CYTOTOXIC AND CYTOSTATIC EFFECTS

OF PHENOXODIOL ON C6, HEPG2, CNE1

AND HT-29 CANCER CELL LINES

TEN YI YANG

MASTERS OF MOLECULAR MEDICINE

INTERNATIONAL MEDICAL UNIVERSITY

MAY 2015

DEDICATION

To my mother,

For her unrelenting belief, sacrifice and dedication;

To my aunt,

For her skepticism which fuels my motivation;

To my high school biology teacher,

For sparking a passion that still remains thus,

And to my girlfriend,

Whom provides a listening ear for when I rant.

i

ABSTRACT

Background: Genistein is a phytoestrogen flavonoids found in soy, legumes and

other food products and is often lauded for its cytotoxic effect on various

cancer cell types. The synthetic sterically modified derivative of genistein,

phenoxodiol has been evaluated in phase II clinical trial in combination with

cisplatin in treating chemo-resistant ovarian cancer. Mechanistic studies show

that phenoxodiol disrupts the plasma membrane electron transport (PMET)

system through the inhibition of surface tNOX protein leading to imbalanced

NAD+ and NADH ratio. The imbalance deteriorates PMET and activates

sphingomyelinase that generates the cytotoxic ceramide that leads to apoptotic

pathways. This study investigates the cytotoxic and cytostatic activities of

phenoxodiol on (a) C6, a rat glioma cell line; (b) HepG2, a human

hepatocellular carcinoma cell line; (c) CNE1, a human highly differentiated

nasopharyngeal carcinoma cell line and (d) HT-29, a human colorectal

adenocarcinoma cell line. Methods used includes cell viability assay, cell cycle

analysis, annexin V-propidium iodide apoptosis test and morphological analysis

through ethidium bromide-acridine orange staining. Results: Results shows that

apoptosis was induced by phenoxodiol for C6 and HepG2 cell lines only and

the IC50 was found to be 1.4 g/mL and 2.0 g/mL for each cell line

respectively. Cell cycle analysis shows that G1/S arrest started after 24 hours

and increased within the next 48 hours.

ii

Morphological analysis shows that the cells portraying typical apoptotic or

necrotic signatures after the treatment of phenoxodiol. Conclusion:

Phenoxodiol is shown to be a prominent anti-cancer agent in treating brain and

liver cancer. Further studies should be carried out to confirm its effects in

animals.

iii

ACKNOWLEDGMENTS

First and foremost, I would like to thank International Medical University for

providing the research grant and facilities usage without which the project will

not be able to proceed.

I wish to express my most sincere gratitude to my supervisors, Dr. Fabian

Davamani and Dr. Ho Ket Li whom both possesses remarkable qualities as a

scientist that I wish to emulate. Their passion, guidance and discipline prove

indispensable to my growth as a fledgling scientist. I am especially grateful to

Dr. Ho for his devotion on assisting me on the project, particularly in

troubleshooting errors and pointing out my mistakes.

I am especially indebted to my fellow postgraduate colleagues, Yew Mei Yeng

and Ng Pei Ying for providing help when I needed it the most especially during

the work with flow cytometer. Ms. Yew and Ms. Ng both taught me on the

operational use of the machine and provided the basis of my protocol on flow

cytometer. A special shout out to Ms. Ng whom also provided extra reagents

when mine ran out which allows the completion of my project.

iv

I would like to extend my thanks to Dr Felicia Chung Fei Lei from the Institute

for Research, Development and Innovation- International Medical University

(IRDI-IMU) for kindly providing the cells used in this project.

Sincere thanks to the laboratory staffs of the IMU research facilities particularly

Ms. Malathi for assisting and guiding me during purchasing of project

consumables and Ms. Yong Lee Mei whom provide assistance on flow

cytometer and several others who kindly provided training for the lab

equipment usage.

And finally, I also place on record, my sense of gratitude to one and all, who

directly or indirectly, have lent their hand in this venture.

v

APPROVAL SHEETS

I, the main supervisor to Ten Yi Yang hereby certify that the dissertation

revisions have been made based on the recommendations by the Dissertation

Examination Committee on 25thOf May 2015.

_____________________________________

Dr. Fabian Amalraj Davamani

Lecturer

School of Human Biology

International Medical University

vi

I certify that an Examination Committee has conducted the final examination of

Ten Yi Yang on his name of degree dissertation entitled "Cytotoxic And

Cytostatic Effects Of Phenoxodiol On C6, HepG2, CNE1 and HT-29 Cancer

Cell Lines". The Committee recommended that the candidate be awarded the

degree of Masters of Molecular Medicine.

_______________________________

Prof Chu Wan Loy

Dean of School of Postgraduate Studies

International Medical University

vii

This Dissertation was submitted to the Senate of the International Medical

University and was accepted by the Senate as having fulfilled the requirements

for the degree of Masters of Molecular Medicine.

_______________________________

Prof Chu Wan Loy

Dean of Postgraduate Studies and Research

International Medical University

Date:

viii

DECLARATION

I hereby declare that the dissertation is based on my original work except for

quotations and citations which have been duly acknowledged. I also declare

that it has not been previously or concurrently submitted for any other degree at

the International Medical University or any other institution.

_______________

(Ten Yi Yang)

ix

TABLE OF CONTENTS

ABSTRACT ........................................................................................................ i

ACKNOWLEDGMENTS ............................................................................... iii

APPROVAL SHEETS ...................................................................................... v

LIST OF FIGURES ........................................................................................ xii

LIST OF ABBREVIATIONS ........................................................................ xv

1 INTRODUCTION ..................................................................................... 1

1.1 Background of study .......................................................................... 1

1.2 Objectives of study ............................................................................. 3

2 LITERATURE REVIEW ......................................................................... 4

2.1 Cancer ................................................................................................. 4

2.1.1 Glioma ........................................................................................... 4

2.1.2 Hepatocellular carcinoma ............................................................. 6

2.1.3 Nasopharyngeal carcinoma ........................................................... 8

2.1.4 Colorectal carcinoma .................................................................... 9

2.2 Phytoestrogens .................................................................................. 11

2.2.1 Genistein ..................................................................................... 12

2.3 Phenoxodiol ....................................................................................... 13

2.3.1 Mechanism of action ................................................................... 14

2.3.2 Other effects of phenoxodiol ...................................................... 18

2.4 Apoptosis ........................................................................................... 19

2.4.1 Apoptosis and cancer drug-discovery ......................................... 23

2.5 Cell cycle ............................................................................................ 25

2.5.1 Cell cycle regulation ................................................................... 27

2.6 Ceramide ........................................................................................... 34

2.6.1 Ceramide and apoptosis .............................................................. 35

2.6.1.1 Ceramide and the extrinsic pathway ................................... 35

2.6.1.2 Ceramide and the intrinsic pathway ................................... 36

2.6.1.3 Ceramide induced apoptotic signals .................................... 38

2.6.2 Ceramide and cell cycle .............................................................. 41

3 MATERIALS AND METHODS ............................................................ 43

x

3.1 Materials ........................................................................................... 43

3.2 Methods ............................................................................................. 46

3.2.1 Preparation of culture media and solutions ................................. 46

3.2.1.1 DMEM .................................................................................... 46

3.2.1.2 PBS .......................................................................................... 47

3.2.1.3 Trypsin-EDTA ....................................................................... 47

3.2.1.4 Phenoxodiol and Cycloheximide .......................................... 47

3.2.1.5 MTT solution ......................................................................... 47

3.2.2 Culture media for cell lines ......................................................... 48

3.2.3 Maintaining and sub-culturing of cells ....................................... 49

3.2.4 Cell seeding ................................................................................. 50

3.2.5 Cell viability assay ...................................................................... 51

3.2.6 Preparation of cell cycle analysis reagents ................................. 53

3.2.6.1 Washing Solution 1 ................................................................ 53

3.2.6.2 Washing Solution 2 ................................................................ 53

3.2.6.3 Staining Solution ................................................................... 54

3.2.7 Cell cycle analysis ....................................................................... 54

3.2.8 Preparation of apoptosis test reagents ......................................... 56

3.2.9 Annexin V-FITC apoptosis test .................................................. 56

3.2.10 Morphological analysis by Acridine Orange (AO) and Ethidium

Bromide (EB) dual staining ....................................................................... 58

3.2.11 Statistical analysis ....................................................................... 59

4 RESULTS ................................................................................................. 60

4.1 Cell viability ...................................................................................... 60

4.2 Flow cytometer cell cycle analysis ................................................... 63

4.3 Annexin V-FITC apoptosis test ....................................................... 67

4.4 EB-AO morphological analysis ....................................................... 70

5 DISCUSSION ........................................................................................... 73

6 CONCLUSION AND FUTURE DIRECTIONS ................................... 84

REFERENCES ................................................................................................ 85

APPENDIX .................................................................................................... 106

Appendix 1: Troubleshooting ................................................................... 106

Appendix 2: Flow cytometry data ............................................................ 121

xi

LIST OF TABLES

Table 2.1: Summary of the CDK and cyclin pair involved in the regulation of

cell cycle phases. ............................................................................................... 28

Table 3.1: List of chemicals used ..................................................................... 43

Table 3.2: List of consumables used ................................................................ 44

Table 3.3: List of apparatuses used .................................................................. 45

Table 4.1: IC50 values of phenoxodiol treated against C6, HepG2, CNE1 and

HT-29 cancer cell lines. .................................................................................... 61

Table 5.1: Summary of IC50 obtained from previously reported cell lines tested

with phenoxodiol (54). * indicates the IC50 obtained from our tested cell lines.

