous cell carcinoma cells. The
International Journal of Biochemistry & Cell Biology, 122:105732.
DOI:10.1016/j.biocel.2020.105732.

Yang, D., Liu, Y., Bai, C., Wang, X. & Powell, C.A. (2020). Epidemiology of lung cancer
and lung cancer screening programs in China and the United States. Cancer Letters,
468:82-87. DOI:10.1016/j.canlet.2019.10.009.
109
Yi, G., Hong, S.H., Son, J., Yoo, J., Park, C., Choi Y., & Koo, H. (2018). Recent
advances in nanoparticle carriers for photodynamic therapy. Quantitative Imaging in
Medicine and Surgery, 8(4):433-443. DOI:10.21037/qims.2018.05.04.

Yonekawa, T. & Thorburn, A. (2013). Autophagy and cell death. Essays in biochemistry,
55:105-117. DOI:10.1042/bse0550105.

Yu, Z., Zhou, P., Pan, W., Li, N., Tang, B (2018). A biomimetic nanoreactor for
synergistic chemiexcited photodynamic therapy and starvation therapy against tumor
metastasis. Nature Communications, 2018;9(5044). DOI:10.1038/s41467-018-07197-8.

Yun, C.W. & Lee, S.H. (2018). The roles of autophagy in cancer. International Journal of
Molecular Sciences, 19(11):3466. DOI:10.3390/ijms19113466.

Zappa, C. & Mousa, S.A. (2016). Non-small cell lung cancer: Current treatment and
future advances. Translational Lung Cancer Research, 5(3):288-300.
DOI:10.21037/tlcr.2016.06.07.

Zargar, A., Chang, S., Kothari, A., Snijders, A.M., Mao, J., Wang, J., Hernández, A.C.,
Keasling, J.D. & Bivona, T.G. (2019). Overcoming the challenges of cancer drug
resistance through bacterial-mediated therapy. Chronic Diseases and Transitional
Medicine, 5(4):258-266. DOI:10.1016/j.cdtm.2019.11.001.

Zhang, C., Qin, W., Bai, X. & Zhang, X. (2020). Nanomaterials to relieve tumor hypoxia
for enhanced photodynamic therapy. Nanotoday, 35:100960.
DOI:10.1016/j.nantod.2020.100960.

Zhang, J., Jiang, C., Figueiró Longo, J.P., Azevedo, R.B., Zhang, H. & Muehlmann, L.A.
(2018). An updated overview on the development of new photosensitizers for anticancer
photodynamic therapy. Acta Pharmaceutica Sinica B, 8(2):137-146.
DOI:10.1016/j.apsb.2017.09.003.

110
Zhang, S., Bai, X. & Shan, F. (2020). The progress and confusion of anti-PD1/PD-L1
immunotherapy for patients with advanced non-small cell lung cancer. International
Immunopharmacology, 80:106247. DOI:10.1016/j.intimp.2020.106247.

Zhang, T., Bao, J., Zhang, M., Ge, Y., Wei, J., Li, Y., Wang, W., Li, M. & Jin, Y. (2020).
Chemo-photodynamic therapy by pulmonary delivery of gefitinib nanoparticles and 5-
aminolevulinic acid for treatment of primary lung cancer of rats. Photodiagnosis and
Photodynamic Therapy, 31:101807. DOI:10.1016/j.pdpdt.2020.101807.

Zhao, X., Shen, R., Bao, L., Wang, C. & Yuan, H. (2020). Chitosan derived glycolipid
nanoparticles for magnetic resonance imaging guided photodynamic therapy of cancer.
Carbohydrate Polymers, 245:116509. DOI:10.1016/j.carbpol.2020.116509.

Zhen, S., Yi, X., Zhao, Z., Lou, X., Xia, F. & Tang, B.Z. (2019). Drug delivery micelles
with efficient near-infrared photosensitizer for combined image-guided photodynamic
therapy and chemotherapy of drug-resistant cancer. Biomaterials, 218:119330.
DOI:10.1016/j.biomaterials.2019.119330.

Zheng, Y., Li, Z., Chen, H. & Gao, Y. (2020). Nanoparticle-based drug delivery systems
for controllable photodynamic cancer therapy. European Journal of Pharmaceutical
Sciences, 144:105213. DOI:10.1016/j.ejps.2020.105213.

Zhou, H., Dong, Y., Ma, X., Xu, J. & Xu, S. (2020). Development of a novel truncated
deguelin derivative possessing nitric oxide donor as a potential anti-lung cancer agent.
Fitoterapia, 146:104670. DOI:10.1016/j.fitote.2020.104670.

