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UNIVERSITI PUTRA MALAYSIA

CYTOTOXIC EFFECT OF GIRINIMBINE ON HEPG2 CELLS

SUVITHA SYAM MOHAN

IB 2011 15

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CYTOTOXIC EFFECT OF GIRINIMBINE ON HEPG2 CELLS

By

SUVITHA SYAM MOHAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Master of Science

September 2011

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DEDICATION

THIS THESIS IS DEDICATED TO

MY BELOVED HUSBAND SYAM MOHAN MY LOVELY SON ADITHYA MOHAN PARENTS AND PARENTS IN LAW

ALL MY TEACHERS AND LECTURERS ALL MY SOULMATES AND KINDHEARTED

FRIENDS AND

TO EVERYONE WHO BELIEVED IN MY ABILITIES AND ALWAYS INSPIRED ME IN MAKING SOME OF MY GOALS COME TRUE

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in

fulfilment of the requirement for the degree of Master of Science

CYTOTOXIC EFFECT OF GIRINIMBINE ON HEPG2 CELLS

By

SUVITHA SYAM MOHAN

September 2011

Chairman: Ahmad Bustamam Abdul, PhD

Faculty: Institute of Bioscience

Girinimbine, a naturally occurring carbazole alkaloid, has been shown to

possess wide range of pharmacological effects. However, to date, there is

no literature evidencing the anticancer effect of this compound in human

hepatocellular carcinoma (HCC). Here, we report that in vitro treatment of

HepG2 cells (HCC cell line) with girinimbine inhibited cell proliferation and

induced cell death in a dose-dependent and time-dependent manner which

were analyzed by MTT and LDH assay (IC50 61 ± 2.3 µM, 56 ± 3.6 µM, and

40 ± 2.7 µM for 24, 48 and 72 h respectively). Girinimbine induced HepG2

cell death was identified by morphological features of apoptosis with the aid

of Hoechst 33342 dye. The DNA analysis of girinimbine treated HepG2 cells

with agarose electrophoresis resulted in significant DNA fragmentation with

an increase in time dependent manner. There was 0.4 units (OD) (p<0.05)

time-dependent increase in caspase-3 activity. Further, girinimbine also

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induced accumulation of cells in G0/G1 phase (approximately 7.5 % (p<0.05)

compared to control cells) in the HepG2 cell cycle progression. The

intracellular level of reactive oxygen species (ROS) in HepG2 cells increased

time-dependently after girinimbine treatment. The initial level of ROS in

HepG2 cells was 105.20 ± 5.26 % which reached 132.70 ± 6.63 % (p<0.05)

after 3 h. The intracellular antioxidant, GSH level after an initial elevation to

30 % to that of control decreased to 20 % after girinimbine treatment at 3 h.

Mitochondrial damage, also increased from by girinimbine treatment as

observed by the loss of mitochondrial membrane potential in flow cytometric

analysis. The loss of mitochondrial membrane potential for control cells was

4.30 ± 0.21 %, while after girinimbine treatment the mitochondrial membrane

potential became 25.30 ± 1.26 % at 3 h. The comet assay revealed a

significant (P<0.05) 5-fold increase than the control upon exposure to 100

µM girinimbine after 3 h incubation. Girinimbine also induces Hsp 70 and

Hsp 90 expression in a dose-dependent manner up to concentration of 100

µM and time -dependent manner after 24 h incubation. However,

pretreatment of antioxidants ascorbic acid and catalase showed decrease in

ROS level, Hsp level and mitochondrial damage, oxidative DNA damage, but

an increase in GSH level. All these events are happening at an early stage

(i.e., at 3 h after girinimbine treatment itself), suggesting the oxidative stress

mechanism in inducing apoptosis in HepG2 cells. Taken together, these

results strongly support the hypothesis that, after exposure, girinimbine

suppressed the growth of HepG2 cells via induction of G0/G1 phase arrest

and oxidative stress mediated apoptosis driven by mitochondrial pathway.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master Sains

