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UNIVERSITI PUTRA MALAYSIA

IN VITRO CYTOTOXICITY AND IN VIVO ANTITUMOUR PROPERTIES OF KENAF SEED OIL TOWARDS LEUKAEMIA

FOO JHI BIAU

IB 2011 12

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IN VITRO CYTOTOXICITY AND IN VIVO ANTITUMOUR PROPERTIES OF

KENAF SEED OIL TOWARDS LEUKAEMIA

BY

FOO JHI BIAU

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

November 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Master of Science

IN VITRO CYTOTOXICITY AND IN VIVO ANTITUMOUR PROPERTIES

OF KENAF SEED OIL TOWARDS LEUKAEMIA

By

FOO JHI BIAU

November 2011

Chairman : Latifah Saiful Yazan, PhD

Faculty : Institute of Bioscience

The current treatments for leukaemia such as chemotherapy and radiation therapy

have prolonged the survival rate. However, the adverse effects of these treatments

are difficult to handle. Thus, there is a need to seek for other remedies, such as the

use of natural products. Natural products such as plants play an important role in the

current cancer treatment. The advantage of using plant-derived anticancer agents is

that the produced adverse effects are lesser as compared to the synthetic drugs. Kenaf

(Hibiscus cannabinus) seed oil (KSO) is a rich source of bioactive phytochemicals

with high anti-oxidative and cancer chemopreventive properties. Nevertheless, the

anti-leukaemia properties of KSO have yet been investigated. This study investigated

the anti-leukaemia properties of KSO in vitro and in vivo. KSO was extracted by

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supercritical carbon dioxide fluid extractor (SFE), and evaluated for cytotoxic

properties on leukaemia (HL-60, WEHI-3B and K562) and normal (3T3) cells by

MTT assay with concentrations ranging from 50 to 800 µg/ml for 72 hours. The

morphological changes of KSO-treated leukaemia cells were observed under an

inverted light microscope and a fluorescence microscope. The cell cycle profile of

KSO-treated leukaemia cells was analysed by flow cytometry. For in vivo, acute

toxicity and anti-leukaemia properties of KSO were determined. Male BALB/c mice

were injected intraperitoneally with WEHI-3B cells and administered orally with

KSO at the dose of 0.5, 1.0 and 1.5 g/kg for 14 days. Upon completion, the blood of

the mice was examined for the expression of cell surface marker of T cell (CD3), B

cell (CD19), monocyte and granulocyte (CD11b) by staining with anti-CD3-FITC,

anti-CD19-PE and anti-CD11b-PE antibodies, respectively. The livers and spleens

were isolated, weighed and photographed. The spleens were processed for

histopathological analysis. The yield of KSO by SFE ranged from 11 to 13% (w/w).

KSO was found to be cytotoxic towards all the leukaemia cells in a dose-dependent

manner with no effects on 3T3 cells even at the highest concentration employed (800

µg/ml). Oil from SFE at 600 bar 40 ºC (KSO V600/40) was the most cytotoxic

towards HL-60, WEHI-3B and K562 cells as compared to other extractions (KSO

V600/60, KSO V600/80 and Soxhlet) with the 50% inhibition concentration (IC50)

values of 178.78±10.52, 189.43±11.63 and 213.33±15.45 µg/ml, respectively. KSO

V600/40-treated leukaemia cells exhibited typical characteristics of apoptosis such as

cellular shrinkage, membrane blebbing, chromatin condensation and nuclear

fragmentation. Cell cycle analysis revealed that KSO V600/40 at IC50 value induced

G1 phase cell cycle arrest and significantly increased (p<0.05) the sub-G1 apoptotic

population in the leukaemia cells. For in vivo, acute toxicity study revealed that KSO

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V600/40 did not cause any mortality in the healthy normal mice even at the highest

dose (5.0 g/kg), suggesting that KSO is non-toxic by oral route. Treatment with KSO