........................................................................................................................... 74

APPENDIX

Table A - 1: Comparison between IC50 values of phenoxodiol against

cycloheximide on C6, HepG2, CNE1 and HT-29 cancer cell lines. ............... 114

xii

LIST OF FIGURES

Figure 2.1: Effects of phenoxodiol on apoptotic pathways and the cell cycle.

Arrow in red represents inhibition while arrow in green represents stimulation

........................................................................................................................... 17

Figure 2.2: A summary of the mammalian cell cycle with its respective

checkpoints. Arrow in red represents inhibition and arrow in green represents

stimulation while arrow in black indicates the approximate time point within

the cell cycle that is regulated by its respective cyclin-CDK complex. ............ 33

Figure 2.3: Ceramide production and metabolism pathway. ........................... 34

Figure 4.1: Treatment of phenoxodiol on (a) C6; (b) HepG2; (c) CNE1; and (d)

HT-29 cell lines for 24 and 48 hours. Cell viability was determined by MTT

assay. * p< 0.05 compared to untreated cells .................................................... 62

Figure 4.2: Cell cycle analysis of the effects of C6 cell line treated with 0

g/mL and 1.4 g/mL phenoxodiol for (a) 24 and (b) 48 hours; (c) shows the

cell cycle differences between 24 and 48 hours treated cells. * p < 0.05

compared to control cells, ** p < 0.05 compared between treated cells time

points. ................................................................................................................ 65

Figure 4.3: Cell cycle analysis of the effects of HepG2 cell line treated with 0

g/mL and 2.0 g/mL phenoxodiol for (a) 24 and (b) 48 hours; (c) shows the

cell cycle differences between 24 and 48 hours treated cells. * p < 0.05

compared to control cells, ** p < 0.05 compared between treated cells time

points. ................................................................................................................ 66

Figure 4.4: Annexin V Propidium Iodide flow cytometry analysis of C6 cells

treated with 0 g/mL and 1.4 g/mL phenoxodiol for 24 and 48 hours. Cells are

divided into four groups; viable, early apoptosis, late apoptosis and secondary

necrosis. * p < 0.05 compared to control cells, ** p < 0.05 compared between

treated cells time points. ................................................................................... 69

Figure 4.5: Annexin V Propidium Iodide flow cytometry analysis of HepG2

cells treated with 0 g/mL and 1.4 g/mL phenoxodiol for 24 and 48 hours.

Cells are divided into four groups; viable, early apoptosis, late apoptosis and

secondary necrosis. * p < 0.05 compared to control cells, ** p < 0.05 compared

between treated cells time points. ..................................................................... 69

Figure 4.6: Morphological analysis at 100x magnification of C6 cell line

treated with 0 g/mL and 1.4 g/mL phenoxodiol for 24 hours and 48 before

staining with 10 g/mL ethidium bromide and acridine orange. ...................... 71

Figure 4.7: Morphological analysis at 100x magnification of HepG2 cell line

treated with 0 g/mL and 2.0 g/mL phenoxodiol for 24 hours and 48 hours

before staining with 10 g/mL ethidium bromide and acridine orange............ 72

xiii

APPENDIX

Figure A - 1: Determination of minimum inhibitory concentration of DMSO

for 24 and 48 hours on (a) C6; (b) HepG2; (c) CNE1 and (d) HT-29 cell lines.

......................................................................................................................... 108

Figure A - 2: Determination of optimum seeding density for C6, HepG2,

CNE1 and HT-29 cell lines for (a) 24 and (b) 48 hours ................................. 110

Figure A - 3: Treatment of cycloheximide on (a) C6; (b) HepG2; (c) CNE1;

and (d) HT-29 cell lines for 24 and 48 hours. Cell viability were determined by

MTT assay. ..................................................................................................... 113

Figure A - 4: Unstained HepG2 cells as example in used for compensation

setup for flow cytometry apoptosis test. Cell population was adjusted so that it

falls within the lower left quadrant which does not contain either stain readings.

......................................................................................................................... 117

Figure A - 5: Annexin-V stained HepG2 cells as example in used for

compensation setup for flow cytometry apoptosis test. Cell population was

adjusted so that it falls within the lower right quadrant which contains only

Annexin V stain readings. ............................................................................... 118

Figure A - 6: Propidium iodide stained HepG2 cells as example in used for

compensation setup for flow cytometry apoptosis test. Cell population was

adjusted so that it falls within the upper right quadrant which contains only

propidium iodide stain readings. ..................................................................... 119

Figure A - 7:Annexin V and propidium iodide stained HepG2 cells as example

in used for compensation setup for flow cytometry apoptosis test. Cell

population were seen in the upper right quadrant containing both stains reading

on successful compensation setup................................................................... 120

Figure A - 8: Cell cycle profile obtained during FACS analysis of C6 cell line

treated with (a) 0 g/mL and (b) 1.4 g/mL phenoxodiol for 24 hours by

plotting cell count against DNA concentration. .............................................. 121

Figure A - 9: Cell cycle profile obtained during FACS analysis of the effects of

C6 cell line treated with (a) 0 g/mL and (b) 1.4 g/mL phenoxodiol for 48

hours by plotting cell count against DNA concentration. ............................... 122

Figure A - 10: Cell cycle profile obtained during FACS analysis of the effects

of HepG2 cell line treated with (a) 0 g/mL and (b) 2.0 g/mL phenoxodiol for

24 hours by plotting cell count against DNA concentration. .......................... 122

Figure A - 11: Cell cycle profile obtained during FACS analysis of the effects

of HepG2 cell line treated with (a) 0 g/mL and (b) 2.0 g/mL phenoxodiol for

48 hours by plotting cell count against DNA concentration. .......................... 123

Figure A - 12: Dot plot representation of Annexin V-PI stained C6 cell line

treated with (a) 0 g/mL and (b) 1.4 g/mL phenoxodiol for 24 hours. ........ 124

Figure A - 13: Dot plot representation of Annexin V-PI stained C6 cell line

treated with (a) 0 g/mL and (b) 1.4 g/mL phenoxodiol for 48 hours. ........ 125

xiv

Figure A - 14: Dot plot representation of Annexin V-PI stained HepG2 cell

line treated with (a) 0 g/mL and (b) 2.0 g/mL phenoxodiol for 24 hours. . 126

Figure A - 15: Dot plot representation of Annexin V-PI stained HepG2 cell

line treated with (a) 0 g/mL and (b) 2.0 g/mL phenoxodiol for 48 hours. . 127

xv

LIST OF ABBREVIATIONS

AIF Apoptosis inducing factor

AO Acridine orange

APC Anaphase-promoting complex

APAF Apoptotic protease activation factor

ATP Adenosine triphosphate

ATR Ataxia telangiectasia and Rad3-related protein

BAX Bcl-2-associated X protein

CAD Caspase-activated DNAse

CDK Cyclin-dependent kinases

cFLIP Cellular FLICE-like inhibitory protein

CTLA-4 Cytotoxic T-lymphocyte-associated protein 4

CXC C-X-C motif chemokine

DED Death effector domain

DISC Death inducing signaling complex

DMEM Dulbeccos Modified Eagle Medium

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

EB Ethidium bromide

EDTA Ethylenediaminetetraacetic acid

ENOX Ecto-NOX

FADD Fas-associated death domain

FLICE FADD-like IL-1-converting enzyme

GCS Glucosylceramide synthase

IAP Inhibitors of apoptosis

JNK c-Jun N-terminal kinase

MAP Mitogen-activated protein

MAPK Mitogen-activated protein kinase

xvi

MOMP Mitochondrial outer membrane permeabilization

MPF M phase-promoting factor

MPT Mitochondrial permeability transition

MTT Thiazolyl Blue Tetrazolium Blue

NADH Nicotinamide adenine dinucleotide

NF- Nuclear factor

NOX NADH-oxidase

NPC Nasopharyngeal carcinoma

PBS Phosphate buffer saline

PIDD p53-induced protein with a death domain

PI3K Phosphoinositide 3-kinase

PIP2 Phosphatidylinositol-4,5-biphosphate

PIP3 Phosphatidylinositol3,4,5trisphosphate

PKC Protein kinase C

PMET Plasma membrane electron transport

PP2A Protein phosphatase 2A

PPAR Peroxisome proliferator-activated receptor

PTK Protein tyrosine kinase

RAIDD Rip-associated protein death domain

TNF Tumor necrosis factor

TRADD TNFR-associated death domain

TRAIL Tumor necrosis factor-related apoptosis-inducing ligand

TNOX Tumor-associated NADH oxidase

VEGF Vascular endothelial growth factor

XIAP X-linked inhibitor of apoptosis protein

1

1 INTRODUCTION

1.1 Background of study

Theres a disturbing continued rising trend of global burden of cancer largely

due to the increased adoption of unhealthy cancer-causing behaviors. In this

project, focus will be made on four types of cancers which are: brain and

nervous system cancer, liver cancer, nasopharyngeal cancer and colorectal

cancer all of which are featured quite prominently in Malaysia. According to

the latest cancer statistics in Malaysia, brain and nervous system cancer is

ranked ninth of the most frequently diagnosed cancer type in males while liver,

nasopharyngeal and colorectal cancer is ranked tenth, fourth and second most

frequently diagnosed cancer types in both gender respectively.