KESAN SITOTOKSIK GIRINIMBINE PADA SEL-SEL HEPG2

Oleh

SUVITHA SYAM MOHAN

September 2011

Pengerusi: Ahmad Bustamam Abdul, PhD

Fakulti: Institut Biosains

Girinimbine adalah carbazole alkaloid semulajadi, telah terbukti memiliki

pelbagai kesan farmakologi. Walau bagaimanapun, sehingga kini, tidak ada

kajian membuktikan kesan antikanser bahan ini dalam hepatocellular

carcinoma (HCC) manusia. Rawatan girinimbine in-vitro, ke atas HepG2 sel-

sel (HCC sel-sel) telah menghalang proliferasi sel dan merangsang

kematian sel mengikut dos dan masa dengan menggunakan analisis kaedah

MTT dan LDH (IC50 61 ± 2.3 μM, 56 ± 3.6 μM, dan 40 ± 2.7 μM untuk 24

jam, 48 dan 72 masing-masing). Girinimbine merangsang kematian sel

HepG2 dikenalpasti melalui ciri-ciri morfologi apoptosis dengan bantuan

pewarna Hoechst 33342. Analisis DNA HepG2 sel yang dirawat dengan

girinimbine dengan elektroforesis agarose menunjukkan pemecahan DNA

yang ketara dengan peningkatan dalam cara bergantung kepada

masa. Terdapat 0.4 unit (OD) (p <0.05) peningkatan dalam aktiviti caspase-

3. Girinimbine juga mencetuskan pengumpulan sel-sel dalam fasa G0/G1

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(kira-kira 7.5% (p <0.05) berbanding dengan mengawal sel-sel) dalam

perkembangan kitaran sel HepG2. Tahap intraselular spesies oksigen reaktif

(ROS) dalam HepG2 sel-sel meningkat bergantung kepada masa rawatan

girinimbine. Tahap awal ROS di HepG2 sel-sel 105.20 ± 5.26% telah

mencapai 132.70 ± 6.63% (p <0.05) selepas 3 jam rawatan. Bagi

antioksidan intraselular, tahap GSH selepas permulaan peningkatan 30%

menurun kepada 20% selepas 3 jam rawatan girinimbine. Kerosakan

mitokondria juga telah meningkat dengan rawatan girinimbine seperti

kehilangan potensi membran mitokondria dalam analisis aliran

sitometrik. Kehilangan potensi membran mitokondria untuk sel-sel kawalan

adalah 4.30 ± 0.21%, manakala selepas rawatan girinimbine potensi

membran mitokondria menjadi 25.30 ± 1.26% pada jam yang ketiga. Kaedah

Comet menunjukkan peningkatan 5 kali ganda yang signifikan (P <0.05)

berbanding dengan kawalan apabila didedahan kepada 100 μM girinimbine

selepas 3 jam pengeraman. Girinimbine juga mendorong HSP 70 dan HSP

90 ekpresi bergantung kepada dos sehingga kepekatan 100 μM dan masa

selepas 24 jam pengeraman. Walau bagaimanapun, prarawatan asid

askorbik antioksidan dan pemangkin menunjukkan penurunan tahap ROS,

HSP dan kerosakan mitokondria, kerosakan oksidatif DNA, kecuali

peningkatan dalam tahap GSH. Semua peristiwa-peristiwa ini berlaku pada

peringkat awal (iaitu, pada 3 jam selepas rawatan girinimbine sendiri),

mencadangkan mekanisme tekanan oksidatif dalam mendorong apoptosis

dalam HepG2 sel-sel. Kesimpulannya, hasil kajian ini sangat menyokong

hipotesis girinimbine yang membantut pertumbuhan sel-sel HepG2 melalui

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induksi G0/G1 fasa tangkap dan diselesaikan dengan tekanan oksidatif

apoptosis yang didorong oleh laluan mitokondria.

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ACKNOWLEDGEMENTS

First of all, I would like to express my most sincere thanks and appreciation

to my supervisor, Dr Ahmad Bustamam Hj Abdul, PhD for his valuable

guidance, suggestions, encouragement and advice throughout my project

and preparation of the thesis.

I would like to extend my gratefulness to my co-supervisor, Professor Dr

Mohamed Aspollah for his support and valuable discussion in making this

research a success. Thank you for your kindness to help when I need it

most.

I am also very grateful to all my friends in UPM and staff of UPM MAKNA

Cancer Research Laboratory. Last but not least, my deepest appreciation to

my parents who have made me who I am today and my brother, who have

always been there for me.