Zhou, Y., Tozzi, F., Chen, J., Fan, F., Xia, L., Wang, J., Gao, G., Zhang, A., Xia, X.,
Brasher, H., Widger, W., Ellis, L.M. & Weihua, Z. (2012). Intracellular ATP levels are a
pivotal determinant of chemoresistance in colon cancer cells. Cancer Research, 72(1).
DOI: 10.1158/0008-5472.CAN-11-1674.

111
APPENDIX A
Letter of permission to work in the laboratory

21 August 2019

TO WHOM IT MAY CONCERN

This letter serves to confirm that Ms AYESHA LOONAT (student No: 201575769) from
Department of Complementary medicine (Homoeopathy) is permitted to carry out her
research project with the Laser Research Centre, Faculty of Health Sciences, University
of Johannesburg. Her project proposal entitled ‘Photodynamic effects of Thuja
occidentalis homeopathic mother tincture on lung cancer cells’ has been approved by
the HDC and is ready to proceed with lab work. She will be provided with all the lab and
research facilities. All the ethical obligations will be followed as per Laser Research
Centre’s Standard Operating Procedures.

Sincerely,

Prof. Heidi Abrahamse

Director: Laser Research Centre
NRF SARChI Chair: Laser Applications in Health
Faculty of Health Sciences
University of Johannesburg
P.O.Box 17011, Doornfontein, 2028
Room 5307, John Orr Building
Tel. (+27) 11 559 6550; Fax. (+27) 11 559 6448
Email: habrahamse@uj.ac.za; www.uj.ac.za/lrc

112
APPENDIX B

Diagrammatic representation of project plan

Title: PHOTODYNAMIC EFFECTS OF T. OCCIDENTALIS HOMEOPATHIC MOTHER

TINCTURE ON LUNG CANCER CELLS

FLOW DIAGRAM

Culture and maintenance of

A549 cells

Dose optimization of T. occidentalis Ø

T. occidentalis Ø dose response on A549 cells

Group I: Control Group II: Dose 1 Group II: Dose 2 Group II: Dose 3
(Untreated) (5 µL) (10 µL) (15 µL)

ATP, LDH, viability and cell

and nuclear morphological
analyses

T. occidentalis Ø mediated PDT

Group I: Group III Group IV Group IV Group IV

2
Untreate Irradiation only Fluency: 5 J/cm Fluency: 5 Fluency: 5
2 2 2
d control Fluency: 5 J/cm + Dose 1 J/cm + Dose 2 J/cm + Dose 3
Wavelength: Wavelength: 660 Wavelength: Wavelength:
660 nm nm 660 nm 660 nm

ATP, LDH, viability and cell

and nuclear morphological
analyses

113
APPENDIX C

Higher Degrees Committee clearance form

114
APPENDIX D

Ethics clearance form

115
APPENDIX E
List of consumables

Product name Supplier/Manufacturer Catalogue

number
96-well clear microplates Sigma-Aldrich 655161
96-well white microplates Sigma-Aldrich 655074
A549 lung cancer cell line ATCC CCL-185
Absolute ethanol Sigma-Aldrich 32221
Amphotericin B Sigma-Aldrich A2942
Biofreezing medium Biochrom GmbH F2270
Cell Titer-Glo® Cell Viability Assay Promega G7570
Coverslips Lasec CG88
Cryogenic vials (2 mL) Corning, The Scientific Group CR430489
CytoTox 96® Non-Radioactive Promega G400
Cytotoxicity Assay
Disposable cell counting slides Invitrogen C10228
Disposable pipette (1 mL) Becton Dickinson BD357522
Disposable pipette (10 mL) Becton Dickinson BD357551
Disposable pipette (2 mL) Becton Dickinson BD357507
Disposable pipette (5 mL) Becton Dickinson BD357543
Eppendorf® microtube (1.5 mL) Sigma-Aldrich Z666505
Eppendorf® microtube (500 μL) Sigma-Aldrich Z666521
Falcon® centrifuge tube (50 mL) Corning, The Scientific Group CR430829
FluoromountTM Aqueous Mounting Sigma-Aldrich F4680
Medium
Foetal Bovine Serum Gibco, Life Technologies 306-00301
Hanks’ Balanced Salt Solution Invitrogen 10-543F
Heavy tin foil Pick n Pay 60010071626
03
Hoechst 33258 Invitrogen H1398
Latex gloves powder free Corning, The Scientific Group EV/40511
Penicillin-Streptomycin Sigma-Aldrich P4333
Phosphate buffered saline Sigma-Aldrich P3744
Roswell Park Memorial Institute 1640 Sigma-Aldrich R8758
Medium
Thuja occidentalis Ø Fusion Homeopathics U4571
Tissue culture flask 175 cm2 Corning, The Scientific Group 423052
Tissue culture flask 25 cm2 Corning, The Scientific Group 4306414
Tissue culture flask 75 cm2 Corning, The Scientific Group 431080
Tissue culture plates 3.4 cm diameter Corning, The Scientific Group 430165
Trypan blue dye Sigma-Aldrich T8154
TrypLE™ Express Invitrogen 12605-028
Universal fit pipette tips (1- 200 μL) Corning, The Scientific Group CR4798
Universal fit pipette tips (100- 1000 Corning, The Scientific Group CR4868
μL)

