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EFFECT OF POTASSIUM KOETJAPATE, A DRIVATIVE OF KOETJAPIC ACID

ISOLATED FROM SANDORICUM KOETJAPE MERR. ON HUMAN COLORECTAL

CANCER.

by

SEYEDEH FATEMEH JAFARI

Thesis submitted in fulfillment of the requirements for the degree of

Doctor of Philosoph y

July 2018

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DEDICATION

This thesis is dedicated to

the angels of my life, my parents, Seyedeh Pouran Hashemi

and

Seyed Sadegh Jafari,

for their sincere love, valuable encouragement and support

throughout my life.

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ACKNOWLEDGEMENT

First and Foremost, praise is to Allah, the Almighty, the most merciful for giving me opportunity, knowledge, patient and strength to undertake this research study.

I would like to express my special thanks to my research supervisor, Assoc. Prof. Dr. Amin Malik Shah Abdul Majid for guiding and supporting me during my research and my sincere thanks and gratitude to Dr. Mohamed Khadeer Ahamed Basheer and Dr. Muhammad Asif, without their help and incredible support throughout my research, this experimental project would have never been successful. I would also like to express my appreciation to my co-supervisor, Prof. Dr. Habibah A. Wahab for her kind support.

I am deeply thankful to IPS (USM) for financial support during my candidature (fellowshipP-FD-0082/11 R), School of P harmaceutical Sciences and EMAN Testing and Research Laboratory, USM, for providing lab facilities.

Special thanks to my colleagues and friends, Dr. Muhammad Adnan Iqbal, Dr. Fouad Saleih R. Al -Suede, Dr Zeyad D. Nassar, Hussein Bahri, Soheila Farahani, Mohammad Ali Sarvghadi, Dr. Aman Malik Shah Abdul Majid, Dr Syed Haroon Khalid, Dr. Loiy Elsir Ahmed Hassan, Armaghan Shafaei, Shamsuddin Sultan Khan, Yasser Tabana, Norshirin Idris, Sa’adiah Mohd Yusoff, Shazmin Kithur Mohamed, Hussein M Baharetha, Mansoureh Nazari, Elham Farsi and Saad Sabbar Dahham for their kind support and sincere help during my research.

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I owe my greatest appreciation to my family at Iran without their continuous support, love and patience I would have never been able to complete this project.

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TABLE OF CONTENT

ACKNOWLEDGEMENT ... ii

TABLE OF CONTENT ... iv

LIST OF TABLES ... x

LIST OF FIGURES ... xi

LIST OF ABBREVIATION ... xviii

ABSTRAK ...xxii

ABSTRACT xxv

CHAPTER 1 - INTRODUCTION 1.1 Cancer ... 1

1.1.1 Colorectal carcinoma ... 3

1.2 Major Signaling Pathways involving in Colon Cancer: ... 6

1.2.1 Wnt/β-catenin Signaling Pathway: ... 6

1.2.2 Notch Signaling Pathway ... 7

1.2.3 P53 Signaling Pathway ... 8

1.2.4 Cell Cycle (pRB/ E2F) Signaling Pathway ... 9

1.2.5 NF-кB Signaling Pathway... 10

1.2.6 Myc/Max Signaling Pathway ... 11

1.2.7 Hypoxia pathway ... 12

1.2.8 MAPK Signaling Pathways ... 14

1.3 Apoptosis ... 16

1.3.1 Apoptotic Pathways ... 17

1.3.1(a) Extrinsic Apoptotic Pathway ... 18

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1.3.1(b) The Intrinsic Pathway of Apoptosis ... 19

1.3.1(c) Perforin/granzyme Pathway ... 20

1.3.1(d) Execution Pathway ... 23

1.3.2 Caspase Family ... 24

1.3.2(a) Caspase-3, -6, -7 ... 25

1.3.2(b) Caspase-8 ... 26

1.3.3 Apoptosis and Colon Cancer ... 26

1.4 Cancer Cell Metastasis ... 29

1.5 Angiogenesis ... 30

1.5.1 Mechanisms of Angiogenesis ... 31

1.5.2 Vascular Endothelial Growth Factor (VEGF) ... 35

1.5.3 Angiogenetic Inhibitors ... 36

1.6 Anticancer Potential of Natural Prod ucts Isolated from Plants ... 38

1.7 Sandoricum koetjape ... 40

1.7.1 Characteristics of Sandoricum koetjape ... 40

1.7.2 Origin and Distribution ... 41

1.7.3 Botanical Description: ... 42

1.7.4 Traditional Medicinal Uses of S.koetjape ... 43

1.7.5 Phytochemistry and Pharmacological Activities of S.koetjape ... 44

1.8 Triterpenoids as Potential Angiogenic and Cancer Inhibitors ... 46

1.9 Pharmacological Activities of Koetjapic acid and Other Active Triterpenoids from S.koetjape’s Bark Extracts ... 49

1.10 Solubility Enhanced Formulations ... 51

1.11 Pharmacokinetic and Bioavailability study ... 52

1.12 Problem Statement and Objectives of Current Project ... 53

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CHAPTER 2 - MATERIAL AND METHODS

2.1 Chemical and Reagents ... 57

2.2 Plant material ... 61

2.3 Extraction and purification of koetjapic acid ... 61

2.4 Formation of potassium koetjapate (KKA) ... 63

2.5 Preparations of Solid Dispersions of Koetjapic Acid by Kneading Method ... 65

2.6 Chemical Characterization ... 65

2.6.1 Solubility studies ... 65

2.6.2 Differential scanning calorimetry studies ... 66

2.6.3 Fourier transform infrared spectroscopy studies ... 66

2.6.4 HPLC analysis ... 66

2.7 Pharmacokinetics and Bioavailability of potassium koetjapate (KKA) in Sprague Dawley Rats: ... 67

2.8 In Vitro Anticancer studies: ... 69

2.8.1 Culture conditions and maintenance of Cell lines ... 69

2.8.2 Harvesting and Counting of Cells ... 70

2.8.3 Assessment of Antiproliferative Effect of KKA by MTT Assay ... 71

2.9 Cell Death Studies ... 71

2.9.1 Determination of Nuclear Condensation Using Hoechst 33342 Stain ... 71

2.9.2 Detection of Mitochondrial Membrane Potential Using Rhodamine 123 Stain ... 72

2.9.3 Caspase Induction Assay ... 72

2.9.4 Human Apoptosis Protein Profiler Array ... 73

2.9.5 Transmission Electron Microsco pe (TEM) ... 75

2.10 Antimetastatic Studies ... 76

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2.10.1 Cell Invasion Assay ... 76

2.10.2 Migration Assay ... 77

2.10.3 Colony Formation Assay ... 77

2.11 HCT 116 Hanging Drop Assay ... 78

2.12 Transcription Factors Controlling 10 Major Cancer Signaling .... 79

2.13 Acute Toxicity Study In Rat ... 80

2.14 In vivo Antitumor Studies ... 81

2.15 Anti-Angiogenic Studies ... 83

2.15.1 Assessment of Cell Viability by MTT Assay... 83

2.15.2 EA.hy926 Cell Migration ... 83

2.15.3 EA.hy926 Cell Invasion ... 84

2.15.4 Tube Formation Assay ... 84

2.15.5 Ex vivo Rat Aortic Ring Assay ... 85

2.15.6 Assessment of VEGF Levels in HCT 116 Cells ... 86

2.15.7 In vivo Matrigel Plug Assay ... 86

2.16 Statistical Analysis ... 87

CHAPTER 3 - RESULTS AND DISCUSSION 3.1 Solubility Studies... 88

3.2 Chemical Characterization of KKA ... 89

3.2.1 FT-IR Spectroscopic Characterization of KKA ... 89

3.2.2 FT-IR Spectroscopic Characterization of Solid Disper sions of ... 92

3.2.3 HPLC Analysis and Optimization of KA and KKA ... 94

3.2.4 Differential Scanning Calorimetry ... 96

3.2.5 Characterization by FT-NMR spectroscopy... 98

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3.3 Pharmacokinetics and Bioavailability of KKA in Sprague Dawley

Rats ... 99

3.4 In vitro Anticancer Activities ... 107

3.4.1 Antiproliferative Effect of Koetjapic Acid Formulations on Cell Proliferation ... 107

3.4.2 Cell Death Studies ... 111

3.4.2(a) KKA Induces Morphological Modifications and Nuclear Condensation in HCT 116 Cells ... 111