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I certify that a Thesis Examination Committee has met on 8-10-2010 to conduct the final examination of Suvitha Syam Mohan on his thesis entitled “Cytotoxic effect of girinimbine on HepG2 cells” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Master of Science. Members of the Examination Committee were as follows: ABDUL RAHMAN OMAR, PhD Professor Department of Veterinary Pathology & Microbiology Faculty of veterinary medicine Universiti Putra Malaysia (Chairman) ROZITA ROSLI, PhD Associated Professor Department of Obstetrics and Gynaecology Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Rasedee Abdullah, PhD Professor Department of Pathology-Microbiology Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) Chow Sek Chuen, PhD Professor (Biomedical Sciences) School of Science Monash University Sunway Campus, Selangor Darul Ehsan Malaysia (External Examiner) ______________________________ HASANAH MOHD. GHAZALI, PhD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of type of degree. The members of the Supervisory Committee were as follows: Ahmad Bustamam Hj Abdul, PhD

Lecturer Institute of Bioscience Universiti Putra Malaysia (Chairman) Mohd Aspollah Bin Hj Md Sukari, PhD

Professor Faculty of Science Universiti Putra Malaysia (Member)

_________________________

BUJANG BIM KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

SUVITHA SYAM MOHAN Date: 22 September 2011

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TABLE OF CONTENTS Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLE LIST OF FIGURES LIST OF ABBREVIATIONS

ii iii v

VIII Ix xi

xIv xv xvii

CHAPTER

1 INTRODUCTION

22

2 LITERATURE REVIEW 27 2.1 Cancer 27 2.1.1 Cancer statistics 28 2.1.2 Hepatocellular carcinoma 29 2.1.3 Treatment of hepatocellular carcinoma 30 2.1.4 Chemotherapy 31 2.2 Natural products or secondary metabolites 32 2.3 Carabazole alkaloids 34 2.3.1 Anticancer carbazole alkaloids 38 2.3.2 Carbazole alkaloids from Murraya

koenigii 40

2.3.3 Girinimbine 42 2.4 Apoptosis and Cell cycle 44 2.5 Caspases 48 3 MATERIALS AND METHODS 52 3.1 Materials 52 3.2 Cell lines and culture condition 53 3.2.1 Cryopreservation 53 3.2.2 Thawing cryopreserved cells 54 3.3 MTT assay 54 3.4 LDH release assay 56 3.5 Microscopic observation of cellular morphology

using phase contrast inverted microscopy 57

3.6 Chromatin condensation asssay 57 3.7 Colourimetric assay of Caspase-3 58 3.8 DNA fragmentation assay 58 3.9 Flowcytometric analysis of DNA cell cycle 59 3.10 Girinimbine treatment on HepG2 cells 60 3.11 Measurement of intracellular reactive oxygen

species (ROS) 60

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3.12 Determination of intracellular glutathione (GSH)

61

3.13 Determinatin of mitochondrial membrane potential (MMP)

62

3.14 Single cell gel electrophoresis (SCGE; the comet assay)

62

3.15 Heat shock protein 70 assay 66 3.16 Heat shock protein 90 assay 67 4 RESULTS 68 4.1 Cytotoxicity of girinimbine in HepG2 cells 68 4.2 Morphological examination by phase contrast

microscopy 74

4.3 Nuclear condensation 76 4.4 Caspase-3 activation 79 4.5 DNA laddering 81 4.6 Flow cytometric analysis of cell cycle and DNA

content 83

4.7 Effect of girinimbine on Reactive oxygen species ROS)

86

4.8 Effect of girinimbine on intracellular level of glutathione (GSH)

89

4.9 Effect of girinimbine on mitochondrial membrane potential

92

4.10 Induction of oxidative DNA damage 95 4.11 Induction of Hsp 70 and Hsp 90 98 5 DISCUSSION 104 5.1 Cytotoxicity by girinimbine: Involvement of

apoptosis and cell cycle arrest 104

5.2 Role of oxidative stress: Oxidative DNA damage, glutathione depletion, stress protein induction and mitochondrial damage

107

6 SUMMARY, CONCLUSION AND FUTURE

RECOMMENDATIONS 116

REFERENCES 119

APPENDICES 136

LIST OF PUBLICATIONS 148

BIODATA OF STUDENT 149