UNIVERSITI PUTRA MALAYSIA

INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA

NORBAITI BINTI MOHD ISA

IB 2013 20

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INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY

BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA

By

NORBAITI BINTI MOHD ISA

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

Science

July 2013

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All material contained within the thesis, including without limitation text, logos, icons, photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless otherwise stated. Use may be vary of any material contained within the thesis for non-commercial purposes from the copyright holder. Commercial use of material may only be made with the express, prior, written permission of Universiti Putra Malaysia. Copyright © Universiti Putra Malaysia

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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

INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY

BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA

By

NORBAITI BINTI MOHD ISA

July 2013

Chairman: Ahmad Bustamam Abdul, PhD

Faculty: Institute of Bioscience

Boesenbergin A (BA) is a chalcone isolated from Boesenbergia rotunda,

containing methoxyl and hydroxyl groups in its structure. These groups have

been reported to exert anti-proliferative, antioxidant and anti-inflammation

activities. However, there has been no literature reported on the biological

activities of BA and its effects on lung cancer. The current in vitro study was

designed to investigate the apoptotic induction of BA in A549 cells, as well

as its antioxidant and anti-inflammatory activities. Human hepatocellular

carcinoma cell (HepG2), human prostate cancer cell (PC3), colon

adenocarcinoma cell (HT-29), non-small cell lung cancer cell (A549) and

normal hepatic cells (WRL-68) were used to evaluate the cytotoxicity of BA

using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

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assay. The antioxidant activity of BA was assessed by the Oxygen Radical

Absorbance Capacity (ORAC) assay and compared to quercetin as a

standard reference antioxidant. The anti-inflammatory assay was performed

by inducing murine monocytic macrophage cell line (RAW 264.7) using 100

U/mL of interferon-γ (IFN-γ) and 5 µg/mL of lipopolysaccharide (LPS) with or

without the presence of BA, and compared concurrently to L-Nitro-Arginine

Methyl Ester (L-NAME) as positive control. The production of nitric oxide

(NO) after inducing the inflammation was then assessed using Griess

reaction. Multiparametric High Content Screening (HCS) assay was

conducted to detect changes in total nuclear intensity (chromatin

condensation and fragmentation), cell permeability, mitochondrial membrane

potential and release of cytochrome c. Caspase assay for caspase-8, -9 and

-3/7 were analyzed using luminescence reader, whereas DNA fragmentation

was detected through gel electrophoresis. To measure the production of

intracellular ROS, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used.

Cell cycle analysis was performed using Flow Cytometer. Expression of

Bax, Bcl2 and Hsp70 protein were detected by using the western blot

analysis.

Toxic effect of BA on different cell types, reported as IC50, yielded 20.22 ±

3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ± 1.19, and 9.324 ± 0.24 µg/mL for

A549, PC3, HepG2, HT-29 and WRL-68, respectively. ORAC results are

reported as the equivalent concentration of trolox that produces the same

level of antioxidant activity as the samples tested at 20µg/mL. BA displayed

low antioxidant activity, when the results of ORAC assay were reported as

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trolox equivalents, where BA (20 µg/mL) and quercetin (5 µg/mL) were

equivalent to a trolox concentration of 11.91 ± 0.23 and 160.97 ± 0.02 µM,

respectively. For the anti-inflammatory assay, the results clearly showed a

concentration-dependent decrease in NO production in the induced RAW

264.7 cells, with a significantly low level being evident even at 25 and 50 µM

of BA concentrations, with a level of 29.69 ± 2.5 and 25.69 ± 2.0 µM of NO

production respectively. The anti-inflammatory activity of BA was significant

between concentration of 25 µM to 50 µM but with no significant cytotoxicity

towards RAW 264.7 cells at 50 µM. The anti cancer activities of BA was

investigated using A549 cell line, as BA was found to be cytotoxic to A549

cells (IC50< 30 µg/mL). The HCS results showed the BA-induced A549 cells

exhibited a concentration-dependent increase in total nuclear intensity, cell

permeability and cytochrome c release, whereas the cells mitochondrial

membrane potential was decreasing (p<0.05), (p<0.01). Luminescence

assay for caspase-8, -9 and -3/7 revealed an elevation of all three caspases

in concentration-dependent manner (p<0.05). A549 cells treated with BA

showed DNA fragmentation at the concentration of 20 and 50 µg/mL of BA.