3.4.2(b) KKA Reduces Mitochondrial Membrane Potential in HCT 116 Cells ... 113

3.4.2(c) KKA Increase Caspase-8, -9 and -3/7 Levels in HCT 116 ... 116

3.4.2(d) Observation of Ultra-Structural Apoptotic Morphology in HCT Cells Treated by KKA Using a Transmission Electromicroscope (TEM) ... 118

3.4.2(e) KKA Regulate the Expression of Multiple Proteins in the Apoptotic Pathways ... 119

3.5 Anti-metastatic Studies ... 122

3.5.1 KKA Inhibits Colon Cancer Cell Invasion ... 122

3.5.2 KKA Inhibits Colon Cancer Cell Migration ... 124

3.5.3 Inhibitory effect of KKA on clonogenicity of HCT 116 cells ... 126

3.6 Potassium Koetjapate Inhibits Tumour Aggregation Property In ... 129

3.7 The Modulatory Effects of KKA on Carcinogenesis Signalling 132 3.8 LD5 0 Value of KKA in SD Rats ... 133

3.9 Antitumor Activity of Potassium Koetjapate ... 134

3.10 Anti-Angiogenic Effect of KKA ... 143

3.10.1 Effect of KKA on Cell Viability of EA.hy926 Cell Line ... 143

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3.10.2 KKA Prevent Blood Vessels Outgrowth in Rat Aortic Ring Assay ... 145 3.10.3 KKA Inhibit EA.hy926 Wound Closure ... 147 3.10.4 KA Inhibits Differentiation of EA.hy926 cells on

Matrigel Matrix ... 149 3.10.5 Inhibition of Vascular Endothelial Growth Factor (VEGF)

Release ... 150 3.10.6 KKA Prevents Matrigel Induced Vasculature in Nude

Mice ... 151 3.11 Discussion ... 155 3.11.1 Develop formulation of KA with improved solubility ,characterization and assess its pharmacokinetic profile.

... 155 3.11.2 Antiproliferative Effect of Koetjapic Acid Formulations on Cell Proliferation ... 156 3.11.3 Pro-apoptotic and Anti-metastatic effects elicited by Potassium koetjapate ... 159 3.11.4 The Modulatory Effects of KKA on Carcinogenesis Signalling Pathways at Transcriptional Level ... 167 3.11.5 Preliminary safety profile and Antitumor Activity of Potassium Koetjapate ... 171 3.11.6 Anti-Angiogenic Effect of KKA ... 173 3.12 Summary ... 176

CHAPTER 4 - GENERAL CONCLUSION

4.1 Conclusion ... 178 4.2 Future Studies ... 181

REFERENCES ... 182 APPENDICES

LIST Of PUBLICATIONS

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LIST OF TABLES

Page

Table 2.1 Chemical and Reagents 56

Table 2.2 Equipments and Apparatus 58

Table 3.1 Solubility Values Of Pure Koetjapic Acid, Potassium Koetjapate (KKA) And The Solid Dispersions Prepared With Various Carriers Via Kneading Method In Water At 25°C

86

Table 3.2 The Selectivity Index (SI) Which Represents IC50 For Normal Cell Line/IC50 For Cancerous Cell Line After 48 Hours Of Treatment

95

Table 3.3 Effect Of KKA On The Expression Of Proteins Involved In Apoptosis

96

Table 3.4 Subcutaneous Tumor Volumes In Different Treatment Groups

97

Table 3.5 Changes In Body Weight Detected In Different Treatment Groups

99

Table 3.6 Pharmacokinetic parameters of KKA in rat plasma after oral administration) (n=6)

100

Table 3.7 The selectivity index (SI) which represents IC50 for normal cell line/IC50 for cancerous cell line after 48 hours of treatment.

101

Table 3.8 Effect of KKA on the expression of proteins involved in apoptosis

113

Table 3.9 Subcutaneous tumor volumes in different treatment groups

128

Table 3.10 Changes in body weight detected in different treatment groups

128

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LIST OF FIGURES

Page Figure 1.1 Adenoma-Carcinoma Sequence, Adapted from

Qiagen (website)

6

Figure 1.2 Schematic Illustration Of Apoptosis. The Three Pathways Of Apoptosis I.E. Extrinsic, Intrinsic And Perforin/Granzyme Pathways. Adopted From (Elmore, 2007)

19

Figure 1.3 TRAIL Death-Receptor Pathway Of Apoptosis 23 Figure 1.4 Steps Involved in Angiogenesis Adopted By

(Welti, Loges, Dimmeler, & Carmeliet, 2013).

35

Figure 1.5 Pictures of S.Koetjape Merr. Leaves, Flowers, Fruits And Bark

44

Figure 1.6 Chemical Structure of Some Terpenoids Isolated From The Bark

46

Figure 1.7 Chemical Structures of ursolic Acid And oleanolic Acid

49

Figure 1.8 Chemical structure of synthetic triterpenoids with strong antiangiogenic activity. Top: CDDO;

middle: CDDO-Me; bottom: CDDO-Im

50

Figure 2.1 Flow Sheet Diagram for Re-Crystallization (Purification) of The Ka

61

Figure 2.2 Conversion of Koetjapic Acid Into Salt of Potassium Koetjapate

85

Figure 3.1 Conversion of Koetjapic Acid Into Salt of Potassium Koetjapate

88

Figure 3.2 FT-IR Spectra (Overlay) of Koetjapic Acid and KKA

88

Figure 3.3 A Chemical Structures of Polyvinylpyrrolidone and Koetjapic Acid(KA) B: FT -IR Spectra (Overlay) of Koetjapic Acid and Solid Dispersions of KA: PVP

89

Figure 3.4 HPLC Chromatograms Showing The Peaks of Koetjapic Acid (a) and Potassium Koetjapate (b)

91

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Figure 3.5 The Thermograms of The Tested Samples 93 Figure 3.6 Chromatograms of blank rat plasma (A), and rat

plasma spiked with 10 µg/ml of KKA (B)

94

Figure 3.7 Calibration curve of KKA 94

Figure 3.8 Chromatograms of KKA in rat plasma at 2 hours after oral administration of 50 mg/kg KKA (A);

rat plasma at 1 hour after intravenous administration of 50 mg/kg KKA (B)

97

Figure 3.9 Mean plasma concentration vs. time profiles (mean± S.E.M, n=6) of KKA after intravenous administration at 50 mg/kg in rat plasma

98

Figure 3.10 Mean plasma concentration vs. time profiles (mean± S.E.M, n=6) of KKA after oral administration at 50 mg/kg in rat plasma

98

Figure 3.11 Photomicrographic images of HCT 116 cells taken under converted phase-contrast microscope at ×200 magnification using a digital camera at 48h.

102

Figure 3.12 Images of HT 29 Cells Taken Under an Inverted Phase-Contrast Microscope at ×200 Magnification Using a Digital Camera At 48h.

103

Figure 3.13(a)

The Photomicrographs Depict the Images of HCT 116 Cells with Hoechst 33342 Stain Taken at 6 and 18 H after the Treatment with KKA. The Arrows Indicate the Clear Signs of Nuclear Condensation Including the Half -Moon (Crescent)-Shaped Apoptotic Nuclei. The Arrowheads Indicate the Apoptotic Cells, Chromatin Dissolution, Breakdown and Fragmentation.

105

Figure 3.13(b)

Graphical Representation of Percentage of Apoptotic Indices. The Apoptotic Index for Each Test Group Was Expressed as A Percentage of The Ratio of Apoptotic Cells Number to The Total Cell Number in 10 Different Fields. Values are Presented as Mean ± Sd (N = 10), *Represents P

< 0.01 and **Represents.

106

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Figure 3.14(a)

The Photomicrographs Demonstrate the Efficacy of KKA in Disruption of The Mitochondrial Membrane Potential. The Mitocho ndrial Membrane Potential in HCT 116 Cells was Evaluated by Visualizing the Uptake of The Lipophilic Cation Dye Rhodamine 123 Into Mitochondria. The Results Showed That the Fluorescence Signal Decreased Drastically With Respect to The Decrease of Mitochond rial Membrane Potential Due to The Treatment With KKA

107

Figure 3.14(b)

Graphical Representation of Percentage of Apoptotic Indices.The Apoptotic Index for Each Test Group Was Expressed as A Percentage of The Ratio Of Unstained Cells Number to The Total Cell Number in 10 Different Fields. Values are Presented as Mean ± SD (N = 10), *Represents P < 0.01 And **Representsp < 0.005.