Phenoxodiol is a synthetic sterically modified genistein derivative, a soy

isoflavone with antitumor effects on various cancer types such as breast,

prostate, lung, liver and gastric cancers. Studies found phenoxodiol to be

efficient over its parent compound and has been explored as a possible curative

against chemo-resistant ovarian cancer when used in combination with cisplatin

or paclitaxel in a phase II clinical trials. At the moment, phenoxodiol is

currently also being evaluated for its side effect when combined with docetaxel

in a phase I/II trial.

2

Phenoxodiol is shown in mechanistic studies to disrupt the plasma membrane

electron transport system leading to imbalanced NAD+ / NADH ratio triggering

sphingomyelinase activation that converts plasma membrane sphingomyelin to

ceramide, an anti-apoptotic agent.

Recent literature suggests that cytotoxicity and cytostaticity effects of

phenoxodiol have been studied extensively in vitro in several cancer cell lines

including ovarian, prostate, breast and leukemia. However, its effects on brain

glioma, hepatocarcinoma, nasopharyngeal carcinoma and colorectal cancer

have not been characterized. We hypothesized that phenoxodiol has a high

potential in inhibiting these cancer types because the molecular target of

phenoxodiol, the plasma membrane tumor specific NADH oxidase is associated

with a majority of cancer types. Besides, genistein (the parent compound of

phenoxodiol) has also been proven to work on ovary, brain, liver,

nasopharyngeal and colon cancer. Hence, it is thought that phenoxodiol will

also be effective against these cancer types with similar mechanism.

3

In this study, the antitumor effect of phenoxodiol on HepG2 (Homo sapiens

hepatocellular carcinoma), HT-29 (Homo sapiens colorectal adenocarcinoma),

CNE1 (Homo sapiens highly differentiated nasopharyngeal carcinoma) and C6

(Rattus norvegicus glioma) were tested via cell viability assay and cell cycle

analysis to evaluate the cytotoxic and cytostatic effect of phenoxodiol.

Apoptosis test and morphological analysis with ethidium bromide and acridine

orange dual staining were used to assess the apoptotic potential of phenoxodiol.

1.2 Objectives of study

The objective of this study is to:

1. determine the inhibitory effect of phenoxodiol on the viability of C6,

HepG2, CNE1 and HT-29 cancer cell lines.

2. evaluate the effect of phenoxodiol on the cell cycle of potential cell

lines.

3. determine the apoptotic effect of phenoxodiol on potential cell lines.

4

2 LITERATURE REVIEW

2.1 Cancer

Cancer, a disease exclusive to multicellular organism, is defined as the aberrant

cellular growth caused by disturbed expression in the genetic level leading to

dysregulation in cellular division and differentiation leading to imbalance in the

cell replication and death ratio which sees favorable a growth in the cancer cell

population (1). Cancer is a common disease worldwide and Malaysia is no

exception, where it was ranked third as the most common mortality cause after

pulmonary disease and septicemia.

2.1.1 Glioma

Gliomas arise from glial cells in the central nervous system encompassing the

spinal cord and the brain but have a higher occurrence in the latter. In Malaysia,

it is the tenth most frequent cancer types in males with a total of 259 cases

reported out of a total of 8123 cases and unlisted in females (2). Gliomas has a

potential to arise from all types of glial cells however, glioblastomas arising

from astrocytes is of the majority of malignant glioma accounting for 82% of

cases (3). Malignant gliomas, although highly aggressive, does not metastasize

and is largely confined to central nervous system (4).

5

Even so, the prognosis for malignant gliomas remains poor with estimated

patient survival duration between 12 to 18 months with the best treatment (5).

Overall, the 5-year relative survival rate is estimated to be at 34% (6).

Progressive genetic destabilization and changes from either naturally occurring

or various environmental factors contributes to the development of malignant

gliomas. Gender-wise, men are more susceptible than women and white

populations are more effected than black populations (7). A number of pre-

existing rare heredity syndromes such as Turcot, Cowden, type 1 and 2

neurofibromatosis, Li-Fraumeni, familial schwannomatosis and tuberous

sclerosis may increase risk of glioma development as well (8). Genome wide

association studies shows that polymorphic variants in the RTEL, TERT,

CDKN2BAS, EGFR, CCDC26 and PHLDB1 genes are associated with glioma

development albeit weakly suggesting the presence of potential multiple

molecular subsets (9).

Surgical resection is usually indicated with the aim of alleviating tumor mass

effect and the extent of surgical intervention is well evidenced to affect patient

survivability (10). Post-surgery, adjuvant radio and chemotherapy is prescribed

with intensity-modulated radiotherapy (11) and alkylating agents (12) as the

preferred methods respectively.

6

Other treatments being explored includes the monoclonal antibody ipilimumab

and bevacizumab which inhibits the immune system downregulator CTLA-4

and VEGF signalling involved in angiogenesis respectively with ipilimumab

showing enhanced patient survivability in randomized trials (13). Recent

discovery of specific cytomegalovirus antigens in glioblastoma multiforme

allows the development of adoptive immunotherapy as new treatment option

(14).

2.1.2 Hepatocellular carcinoma

Liver cancer originates from hepatocytes and it is associated primarily with

hepatitis B or C viral infection (15). Following viral infection, the

hepatocellular innate immune pathways is activatedby the release of CXCL10

chemokine which leads to the recruitment of inflammation-causing immune

effector cells designed to eliminate viral particles through binding and

activation of CXCR3 receptor found on these cells (16). However, inadequate

viral elimination in up to 85% of patients with acute viral infection leads to a

persistent presence of proinflammatory immune cells in the liver (17) and

subsequently causes nearby tissue destruction that links to hepatocarcinoma and

various liver diseases. Other causes are through chronic hepatic inflammation

due to various non-viral causes.

7

Malaysia cancer statistics shows liver cancer as the tenth most frequent cancer

type diagnosed, contributing to 605 cases or 3.3% of total cancer cases reported

in Malaysia and of all the cases, 443 cases are reported in men (2).

Conventional curative treatment type for liver cancer is effective with up to

75% 5-year survival rates (18) which includes surgical resection of liver, liver

transplantation and local ablation therapy using chemical or thermal ablation

methods. Even though the rate of effectiveness is promising, the low eligibility

of patients (less than 20%) due to reasons such as donor shortage, dysfunctional

liver or advanced hepatocarcinoma stage means the majority of liver cancer

patients are only able to opt for palliative or symptomatic treatment with a

much lower survival rate and duration (19). New treatment options are being

explored for the treatment of hepatocarcinoma such as oncolytic viral therapy

with a genetically modified poxvirus JX-594 that shows promising result in

phase I and II clinical trials (20,21).

8

2.1.3 Nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) originates from the nasopharynx which is

the top part of pharynx lying just behind the nose. In Malaysia, there is high

risk of nasopharyngeal cancer particularly in Chinese and Malay population,

contributing to a total of 940 cases and 5.2% of the overall cancer cases in

Malaysia (2) and of the total cases, 685 cases are male patients.

Genetics and environmental factors may play a role in influencing the unique

geographical incidence pattern of NPC. Probable environmental carcinogens

has been found to cause the loss of alleles on chromosome 3 and 9 short arms

which leads to inactivation of tumor suppressor genes such as p14, p15 and p16

(22).The exact carcinogens in question is yet to be pinpoint but it is speculated

that salted consumables particularly Chinese salted fish leads to NPC

development (23).

Local control of nasopharyngeal carcinoma using surgical method is not viable

as it is highly prone to metastasis to the cervical lymph node partly due to its

anatomical spot (24). Hence, NPC is mainly treated using radiotherapy.

Technological advances improve the conventional two-dimensional techniques

used to deliver radical radiotherapy.

9

When compared with two-dimensional methods, techniques such as three-

dimensional conformal and intensity-modulated radiotherapy are reported to be

superior in treating nasopharyngeal carcinoma (25). Furthermore, emerging

new techniques such as Tomotherapy and RapidArc radiotherapy improves the

dosimetric efficacy in late-stage NPC treatment (26). Radiotherapy in NPC

treatment may be effective in controlling the primary tumor (27), however, a

regimen of chemotherapy such as Cisplatinin combination with other agents

such as 5-flurouracil or with docetaxel and capecitabine is still recommended to

be prescribed together as radiotherapy alone does not inhibit lymph node

metastasis of NPC to distant sites (28).

2.1.4 Colorectal carcinoma

Colorectal cancer is the formation of cancerous tumor in parts of the large

intestine like the colon or the rectum (29). The risk of colorectal development is

approximately 5.0% in men and slightly lower (4.7%) in women with

diagnosed men having 30% to 40% higher mortality rate than women (30).

Progressively higher incidence is contributed by risk factor such as age, gender

and ethnicity (31). The exact correlation between incidence and risk factors are

yet to known but it is speculated that complex sex hormones interactionsand

socioeconomic status may play a role (32).