116
APPENDIX F

List of equipment

Product name Supplier/Manufacturer Catalogue

number
Barnstead™ Smart2Pure™ Water Thermo Scientific 50129688
Purification System
Biological Safety Cabinet Airvolution -
Countess™ Automated cell counter ThermoFischer AMQAX1000
Digital camera OLYMPUS SC30
FieldMate Power meter National Laser Centre 125JO7R
Fluorescent microscope Carl Zeiss Axio observer
Z1
Heracell™ 150i CO2 Incubator Thermo Scientific 51026280
Heraeus™ Fresco™ 17 Thermo Scientific 75002402
Microcentrifuge
Heraeus™ Multifuge™ X1 Thermo Scientific 75004250
Centrifuge Series
Inverted light microscope Wirsam Olympus
CKX41
Laser (660 nm) National Laser Centre FC-655-300-
MM2-SMA-
1340
TW 20 Water Bath Labotec Julabo TW 20
Victor3™ multilabel plate reader Perkin Elmer 1420

117
APPENDIX G
Calculations

G1. Calculation to determine the total amount of cells in suspension

A Countess™ Automated Cell counter was used to determine the number of viable cells
per mL. The total number of cells in suspension was calculated as follows:

𝑻𝒐𝒕𝒂𝒍 𝒄𝒆𝒍𝒍𝒔 𝒊𝒏 𝒔𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏 = 𝑻𝒐𝒕𝒂𝒍 𝒄𝒆𝒍𝒍𝒔 𝒑𝒆𝒓 𝒎𝑳 × 𝑺𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏 𝒗𝒐𝒍𝒖𝒎𝒆 (𝒎𝑳)

G2. Calculation to determine seeding ratios in flasks

Once flasks reached confluency, cells were detached and seeded in the next culture
flask as follows:

1) Determine the seeding ratio:

𝑻𝒐𝒕𝒂𝒍 𝒄𝒆𝒍𝒍𝒔 𝒊𝒏 𝒔𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏
𝑪𝒆𝒍𝒍 𝒔𝒆𝒆𝒅𝒊𝒏𝒈 𝒓𝒂𝒕𝒊𝒐 =
𝑷𝒓𝒐𝒑𝒐𝒔𝒆𝒅 𝒔𝒆𝒆𝒅𝒊𝒏𝒈 𝒅𝒆𝒏𝒔𝒊𝒕𝒚

2) Determine the volume of cells to seed from cell suspension:

𝑺𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏 𝒗𝒐𝒍𝒖𝒎𝒆 (𝒎𝑳)
𝑽𝒐𝒍 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒕𝒐 𝒔𝒆𝒆𝒅 𝒇𝒓𝒐𝒎 𝒄𝒆𝒍𝒍 𝒔𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏 (𝒎𝑳) =
𝑪𝒆𝒍𝒍 𝒔𝒆𝒆𝒅𝒊𝒏𝒈 𝒓𝒂𝒕𝒊𝒐

G3. Calculation to determine cell seeding density for culture plates

The following calculation can be used to obtain a seeding density of 5 × 10 5 cells per
culture plate:

1) Determine the seeding ratio:

𝑻𝒐𝒕𝒂𝒍 𝒄𝒆𝒍𝒍𝒔 𝒊𝒏 𝒔𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏
𝑪𝒆𝒍𝒍 𝒔𝒆𝒆𝒅𝒊𝒏𝒈 𝒓𝒂𝒕𝒊𝒐 =
𝑷𝒓𝒐𝒑𝒐𝒔𝒆𝒅 𝒔𝒆𝒆𝒅𝒊𝒏𝒈 𝒅𝒆𝒏𝒔𝒊𝒕𝒚

2) Determine the volume of cells to seed from cell suspension:

𝑺𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏 𝒗𝒐𝒍𝒖𝒎𝒆 (𝒎𝑳)