108

Figure 3.15(a)

Induction of Caspase Activity in HCT 116 Cells Treated with KKA and Betulinic Acid (5 Μm).

The Cells were Stained With The Green Fluorescent FAM-VADFMK Dye After 8h Of Treatment. The Photomicrograph Shows The Fluorescence-Emitting Cells. The Images Were Taken By EVOS Fluorescence Microscope At 20×

Magnification.

109

Figure 3.15(b)

Graphical Representation of The Per Cent Induction of Caspase Activity in Representative Group of Cells. Values are Presented as Mean ± SD (N = 10), *Represents P < 0.01 And**Represents P < 0.005.

110

Figure 3.16 Ultrastructural Micrographs by TEM Reveal The Apoptotic Property of KKA. Ultra -Structural Micrograph of HCT After 24 H Treatment With 3.5 and 7 µm/Ml of the KKA. A Indicates Untreated Cell That Received Distilled Water; It Showed Normal Cell Morphology With Intact Cell Membrane. The Treated Cells With 3.5 and 7 µm/Ml of The KKA Showed Related Apoptotic Morphological Changes Such as Chromatin Condensation, Fragmentation, Blebbing of Cell Membrane and Formation Apoptotic Bodies (B - E). Photos Were Taken at 1600x Magnification (Scale Bar 5 µm).

111

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Figure 3.17 Effect of KKA Treatment on The Expression of Multiple Proteins Involved In The Cell Death Cascade. Heatmap Represents Signal Intensities of Each Protein in A Control and KKA Treatment Group. Red Band in Cluster Diagram Shows Up- Regulation While Green Band Indicate Down - Regulation of Protein. Values Indicated Are Mean

± SD of Two Independent Experiments (N = 4 For Each Protein). * = P < 0.05, ** = P < 0.01 And

*** = P < 0.001 Respectively

112

Figure 3.18 Photomicrographic Illustration of The Anti - Invasive Activity KKA Against HCT 116 Cells.

The Cells Were Counted and The Results Are Reported as The Average Percentage of Three Independent Experiments (N = 3) in Term of Invasion Inhibition Compared to The Negative Control (Distilled Water). Values Shown are Mean ± SD. *** Indicates P < 0.001: Significant Difference of Invasion Inhibition at Different Concentrations (2.5(A) And 5(C) µg/Ml) And Positive Control 5-FU(D) Compared To The Negative Control , Distilled Water,(B).

114

Figure 3.19 Wounds (Arrows) Of HCT Cells Treated With The Concentration Of 2.5 And 5 µm/Ml Of KKA And 5 µm/Ml Of 5-FU. At Zero, 6 And 18 H, And Negative Control (Distilled Water). Both Concentration Of KKA Show Significant Inhibition At 6 And 18 H Compared To The Negative Control (Distilled Water) Which Shows Complete Wound Closure At 18 H. Photos Were Taken At 4x Magnification (Scale Bar 1000 µm).

116

Figure 3.20 Percentage Of Inhibiting Migration Of HCT Cells After 6h And 18 H Treatment KKA Prevented The Migration Of HCT Cells Significantly At Two Selected Doses Compared To The Negative Control (Distilled Water). (*** P < 0.001).

Results Are Means ± SD Of Three Experiments.

117

Figure 3.21 (a) Effect of koetjapic acid, potassium koetjapate and betulinic acid on colony formation of HCT 116 cells. The picture clearly depicts the dose - dependent inhibition of HCT 116 colonies.

(b) Graphical representation depicts percentage of plating efficiency of representative test groups.

Plating efficiency was determin ed by the percent ratio of number of colonies developed to the number of cells initially seeded.

119

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(c) Graphical representation illustrates the percentage of surviving fraction obtained after the treatment with koetjapic acid, potassium koetjapate and betulinic acid. The percent surviving fraction of HCT 116 colonies were decreased with increasing concentration of potassium koetjapate.

Figure 3.22 The Comparative Effects Of Potassium Koetjapate On in vitro HCT 116 Tumour In Hanging Drop Assay

122

Figure 3.23 Treatment With KKA (7 Μm/Ml) Significantly Altered The Expression Of Multiple Cell Signalling Pathways In HCT 116 Cells.

Significant Decrease In The Activity Of Notch, Wnt, Hypoxia, MAPK/ERC And MAPK /JNK, And Significant Up-Regulation Of Transcription Factor For Cell C ycle (Prb-E2F) Pathway Was Observed. While No Significant Changes Detected In C-Myc,, P53, NF-Kb, And TGF-Β Pathways. Error Bars Indicate Standard Deviations From Mean. *P < 0.05, **P < 0.01 And

***P < 0.001

123

Figure 3.24 In Vivo Antitumor Efficacy Of KKA Determined Using A Human Tumor Xenograft Model In Athymic Nude Mice Bearing HCT-116 Tumors At Day 21 Post- Inoculation.

127

Figure 3.25 Graphical Illustration Of Antitumor Effect Of KKA In An Ectopic Xenograft Colon Cancer Model. Values Shown Are Mean ± SD (N = = 5 – 6 Per Group). Tumor Size In The Treated Group Was Compared To That In Negative Control Group. *P Values < 0.05 Show Significant Inhibition Of Tumor Growth.

129

Figure 3.28 Effect Of KKA And KA On Cell Viability Of EA.Hy926 Cell Line. Values Exposed Are Mean

± SD Of Three Independent Experiments (n = 3)

132

Figure 3.29 Antiangiogenesis Effect Of Of KKA In The Rat Aortic Ring Model. A = NC B = 12 µg/Ml, C = 25 µg/Ml, D = 50 µg/Ml, E = Betulinic Acid 20 µg/Ml F=100 µg/Ml Of KKA

133

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Figure 3.30 Average Lengths Of Blood Vessels In KKA Treatment And Control Groups Values Shown Are Mean ± SD Of Three Independent Experiments (n = 3). * = p < 0.05, ** = p < 0.01 And *** = P < 0.001 Show Significant Different In Growth Inhibition Activity Of KKA And PC (BA, 20 µg/Ml) Treated Groups With Negative Control (Distilled Water).

134

Figure 3.31 Micrograph Illustration Of Anti -Migratory Effect Of KKA Against EA.Hy926 Cells. Photos Were Taken At 4 × Magnification

135

Figure 3.32 Graphical Representation Of The Dose And Time - Dependent Inhibitory Effect Of KKA On Migration Of EA.Hy926 Base On The Average Distance Traveled By The Cells . ± SD (N = 3). *

= P < 0.05

136

Figure 3.33 Images of EA.hy926 Matrigel tube formation assay. KA inhibits matrigel tube formation.

EA.hy926 were treated with (A) 10 µm/ml ,KKA (B) 30 µm/ml , KA (C) 30 µm/ml ,KKA (D) negative control(distilled water).

137

Figure 3.34 The Dose response relationship of KA on tube formation assay, Values shown are mean ± SD of three independent experiments (n = 3). * = p <

0.05, ** = p < 0.01 and *** = p < 0.001 show significant activity of KKA and KA treated groups on EA.hy926 tub formation inhibition ,compare to negative control (distilled water).

137

Figure 3.35 Effect Of KKA On The Release Of VEGF-A From Human Colon Cancer HCT 116 Cells. Significant Decrease In The Secretion Of VEGF Was Detected In All The Treatment Groups. Values Shown Are Mean ± SD Of Three Independent Experiments (N = 3). Ns = * = P < 0.05, ** = P <

0.001 And *** = P < 0.001 Respectively.

138

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Figure 3.36 In Vivo Anti-Angiogenic Activities Of KKA Determined Using A Human Tumor Xenograft Model In Athymic Nude Mice Bearing HCT -116 Tumors At Day 21 Post-Inoculation. KKA At Different Doses Of 50 (B And B), 100 (C and c) And 200 Mg/Kg (E and e)) Strongly Inhibited Vascularization In Matrigel Plugs Implanted In Nude Mice In Compare To Negative Control Treated Group (A and a). Comparable With Imatinib As PC At The Dose Of 100 Mg/Kg. (D and d).