10

There is a high colorectal cancer incidence reported in Malaysia, being the

second most frequent cancer types in both genders (2) contributing 2246 cases

and 12.3% of all cancer cases being diagnosed in the most recent report

published . Additionally more males (1185 cases) are being diagnosed with

colorectal cancer than females (1011 cases).

Prognosis for localized colorectal cancers is good with a 90.3% 5-year relative

survival rate which declines to 12.5% if the cancer metastasized to other organs

(6). Treatment option for colorectal cancer varies depending on the stage and

location of cancer. Mainstay treatment for colorectal cancer is the surgical

resection of tumour supplemented with chemotherapy treatments such as 5-

flurorouracil and leucovorin combination that significantly improves

survivability in a phase III trial in treatment of end-stage colorectal cancer (33)

or radiotherapy.

11

2.2 Phytoestrogens

Phytoestrogens are biochemically heterocyclic phenols which are a type of

flavonoids with a similar structure and or function to endogenous mammalian

oestrogens hence they exhibit either a similar activity to oestrogens or a weak

antioestrogen-like property (34) and can be further classified into flavones,

isoflavones, coumestans and ligands (35). The isoflavone genistein is one of

such phytoestrogen. Found predominantly from soy and legumes food products

(36), genistein is metabolized into genistein deriatives such as dihydrogenistein

or 6-hydroxy-O-desmethylangolensin by the gut microflora through

conjugation with glycoside that subsequently exerts oestrogenic and

antioxidative effect (37).

12

2.2.1 Genistein

Genistein is often lauded for its anti-cancer properties particularly in prostate

cancer (38). Although like other flavonoids, genistein also possesses many

health benefits as mentioned previously. Genistein is found to cause

perturbation of cell cycle progression mostly in the G2/M phase leading to

disrupted cancer development in several cancer types such as breast, prostate,

lung, liver and gastric cancers (39-42). Mechanistic studies shows genistein

inhibits protein tyrosine kinase (PTK) (43) which is deregulated in cancer cells

leading to consecutive activation of PTK-mediated signalling pathways leading

to uncontrolled cellular growth and proliferation (44). Sakla et al. also reported

similar tyrosine kinase inhibition action through ER-dependent mechanism

leading to down regulated expression of HER2 protein (45), a growth factor

signal regulator found overexpressed in aggressive and chemoresistant breast

cancer(46) and involved in tumorigenesis (47). Furthermore, genistein is found

to supress the transcription factor nuclear factor (NF-) (48) which is found

consecutively activated in many tumor types which causes expression of anti-

apoptotic oncogenes (49). Genistein is also demonstrated to inhibit the enzyme

topoisomerase I and II (50,51), 5- reductase (52) and the signal transductor

protein histidine kinase (53) all of which contributed to the anti-cancer

properties of genistein.

13

2.3 Phenoxodiol

Phenoxodiol (2H-1-benzopyron-7-0,3-(hydroxy phenyl)) is a synthetic steric-

modified derivative of genistein and it is found that phenoxodiol offers

substantially improved bio-availability, lowered metabolism rate and antitumor

potency over its parent compound genistein (54).

The underlying foundation behind phenoxodiol development is as a chemo-

sensitizer where it is found to reincur sensitivity in late stage tumor cells

resistant to docetaxel and platinum-based drugs (55). Phenoxodiol is also being

explored as a monotherapy where in vitro studies shows phenoxodiol

effectively inhibits ovarian cancer cells and has progressed to Phase III clinical

trial conducted by Marshall Edwards, Inc (now MEI Pharma) as orally

administered phenoxodiol in recurrent ovarian cancer patients. However there

is no statistically significant improvement was observed in primary or

secondary endpoint (56). A revised study has been conducted for the evaluation

of phenoxodiol against chemo-resistance ovarian cancer in combination with

cisplatin or paclitaxel is currently in Phase II clinical trial which shows that the

combination of cisplatin and phenoxodiol was well tolerated and effective

against chemo-resistant ovarian cancer (57). Currently, phenoxodiol is

evaluated together with docetaxel for side effects in a phase I/II trial for

treatment against advanced stage recurrent ovarian, fallopian tube or

gastrointestinal carcinoma (58).

14

Because phenoxodiol is an analogue of genistein, phenoxodiol shares similar

cytotoxic mechanism. Likewise, phenoxodiol is found to disrupt the cell cycle

and causes checkpoint arrest, however unlike genistein which found to induces

G2/M phase arrest and G1/S phase arrest in murine fibroblast and melanoma

cells (59), phenoxodiol is found to promote G1/S phase arrest in various cancer

cell types (60,61). Mechanistic studies characterize the anti-tumor properties

exerted by phenoxodiol on various cancer cell lines. It has been shown that

phenoxodiol induces the caspase-dependent apoptotic pathway in prostate and

ovarian cancer cell lines through degradation of XIAP and cFLIP anti-apoptotic

proteins (62,63) of which are triggered through ceramide accumulation that

leads to downstream cascade of events.

2.3.1 Mechanism of action

The hydrophobic characteristics of phenoxodiol cause its tendency to partition

within the cellular membrane. Cellular membranous systems such as the

mitochondrial membrane contain electron transport system that generates

membrane potential which drives the production of ATP through oxidative

phosphorylation. The electron shuttler, ubiqinone is involved in mediating

redox cycling in the mitochondrial electron transport chain through the

shuttling of electrons between complexes. Mounting evidence suggested the

presence of a similar system in the plasma membrane (64) involved in the

production of glycolytic ATP through oxidation of cytosolic NADH (65).

15

This suggested that lipophilic compounds like phenoxodiol may act primarily

on the plasma membrane electron transport (PMET) leading to cellular redox

imbalance.

The perturbation of intracellular redox homeostasis such as the ratio of

NADH/NAD+, balance of glutathione and ubiquinone content and state of

redox affects cellular function such as viability and proliferation (66) thus

ultimately leads to apoptosis (67). Phenoxodiol was reported to have a high

affinity to purified recombinant cell surface NADH-oxidase (NOX) leading to

truncated hydroquinone oxidation and the catalyzation of interchanging activity

between protein disulfide and thiol (68). The cell surface ECTO-NOX protein,

designated as tNOX due to its tumor-specificity, upon inhibition by

phenoxodiol causes inhibition of growth followed by apoptosis in transgenic

mice embryonic fibroblasts expressing tNOX gene but not in wild-type mice.

Cytotoxic specificity of phenoxodiol on tumor cells may be accounted by its

ineffectiveness on the non-tumor specific consecutive form of enzyme in

question (CNOX) (68). The correlation between the chemosensitizing effect of

phenoxodiol and tNOX was also explored extensively and was demonstrated to

improve cisplatin and paclitaxel sensitization of platinum resistant HeLa cells

tNOX activity and proliferation (69). More interestingly, tNOX which was

previously demonstrated to have prion like properties (70) imparts phenoxodiol

chemosensitized conformation to subsequent tNOX molecules in the absence of

phenoxodiol (69).

16

Inhibition of tNOX by phenoxodiol causes an imbalanced ratio of NAD+ and

NADH which subsequently leads to deterioration of PMET. The accumulation

of excessive cytosolic NADH activates the plasma membrane

sphingomyelinase leading to ceramide generation from metabolic hydrolysis of

complex sphingolipids like sphingomyelin and cerebrosides (71) and

concurrently decreases sphingosine-1-phosphate levels from the inhibition of

sphingosine kinase at which both events contributed to potential G1 arrest and

subsequent apoptosis (72). A summary of the effects of phenoxodiol on

apoptotic pathways and cell cycle is seen in Figure 2.1. Furthermore, the

reduced coenzyme Q10 level releases sphingomyelinase inhibition (73). To

further support the statement, sphingosine kinase 1 activity in osteosarcoma cell

line is shown to be synergistically reduced when treated with phenoxodiol and

doxorubicin which subsequently increases cellular ceramide level and

triggering cell death (74) plus, phenoxodiol is reported to increase ceramide

level by around 2.3-fold in multidrug resistant tumor cell lines (75).

17

Figure 2.1: Effects of phenoxodiol on apoptotic pathways and the cell cycle.

Arrow in red represents inhibition while arrow in green represents stimulation

18

2.3.2 Other effects of phenoxodiol

Anti-tumor effects of phenoxodiol may not be limited to directly exerting

cytolytic effects via induction of apoptosis on cancer cells. Phenoxodiol is

reported to exhibit anti-angiogenic effect by inhibiting proliferation and

migration of endothelial cells while simultaneously reduces the formation of

capillary tube and decreases matrix metalloproteinase 2 expression (76). A

similar observation was found in endothelial cells treated with exogenous

ceramide analogs which its molecular mechanism was attributed to the

decreased cyclin D1 expression through upregulation of CAV-1 expression

which represses cyclin D1 promoter activity (77) and inhibition of ERK1/2

phosphorylation (78).

Phenoxodiol is also shown to improve immunomodulation through

enhancement of natural killer cells lytic function (79). This phenomenon can be

attributed to ceramide triggering of prosurvival NF- pathway through

activation of calpain which cleaves the NF- inhibitor p105 (80) that

suggested a negative feedback regulation of ceramide. The activation of NF-

pathway not only leads to transcriptional activation of various prosurvival

oncogenes but also genes related to innate and adaptive immune response (81).