Production of ROS was also elevated in a concentration-dependent manner,

indicated by the increase of DCF fluorescence intensity in treated cells

(p<0.05). Protein analysis by western blot showed an increase of Bax, whilst

Bcl2 and Hsp70 protein were subsequently decreased due to time-

dependent treatment (p<0.05). Cell cycle analysis by flow cytometer showed

a significant concentration-dependent increase in the sub G1 phase

(p<0.05). A slight increase of cell arrest was observed in S phase but it was

not statistically significant (p>0.05), which confirmed cell cycle arrest was not

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induced in treated A549 cells. Collectively, results presented in this study

demonstrate that BA inhibited the proliferation of non-small lung cancer in

vitro, leading to a possible induction of apoptosis, which was later confirmed

to be induced through the extrinsic and intrinsic pathways. Moreover the role

of free radicals was significantly found to be elevated with concomitant

decrease in Hsp70. This compound also showed low antioxidant capacity

and significant anti-inflammatory activity. BA can be a potential drug to treat

lung cancer and inflammation.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PENGARUHAN APOPTOSIS DI DALAM SEL A549 IN VITRO OLEH

BOESENBERGIN A YANG DIASINGKAN DARIPADA BOESENBERGIA

ROTUNDA

Oleh

NORBAITI BINTI MOHD ISA

Julai 2013

Pengerusi: Ahmad Bustamam Abdul, PhD

Fakulti: Institut Biosains

Boesenbergin A (BA) ialah sejenis calkon daripada Boesenbergia rotunda

yang mengandungi kumpulan methoxyl dan hydroxyl di dalam strukturnya.

Kumpulan ini pernah dilaporkan mengandungi aktiviti anti-proliferatif,

antioksida dan anti-radang. Walaubagaimanapun, belum terdapat sorotan

literatur yang melaporkan tentang aktiviti biologi BA dan kesannya terhadap

kanser paru-paru. Kajian in vitro kini direka bertujuan untuk menyiasat

pencetusan apoptosis oleh BA pada sel A549, juga aktiviti antioksida dan

anti-radangnya. Sel kanser hati manusia (HepG2), sel kanser prostat

manusia (PC3), sel kanser kolon (HT-29), sel kanser paru-paru bukan kecil

(A549) dan sel hati normal (WRL-68) telah digunakan untuk menilai

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sitotoksisiti BA dengan menggunakan asai MTT. Aktiviti antioksida BA dinilai

melalui asai ORAC lalu dibandingkan dengan quercetin yang bertindak

sebagai anti oksida rujukan standard. Asai anti-radang telah dilaksanakan

dengan meransang sel murine monocytic macrophage (RAW 264.7)

menggunakan 100 U/mL interferon-y (IFN-y) dan 5 µg/mL

lipopolysaccharide (LPS) dengan atau tanpa kehadiran BA, dan

dibandingkan dengan L-Nitro-Arginine Methyl Ester (L-NAME) yang

bertindak sebagai kawalan positif. Penghasilan nitric oxide (NO) selepas

meransang keradangan kemudiannya dinilai menggunakan reaksi Griess.

Multiparametrik High Content Screening (HCS) dijalankan bagi mengesan

perubahan dalam intensiti nuklear seluruh (kondensasi nuclear dan

fragmentasi), ketembusan sel, potensi membran mitokondria dan pelepasan

sitokrom c. Asai caspase untuk caspase-8, -9 dan -3/7 telah dianalisis

menggunakan pembaca luminesen, sementara fragmentasi DNA dikesan

melalui kaedah gel elektroforesis. Bagi mengukur penghasilan ROS

intraselular, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) digunakan. Kitaran

sel analisis pula dilaksanakan dengan menggunakan flow cytometer.

Ekspresi protein Bax, Bcl2 dan Hsp70 dikesan menggunakan analisis

western blot.