140

Figure 3.37 H&E Stained Cross-Sections Taken From Matrigel Plugs Implanted Subcutaneously In Representative Groups Of Animals. Arrows Indicate Prompt And Well -Developed Blood Vessels In The Control Group(A) In Compare With Less Blood Vessels In Treated Groups: B (KKA, Dose100 Mg/Kg) C: (PC, Imatinib 100 Mg/Kg), D: (KKA, 200 Mg/Kg).

141

Figure 3.38 Graphical Representation Of The Effect Of KKA On The Mean Blood Vessel Count In Matrigel Sections. (*** = P < 0.001, N = 6, Values Are Mean ± SEM Of 10 Low Power Microsco pic Fields).

141

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LIST OF ABBREVIATION

APC gene Adenomatous polyposis coli gene

LDH Lactate dehydrogenase

AIF Apoptosis inducing factor

Apaf-1 Apoptotic protease activating factor 1 BAG-1

BAK Bax

Bcl-2 family anti-apoptotic protein Bcl-2 homologous antagonist killer Bcl-2 Associated-X Protein

Bcl-2 Bid Bim CAM

B-cell lymphoma-2

BH3 interacting-domain death agonist Bcl-2-interacting mediator of cell death Chick embryo chorioallantoic membrane

CC Column chromatography

CCRF-CEM CDDO CDDO-Me CDDO-Im

Human lymphoblast leukemia cell line 2-cyano-3,12-dioxoolean-1,9-dien-28-oate 2-cyano-3,12-dioxoolean-1,9-dien-28-oate methyl

2-cyano-3,12-dioxoolean-1,9-dien-28-oic imidazolide

cFLIP Cellular FLICE-like inhibitory protein cIAP Cellular inhibitor of apoptosis

CL-6 Cox2

Cholangiocarcinoma cells Cyclooxygenase-2

CRC CTL

Colorectal carcinoma cytotoxic T lymphocytes Cyt c

DCC

Cytochrome c

Deleted in Colorectal Carcinoma DCFH-DA 2′,7′-Dichlorofluorescein diacetate

DcRs Decoy receptors

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DISC Death inducing signalling complex

DIABLO Direct inhibitor of apoptosis-binding protein with low pI

DMSO

DNA

Dimethyl sulfoxide Deoxyribonucleic acid DPPH

DSC EC

2,2 Diphenyl-1-picrylhydrazyl

Differentialtial scanning calorimetry Endothelial cell nmr

ECM Extracellular matrix

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor ERK ½

EMT EMSA

Extracellular-signal-regulated kinases Epithelial–mesenchymal transition Electrophoretic mobility shift assay

FADD Fas-associated death domains

Fas (CD95/Apo1) First apoptotic signal/ Cluster of differentiation 95/ Apoptosis antigen 1

FasL Fas Ligand

FBS Foetal bovine serum

FLIP FLICE like inhibitory protein FRAP

FT-NMR

Ferric reducing antioxidant power

Fourier transform-nuclear magnetic resonance GC-MS Gas chromatography mass spectrometer

Hep-2 Human laryngeal cancer cell line HIF-1α Hypoxia inducible factor-1α

HREs Hypoxia response DNA elements

HSP Heat shock proteins

IAPs IGF2

IGFBP-2

Inhibitors of apoptosis proteins Insulin-like growth factor 2 receptor

Insulin like growth factor binding protein 2

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IGFBP-6 Insulin like growth factor binding protein 6

IκΒ Inhibitory κβ

K562 KA KKA

Human erythroleukemia cells Koetjapic acid

Potassium koetjape

KB Oral carcinoma cells

LD5 0

LEF1

Lethal dose to kill 50% of animals Lymphoid enhancing factor-1 MAPK Mitogen activated protein kinases MCF-7 Breast adenocarcinoma cell line

MMP Matixmetalloproteases

MOH Ministry of health

MOMP Mitochondrial outer membrane potential MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl

tetrazolium bromide

MVD Microvessels density

NCR National cancer registry

NF-κB NMR

Nuclear factor-κB

Nuclear magnetic resonance

NuMA Nuclear protein

PBS Phosphate buffer saline

PIGF Placental growth factor

pRb Retinoblastoma protein

PS PVP

Phosphatidylserine Polyvinylpyrrolidone Smac

Rpm

RPMI-1640

Second mitochondria-derived activator of caspases

Revolutions per minute

Roswell Park Memorial Institute-1640

ROS Reactive oxygen species

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SD Sprague Dawley SI

SIMPs

Selectivity index

Soluble intermembrane proteins

TCF T-cell factor

TGF TGI

Transforming growth factor Tumor growth inhibition

TLC Thin layer chromatography

TNF-R Tumour necrosis factor cell surface death receptors

TRAIL Tumor necrosis factor-related apoptosis inducing ligand

TRAILR1 (DR4) Death receptors 4 TRAILR2 (DR5) Death receptors 5 TS

USM

Tumor spheroid

Universiti Sains Malaysia

VEGF Vascular endothelial growth factor VEGFR

WNT

VEGF receptors Wingless integrated

xIAP X-linked inhibitor of apoptosis

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KESAN KALIUM KOETJAPAT, SATU TERBITAN ASID KOETJAPIK YANG DIASINGKAN DARIPADA SANDORICUM KOETJAPE MERR. KE ATAS KANSER KOLOREKTAL MANUSIA.

ABSTRAK

Dalam kajian ini, usaha dilakukan untuk menambahbaik pelarutan air acid koetjapik (KA) dan mengkaji efikasi anti - kanser kolonnya, menggunakan metod-metod ‘in vitro’ dan ‘in vivo’. Garam kalium KA iaitu, kalium koetjapat disediakan melalui metod separa -sintetik. Aktiviti - aktiviti antikanser kalium koetjapat dibandingkan dengan aktiviti sebatian asal iaitu, KA. Asai viabiliti sel MTT digunakan untuk mendapatkan dan membandingkan nilai IC5 0 kedua-dua sebatian. Kesan-kesan pro-apoptotik kalium koetjapat dinilaikan dengan menggunakan asai kaspas -kaspas (3/7, 8 dan 9), pewarna fluresen Hoechst 33342 dan Rhodamine 123. ‘Profiler array’ protiom apoptosis manusia digunakan untuk mengenalpasti sasaran protin yang bertanggungjawab bagi induksi apoptosis. Tambahan lagi, kesan antitumor in vitro kalium koetjapat telah dikaji menggunakan asai

‘titik tergantung’. Tiga dos kalium koetjapat (25, 50, dan 100 mg/kg berat badan) telah dikaji dalam model m encit bogel ‘athymic’ untuk mengkaji efikasi in vivo anti-tumor kalium koetjapat. Dalam kajian ini pelbagai formulasi KA telah disediakan. Kajian pelarutan menunjukkan bahawa derivatif KA iaitu, kalium koetjapat, mempunyai pelarutan air lebih baik daripada dispersi pepejal KA. Kajian antikanser in vitro menunjukkan bahawa kalium koetjapat mempunyai aktiviti sitotoksik lebih baik daripada KA dan kompleks dispersi pepejalnya, terhadap titisan sel HCT 116. asai

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pewarna fluresen menunjukkan bahawa kalium koetjapat mempunyai sifa t menginduksi apoptosis. Ia menginduksi kondensasi kromatin dan menurunkan potensi membran mitokondria secara kebergantungan dos.

Tambahan lagi, ia menaikkan tahap kaspas dalam sel -sel HCT 116.