19

Phenoxodiol is found to inhibit the catalytic activity DNA topoisomerase II, a

key enzyme in regulating untangling of over-wounded DNA through

stabilization of the cleavable complex (82). The interaction between ceramide

and DNA topoisomerase II has not been elucidated, however, ceramide 1-

sulfates 1 and 2 isolated from Japanese Bryozoa Watersipora cucullata is found

to be a potent inhibitor of DNA topoisomerase I (83).

2.4 Apoptosis

Apoptosis or programmed cell death is a term used to characterize a type of

distinct cell death with a specific morphology and biochemical processes.

Apoptosis is a natural occurrence in cell senescence and is essential in tissue

cell population homeostatic maintenance. However, apoptosis can also be

induced by external stimuli such as immune reactions or cellular stress as a

defensive mechanism (84).

Apoptosis can be activated through two pathways, the intrinsic and the extrinsic

pathways which interlinked with one another with molecules from one pathway

influencing the other (85). A more unconventional pathway involves T-cells

activation of perforin-granzyme for triggering cell death. These pathways are

known as the caspase-dependent pathway as it involves the endoprotease

caspase responsible for the hydrolysis of peptide bonds at aspartic acid residues

during cell disassembly into apoptotic bodies (86).

20

The extrinsic pathway involves binding and activation of TNF superfamily of

cell death receptor like Fas and tumor necrosis factor (TNF)-1 by its respective

death ligand, the Fas ligand (FasL) and TNF-2 on the plasma membrane

resulting in trimerization and death effector domain (DED) clustering of

receptor in addition to recruitment of adapter proteins like Fas-associated death

domain (FADD) or TNFR-associated death domain (TRADD) (87) which in

turns recruits procaspase-8 monomeric protein to form death inducing signaling

complex (DISC) (88). The self-cleavage of oligermerized procaspase-8 within

DISC activates caspase-8. Caspase-8 activation then drives various downstream

procaspases which varies according to cell types. Type I cells which consists of

several lymphoid cell lines are able to directly activate downstream procaspases

such as procaspase-3 as caspase-8 is sufficiently activated. In other cell types,

known as type II cells, the weakly activated caspase-8 are unable to directly

activate procaspase-3 but able to activate mitochondrion-mediated pathway

through the truncation activation of the proapoptotic protein Bid into tBid

which causes the release of apoptotic molecules such as cytochrome c and

apoptosis-inducing factor from the mitrochondria (89). Procaspase-8 can also

be activated independent of neither FADD interaction nor DISC formation

through cytochrome c-dependent pathway where the release of cytochrome c

triggers the activation of caspase-6 which in turns activates procaspase-8 (90).

21

A lesser known procaspase-10 mediated extrinsic pathway is similar to that of

procaspase-8 and responsible mainly for lymphoid cells apoptosis. In Fas- and

TNF- death ligand-receptor apoptosis, caspase-10 is shown to function

independently from caspase-8 from the occurrence of apoptosis in capsase-10-

overexpressed but caspase-8 deficit cells (91). Caspase-10 is also found to

cleave substrate differently than of caspase-8 (92) which may indicates each

possesses a unique role in initiation of apoptosis. Caspase-10 was also much

more frequently under-expressed than caspase-8 in several carcinoma cell lines

which may suggest a role of caspase-10 in cancer onset (93).

The highly conserved rudimentary caspase-2 dependent extrinsic pathway also

functions similarly to that of caspase-8. Likewise, following the binding and

activation of death ligand and corresponding receptors from apoptotic stimuli

(94), the recruitment of Rip-associated protein death domain (RAIDD) binds

and oligomerizes with p53-induced protein with a death domain (PIDD) to

form PIDDosome complex (95) followed by self-cleavage activation of

procaspase-2 monomers. It is speculated that the active caspase-2 then cleaves

the proapoptotic Bid causing membrane permeabilization of mitochondria

leading to release of proapoptotic proteins (96) which is further elaborated

below.

22

The intrinsic pathway or the mitochondrion-mediated pathway is activated in

the presence of non-receptor stimuli such as cellular stress or absence of certain

biochemicals leads to mitochondrial inner membrane changes resulting in

mitochondrial permeability transition (MPT) pore opening allowing the release

of the pro-apoptotic proteins cytochrome c, Smac/DIABLO and HtrA2/Omi

serine proteases (97). The release of cytochrome c activates cytosolic caspase-6

and apoptotic protease activation factor (Apaf)-1 and procaspase-9 leads to the

formation of apoptosome (98) which in turns activates procaspase-3 and 7. A

positive feedback pathway occurs between the activated caspase-3 and

procaspase-9. Caspase activity is also enhanced through the inhibition of

inhibitor of apoptosis proteins by Smac/DIABLO and HtrA2/Omi (99). In late

apoptosis, apoptosis inducing factor (AIF), endonuclease G and caspase-

activated DNAse (CAD) are released from the mitochondria. All of which

translocates to nucleus causing fragmentation of DNA where AIF and

endonuclease G performs in a caspase-independent fashion (100) but a

cleavage by caspase-3 is required for the activation of CAD (101). The

apoptotic events described are tightly regulated by the Bcl-2 protein family

(102) which in turn regulated by the tumor suppressor protein p53 (103).

23

Caspase-2, -8, -9 and -10 is known as the initiator apoptotic caspases, as they

activate several apoptosis executioner caspases such as caspase-3, -6 and -7

(104). The executioner caspases, upon activation cleaves several vital proteins

such as DNA repair proteins such as poly ADP ribose polymerase (PARP),

DNA-dependent protein kinase (DNA-PK) and U1-70kD and subsequently

leads to DNA degradation, lamin A and fodrin which are found in nuclear and

cytosolic skeleton causing chromatins condensation and nuclear membrane

decomposition and eventually results in the formation of apoptotic body (105).

2.4.1 Apoptosis and cancer drug-discovery

In cancer drug discovery, restoration of the apoptotic pathway is an effective

method in treating cancer. This is because tumor cells are under constant stress

and marked for removal but sustained due to aberration in apoptotic pathways.

In tumor cells, apoptosis is evaded through manipulation of the Fas-mediated

apoptosis. Down-regulation of Fas expression or deregulation of key

components in Fas-mediated apoptotic pathway (106) is a common tumor

hallmark in several tumor cell lines.

24

Additionally, some carcinomas such as brain (107) and ovarian (108) cancers

were found to overexpressed FasL. This seemingly counter-intuitive way is

actually a self-defensive mechanism employed by tumor cells to induce

immune privilege in tumor site not unlike specialized organs such as the brain,

testes and eyes. FasL expression in tumor cells allows the crosslinking of Fas

receptor expressed on the surface of tumor invading cytotoxic T-cells that

subsequently leads to apoptosis of the T-cells (109). The simultaneous down-

regulation and up-regulation of Fas and FasL in tumor cells prevents apoptosis

and invoking immune response.

A drug-induced apoptotic mode of death is much more preferred over necrosis

as apoptosis does not induce inflammatory response. At a necrotic site,

leukocytes consisting of neutrophils will infiltrate the site rapidly which is

followed subsequently by monocytes accumulation (110) which will cause

further destruction of normal tissues surrounding the necrotic site and

subsequent fibrosis. In an in vivo condition, apoptotic cells maintain membrane

integrity and do not release proinflammatory cytokines. Before the cells

disintegrate, adjacent phagocytes engulf the cells thus preventing the lyses of

apoptotic cells. Furthermore, production of inflammatory meditators IL-10 or

TGF- that inhibits inflammation by macrophages can be stimulated by

apoptotic cells (111,112). However, this may not always be the case for

apoptotic cells as there are reports of stimulated apoptotic cells causing intense

inflammation in mice (113).

25

The unpredictable nature of apoptosis can be attributed to the clearance rate by

phagocytes as over time, if apoptotic cells are unable to be ingested by

phagocytes in time, they undergoes secondary necrosis where the membrane

became macromolecules permeable (114) and thus causing the release of

intracellular contents with part of it being proinflammatory cytokines which

triggers host inflammatory responses. Secondary process will also occurs in

vitro where under the absence of phagocytes, apoptotic cells will ultimately

swell and lyses, a phenomenon that may occur with phenoxodiol treated cells at

longer time.

2.5 Cell cycle

The most basic nature of a cell is growth and proliferation which is an

important process in tissue and organ development and also repair and replace

cells loss due to injury. When cells undergo division, two consecutive processes

ultimately happen, which are the replication of DNA and chromosomal

segregration into daughter cells that can be subdivided further in various stages

collectively known as the cell cycle. A cell cycle is consists of four different

stages, G1, S, G2 and M which morphologically can be divided into interphase

(G1, S, G2) and mitosis (M) phase that consist of prophase, metaphase,

anaphase and telophase.