Kesan toksik BA terhadap jenis sel yang berlainan, dilaporkan sebagai IC50,

masing-masing bernilai 20.22 ± 3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ±

1.19, dan 9.324 ± 0.24 µg/mL untuk A549, PC3, HepG2, HT-29 and WRL-

68. Keputusan ORAC dilaporkan sebagai kepekatan setaraf trolox yang

menghasilkan aktiviti anti oksida yang sama paras dengan sampel yang diuji

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pada 20 µg/mL. BA menunjukkan aktiviti anti oksida yang rendah apabila

keputusan ORAC dilaporkan sebagai setaraf trolox, di mana BA (20 µg/mL)

dan quercetin (5 µg/mL) adalah setaraf kepada kepekatan trolox iaitu 11.91

± 0.23 dan 160.97 ± 0.02 µM. Bagi asai anti-radang, keputusannya jelas

menunjukkan peningkatan secara kebergantungan-kepekatan dalam

penghasilan NO dalam sel RAW 264.7 yang diransang, dengan paras

rendah BA secara siknifikan menjadi bukti walau pada kepekatan 25 dan 50

µM, dengan penghasilan NO masing-masing pada paras 29.69 ± 2.5 dan

25.69 ± 2.0 µM. Aktiviti anti-radang BA adalah siknifikan antara kepekatan

25 µM hingga 50 µM tetapi tanpa sitotoksisiti yang siknifikan terhadap sel

RAW 264.7 pada 50 µM. Aktiviti anti-kanser BA yang selanjutnya disiasat

dengan menggunakan sel A549 memandangkan BA didapati bersifat

sitotoksik terhadap sel A549 (IC50<30 µg/mL). Keputusan HCS menunjukkan

bahawa sel A549 yang diransang oleh BA mempamerkan peningkatan

secara kebergantungan-kepekatan dalam intensiti nuklear seluruh,

ketembusan sel dan pelepasan sitokrom c, manakala potensi membran

mitokondria pula menurun (p<0.05), (p<0.01). Asai luminesen untuk

caspase-8,-9 dan -3/7 mendedahkan peningkatan bagi ketiga-tiga caspase

secara kebergantungan-kepekatan (p<0.05). Sel A549 yang dirawat dengan

BA menunjukkan fragmentasi DNA pada kepekatan 20 dan 50 µg/mL

(p<0.05). Penghasilan ROS ditingkatkan juga secara kebergantungan-

kepekatan, diindikasi oleh peningkatan intensiti fluoresen dalam sel yang

dirawat (p<0.05). Analisis protein oleh western blot menunjukkan

peningkatan Bax, sementara protein Bcl2 dan Hsp70 menurun berikutan

rawatan secara kebergantungan-masa (p<0.05). Analisis kitaran sel oleh

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flow cytometer menunjukkan peningkatan secara kebergantungan-

kepekatan pada fasa sub G1 (p<0.05). Sedikit peningkatan pada

penangkapan sel dapat dilihat pada fasa S, akan tetapi ia tidak siknifikan

secara statistik (p>0.05), yang membuktikan bahawa penangkapan sel tidak

dicetuskan oleh BA pada sel A549 yang dirawat. Secara keseluruhan,

keputusan yang dipersembahkan dalam kajian ini menunjukkan bahawa BA

menghalang proliferasi ke atas sel kanser paru-paru bukan-besar secara in

vitro, membawa kepada pendorongon apoptosis, yang selanjutnya disahkan

didorong melalui laluan ekstrinsik dan intrinsik. Tambahan pula, peranan

radikal bebas didapati secara meningkat secara siknifikan dengan dituruti

penurunan Hsp70. Kompaun ini juga menunjukkan kapasiti antioksida yang

rendah dan aktiviti anti-radang yang siknifikan. BA berpotensi menjadi ubat

untuk merawat kanser paru-paru dan keradangan.

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ACKNOWLEDGEMENTS

First and foremost, I would like to express tremendous gratitude, respect and

admiration for my supervisor, Ahmad Bustamam Hj Abdul, PhD. Throughout

my studies, I have learned from his wisdom and experience, and benefited

from his continuous guidance and support. His enthusiasm and commitment

to this research project is deeply appreciated and undoubtly invaluable. He

has been the most wonderful mentor and teacher. My utmost appreciation

also goes to Prof. Dr. Rasedee Abdullah and Assoc. Prof. Dr. Siddig Ibrahim

Abdelwahab, that without their continuous support, encouragement, help,

and advice, I was not be able to continue and complete this thesis.

I also want to thank Assoc. Prof. Dr. Syam Mohan for his help and support.

He’s not only a senior, but also a brother to me. Special thanks to all my

fellow colleagues and staffs at UPM Makna Cancer Research Laboratory.

I also wish to thank my mother, Puan Norkiah Adnan for her unconditional

love and support.