Keputusan protin apoptosis ‘array’ menunjukkan bahawa kalium koetjapat mempengaruhi aktiviti beberapa protin. Ia menurun-aturkan ekspresi beberapa protin anti-apoptotik dan regulator negatif apoptosis termasuk Bcl-2, HSP60, HSP90 dan IGF-1 dalam sel-sel HCT 116 dengan penaik- aturan protin-protin TRAILR-1 dan TRAILR-2, CD40, IGFBP-6, p27, kaspas 3 dan kaspas 8. Tambahan lagi, kalium koetjapat menunjukkan kesan antimetastatik terhadap sel HCT 116 dalam asai -asai in vitro. Keputusan-keputusan ini mungkin disebabkan oleh penurun -aturan laluan- laluan signal Wnt, Notch, Hypoxia, MAPK/ERC dan MAPK/JNK dalam sel-sel HCT 116, bersama dengan penaik -aturan faktor transkripsi untuk laluan-laluan putaran sel (pRb -E2F). KKA juga menghalang proses angiogenesis in vitro dengan menghalang proses -proses penembusan, penghijrahan dan pem bentukan dan pembentukan tiub sel -sel endothelium . Kajian toksisiti akut menunjukkan bahawa kalium koetjapat mempunyai LD5 0 lebih daripada 2000 mg/kg dalam tikus betina SD. Keputusan kajian tumor ‘spheroid’ menunjukkan bahawa kalium koetjapat mempunyai kebergantungan dos potensi antitumor dan data ini berkait dengan keputusan kajian tumor in vivo. Kalium koetjapat menunjukkan perencatan poten pembiakan tumor (68.15%, 82.35% dan 92.76%, pada 25, 50 dan 100mg/kg, secara berurutan). Keseluruhannya, keputusan kajian ini

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menunjukkan bahawa kalium koetjapat mempunyai aktiviti anti -kanser terhadap kanser kolorektal.

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EFFECT OF POTASSIUM KOETJAPATE, A DRIVATIVE OF KOETJAPIC ACID ISOLATED FROM SANDORICUM KOETJAPE

MERR. ON HUMAN COLORECTAL CANC ER.

ABSTRACT

In the present study an attempt was made to enhance the aqueous solubility of KA and to study its anti -colon cancer efficacy using in vitro and in vivo methods. Potassium salt of KA i.e., potassium koetjapate was prepared by semi-synthetic method. Anticancer activities of potassium koetjapate were compared with the native compound i.e., KA. MTT cell viability assay was used to obtain and compare the IC5 0 values of both the compounds. Pro-apoptotic effects of potassium koetjapate were assessed using caspases (3/7, 8 and 9), Hoechst 33342 and Rhodamine 123 fluorescent staining assays. Human apoptosis proteome profiler array was used to identify the protein targets responsible for the induction of apoptosis. Furthermore, in vitro antitumor effects of potassium koetjapate were studied using hanging drop assay. Three doses of potassium koetjapate (25, 50, and 100 mg/kg body weight) were tested in athymic nude mice model to study the in vivo anti-tumorigenic efficacy of potassium koetjapate. In this s tudy, various formulations of KA were prepared. Solubility studies revealed that resultant KA derivative i.e.

potassium koetjapate had better aqueous solubility than the solid dispersions of KA. In vitro anticancer studies revealed that potassium koetjapate has better cytotoxic activity than KA and its solid dispersion complex towards HCT 116 cell line. Fluorescent staining assays showed

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that potassium koetjapate has apoptosis -inducing nature. It induced chromatin condensation and decreased mitochondrial me mbrane potential in a dose-dependent manner. Furthermore, it increased the levels of caspases in HCT 116 cells. The results on apoptosis protein array show that potassium koetjapate modulated the activity of multiple proteins. It down-regulates the expression of multiple anti -apoptotic proteins and negative regulators of apoptosis including Bcl -2, HSP60, HSP90 and IGF- 1 in HCT 116 cells with concomitant up -regulation of TRAILR -1 and TRAILR-2, CD40, IGFBP-6, p27, Caspase 3 and caspase 8 proteins.

Furthermore, potassium koetjapate showed antimetastatic effect towards HCT 116 cells in a series of in vitro assays. These results are probably due to down regulation of Wnt , Notch, Hypoxia, MAPK/ERC and MAPK/JNK signalling pathways in HCT 116 cells coupled with t he up-regulation of transcription factor for cell cycle (pRb -E2F) pathways. Moreover, KKA inhibited angiogenesis in vitro by stopping endothelial cells neovascularization, migration, tube formation and VEGF release. Acute toxicity studies reveal that potas sium koetjapate has LD5 0 more than 2000 mg/kg in female SD rats. Results of spheroid tumor studies show that potassium koetjapate has dose -dependent antitumor potential and this data correlates with the outcomes of the in vivo tumor studies. Potassium koetjapate showed potent inhibition of tumor growth (68.15%,82.35 % and 92.76% at 25, 50 and 100mg/kg, respectively). Altogether, outcome of present study shows that potassium koetjapate has good anti -cancer activity towards colorectal cancer.

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CHAPTER 1 INTRODUCTION

1.1 Cancer

A major global health issue, cancer is described as unrestrained growth of cells leading to the invasion of local tissues and tumor metastasis (1). Cancers are categorized by the type of cells from which the tumors originate including carcinoma, sarcoma, blastoma, germ cell tumor, lymphoma, leukemia and adenoma (benign tumor of glandular origin), adenocarcinoma (malignant adenoma). Cancers de rived from epithelial cells that include common cancers such as most types of breast, prostate, lung and colorectal cancers are subsumed under carcinoma while sarcoma cancer types occurring in connective tissues such as fat, bone, nerve, and cartilage. Conditions such as leukemia and lymphoma start off from hematopoietic whereas germ cell tumors which are mostly existing in the testicle or the ovary result from pluripotent cells. Blastoma cancers originate from immature “precursor” cells or embryonic tiss ue (2). In 2008, lung, breast and colorectal cancers were reported to be the three most commonly diagnosed cancers, while in case of cancer -associated mortalities worldwide, lung, stomach, and liver cancers were found to be the most common cancer types (3) Presently more than 200 kinds of cancer have been recognized which are the second major cause of death worldwide, overtaken only by heart disease (4). In 2015, cancers of breast, colorectal, lung and prostate were estimated to be the most common causes of cancer-related mortalities (5). According a recent report, cancer is now

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the leading cause of death in 21 states in the USA, cancer is currently the primary cause of death, due to exceptionally large reductions in deaths from heart diseases. Although cancer -related death rate has decreased by 23% since 1991, or more than 1.7 mill ion deaths were prevented up to 2012, death rates are increasing for cancers of the pancreas, liver, and uterine corpus . In developing countries, there are more new cases than what is documented in the developed countries (5,600,000 vs. 7,100,000 cases respectively(6) . Cancer was found to be the third most common reason for deaths overtaken by cardiovascular and septicaemia diseases in Malaysia according to the Malaysian National Cancer Registry report in 2007.

According to another report, three most commonly diagnosed cancers in Malaysia were breast, cervical and colorectal cancers followed by bone marrow, lung, lymph node and liver cancers respectively while five most commonly diagnosed cancers in the male population were lung, colorectal, nasopharynx, prostate and lymphoma respectively. W hereas in the female population, cancers of breast, colorectal, cervix, ovary and lung were at the top list (7). A range of factors may result in an increase in the risk of cancer. The causative factors involved in the progress of cancer include smoking, obesity, exposure to chemicals, oxidative stress, and radiation as external factors while inherited mutations (hyper-activation of oncogenes, and inhibition of tumor-suppressing genes), metabolic deregulations, hormone imbalance, and dysfunction of immune system are considered as the main internal factors (8) . Surgery, chemotherapy and radiotherapy are the most common treatments of cancer n owadays (9).