26

In G1 phase, the cells prepares for DNA synthesis through increased production

of mRNA and proteins involved in DNA replication. . It is found that some

cells like Xenopus embryos (115) and cancer cells (116) are able to bypass G2

phase completely and enters mitotic phase after the replication of DNA. In the

M phase, chromosomal condensation occurs by condensing the replicated

DNA, packaged in elongated chromosomal form into a more compacted form

for segregation. Subsequently, breakdown of the nuclear envelope leads to

attachment of sister chromatids to the microtubules of mitotic spindles where it

will be aligned at the equator during metaphase. In anaphase, separation of

sister chromatids to opposite pole of spindle occurs where decondensation and

intact nuclei is reformed. Finally, cytokinesis occurs where the cells are divided

through cytoplasmic division (117). An additional phase, known as G0 phase is

used to describe stagnant cells that are not actively dividing but with potential

for division (115) which consist of the majority of non-proliferating cells in our

body.

27

2.5.1 Cell cycle regulation

Cell cycle phase transition is regulated by a family of serine/threonine protein

kinases known as the cyclin-dependent kinases (CDK) which is activated at

various specific points within the cell cycle. There are nine identified CDKs

and five are activated in cell cycle. CDK2, 4 and 6 is activated during G1 phase,

CDK2 in S phase and CDK1 that is activated in both G2 and M phase. All

CDKs is activated by CDK7 in combination with cyclin H known as CDK

activating kinase. The full activation of CDK activity besides binding of cyclin

requires phosphorylation at threonine and tyrosine residue by CDK activation

kinase which induces conformational changes that enhances cyclin binding

(118).The role of the remaining CDK in cell cycle progression is yet to be

determined (119). The activation of CDKs requires their interaction with cyclin

to phosphorylate the downstream proteins (120) that allows the progression of

cell cycle.

As CDK is regulated by cyclin, the level of CDK remains constant in contrast

to cyclin level which allows periodic activation of CDK (121). The activation

of CDK at different cell cycle phases requires different cyclins as summarized

in Table 2.1 below. Cyclin D family (Cyclin D1-3) is involved in binding to

CDK4 and 6 and the CDK-Cyclin D complex is involved in cell cycle

progression into G1 phase (122).

28

Expression of cyclin D is not consecutive but rather driven by growth factor

stimulation (123), unlike other cyclins which is expressed periodically. Cyclin

E is another cyclin involved in G1 associates with CDK2 which allows

progression from G1 into S phase (124). Cyclin A forms an essential complex

with CDK2 in S phase progression (125) and with CDK1 to promote entry into

mitosis phase. Further regulation in the mitosis phase is done by CDK1-cyclin

B complex (126). Out of the sixteen identified cyclin proteins, not all are

involved in cell cycle regulation (127,128). Some cyclins are involved in

ubiquitination meditated proteolysis at end of each cycle phase (129).

CDK Cyclin Cell cycle phase

involved

CDK4 D1, D2, D3 G1 CDK6 D1, D2. D3 G1

CDK2 E G1/S transition

CDK2 A S

CDK1 A G2/M transition

CDK1 B Mitosis

CDK7 H All (As CDK activating

kinase)

Table 2.1: Summary of the CDK and cyclin pair involved in the regulation of

cell cycle phases.

29

Cell cycle quality control are in place namely restriction points and checkpoints

to ensure the correct cell cycle progression which upon blocking of early cell

cycle events such as inhibition of DNA synthesis, later events such as mitosis

and cytokinesis will be halted.

Cell cycle in eukaryotic cells is safeguarded at three checkpoints which is at the

boundary between G1 and S phase, G2 and mitosis phase and metaphase and

anaphase. Should the condition for cell division is unmet, cell cycle progression

will be halted at these checkpoints. These checkpoints are made out of

accelerators and brakes that control progression of cell cycle. Surveillance

mechanisms are in place to detect conditions and send inhibitory signals should

the condition is sensed to be unfavorable which are essential for cell

survivability under hostile environment.

The G1 checkpoint in mammalian cells is known as the restriction point.

Restriction point can be summarized as the point of no return where when the

cell passes this point, it became committed towards cell cycle progression

and doesnt require stimulation from proliferation stimulants (130). Cyclin D

will form a complex with G1 phase CDKs and inhibits Rb that is involved in

negative regulation of cyclin A and E which are involved in synthesis of DNA

plus accumulation of cyclin B, through binding and inactivation of E2F

transcription factor and inhibits ribosomal RNA gene transcription leading to

cell growth inhibition (131).

30

As mentioned previously, should a cell pass the restriction point (for

mammalian cells) or START (for budding yeast cells) it will be committed to

the cell cycle. The irreversible transition is due to positive feedback in the

CDK-cyclin cell cycle control system. Mammalian cells that does not

undergoes cell division remains in the G1 or G0 phase as the CDKs and cyclin

are kept inoperative through three means: suppression of cyclin genes

transcription by Rb protein, rapid degradation of cyclin by APCCDH1 and

inhibition by p27 all of which can be inactivated by the CDK-cyclin complex

phosphorylation. The balance between the antagonists and CDK-cyclin creates

two irreversible states in the cell cycle: the G1 state and S-G2-M state. During

the restriction point period, the G1 CDK-cyclin complex (CDK4 and 6 with

cyclin D) removes the antagonist through phosphorylation of Rb and p27 hence

tipping the scales in favour of an irreversible transition to the S-G2-M state. The

state of commitment is irreversible as upon passing the restriction point, cyclin

D are not required for cell commitment as S and M phase CDK-cyclin complex

will maintain the inhibition of their antagonists until the end of mitosis where

all S and M phase cyclins are lost and thus removing the inhibition on

antagonists which sees the cells maintained in G1 phase (132).

The activity of CDK1-cyclin B complex or M phase-promoting factor (MPF) is

essential for cell cycle progression into mitosis phase. Complete undamaged

DNA replication is required before chromosomal condensation and subsequent

nuclear division can occur in mitosis.

31

Should DNA damage is detected, the cell will be arrested in between S and G2

phase by inactivation of MPF through Wee1 phosphorylation of tyrosine and

threonine residues within the catalytic site into preMPF thus allowing time for

DNA repair. The transition from G2 into M phase is continued by Cdc25C

dephosphorylation of preMPF into MPF. Hence, the G2 checkpoint is guarded

by positive feedback of MPF which inhibits Wee1 and activates Cdc25C

concurrently. The transition from G2 to M phase is blocked by DNA damage

through activation of checkpoint kinase (Chk) 1 and/or 2 which phosphorylates

Cdc25C that subsequently causes binding to protein 14-3-3 in the cytoskeleton

and sequestered away in the cytosol thus prevents the conversion of preMPF to

MPF. Additionally, Chk2 phosphorylation of Cdc25C reduces its catalytic

activity (133). In incomplete DNA replication or DNA damage, persistent

presence of single stranded DNA is detected by ataxia telangiectasia and Rad3-

related protein (ATR) in junction with ATRIP and leads to activation of Chk1

by phosphorylation. The sequestration of Cdc25 and p53 stabilization by

activated Chk1 leads to arrest between the S and G2 phase, activation of DNA

repair enzymes or apoptotic signaling.

32

At the end of metaphase, all chromosomes should be attached by its

kinetochores to the bipolar mitotic spindle before sister chromatids separation

in anaphase can happen. This event is safeguarded by spindle or metaphase

checkpoint. If presence of free kinetochores is detected such as through

treatment with microtubule depolymerizing drugs such as nocodazole,

vinblastine or podophyllotoxin, mitosis is blocked (134).

Early events in mitosis such as the breaking down of nuclear envelope,

followed by assembly of spindle fibres and chromosomal alignment are

promoted by the CDK1-cyclin B complex or MPF but inhibit later mitotic

events. The activation of Cdc20-APC complex is also promoted by MPF action

which initiates the separation of sister chromatids in anaphase through the

destruction of securin or precocious dissociation of sister chromatids (Pds) 1 in

budding yeast cells. Securin inhibits the protease separase or Esp1 in yeast cells

throughout most part of cell cycle which upon removal of securin triggers

separase activation leading to degradation of the cohesin proteins holding the

sister chromatids together, hence leading to the first stage of anaphase. The

Cdc20-APC complex also targets and degrades the cyclin B component in the

MPF complex in a negative feedback fashion, hence as the cell cycle

progresses, the activities of MPF and Cdc20-APC complex will sequentially

rises and fall and eventually quenched at the end of mitosis with the loss of

cyclin B and the cells reenters G1 phase.

33

The spindle checkpoint blocks progression of mitosis as free kinetochores will

activate Mad2 protein which binds and inhibits Cdc20 thus preventing from

degrading securin and mitotic cyclin and causes arrest in metaphase. Post

mitosis, the daughter cells reenters the G1 state.

Figure 2.2: A summary of the mammalian cell cycle with its respective

checkpoints. Arrow in red represents inhibition and arrow in green represents

stimulation while arrow in black indicates the approximate time point within

the cell cycle that is regulated by its respective cyclin-CDK complex.

34

2.6 Ceramide

As mentioned previously, the core mechanism of phenoxodiol-induced

apoptotic cell death is due to the accumulation of ceramide. Ceramides consists

of a family of lipid molecules. Composed of portion of sphingosine and fatty

acid, these sphingolipids is a major structural element found in biomembranes

which together with phosphocholine or phosphoethanolamine forms

sphingomyelin, an important lipid in lipid bilayer. It was later found that the

role of ceramide extended beyond structural roles by exhibiting a diverse effect

on cellular signaling and cell function regulation, one of such is the induction

of signaling cascade that potentiates apoptosis. Generation of ceramide is

through three intrinsic pathways de novo synthesis pathway, hydrolysis of

sphingomyelin and salvage pathway which is summarized in Figure 2.3.