𝑽𝒐𝒍 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒕𝒐 𝒔𝒆𝒆𝒅 𝒇𝒓𝒐𝒎 𝒄𝒆𝒍𝒍 𝒔𝒖𝒔𝒑𝒆𝒏𝒔𝒊𝒐𝒏 (𝒎𝑳) =
𝑪𝒆𝒍𝒍 𝒔𝒆𝒆𝒅𝒊𝒏𝒈 𝒓𝒂𝒕𝒊𝒐
118
G4. Laser irradiation calculations

To obtain a fluency of 5 J/cm2, the duration of laser irradiation at a wavelength of 660nm

was calculated using the following formulae:

𝑭𝒍𝒖𝒆𝒏𝒄𝒚 (𝑱⁄𝒄𝒎𝟐 ) = 𝒕𝒊𝒎𝒆(𝒔) × [𝒑𝒐𝒘𝒆𝒓 𝒐𝒖𝒕𝒑𝒖𝒕 (𝑾)⁄𝑺𝒖𝒓𝒇𝒂𝒄𝒆 𝒂𝒓𝒆𝒂 (𝒄𝒎𝟐 )]

1) A power meter (FieldMate) was used to determine the power output (W) of the
660nm laser at bench level in the dark. The power output for the 660nm laser
was 87.6 mW.

2) Calculate the intensity of the laser:

𝑷𝒐𝒘𝒆𝒓 𝒐𝒖𝒕𝒑𝒖𝒕 (𝒎𝑾)

𝑰𝒏𝒕𝒆𝒏𝒔𝒊𝒕𝒚 (𝒎𝑾⁄𝒄𝒎𝟐 ) =
𝑺𝒖𝒓𝒇𝒂𝒄𝒆 𝒂𝒓𝒆𝒂 (𝒄𝒎𝟐 )

𝑷𝒐𝒘𝒆𝒓 𝒐𝒖𝒕𝒑𝒖𝒕 (𝒎𝑾) × 𝟒

=
𝝅(𝒅𝒊𝒂𝒎𝒆𝒕𝒆𝒓)𝟐
𝟖𝟕. 𝟔 𝒎𝑾 × 𝟒
=
𝝅(𝟑. 𝟒 𝒄𝒎)𝟐

= 𝟗. 𝟔𝟓 𝒎𝑾/𝒄𝒎𝟐

3) Convert the intensity (mW/cm2) to (W/cm2):

𝟐)
𝑰𝒏𝒕𝒆𝒏𝒔𝒊𝒕𝒚 (𝒎𝑾⁄𝒄𝒎𝟐 )
𝑰𝒏𝒕𝒆𝒏𝒔𝒊𝒕𝒚 (𝑾⁄𝒄𝒎 =
𝟏𝟎𝟎𝟎
𝟗. 𝟔𝟓 𝒎𝑾/𝒄𝒎𝟐
=
𝟏𝟎𝟎𝟎
= 𝟗. 𝟔𝟓 × 𝟏𝟎−𝟑 𝑾/𝒄𝒎𝟐

119
4) Calculate the time needed to deliver a fluence of 5 J/cm 2 to a culture plate
requiring irradiation:
𝑭𝒍𝒖𝒏𝒆𝒄𝒚 (𝑱⁄𝒄𝒎𝟐 )
𝑻𝒊𝒎𝒆 (𝒔) 𝒑𝒆𝒓 𝒑𝒍𝒂𝒕𝒆 =
𝑰𝒏𝒕𝒆𝒏𝒔𝒊𝒕𝒚 (𝑾⁄𝒄𝒎𝟐 )

𝟓 𝑱/𝒄𝒎𝟐
=
𝟗. 𝟔𝟓 × 𝟏𝟎−𝟑 𝑾⁄𝒄𝒎𝟐

= 𝟓𝟏𝟖. 𝟏𝟑 𝒔

5) Convert time in seconds to minutes (min) and seconds (s):

𝟓𝟏𝟖. 𝟏𝟑 𝒔
𝑻𝒊𝒎𝒆 (𝒎𝒊𝒏𝒖𝒕𝒆𝒔 𝒂𝒏𝒅 𝒔𝒆𝒄𝒐𝒏𝒅𝒔) =
𝟔𝟎
= 𝟗 𝒎𝒊𝒏 𝒂𝒏𝒅 𝟑 𝒔 𝒑𝒆𝒓 𝒑𝒍𝒂𝒕𝒆

120
APPENDIX H

Certificate of analysis for T. occidentalis Ø

121
APPENDIX I

Certificate of analysis for A549 cells

122
123
APPENDIX J
Product sheet (A549 cells)