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1.1.1 Colorectal carcinoma

Among various types of cancer, colorectal cancer (CRC), a malignant tumor of large intest ine, ranks third in the world as a lethal and metastatic carcinoma while the incidence and death rates of CRC has decreased by around 3% per year in both men and women from 2003 up to the end of 2012(5). Still, CRC is a significant cause of mortality in both men and women worldwide. According to a survey, every year more than 945 000 people develop colon cancer out of which around 492 000 patients die (10). in the Malaysian Peninsula, CRC is the most prevalent type of cancer in men and the third most common cancer in women According to the National Cancer Registry Report 2003 -2005. The Age-Standardized Rate (ASR) was highest among Chinese men, in whom it was more than two times that of Indian and Malay men. Chinese women also had an ASR, which was more than twice that of Indian and Malay women. Hypertension, obesity, abnormal blood lipids, and high fasting blood glucose are considered as main metabolic risk factors for colorectal cancer (11) . The growth in colorectal tumor is due to the mutational activation of oncogenes coupled with the mutational inactivation of tumor suppressor genes . Hence, it is described as a multi-step disease that converts normal epithelial cells of the colon into invasive carcinoma (Figure 1.1). An ordered series of events, recognized as the “Adenoma-Carcinoma Sequence”, causes the development of colorectal neoplasms. There must be at least four mutated genes to form malignant tumors affected by few further chan ges leading to benign tumorigenesis. The histopathologic changes occur due to genetic as well as environmental factors. The most important environmental factors

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include pathogen invasion, toxins, generation of ROS (Reactive Oxygen Species) and stress conditions. Currently, the inherited and somatic genetic deficiencies contributing to the development of colorectal carcinogenesis have been discovered such as sustainable changes in COX2 (Cyclooxygenase-2), KRas, Ctnn-Beta (Catenin-Beta), APC (AdenomatousPolyposisColi),SMAD4 (Smaand MAD (MothersAgainstDe capentaplegic), p53, TGF-BetaR2 (Transforming Growth Factor -Beta Receptor-Type II), BAX (Bcl2 Associated-X Protein), E2F4 (E2F Transcription Factor-4) and MMR (Mismatch Repair) genes like, MSH2 (MutS Homolog-2), MSH3 (MutS Homolog-3), MSH6 (MutS Homolog-6), MLH1 (MutL Homolog-1) and MMP (Matrix Metalloproteinase)-1/2/7/9/11/12/14, loss of the 18q21 gene and microsatellites instability (12-14). At the beginning, when the normal colonic epithelia (Stage-0) is subject to unfavorable conditions such as pathogenic invasions, APC and Ctnn -Beta mutations, toxins and generation of ROS; it is changed into Dysplastic ACF (Aberrant Crypt Foci) or Dysplastic Adenoma or Dysplast ic Epithelia (Stage-I) (15). An increase in microsatellite repeat sequences along with KRas and COX2 mutations play a major role in transforming Dysplastic epithelia to early adenoma phase (Stage-II). KRas mutations occur in 39% of human colorectal cancers such as KRas mutations combined with MSH2 (deletion) mutations DCC (point mutation in 70% of colon cancers) and MLH1 (substitution, deletion and hypermethylation of CpG sites), which lead to the transition fr om early adenoma to late adenoma (Stage -III). Lastly, transitions from late adenoma to increasingly tumor metastasis through colorectal carcinoma (Stage -IV))

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comprise the less frequently targeted genes like BAX via frameshift mutations which happen in over 50% of colon adenocarcinomas along with deletion in E2F4, MSH3 and MSH6 fallowed by gene modifications of p53 and TGF-BetaR2, MMP1/MMP2/MMP7/MMP9/MMP11/MMP12 (over - expression) and SMAD4 mutations (lack of alleles on chromosomes 17 and 18 in polyploid colo rectal tumors). Particularly, point mutation on p5 3 gene is the main reason for causing 50% of colorectal cancers whereas SMAD4 mutations (aneuploid/polyploid) are linked with metastatic carcinoma (16, 17).

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Figure 1.1: Adenoma-Carcinoma Sequence, Adapted from Qiagen (18)

1.2 Major Signaling Pathways involving in Colon Cancer:

1.2.1 Wnt/β-catenin Signaling Pathway:

The Wnt signal transduction pathway is an old pathway that is evolutionarily conserved and has a key role in the development of the embryo such as cell migration, cell polarity, along with a major role in cell to cell signaling (19, 20). Mutations in the Wnt pathway have a fundamental

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role in cancer development, especially in colon cancer. The Wnts consist of a large family of nineteen glycoproteins in humans. So far, major signaling branches which are downstream of the Fz receptor have been identified such as canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin independent pathway. The Planar Cell Polarity and the Wnt/Ca2+

pathways are two branches of Wnt/β-catenin dependent pathway which are being actively dissected at the molecular and biochemical levels (21).

Around 90% of all colon cancer cases are related to the APC tumor suppressor gene mutations, which trigger Wnt/β-catenin accumulation (22).

β-catenin and TCF/LEF transcription factors combine and consequently boost the expression of c-Myc, cyclin D1 and MMP genes involved in carcinogenesis and tumor angiogenesis (23) . Therefore, a possible objective in curing different types of cancer, i.e. colon cancer, can be the down - regulation of the Wnt pathway.

1.2.2 Notch Signaling Pathway

Notch signaling pathway can promote or suppress cell proliferation, differentiation, death, and fate specification. This pathway significantly interferes in some physiological programs such as apoptosis, adhesion, migration and angiogenesis throughout adult tissue renewal leading to the development of the organism. Because of its essential role in many important processes, abnormal gain or loss of pathway components has been directly linked to multiple human diseases including cancer (24, 25).

Notch can be an oncogene or a tumor suppressor gene based on factors such as the timing, cell type, signal strength, and the n ormal function of certain

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tissues (26) . Various types of cancer including colon, melanoma, renal, breast, pancreas, and lung cancers have Notch pathway signaling (27, 28).

Some studies have shown strong relations between Notch and Wnt pathways in colon cancer which strengthen the hypothesis that Notch signaling might be in a downstream of Wnt (29-31). Thus, Notch signaling pathway can be used in combination with Wnt inhibitors as a potential treatment of colorectal carcinoma.

1.2.3 P53 Signaling Pathway

The P53 is a nuclear protein, which is known as “the guardian of the genome” because of its role in the detection of genetic damage and in triggering the genetic repair mechanisms (32). It can also trigger apoptosis in case of irreparable DNA damage. When P53 is mutated, it will not be able to perform its function as frequency (>50%) of mutation in the P53 gene is the most in human cancer. This indicates that the P53 tumor suppressor gene has an a key role in cancer prevention is played by the P53 tumor suppressor gene through the cell cycle arrest mechanism which may result in inducing apoptosis (33). Mutations in the P53 suppressor gene are common in all cancers, which usually occur in the central DNA -binding core domain. Mutations mostly hamper the protein’s ability to attach to its target DNA strands, thus preventing transcriptional activation of the genes.

The P53 protein controls cell death using different mechanisms that result in the regulation of genes entangl ed in both the extrinsic and intrinsic pathways of apoptosis either via transcriptional -independent or transcriptional-dependent mechanisms (34). Therefore, increasing the

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amount of P53 could be a promising strategy in the treatment of cancers with lower side effects. Recently, small molecules such as small peptides have been used to reactivate the suppressed wild -type P53 or to return the mutant into a wild-type P53 (35).

1.2.4 Cell Cycle (pRB/ E2F) Signaling Pathway

Retinoblastoma (Rb) is a malignancy of the developing retina, which happens in children and is considered to be the most common malignancy of the eye in children (36). The retinoblastoma tumor suppressor (pRB) plays an important role in cell cycle processes and apoptosis. The pRB gene is mutated in around 50% of all human tumors . Moreover, genes encoding upstream regulators of pRB have been found to be mutated in the remaining 50% of all human tumors. About 60% of affected individuals have unilateral Rb while around 40% have bilateral Rb Heritable retinoblastoma is an autosomal dominant vulnerability to Rb RB1 was the first tumor suppressor gene cloned. Recently, it has been reported that pRB can bind with a series of transcription factors such as E2F and form dimmers tha t control the expression of several downstream effector genes involved in cell cycle control, mitosis, DNA repair and apoptosis (37). It appears that RB1 interacts with more than 100 cell proteins resulting in regulation of the critical G1 to S-phase transition in the cell cycle. It has also b een found that the Rb/E2F complex has the main role in maintaining G1 arrest in connection with the p21/p27 family of cdk inhibitors (38).

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1.2.5 NF-кB Signaling Pathway

The nuclear factor κB enhancer binding protein (NF-κB) family of transcription factors, control the expression of a lar ge group of genes engaged in diverse cellular processes including the inflammation, immunity, migration, adhesion, cell growth, apoptosis and cell survival.