Not to forget my lovely housemates: Suzita, Norliana and Nabila for lending

your ears and sharing your ideas.

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APPROVAL

I certify that a Thesis Examination Committee has met on 4th July 2013 to conduct the final examination of Norbaiti binti Mohd Isa on her Master of Science thesis entitled “INDUCTION OF APOPTOSIS IN A549 CELLS IN VITRO BY BOESENBERGIN A ISOLATED FROM BOESENBERGIA ROTUNDA” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U. (A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master of Science. Members of Thesis Examination Committee were as follows: Md. Zuki Abu Bakar, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Hazilawati Hamzah, PhD Senior Lecturer Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) Sharida Fakurazi, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Vijayapandi Pandy, PhD Associate Professor Faculty of Medicine Universiti Malaya (External Examiner) ________________________________ NORITAH OMAR, PhD Assoc. Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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This thesis submitted to the Senate of University Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: Ahmad Bustamam Hj Abdul, PhD

Senior Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Rasedee Bin Abdullah, PhD

Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Siddig Ibrahim Abdelwahab, PhD

Associate Professor Medical Research Centre Jazan University Kingdom of Saudi Arabia (Member)

_________________________ BUJANG BIN 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.

NORBAITI BINTI MOHD ISA

Date: 4th July 2013

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TABLE OF CONTENTS Page

ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

ii vi x xi xiii xviii xix xxi

CHAPTER

1

INTRODUCTION

1

2 LITERATURE REVIEW

7

2.1 Natural Products

7

2.2 Drug Discovery from Plant-Derived Substances

8

2.3 Plant Derived Antioxidant Compounds

10

2.4 Plant Derived Anti Inflammatory Compounds

11

2.5 Plant Derived Anti Cancer Compounds

12

2.6 Natural Compounds from Local Medicinal Plants

13

2.7 Zingiberaceae

14

2.8 Boesenbergia Rotunda

15

2.8.1 Biological Activities of B. rotunda

17

2.9 Disorders and diseases

18

2.9.1 Oxidative stress

18

2.9.2 Inflammation

20

2.9.3 Cancer

21

2.10 Biology of Tumour

24

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2.11 Lung Cancer

24

2.12 Non Small Cell Lung Cancer

26

2.13 A549 Cell Line

27

2.14 Cell Death

27

2.14.1 Necrosis

28

2.14.2 Apoptosis

29

2.14.3 Morphological and biochemical characteristics of apoptosis

30

2.14.4 Apoptosis Pathways

31

2.15 Caspases

34

2.16 Bcl2 Family

37

2.17 Hsp70 Protein 2.18 Reactive Oxygen Species (ROS)

38

39

2.19 Cell Cycle

39

3 MATERIALS AND METHODS

43

3.1 Isolation of boesenbergin A

43

3.2 In vitro cytotoxicity effects of boesenbergin A 3.2.1 Cell Culture Condition 3.2.2 Cell Viability Assay

43

43

43

3.3 ORAC Antioxidant Activity Assay

44

3.4 Anti Inflammatory Assay 45 3.4.1 Cell Culture and Stimulation

45

3.4.2 Nitrite Determination Using the Griess Assay

46

3.4.3 RAW 264.7 Cell Viability

47

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3.5 Multiparametric High Content Screening (HCS) Assay

47

3.6 Caspase -8, -9 and -3/7 assay 3.7 DNA Laddering

49

49

3.8 Measurement of reactive oxygen species generation

50

3.9 Protein Detection by Western Blotting 3.9.1 Extraction of Whole Protein from the Cell

51

51 3.9.2 Western Blotting Analysis

3.10 Cell Cycle Analysis 3.11 Statistical Analysis

51

53

54

4 RESULTS

55

4.1 Cytotoxic Assay

55

4.2 Orac Antioxidant Activity Assay

58

4.3 Effect of Boesenbergin A on NO production and RAW 264.7 cell viability

59

4.4 Multiparametric High Content Screening (HCS) Assay

61

4.5 Caspase-8, -9 and -3/7 Assay

4.6 DNA Laddering

64

65

4.7 Boesenbergin A induces ROS formation

66

4.8 Western Blot Analysis 67

4.9 Flow Cytometric Analysis of Cell Cycle 71 5

DISCUSSION

74

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6 SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE RESEARCH

84

REFERENCES APPENDICES BIODATA OF STUDENT LIST OF PUBLICATIONS

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