Figure 2.3: Ceramide production and metabolism pathway.

35

2.6.1 Ceramide and apoptosis

2.6.1.1 Ceramide and the extrinsic pathway

The hydrophobicity of ceramide ensures ceramide is always partitioned within

the bilayer membrane at its site of production and exerting its function from

within. Known as the ceramide-enriched membrane platforms (135), these

regions serves to cluster the death receptors, TRAILR2 and CD95 and upon

activation amplify downstream apoptotic signaling events through facilitation

of DISC complex formation (136). TRAIL and TNF-induced apoptosis

associates with activation of neutral and acid sphingomyelinase which leads to

increased ceramide formation (137). Additionally, in several tumor types such

as glioblastoma (138)and prostate cancer (139), the down regulation of FLICE

inhibitory proteins through inactivation of Akt pathway, removes caspase-8

inhibition and subsequently promotes apoptosis.

36

2.6.1.2 Ceramide and the intrinsic pathway

As mentioned previously, the extrinsic and intrinsic apoptotic pathway are not

mutually exclusive of one another, cross over can happen from extrinsic to

intrinsic pathway via FLIP inhibition, caspase-8 and truncated-BID activation.

The proapoptotic nature of ceramide is largely attributed to the orchestration of

a myriad of downstream signaling pathway that eventually causes the release of

pro-apoptotic proteins from the mitochondria a la the intrinsic pathway.

However, to do that, it must first reach the mitochondria which are hindered by

its hydrophobic nature of ceramide. To overcome, upon generation within the

membrane bilayer, ceramide platforms are formed that infold into the cytosol

and fuses with mitochondria. With that, a commute pathway is formed that

allows the direct transfer of ceramide from plasma membrane to mitochondria

leading to accumulation of ceramide at mitochondria and subsequent apoptosis

(140). Other methods that causes mitochondrial accumulation of ceramide

includes the production of ceramide via de novo or the salvage pathway by

mitochondria-associated membrane (141) and the localization of key ceramide

production enzymes such as ceramide synthase, neutral sphingomyelinase and

ceramidase in mitochondria for in situ production of ceramide (142-145).

37

The key event of ceramide apoptotic signaling is the induction of mitochondrial

outer membrane permeabilization (MOMP) from ceramide channel formation.

The level of mitochondria accumulated ceramide is shown to directly correlate

with MOMP (146). The induction of MOMP allows the leakage of

mitochondria apoptotic proteins such as cytochrome c and intermembraneous

proteins that have a molecular mass lesser than approximately 60kDa (147).

Ceramide alone is insufficient to induce MOMP but rather together with the

proapoptotic Bcl-2 protein, Bax in synergistically causes the permeabilization

of mitochondria outer membrane (148) through the formation of ceramide-rich

macrodomains essential for BAX insertion, oligomerization and pore formation

(149).

Additionally, ceramide also causes a transient increased in pH intracellularly

leading to essential conformational changes in BAX (150) and allowing BAX

translocation from the cytosolic 14-3-3 proteins to mitochondria via JUN N-

terminal kinase activation which is a downstream process from p38 MAPK

activation and Akt down regulation of ceramide (151). The collective activation

of protein phosphatase 2A (PP2A) and the endolysosome protease cathespin D

plus the inactivation of Akt by ceramide all together contributes to the

activation of glycogen synthase kinase 3 (152,153) which also induces MOMP

through the activation of caspase-2 and caspase-8 which leads to the cleavage

of BID to form tBID that translocate to mitochondria (154).

38

In addition to the activation of protein phosphatase 2A, caspase-2 activation

and mitochondria apoptosis is also induced by ceramide through the down

regulation of BCL-2, a pro-survival mitochondrial protein that blocks

apoptosis, overloading of calcium ions and apoptosis receptors (155). Another

effect of ceramide on mitochondrial function is the activation of protein kinase

C which likewise leads to release of cytochrome c and activation of caspase-9

(156).

2.6.1.3 Ceramide induced apoptotic signals

The downstream apoptotic cascade orchestrated by ceramide is due to the

induced apoptotic signals in upstream pathways. Akt, a serine/threonine-

specific protein kinase is one of the major pathways being down regulated by

ceramide (157). Akt pathway is closely associated with tumorigenesis and

frequently altered in most cancer types and indicates poor prognosis and

chemoresistance (158). Activation of Akt occurs through growth factors

binding to a plasma membrane tyrosine kinase receptor which activates PI3K

that converts phosphatidylinositol-4,5-biphosphate (PIP2) to

phosphatidylinositol-3,4,5-trisphosphate (PIP3). Cytosolic Akt is then

translocated to the plasma membrane in an event triggered by PIP3 where it

will be activated via phosphorylation by phosphoinositide-dependent protein

kinase 1 and mTOR complex 2 (159).

39

Ceramide inhibits Akt pathway via three methods. The atypical PKC activity

is found to be stimulated by the active ceramide form, C6 ceramide which

causes increased association of PKC with Akt (160), in addition, the binding

of PIP3 to Akt PH domain is stifled by PKC through phosphorylation on PH

domain Thr34 which effectively blocks Akt translocation to the plasma

membrane to be activated (161). As mentioned, another direct downstream

signal of ceramide is the activation of PP2A (157). In various cancer types, the

activation of PP2A correlates with inhibited Akt signaling (162). The activation

of PP2A can occur directly and indirectly by ceramide where the association

between inhibitor 2 of PP2A and PP2A is reduced which resulted in indirect

PP2A activation (163). Likewise, Akt phosphorylation can be reduced by the

activation of p38 from ceramide action as observed in HL-60 cells (151).

The MAP kinases p38 and JNK that is involved in cell growth and survivability

is also found to be regulated by both exogenous (164) and endogenous

ceramide (73). Both of these MAP kinases are activated by ceramide through

upregulated transcriptional expression of thioredoxin-interacting protein which

in-turn diminishes thioredoxin activity thus removing the inhibition of

apoptosis signal regulating kinase 1 leading to downstream activation of both

p38 and JNK (164).

40

Activation of p53 is also being promoted by ceramide action (165). The

activation of PP2A also leads to inhibition of the anti-apoptotic Bcl-2

phosphorylation and subsequent increased binding between p53 and Bcl-2

therefore inhibits Bcl-2 and thus leading to apoptosis (166). The accumulation

of p53 also leads to increased pro-apoptotic Bax level and the decreased in the

anti-apoptotic Bcl-2 level in neuroblastoma cells, suggesting the regulation of

Bax/Bcl-2 ratio by p53 in ceramide-induced apoptosis as one the many

apoptotic mechanism of ceramide (167).

Finally, ceramide is shown to downregulates prosurvival IAP survivin involved

in cell proliferation and division, metastasis and angiogenesis in tumor cells

(168). The expression of survivin at the transcriptional level is inhibited by

ceramide. The downregulation of survivin synergistically enhanced cell death

from the intrinsic apoptosis pathway and increased p53 and Bax all of which is

meditated by ceramide action.

41

2.6.2 Ceramide and cell cycle

As shown in Figure 2.1, ceramide regulation of cell cycle involves the

modulation of cell cycle inhibitors. During the cell cycle G1 to S phase

transition, complexes are formed between cyclin D and E with CDK2, 4 and 6

to phosphorylate Rb. Ceramide is found to upregulate p21 leading to activation

of Rb through decreased expression of cyclin E and D1 and CDK 2 and 7

activity (169) then inactivates it through degradation in a negative feedback

fashion to reverse the cell cycle checkpoint arrest (170).

The mechanism of cell cycle arrest can be partly attributed to activation of

peroxisome proliferator-activated receptor- (PPAR) (169) to which a reversal

in CDK7 suppression is found when treated with PPAR antagonist. PPAR

family of membranous receptor proteins functioning as transcriptional factors

in regulating genes involved in cell differentiation and metabolism. The

activation of PPAR causes growth arrest in various cancer cell types (171).

Exogenously added ceramide is found to stimulate PPAR which subsequently

forms a heterodimer with retinoid X receptor to bind and activate specific

regions of DNA. Expression of the CDK inhibitor, p21 is also found to be

upregulated by PPAR (172) which as a consequence sees the accumulation of

dephosphorylated Rb and CDK2 association with p21 (173). In addition, p21

activity is also found to be regulated by ceramide through p53 induction

leading to downstream activation of p21 (174).

42

The upregulation of protein phosphatase activity by ceramide leads to CDK2

inhibition which is reversible upon treated with protein phosphatase antagonists

(175) as PP2A meditates p27 expression via Akt dependent and independent

pathways. Finally, the inhibition of Akt pathway by ceramide also

synergistically enhance p27 action through stabilization of the CDK inhibitor

which all together contributes to the cell cycle arrest in G1 phase meditated by

ceramide action.

Accumulation of ceramide also leads to G2 arrest as seen in rhabdomyosarcoma

cells from the increased expression of p21 and downregulation of cyclin D

(176). Ceramide is found to inhibit MDM2 which binds and meditates

proteosomal degradation of p21. MDM2 also regulates p53 in a negative

fashion, leading to decreased p21 levels. Consistent with these findings,

overexpression of MDM2 diminishes G2 arrest meditated by ceramide

upregulation of p21. Suppression of survivin expression associated with

transition between G2 and M phase is also found to be meditated by ceramide

which similarly leads to arrest in G2 phase (177,178).