Deregulated NF-κB has been linked to a variety of human diseases, particularly cancers. The oncogen ic role of NF-κB seems to be mediated through its anti-apoptotic function, particularly through induction of Bcl - XL. The NF- κB family consists of five DNA binding proteins as follows:

c-Rel, NF- κB 1/p50 NF- κB2/p52, RelA (p65) and RelB, which function as various homodimers and heterodimers. A highly conserved 300 - aminoacid-long N-terminal Rel homology domain (RHD) which is a common domain in all five NF- κB proteins functions to dimerization, DNA binding, interaction with the inhibitors of NF - κB as well as nuclear translocation (39, 40). It was reported in several studies that NF -кB target over 200 different genes such as Myc, Rel, and Cyclin D1 -4 which are engaged in cell cycle regulation, Bcl -2, Bcl-Xl, A1/Bf-1 which function in the apoptosis process, VEGF gene which has a critical role in angiogenesis process leading to the belief in the oncogenic potential of “normal” NF -kB (39, 41, 42). It was also found that NF -kB signaling system has an important role in bridging inflammation and cancer (39). Some new studies reported on the identification of cancer -associated mutations in upstream components of the IkB kinase -NF-kB (IKK-NF-kB) signaling system that can result in cell autonomous activation of NF -kB in multiple myeloma (43, 44) . Recently, Kojima and his group have reported that LPS increases

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COX-2 expression in a certain colon carcinoma cell line through NF -kB which is continuously activated in colorectal carcinoma tissue samples.

They also demonstrated that NF-kB is constitutively activated in colorectal carcinoma tissues by using an electrophor etic mobility shift assay (EMSA) andimmuno-histochemical staining. In addition, they found that NF -kB activation is closely related to cancer progression (45) . New recent studies suggest that activation of NF -κB appears to perform an essential role in cancer development as it was reported that NF -κB is obviously activated in 50% of CRC patients and those with colitis associated tumors and later it has been established by mouse model studies that NF -κB functions criticality in the development of Colitis -associated cancer (CAC) (40).

Recently researchers tried to synthesize compounds that target this pathway such as cinnamaldehyde which was reported as an apoptosis inducer agent acting via mitochondrial pathway, and hence it has been found as a potent NF-кB pathway inhibitor. The essential roles taken by this pathway in apoptosis inhibition and tumor maintenance suggest that inhibitors of the pathway would be effective anti -cancer agents(45-47)

1.2.6 Myc/Max Signaling Pathway

Numerous studies demonstrated that a mut ated version of Myc, which is constitutively expressed, leads to the unregulated expression of many genes, which result in diverse cancers. Myc has been reported to be hyper-activated in 70% of all human cancer cases including colon, breast and lung cancers while it acts as an angiogenesis switch as well (48, 49).

On the other hand, Myc/Max heterodimers induce intracellular transduction

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pathways which are critical for induction of apoptosis (49) . It has been found that Myc is activated via various mitogenic signals such as MAPK/ERK pathway(50). A notable tumor shrinking in transgenic mice was achieved by suppressing of the Myc/Max which resulted in targeting this pathway(51). Another study demonstrated that c-Myc maintains embryonal rhabdomyosarcoma (ERMS) transformed phenotype and radio - resistance by safeguarding cancer cells from radiation -induced apoptosis and DNA damage, while stimulating DNA repair induced by radiation. The findings suggest that c-Myc targeting can be an effective treatment in cancer therapy (48).

1.2.7 Hypoxia pathway

Aerobic energy metabolism processes such as oxidative phosphorylation needs oxygen in eukaryotic cell while low oxygen environments activate the hypoxia signaling pathways. Hypoxia signaling dysfunction normally happens in conditions such as tumor angiogenesis and chronic inflammation. Solid tumors frequently consist of hypoxic regions. The cells in the core of tumor which is located too far away from blood vessels become hypoxic. Being related to higher invasion risk and metastasis, intratumoral hypoxia is more robust to chemotherapy and radiation resulted in more patient mortality (52). As a transcription factor, hypoxia inducible factor (HIF) causes most of the response in hypoxia pathways. HIF-1 complex is a heterodimers protein comprising of an exclusive subunit that is tightly expressed (HIF -1α) having 3 isoforms of HIF-1α, HIF-2α, and HIF-3α and a regular constitutively-expressed beta

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subunit (HIF-1β). The presence of oxygen provokes prolylhydroxylas es to hydroxylate HIF, resulting in the polyubiquitination and degradation of HIF while under low oxygen conditions, prolylhydroxylase inhibition leads HIF to accumulate which in its turn activates transcription of about 100 genes encoding proteins that mediate some major biological processes such as angiogenesis, wound healing, invasion and metastasis, human metabolism, chondrocyte survival in bone growth plates, autophagy and cell death. Increased HIF -1α or HIF-2α levels are found in human colon, breast, prostate and lung carcinomas, and are linked with increased patient mortality. Some experimental data shows that ham pering HIF-1 signaling blocks tumor growth in mouse models (53, 54). Research has indicated that in normal oxygen pressure, NF-κB (nuclear factor κB) directly modulates HIF-1α expression. Investigation of siRNA (sma ll interfering RNA) as for individual NF-κB members displayed differential effects on HIF -1α mRNA levels, implying that NF-κB can control expression of normal HIF -1α VEGF-A and Ang-2 genes which are associated with extreme tumor angiogenesis and metastasis(52, 54). Another study has shown that HIF -1α and its downstream target miR -210 is provoked by hypoxia blocking Bcl -2 expression and increasing autophagy, hence this triggers resistance to radiotherapy in colon can cer cells. (55). The critical role of HIF in regulating the expression of multiple genes involved in tumor metabolism and angiogenesis makes it a potential target in cancer therapy. Hence, greater anticancer effects may be achieved by obstruction of HIF -1.

Therefore, HIF inhibitors including the Klugine, Betulonic acid, phenethyl

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isothiocyanate, taxol, and Acriflavine are still investigated for their anti - cancer properties (53, 56).

1.2.8 MAPK Signaling Pathways

MAPK pathway is a term used for referring to a three kinases module activated by phosphorylating one another se quentially as a reaction to different types of stimuli including neurotransmitters, cellular stress, cytokines, growth factors and cell adherence stimuli including neurotransmitters, cellular stress, cytokines, growth factors and cell adherence(57). This pathway uses one of the most generic singling designs discovered in biological signal transductions. Mitogen -activated protein kinases (MAPKs) are serine/threonine -specific protein kinases that were broadly used during evolution in many physiological processes such as gene expression, mitosis, growth control, cellular adaptation to chemical, physical stress, metabolism, motility, cell differentiation and survival, inflammation and apoptosis in all eukaryotic cells. There are 14 MAPKs in mammals, which have been divided into seven groups. Conventional MAPKs consist of the extracellular signal regulated kinases 1/2 (ERK1/2), p38 isoforms (α, β, γ, and δ), c-Jun amino (N)-terminal kinases1/2/3 (JNK1/2/3), and ERK5(58) . The MAPK/JNKs and MAPK/ERKs are involved in growth factor signaling, regulati on of mitosis, migration and apoptosis while MAPK/p38 play an important role in inflammation (59). It was found that de-regulation of the above pathway resulted in various diseases such as cancer, immunologica l disorders, degenerative and inflammatory syndromes. As reported, MAPKs down regulate over 170

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tumor suppressor genes including Tob1, JunD and Ddit3, which suppress cell growth and proliferation (60). Phosphorylation activates th e MAPK pathway through a MAPKK/MKK (MAPK kinase), and that is phosphorylated by a MAPKKK/MKKK (MAPKK kinase). It was also found that MAPKs are disabled by several phosphatases including a preserved family of phosphatases named MAP kinase phosphatases (MKPs ). These types of enzymes can hydrolyze the phosphate from phosphotyrosine and the phosphothreonine remains. The deletion of each phosphate group extremely reduces MAPK activity essentially giving up signaling. It has been found that some types of tyrosine phosphatases take part in inactivating MAP kinases including phosphatases such as STEP, HePTP, and PTPRR in mammals (61, 62).

Most inducers of the MAPK pathway begin signaling via activating receptors in the cell membrane, which result in activation of MAPKKK typical through a small GTPase. There is a large number of known MAPK agents including mostly of protein kinases, transcription factors, and cytoskeletal proteins. When MAPK is activated it can transfer from the cytoplasm to the nucleus, in which it controls gene transcription via impacting the structure of chromatin and transforming transcription factors activity (57, 62). It was reported that the activation of MAPK/ERK pathway provoked cell cycle arrest and apoptosis and hence could be a n effective therapeutic target of different types of cancer such as osteosarcoma and pancreatic cancer therefore serious attempts were made to produce inhibitors of the ERK and JNK pathways and examine them in clinical trials (58, 63).

