COPYRIGHTpsasir.upm.edu.my/20370/1/IB_2011_12_ir.pdf · the mice was examined for the expression of...
Transcript of COPYRIGHTpsasir.upm.edu.my/20370/1/IB_2011_12_ir.pdf · the mice was examined for the expression of...
© COPYRIG
HT UPM
UNIVERSITI PUTRA MALAYSIA
IN VITRO CYTOTOXICITY AND IN VIVO ANTITUMOUR PROPERTIES OF KENAF SEED OIL TOWARDS LEUKAEMIA
FOO JHI BIAU
IB 2011 12
© COPYRIG
HT UPM
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
© COPYRIG
HT UPM
iii
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
© COPYRIG
HT UPM
iv
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
© COPYRIG
HT UPM
v
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.
© COPYRIG
HT UPM
vi
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.
© COPYRIG
HT UPM
vii
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
© COPYRIG
HT UPM
viii
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.
© COPYRIG
HT UPM
ix
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.
© COPYRIG
HT UPM
x
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.
© COPYRIG
HT UPM
xi
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
© COPYRIG
HT UPM
xii
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:
© COPYRIG
HT UPM
xiii
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
© COPYRIG
HT UPM
xiv
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
© COPYRIG
HT UPM
xv
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