V600/40 at 1.0 and 1.5 g/kg increased the population of T cells, but decreased the

population of immature monocytes and granulocytes in the blood of WEHI-3B

injected BALB/c mice (WEHI-3B/BALB/c mice). Spleen and liver weight of WEHI-

3B/BALB/c mice decreased after the treatment with KSO V600/40. Moreover,

infiltration of leukaemic cells into the splenic red pulp reduced after the treatment,

indicating that KSO V600/40 reduced the severity of leukaemia in WEHI-

3B/BALB/c mice. In conclusion, KSO V600/40 showed cytotoxic effect via the

induction of G1 phase cell cycle arrest and apoptosis in the leukaemia cells, and

reduced the severity of leukaemia in WEHI-3B/BALB/c mice.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PENGENALPASTIAN KESAN SITOTOKSIK IN VITRO DAN ANTITUMOR

IN VIVO MINYAK BIJI KENAF TERHADAP LEUKEMIA

Oleh

FOO JHI BIAU

November 2011

Pengerusi : Latifah Saiful Yazan, PhD

Fakulti : Institut Biosains

Rawatan untuk leukemia yang ada pada waktu ini seperti kemoterapi dan terapi

radiasi telah meningkatkan tahap kehidupan. Walau bagaimanapun kesan sampingan

daripada rawatan berkenaan adalah sukar ditangani. Oleh itu, adalah penting untuk

mendapatkan kaedah rawatan yang lain seperti penggunaan bahan semulajadi. Bahan

semulajadi seperti tumbuh-tumbuhan memainkan peranan yang penting dalam

rawatan kanser pada masa kini. Kelebihan penggunaan agen antikanser berasaskan

tumbuh-tumbuhan adalah dapat mengurangkan kesan sampingan berbanding ubatan

sintetik. Minyak biji kenaf (Hibiscus cannabinus) (KSO) merupakan sumber yang

kaya dengan fitokimia bioaktif dengan ciri anti-pengoksidaan yang tinggi dan

pencegahan kanser. Namun demikian, ciri anti-leukemia KSO masih belum dikaji.

Kajian ini dilakukan bagi menentukan kesan anti-leukemia KSO in vitro dan in vivo.

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KSO diekstrak menggunakan pengekstrak cecair karbon dioksida superkritikal (SFE)

dan diuji kesan sitotoksik ke atas sel leukemia (HL-60, WEHI-3B dan K562) dan sel

normal (3T3) pada kepekatan dalam julat 50 ke 800 µg/ml selama 72 jam. Perubahan

morfologi sel leukemia yang dirawat dengan KSO diperhatikan di bawah mikroskop

keterbalikan dan mikroskop pendarflour. Profil kitaran sel leukemia yang dirawat

dengan KSO dianalisis dengan sitometri aliran. Bagi kajian in vivo, ciri ketoksikan

akut dan anti-leukemia KSO ditentukan. Mencit BALB/c jantan disuntik dengan sel

WEHI-3B secara intraperitoneal dan KSO diberi secara oral pada dos 0.5, 1.0 dan 1.5

g/kg selama 14 hari. Selepas tamat, darah mencit dikaji untuk kehadiran penanda

permukaan sel T (CD3), sel B (CD19), monosit serta granulosit (CD11b) dengan

pewarnaan dengan anti-CD3-FITC, anti-CD19-PE dan anti-CD11b-PE, masing-

masing. Hati dan limpa mencit diasing, ditimbang dan diambil gambar. Limpa

diproses untuk analisis histopatologi. Hasil pengekstrakan KSO daripada SFE adalah

