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UNIVERSITI PUTRA MALAYSIA
NURUL HUSNA SHAFIE
IB 2013 15
ANTI-CANCER ACTIVITY AND MECHANISM OF ACTION OF RICE BRAN PHYTIC ACID ON COLON CANCER IN VITRO AND IN VIVO
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ANTI-CANCER ACTIVITY AND MECHANISM OF ACTION OF RICE
BRAN PHYTIC ACID ON COLON CANCER IN VITRO AND IN VIVO
By
NURUL HUSNA SHAFIE
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirements for the Degree of Doctor of Philosophy
May 2013
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirements for degree of Doctor of Philosophy.
ANTI-CANCER ACTIVITY AND MECHANISM OF ACTION OF RICE
BRAN PHYTIC ACID ON COLON CANCER IN VITRO AND IN VIVO
By
NURUL HUSNA SHAFIE
May 2013
Chairman : Norhaizan Mohd Esa, Ph.D
Institute : Biosciences
Colorectal cancer or colon cancer is a major neoplastic disease affecting men and
women worldwide. Since Burkitt’s pioneering research that pointed to inverse
relationship between colon cancer risk and consumption of fiber-rich foods, many
epidemiological and laboratory animal studies have tested this hypothesis. The
protective effects of dietary fiber on colon cancer development depend on the nature
and source of fiber. Rice bran is one of the richest sources of dietary fiber and
contains phytonutrients, including phytic acid, known to possess various medicinal
properties. Phytic acid, or inositol hexaphosphate (IP6), is a polyphosphorylated
carbohydrate, that has been suggested to play a significant role in the inhibition of
colorectal cancer. Thus, our attention was drawn to the possibility to utilize the local
source of phytic acid in identifying non-toxic anti-cancer agents that can potentially
lead to the development of better treatments for colorectal cancer.
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In particular, the present study was aimed at investigating the anti-cancer effect of
IP6 extracted from rice bran, in colon cancer model in vitro and in vivo. It began with
the investigation of the inhibitory effect and associated mechanisms of rice bran IP6
on human colorectal cancer cell line, HT-29. IP6 induced marked growth inhibition
in a dose and time dependent manner as evaluated by the MTT proliferation assay
(IC50 = 12 μg/mL). Indeed, IP6 also did not cause any cytotoxicity towards normal
3T3 cells. Cell cycle progression studies were performed by flow cytometric analysis
following propidium iodide (PI) staining of the cells. IP6 treatment (9.5, 12.0 and
14.5 μg/mL IP6) for 24, 48 and 72 hours, resulted in significant accumulation of
G0/G1 phase cells (63 – 65 %) compared to control (50.53 %) (p<0.05). Together,
these results suggested that IP6 causes the inhibition of cell growth through G0/G1
phase arrest in the cell cycle progression of HT-29 cells.
Additionally, investigation of the ability of IP6 to induce colon tumor cell apoptosis
was carried out. It was proven that IP6 significantly induced early apoptotic cell
death (30 %) in a dose- and time dependent manner compared to control cells
showing <1 % of cell death as confirmed by Annexin V assay (p<0.05). Quantitative
real-time polymerase chain reaction (qRT-PCR) analysis was performed next
followed by Western blotting to further determine apoptosis at the molecular level.
Interestingly, IP6 showed an extensive significant reduction of anti-apoptotic Bcl-xl
and a coherent increment of pro-apoptotic Bax at the mRNA and protein level. These
results showed that IP6 caused a marked increase in apoptosis, which was
accompanied by significantly increased mRNA and protein levels of caspase 3 and
caspase 8 (p<0.05). These molecular alterations provide an insight into the apoptotic
death of human colon cancer, HT-29 cells, elicited by IP6.
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The second part of this study in which the hypothesis whether IP6 extracted from rice
bran had any effect on colon carcinogenesis was further investigated in an animal
model of experimental colon cancer. Male Sprague-Dawley, weanling rats were
divided into 5 groups with 12 rats in each group. Rats received two intraperitoneal
(i.p.) injections of azoxymethane (AOM) in saline (15 mg/kg body weight) over a 2-
week period for colon cancer induction. The IP6 treatments were given in three
concentrations: 0.2 % (w/v), 0.5 % (w/v) and 1.0 % (w/v) via drinking water and the
treatment were divided into two termination timelines. For the first termination, 6
rats from each group were killed after 8 weeks of IP6 treatment. The colons of these
rats were analyzed for detection and quantification of aberrant crypt foci (ACF), an
early biomarker of colon cancer. Analysis of ACF incidence demonstrated that
administration of IP6 extracted from rice bran significantly reduced the total number
of ACF (p<0.05). Furthermore, IP6 also significantly reduced the number of
dysplastic and non-dysplastic ACF in a dose-dependent manner.