124
125
126
APPENDIX K

Graph values, standard error and statistical significance

𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑫𝒆𝒗𝒊𝒂𝒕𝒊𝒐𝒏 (𝑺𝑫)

𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑬𝒓𝒓𝒐𝒓 (𝑺𝑬) =
√𝑺𝒂𝒎𝒑𝒍𝒆 𝑺𝒊𝒛𝒆 (𝒏)

M1. ATP assay

ATP TO
C D1 D2 D3
2540812 1455735 514832 98915
2101956 1348004 316799 114120
1252028 823839 333999 65634
1301641 663834 274317 67503
1060852 286918 816556 283547
1498629 267620 759960 277534
MEAN 1625986 807658 502744 151209
SD 573427 509065 236649 101893
SE 234101 207825 96612 41598
p-value 0.003 0.000 0.000

ATP TO-PDT
C L D1 + L D2 + L D3 + L
2525358 2102333 614299 518456 87773
2617004 2633411 657233 524341 84609
2034681 2121635 518391 137288 102944
1983846 2549857 480957 140037 94078
1539588 1488161 953343 192646 43030
2085457 1763350 719351 173846 4452
MEAN 2130989 2109791 657262 281102 69481
SD 393571 441470 169548 187303 37991
SE 160675 180229 69218 76466 15510
p-value 0.756 0.000 0.000 0.000

127
M2. LDH assay

LDH TO
C D1 D2 D3
0.488 0.48 0.496 0.494
0.449 0.51 0.538 0.579
0.447 0.536 0.553 0.639
0.428 0.628 0.658 0.638
0.444 0.47 0.442 0.62
0.446 0.554 0.54 0.666
MEAN 0.45 0.53 0.538 0.606
SD 0.01995 0.05782 0.0716 0.0619
SE 0.00332 0.02147 0.028 0.0123
p-value 0.031 0.018 0.000

LDH TO-PDT
C L D1 + L D2 + L D3 + L
0,271 0.243 1.548 1.278 1.052
0.301 0.289 1.343 1.207 1.16
0.306 0.341 1.004 1.267 1.33
0.279 0.225 1.007 1.405 1.424
0.22 0.236 0.848 1.126 1.269
0.235 0.269 0.622 1.067 1.18
MEAN 0.268 0.267 1.062 1.225 1.236
SD 0.03888 0.04305 0.3349 0.1201 0.1327
SE 0.0142 0.01692 0.0975 0.0479 0.0402
p-value 0.805 0.000 0.000 0.000

128
M3. Trypan blue assay

Trypan blue TO
C D1 D2 D3
84 50 38 16
77 46 39 18
88 57 35 23
91 55 30 28
83 51 28 18
84 52 30 17
MEAN 84.5 51.833 33.333 20
SD 4.7645 3.8687 4.6332 4.6043
SE 1.9426 1.5374 1.6525 1.7062
p-value 0.000 0.000 0.000

Trypan blue TO-PDT

C L D1 + L D2 + L D3 + L
95 96 46 20 12
98 95 48 19 8
97 97 48 23 13
97 97 45 25 13
97 95 49 23 13
96 97 50 20 10
MEAN 96.6667 96.1667 47.667 21.667 11.5
SD 1.0328 0.98319 1.8619 2.3381 2.0736
SE 0.26411 0.40004 0.6854 0.8962 0.8408
p-value 0.605 0.000 0.000 0.000

Trypan blue TO-PDT

C D1 D2 D3 D4 D5 D6 D7 D8 D9
97 91 95 92 94 95 88 74 29 24
97 91 93 91 93 93 85 74 9 17
MEAN 97 91 94 91.5 93.5 94 86.5 74 19 20.5
SD 0 0 1.414 0.707 0.707 1.414 2.121 0 14.142 4.949
SE 0 0 0.289 0.144 0.144 0.289 0.433 0 2.887 1.010
p-
value 0.425 0.701 0.470 0.657 0.701 0.131 0.002 0.000 0.000

129
APPENDIX L

Biosafety clearance from ATCC to receive cells

130
APPENDIX M

Plagiarism report

ORIGINALITY REPORT

19 %
SIMILARITY INDEX
12%
INTERNET SOURCES
15%
PUBLICATIONS
5%
STUDENT
PAPERS

PRIMARY SOURCES

www.mdpi.co
m1 1
Internet Source %

Submitted to University of Johannsburg

2 1
Student Paper %

www.hindawi.co
m3 1
Internet Source %

www.laser-
therapy.us 4 1
Internet Source %

"INVITED ABSTRACTS", Journal of

1
Thoracic 5 %
Publication

131