di antara 11-13% (berat/berat). KSO didapati sitotosik terhadap kesemua sel

leukemia secara bersandar dos tanpa memberi kesan terhadap sel normal 3T3

walaupun pada kepekatan yang paling tinggi (800 µg/ml). Minyak yang terhasil

daripada pengekstrakan SFE pada 600 bar 40 ºC (KSO V600/40) memberikan kesan

sitotoksik yang paling tinggi terhadap sel HL-60, WEHI-3B dan K562 berbanding

estrak yang lain (V600/60, V600/80 dan Soxhlet) dengan nilai IC50 178.78±10.52,

189.43±11.63 dan 213.33±15.45 µg/ml, masing-masing. Sel leukemia yang dirawat

dengan KSO V600/40 menunjukkan ciri-ciri apoptosis seperti pengecutan sel,

blebbing, kondensasi kromatin dan fragmentasi nukleus. Analisis kitaran sel

menunjukkan KSO V600/40 pada nilai IC50 mengaruh penahanan kitaran sel pada

fasa G1 dan meningkatkan populasi apoptosis sub-G1 secara signifikan (p<0.05)

pada sel leukemia tersebut. Secara in vivo, kajian ketoksikan akut menunjukkan KSO

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V600/40 tidak menyebabkan sebarang kematian pada normal mencit walaupun pada

dos yang paling tinggi (5.0 g/kg). Ini menyarankan bahawa KSO adalah tidak toksik

apabila diberi secara oral. Rawatan dengan KSO V600/40 pada 1.0 and 1.5 g/kg

meningkatkan populasi sel T, tetapi menurunkan populasi monosit dan granulosit

yang tidak matang dalam darah mencit WEHI-3B/BALB/c. Berat limpa dan hati

mencit WEHI-3B/BALB/c menurun selepas rawatan dengan KSO V600/40. Sebagai

tambahan, infiltrasi sel leukemia ke dalam pulpa merah limpa menurun selepas

rawatan, menunjukkan KSO V600/40 mengurangkan kesan teruk leukemia pada

mencit WEHI-3B/BALB/c. Secara kesimpulannya, KSO V600/40 mengaruh

penahanan kitaran sel pada fasa G1 dan apoptosis pada sel leukemia, dan

mengurangkan keterukan leukemia pada mencit BALB/c yang diaruh WEHI-3B.

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ACKNOWLEDGEMENT

“Rome was not built in a day” – so goes the famous saying, and the completion of a

project of any scale would most definitely not be accomplished with the efforts of

one individual alone. Therefore, I would like to take this opportunity to express my

deepest gratitude to several individuals, who lent their support in various ways

throughout the duration of my study.

First and foremost, I would like to express my deepest gratitude to my supervisor, Dr.

Latifah Saiful Yazan for her valuable guidance, suggestions, encouragement and

believing in my research capabilities throughout my project and preparation of the

thesis.

I would like to extend my sincere gratitude to my co-supervisor, Professor Maznah

Ismail for her support and valuable discussion in making this research a success.

Thank you for your kindness to help when I need it most.

Not to be forgotten to express my sincere thanks to Associate Professor Dr. Johnson

Stanlas, Associate Professor Dr. Siddig Ibrahim Abdelwahab, Dr. Syam Mohan and

Dr. Teo Guan Young for your generosity to help throughout the study. The ideas and

guidance are appreciated.

I am very grateful to all science officers, assistants and technicians in Institute of

Bioscience, Universiti Putra Malaysia, Mrs. Siti Muskinah, Ms. Norsha, Mr. Kim

Wei, Mr. Abidin, Mrs. Mastura, Ms. Siti Aisyah, Mrs. Lina, Mrs. Nancy, Mrs.

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Norhafiza, Ms. Normahfuzah, Dr. Tan Sheau Wei and Mrs. Tommini; Staff of

Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra

Malaysia, Mrs. Amrina, Mrs. Normah, Mrs. Juita, and Mrs. Zamzarina. Thank you

very much for your guidance, kind assistance, informative description about the

equipment, chemicals in the laboratory as well as the methods of handling and

operating them.

Special thanks go to my group members and friends Mr. Hisyam, Mrs. Noreen, Mr.