For the second termination, the other 6 rats in each group were killed after 16 weeks
of IP6 treatment. Colons of these rats were assessed for tumor incidence. It was
shown that administration of phytic acid in the drinking water, significantly
suppressed the total number of tumor incidences compared to the control (p<0.05). In
another case, deregulation of the Wnt/β-catenin signaling pathway has been
implicated in colorectal tumorigenesis resulting in accumulation of β-catenin, a
major mechanism in the AOM-induced colon carcinogenesis model. Indeed,
expression of enzymes associated with inflammation, such as inducible
cyclooxygenase-2 (COX-2), has been shown to play a role in colon tumor
progression. Therefore, the potential of IP6 in targeting key components of the
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Wnt/β-catenin signaling pathway and COX-2 as a rational for cancer drug discovery
was demonstrated. Colon tumors were further analyzed for β-catenin and COX-2
expression at mRNA and protein level by qPCR and Western blot respectively. It
was shown that administration of IP6 significantly down-regulated β-catenin and
COX-2 expression in colon tumors as compared with control (no IP6 treatment)
(p<0.05). Therefore, it can be suggested that β-catenin and COX-2 could be potential
targets for colon cancer chemoprevention.
Collectively, results presented in this study demonstrated that IP6 extracted from rice
bran inhibited the proliferation of colon cancer in vitro selectively by arresting the
cell cycle and thus leading to programmed cell death, which was later confirmed to
be through the regulation of pro- and anti-apoptotic proteins and caspase dependent
pathways. Moreover, the study in vivo with the AOM induced rat colon
carcinogenesis model clearly showed that IP6 had inhibited the development of colon
cancer through the suppression of ACF, leading to reduction in tumor incidence,
which was via the down-regulation of β-catenin and COX-2 expression.
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Abstrak tesis yang telah dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan ijazah Doktor Falsafah
AKTIVITI ANTI-KANSER DAN MEKANISME TINDAKAN ASID FITIK
DEDAK BERAS KE ATAS KANSER KOLON IN VITRO DAN IN VIVO
Oleh
NURUL HUSNA SHAFIE
Mei 2013
Pengerusi : Norhaizan Mohd Esa, Ph.D
Institut : Biosains
Kanser kolon atau usus besar ialah antara penyakit neoplasia yang utama di kalangan
lelaki dan wanita di seluruh dunia. Sejak bermulanya penyelidikan kanser Burkitt’s
yang membuktikan hubungan terbalik di antara risiko kanser kolon dan penggunaan
makanan kaya serat, banyak kajian epidemiologi dan makmal haiwan telah menguji
hipotesis ini. Perbandingan antara serat diet dengan kejadian dan kadar kematian
kanser kolon menyokong hipotesis di mana serat diet, terutamanya serat daripada
sumber bijirin dapat menghalang penyakit kanser kolon. Sebagai contoh, dedak beras
juga merupakan salah satu daripada bahan yang kaya dengan serat dan mengandungi
pelbagai fitonutrien dan fitokimia termasuk asid fitik yang berpotensi sebagai bahan
ubatan. Asid fitik atau juga dikenali sebagai inositol heksafosfat ialah sejenis
karbohidrat polifosfat, telah dibuktikan mempunyai peranan penting dalam
mencegah dan merawat kanser kolon. Oleh itu, ianya telah menarik perhatian kami
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untuk menggunakan asid fitik dari sumber tempatan yang tidak toksik dan
dikembangkan potensinya sebagai rawatan penyakit kanser kolon.