43

3 MATERIALS AND METHODS

3.1 Materials

Name Brand Product ID

Annexin V-FITC early

apoptosis detection kit

CELL SIGNALING

TECHNOLOGY INC, USA

6592

Cycloheximide ACROS ORGANICS, USA 35742-0010

Dimethyl sulfoxide (DMSO) SIGMA-ALDRICH, USA 472301

Dulbeccos Modified Eagle Medium (DMEM)

SIGMA-ALDRICH, USA D7777

Ethylenediaminetetraacetic

acid (EDTA)

SIGMA-ALDRICH, USA E9617-1-0250

Fetal bovine serum (FBS) SIGMA-ALDRICH, USA F7524

Hydrochloric acid ACROS ORGANICS, USA 12463-0010

Penicillin-streptomycin

antibiotic

GIBCO, LIFE

TECHNOLOGIES, USA

15140-122

Phenoxodiol SIGMA-ALDRICH, USA D7446

Phosphate buffer saline (PBS) AMRESCO, USA E404

Propidium iodide SIGMA-ALDRICH, USA 81845

RNAse A SIGMA-ALDRICH, USA R6148

Sodium azide SIGMA-ALDRICH, USA S2002

Sodium bicarbonate AMRESCO, USA

E404

Sodium hydroxide SIGMA-ALDRICH, USA 221465

Thiazolyl Blue Tetrazolium

Blue (MTT)

SIGMA-ALDRICH, USA M5655

Triton X-100 SIGMA-ALDRICH, USA X100

Tryphan blue solution 0.4% SIGMA-ALDRICH, USA T8154

Trypsin-EDTA solution 10X SIGMA-ALDRICH, USA 59418C

Table 3.1: List of chemicals used

44

Name Brand Product ID

6-well plates JET BIOFIL, USA TCP011006

96-well plates THERMOSCIENTIFIC,

USA

163320

Bottle-top vacuum filter SIGMA-ALDRICH, USA 430049

Centrifuge tubes (15 mL) GREINER BIO-ONE, USA 188271

Centrifuge tubes (50 mL) THERMOLINE

SCIENTIFIC, AUSTRALIA

TL32103

Culture flask (25cm2) THERMOSCIENTIFIC,

USA

EW-01932-41

Microcentrifuge tubes AXYGEN SCIENTIFIC,

USA

MCT-150-C

Serological pipettes (5 mL) JET BIOFIL, USA GSP010005

Serological pipettes (10 mL) JET BIOFIL, USA GSP010010

Sterile petri dishes THERMOLINE

SCIENTIFIC, AUSTRALIA

CCD009015

Pipette tips GREINER BIO-ONE,

BELGIUM

771290 (0.5-20L)

739261 (10-200L)

877270 (200-1000L)

Table 3.2: List of consumables used

45

Name Brand Product ID

Autoclave system HIRAYAMA, JP HV-50

Centrifuge EPPENDORF,

GERMANY

5810

Class II biosafety

cabinet

ESCO

TECHNOLOGIES INC,

USA

SC2-4A1

CO2 incubator NEW BRUNSWICK

SCIENTIFIC.

GERMANY

170-200

Flow cytometer BD BIOSCIENCE, USA BDFACSCALIBUR

Fluorescence

microscope

NIKON

CORPORATION,

JAPAN

ECLIPSE-TI

Hemocytometer GRALE HDS, AUS 4514

Inverted light

microscope

LEICA

MICROSYSTEMS,

USA

DM13000B

Microcentrifuge EPPENDORF,

GERMANY

MINISPIN

pH meter HANNA

INSTRUMENTS, USA

HI 110

Pipettes EPPENDORF,

GERMANY

RESEARCHPLUS

Refrigerated centrifuge EPPENDORF,

GERMANY

5804R

Ultrapure water system EMD MILIPORE,

CANADA

SYNSVR0WW

Vortex FINEPCR, KOREA FINEVORTEX

Water bath MEMMERT,

GERMANY

WMB10

Table 3.3: List of apparatuses used

46

3.2 Methods

3.2.1 Preparation of culture media and solutions

3.2.1.1 DMEM

Thirteen point five gram of DMEM in powder form, containing 4500 mg/L

glucose and L-glutamine and 110 mg/L sodium pyruvate was weighted out and

together with 3.7 g of sodium bicarbonate were dissolved using ultrapure water

to make approximate 800 mL of media. pH of media was adjusted to between

7.1 to 7.3 using a pre-calibrated pH meter. Media was subsequently top up with

ultrapure water to make 1 L volume. Under aseptic condition, media was filter

sterilized with an aid of a vacuum pump into sterile pre-autoclaved bottles. 10

mL of filtered media was aliquot out into a sterile petri dish and incubated for 3

days in a CO2 incubator at 37oC and 5% CO2 to validate sterility by noting

media appearance such as cloudiness, colour changes or presence of microbes

under microscopic observation or naked eyes. Only sterility verified media will

be used for cell culturing and experiments. Prepared DMEM media was stored

in 4oC temperature.

47

3.2.1.2 PBS

One tablet of PBS containing 10mM phosphate buffer, 137mM sodium

chloride, and 2.7mM potassium chloride was dissolved in 100 mL of ultrapure

water before being autoclaved at 121oC for 15 minutes. Prepared PBS was

stored in 4oC temperature.

3.2.1.3 Trypsin-EDTA

One part of 10X Trypsin-EDTA solution containing 0.5% trypsin and 0.2%

EDTA was diluted with nine part of sterile PBS to make 1X Trypsin-EDTA

solution. Prepared Tryspin-EDTA solution was stored in -20oC temperature.

3.2.1.4 Phenoxodiol and Cycloheximide

Analytical grade phenoxodiol and cycloheximide was weighted and dissolved

in DMSO to make a stock concentration of 10 mg/mL which was further

diluted pre-usage in experiments.

3.2.1.5 MTT solution

MTT powder was weighted and dissolved in sterile PBS to make a stock

concentration of 5 mg/mL in dark.

48

3.2.2 Culture media for cell lines

The initial cultures for HepG2 (Homo sapiens hepatocellular carcinoma), HT-

29 (Homo sapiens colorectal adenocarcinoma), CNE1 (Homo sapiens highly

differentiated nasopharyngeal carcinoma) and C6 (Rattus norvegicus glioma)

cell lines used in this study were obtained from from IMUs Institute for

Research Development and Innovation (IRDI) cell bank (Kuala Lumpur,

Malaysia) and were cultured in vitro using sterilized high glucose DMEM

supplemented with 3.7 g/L sodium bicarbonate, 10% FBS and 1% Pen-Strep

antibiotic solution containing 100 units/mL penicillin and 100 units/mL

streptomycin hereby designated as complete DMEM.

The basal medium DMEM is a modified version of Basal Medium Eagle

(BME) with a four-fold increased concentration of amino acids and vitamins.

The high glucose content with 4500 mg/L is shown to improve cultivations of

various cell lines (179). The supplementation of FBS is required as to provide

the essential proteins and growth factors for optimum cell growth (180) while

sodium bicarbonate acts as a buffering system in the presence of artificial CO2

for pH maintenance (179). The use of antibiotic is to suppress growth of

microbial contaminants (181).

49

3.2.3 Maintaining and sub-culturing of cells

All cell lines were seeded and cultured in 25cm2 cell culture flask and

incubated in a CO2 incubator producing an artificial humidified environment

with 5% CO2 at 37oC. The cultures were maintained at exponential phase by

ensuring a cell confluence of less than 90%. Cultures having 80% and above

confluence will have its extra cells discarded where in brief, old media is

discarded and the cells were washed with 1 mL of sterile PBS by gently

swirling the flask as to prevent air bubbles formation. PBS was subsequently

discarded and 1 mL of 1X Trypsin-EDTA solution was added to flask and

incubated in CO2 incubator for approximately 5 minutes at 37oC and 5% CO2.

The flask was tapped gently to further ease detachment of cells and observed

under an inverted light microscope to verify cells detachment. Approximately

500 L of detached cells was discarded and the remaining was resuspended in

10 mL of fresh complete DMEM. The flasks were then further incubated for

three to four days at 5% CO2 at 37oC. Old flasks will be reused up to a month

before sub-culturing to a new flask where similarly, cells were collected by

trypsinization method and resuspended in 1 mL of complete DMEM before

transferring to new flask. The final volume of the flask was brought up to

approximately 10 mL by adding 9 mL of fresh culturing media. All procedures

was performed aseptically in a Class II Biosafety Cabinet and all media and

solution used were pre-warmed to 37oC in a water bath before used.

50

3.2.4 Cell seeding

Culture was first allowed to achieve a confluence level around 80-90%.

Following that, the cells were seeded into a sterile 96-well or 6-well plate. The

old media were discarded and the cells were washed with 1 mL of sterile PBS.

PBS was then discarded and replaced with 1 mL of 1X Trypsin-EDTA solution

before incubating in a humidified CO2 incubator for approximately 5 minutes at

37oC and 5% CO2. The flask was tapped gently to detach cells and

subseque