Zulfahmi, Mrs. Norsyafini, Mrs. Aina, Ms. Armania, Mr. Wei Keat, Mr. Chee Wun,

Ms. Sandy Mun Waei, Ms. Aisyah, Mr. Yusri, Ms. Su Hua and Ms. Huan Yong who

have helped me directly and indirectly for the past three years. Thank you for willing

to share with me your knowledge. Learning from you all is one of the most beautiful

things happened in my life.

I would also like to acknowledge the financial support from Ministry of Plantation

Industries and Commodities Malaysia (research grant) and the Universiti Putra

Malaysia (Graduate Research Fellowship) for my study.

Last but not least, my deepest appreciation to my parents who have made me who I

am today and my siblings who have always been there for me.

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I certify that a Thesis Examination Committee has met on 24 November 2011 to

conduct the final examination of Foo Jhi Biau on his thesis entitled “In vitro

Cytotoxicity and In Vivo Antitumour Properties of Kenaf Seed Oil Towards

Leukaemia” 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 the Thesis Examination Committee were as follows:

Huzwah binti Khazaai, PhD Senior Lecturer

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Norhaizan binti Mohd Esa, PhD Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Internal Examiner)

Sharida binti Fakurazi, PhD Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Internal Examiner)

Nor Fadilah Rajab, PhD Associate Professor

Faculty of Health Sciences

Universiti Kebangsaan Malaysia

(External Examiner)

SEOW HENG FONG, PhD Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 25 January 2012

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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 Master of Science. The

members of the Supervisory Committee were as follows:

Latifah Saiful Yazan, PhD Senior Lecturer

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Maznah Ismail, PhD Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(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.

FOO JHI BIAU

Date: 24 November 2011

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TABLE OF CONTENTS

Page

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL xi

DECLARATION xiii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 5

2.1 Leukaemia 5

2.1.1 Acute myeloid leukaemia 6

2.1.2 Acute lymphocytic leukaemia 8

2.1.3 Chronic myeloid leukaemia 9

2.1.4 Chronic lymphocytic leukaemia 10

2.2 Leukaemic cell line 11

2.2.1 Human promyelocytic leukaemic cells 11

2.2.2 Murine myelomonocytic leukaemic cells 12

2.2.3 Human chronic myelogenous leukaemic cells 14

2.3 Supercritical carbon dioxide fluid extraction 15

2.4 Hibiscus cannabinus (Kenaf) 18

2.4.1 Yield of kenaf seed oil 20

2.4.2 Chemical composition of kenaf seed oil 21

2.5 Apoptosis 25

2.6 Cell cycle 29

3 MATERIALS AND METHODS 32

3.1 Experimental design 32

3.2 Materials and equipment 33

3.2.1 Kenaf seeds 33

3.2.2 Cell culture 33

3.2.3 Cytotoxicity study 34

3.2.4 Morphological study 34

3.2.5 Cell cycle analysis 34

3.2.6 Animal and induction of leukaemia 34

3.2.7 Determination of the expression of white blood cells

surface markers

35

3.2.8 Haematoxylin and eosin staining 35

3.3 Methods 36

3.3.1 Kenaf seed oil extraction 36

3.3.2 In vitro study 37

3.3.3 In vivo study 41

3.4 Statistical analysis 47

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4 KENAF SEED OIL FROM SUPERCRITICAL CARBON

DIOXIDE FLUID EXTRACTION INDUCED G1 PHASE

CELL CYCLE ARREST AND APOPTOSIS IN

LEUKAEMIA CELLS

48

Copyright permission/Acceptance letter 69

5 KENAF SEED OIL FROM SUPERCRITICAL CARBON

DIOXIDE FLUID EXTRACTION INHIBITS THE

PROLIFERATION OF WEHI-3B LEUKAEMIA CELLS IN

VIVO

71

Copyright permission/Acceptance letter 92

6 GENERAL DISCUSSION 93

7 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATION FOR FUTURE RESEARCH

97

6.1 Summary and general conclusion 97

6.2 Recommendation 99

REFERENCES 100

APPENDICES 113

BIODATA OF STUDENT 116