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1.3 Apoptosis

The apoptosis process was first described in 1972 as a distinct form of cell death in term of morphology, whereas some characteristics of this phenomenon were explained earlier (64). So far apoptosis has been accepted as the most important type of genetically determined or

“programmed” death of cells that is involved in cell disposal. Nevertheless, there are other types of programmed cell death which are also defined or will be discovered in the future (65, 66). Generally, organisms having multi-cells use two major methods for cell disposa l: necrosis and apoptosis.

Necrosis can be caused due to the breaking apart of the plasmatic membrane as a result of the formation of a swelling process. In contrast, in apoptosis, chromatin is condensed followed by fragmentation and forming apoptotic remains quickly engulfed by the macrophages hence this process does not induce any inflammatory reaction (67). Apoptosis can be activated by a variety of stimuli such as changes in the concentration of growth factors, ionizing radiations, heat shock and other cellular stress, infection by bacterial or viral particles, genetic mutations and damage of DNA (64, 67). Generally, cleavage of proteins, caspase cascade activation, changes in cellular bioenergetics and membrane potential alo ng with expression of cell surface proteins which cause the early detection of apoptotic cells followed by DNA fragmentation are considered as the main features of apoptosis. Mitochondria perform a main role in mediating apoptosis by releasing pro-apoptotic proteins such as cytochrome c, Smac, Omi and AIF into the cytosol. Excessive apoptosis can result in the progress of acquired immune deficiency syndrome (AIDS), renal damage, neurodegenerative

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disorders and cardiac ischemia. On the other hand, reduced ap optosis can result in development of cancer and autoimmune diseases (68, 69) .

1.3.1 Apoptotic Pathways

Apoptosis may be caused in mammals through three pathways based on apoptosis regulators and the location where they act: the first pathway is extrinsic pathway, started by the ligation of death receptors before the activation of caspase-8 and processing extracellular death -inducing; the second pathway is called intrinsic pathway, started by cell stress before the activation of caspase-9, and the third type of pathway is called Granzyme/Perforin pathway, which can trigger members of the caspase family by processing of caspase zymogens. Nevertheless, granzyme A functions in a caspase-independent way. Ultimately, the apoptosis pathways lead to the execution pathway ending in cytomorphological features of apoptosis suc cell h as chromatin condensation, shrinka ge, cytoplasmic blebs formation prior to phagocytosing the apoptotic bodies (64). A summarized schematic image of apoptosis pathways is illustrated in Figure 1.2.

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Figure 1.2: Schematic illustration of apoptosis. The three pathways of apoptosis i.e. extrinsic, intrinsic and perforin/granzyme pathways.

Adopted from (64).

1.3.1(a) Extrinsic Apoptotic Pathway

The extrinsic pathway or death receptor -mediated pathway is triggered by binding of extra - cellular ligands with a family of tumor necrosis factor death receptors whic h are located in the cell membrane. Fas (fibroblast associated antigen) and tumour necrosis factor receptor (TNF - R) are considered as typical death receptors which are involved in this pathway. These receptors contain an extracellular cysteine -rich site in order to bind the ligand, and an intracellular site for signal conduction.

FasL/FasR, TNF-alpha/TNFR1, Apo2L/DR4/DR5 and Apo3L/DR3 are considered as the most prominent ligands and their corresponding receptors

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of this pathway. Binding of Fas ligand to FA DD (Fas-associating protein with death domain) and the binding of TNF ligand to TNF receptor lead to the binding of the adapter protein TRADD with complex of FADD and RIP(64, 70) . Subsequently FADD binds to procaspase -8 through dimerization of the death effector domain leading to formation of death - inducing signaling complex (DISC). Next, DISC activates downstream caspases 3 or other executioner caspases resulting in destruction of cellular targets and apoptosis. Moreover, in certain cell types, BH3 -only protein (Bid) is activated by caspase -8 resulting in truncated Bid (tBid).

Subsequently, tBid moves to the mitochondria and activates cytochrome c release leading to activation of caspase -9 and caspase-3(67). The whole process is described in Figure 1.3.

1.3.1(b) The Intrinsic Pathway of Apoptosis

The intrinsic pathway of apoptosis is initiated via non -receptor mediated mitochondrial-stimuli that act directly on targets inside the cell by producing intracellular signals which can be either positive or negative.

The lack of certain growth factors, c ytokines and hormones create negative signals followed by prevention of apoptosis. On the contrary, loss of apoptotic suppression leads to activation of apoptosis via positive signals such as radiation, oxidative stress, hypoxia, viral infections, and toxi ns which result in Bax/Bak insertion into mitochondrial membrane. The change in mitochondrial transmembrane by Bax/Bak is followed by loss of the mitochondrial transmembrane potential and releasing of pro -apoptotic proteins such as Cytochrome c into the cytosol from the intermembrane

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space(64, 71) . Moreover, BH3-only proteins, such as Bid and Bim are involved in homo-oligomerisation of Bax or Bak which induce their pro - apoptotic function. Subsequently, Cytochrome c binds to the Apaf1 and (d)ATP causing the connection of pro -caspase-9 to the complex and forming the Apoptosome. Activated caspase -9 in turn induces caspase -3 and triggers proteolytic cascade. In contrast, anti -apoptotic Bcl-2 family members, for example Bcl -2 and Bcl-XL, inhibit cytochrome c release, probably via inhibition of Bax and Bak. Furthermore, mitochondria release many other polypeptides such as AIF, endonuclease G, second mitochondrial activator of caspases (Smac/Diablo) which can promote caspase activation via suppressing the inhibitory effects of anti -apoptosis proteins (IAPs) while AIF and Endo G create DNA damage and condensation(67, 72). In case of apoptosis initiation via chemotherapeutic agents, the mitochondrial pathway is more important than the death - receptor pathway for example caspase 9 –deficient cells and Apaf-1–

negative thymocytes are resistant to chemotherapeutic agents, however they can be stimulated into apoptosis via Fas, TRAIL, or TNF (73).

1.3.1(c) Perforin/granzyme Pathway

Granzyme B (Gzm B) is a caspase -like serine protease that is released by cytotoxic T lymphocytes (CTL) and natural ki ller (NK) cells to kill virus-infected and tumor cells. Therefore, granzyme B plays a significant role in human pathologies such as anti -viral immunity and tumor immune surveillance. The serine proteases granzyme A and granzyme B are the most important com ponent within the granules which

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have been examined in recent years (64, 74). Although caspase 3 was the first substrate to be identified for Gzm B, other reports have shown that Gzm B can stimulate several members of the caspase family of cysteine proteases by proteolytic processing of their substrates (75) . Gzm B is able to cleave proteins at critical aspartate residues, thereby stimulating pro - caspase-10 which cleaves ICAD (Inhibitor of Caspase Activated DNAse).

Additionally, Gzm B uses the mitochondrial pathw ay to improve the death signal through specific cleavage of Bid where this protein stimulates the release of mitochondrial cytochrome c into the cytosol (76, 77) . Goping and her colleagues also have shown that Gzm B can directly stimulate caspase-3 resulting in the release of pro -apoptotic proteins that suppress caspase inhibition and direct induction of the execution phase of ap optosis which suggest that both the mitochondrial pathway and direct stimulation of caspase-3 are essential for granzyme B -induced killing (75). Other research indicates that death receptor s and caspases do not play any role in the apoptosis of activated T helper 2 cells induced by T cell receptors because obstructing their ligands does not have any effect on apoptosis. In contrast, adapter proteins with death domains, Fas -Fas ligand interaction, and caspases are involved in apoptosis and regulating cytotoxic T helper 1 cells whereas granzyme B does not have any impact. Also granzyme A has a major role in cytotoxic apoptosis induced by T cells and stimulation of pathways that are independent from caspases. Granzyme A causes DNA disintegration using DNAse NM23 -H1. The nucleosome assemblage protein SET inhibits the NM23 -H1 gene. Granzyme A protease cuts the SET complex and, therefore prevents the inhibition of NM23 -H1, causing

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apoptosis via degradation of DNA;; hence inactivation of this complex by granzyme A probably results in apoptosis through hindering the DNA maintenance and chromatin structure stability (64, 78).

Figure 1.3: TRAIL death-receptor pathway of apoptosis.

Rujukan

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