Secara khususnya, kajian ini adalah bertujuan untuk mengkaji keberkesanan asid fitik
yang diekstrak daripada dedak beras sebagai anti-kanser menggunakan model kanser
kolon in vitro dan in vivo. Kajian ini dimulakan dengan penyelidikan kesan
perencatan pertumbuhan dan mekanisme tindakan yang terlibat oleh asid fitik (IP6)
pada sel kanser kolon manusia (HT-29). IP6 telah dibuktikan dapat menghalang
pertumbuhan sel kanser kolon (HT-29) bergantung pada dos dan masa yang dinilai
menggunakan asai proliferasi MTT (IC50 = 12 μg/mL). Malahan dapat dibuktikan
bahawa asid fitik tidak mempunyai kesan toksik terhadap sel normal, 3T3. Selain itu,
kajian terhadap proses kitaran sel telah dijalankan menggunakan pewarna
‘propidium’ iodida diikuti dengan analisis sitometri aliran. Selepas perawatan dan
pendedahan asid fitik ke atas sel kanser kolon (HT-29) dengan dos-dos tertentu (9.5,
12.0 and 14.5 μg/mL IP6) selama 24, 48 and 72 jam, terbukti bahawa asid fitik
menyebabkan pengumpulan sel pada fasa G0/G1 sebanyak 63 – 65 % berbanding
dengan kawalan atau sel tanpa rawatan dengan asid fitik hanya sebanyak 50.53 %
(p<0.05). Oleh itu, dapat dibuktikan bahawa asid fitik (IP6) merencatkan
pertumbuhan sel kanser kolon (HT-29) melalui penahanan kitaran sel, iaitu pada fasa
G0/G1.
Selain itu, penyelidikan ke atas asid fitik dalam mengaruh proses apoptosis terhadap
sel kanser kolon telah dijalankan. Ini telah dibuktikan di mana IP6 menyebabkan
peningkatan sel apoptosis awal sebanyak 30 % berbanding dengan kawalan hanya
< 1 % sel yang mati dan ini bergantung pada dos dan masa seperti yang ditunjukkan
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oleh asai Annexin V (p<0.05). Analisis qRT-PCR seterusnya dijalankan dan diikuti
dengan ‘Western blot’ untuk kajian yang lebih mendalam dalam membuktikan
proses apoptosis oleh IP6, iaitu pada peringkat molekul. Menariknya, IP6
menunjukkan kesan ‘down’-modulasi atau pengurangan yang nyata ke atas anti-
apoptosis Bcl-xl dan pada masa yang sama meningkatkan atau ‘up’-regulasi ke atas
pro-apoptosis Bax pada peringkat mRNA dan protein. Semua data ini menunjukkan
keberkesanan IP6 dalam meningkatkan sel apoptosis, dan turut disokong dengan
penemuan peningkatan aktiviti ‘caspase’ 3 and ‘caspase’ 8 setelah sel kanser kolon
(HT-29) dirawat dengan asid fitik (IP6). Perubahan peringkat molekul ini
menyediakan penemuan yang mendalam terhadap kesan asid fitik (IP6) dalam
mengaruh apoptosis ke atas sel kanser kolon (HT-29).
Bahagian kedua penyelidikan ini ialah kajian ke atas kesan asid fitik (IP6) yang
diekstrak daripada dedak beras terhadap proses ‘karsinogenesis’ atau tumor kolon
yang diuji menggunakan model haiwan yang diaruh dengan kanser kolon. Pada
peringkat awal, tikus jantan Sprague-Dawley dibahagikan kepada 5 kumpulan
dengan 12 ekor bagi setiap kumpulan. Tikus-tikus tersebut menerima dua kali
suntikan azoksimetana (AOM) dalam ‘saline’ (15 mg/kg body weight) pada dua
minggu berturut-turut bagi tujuan pengaruhan kanser kolon. Rawatan dengan asid
fitik (IP6) adalah berdasarkan tiga dos yang berbeza: 0.2 % (w/v); 0.5 % (w/v) dan
1.0 % (w/v) melalui air minuman dan dibahagikan kepada dua kali masa penamatan.
Untuk penamatan pertama iaitu selepas 8 minggu rawatan dengan asid fitik, 6 ekor
tikus dibunuh dan kolon atau usus tikus tersebut diambil dan dianalisa untuk tujuan
pengesanan dan pengiraan ‘aberrant crypt foci’ (ACF), iaitu penanda awal kanser
kolon. Analisis terhadap kejadian pembentukan ACF ini membuktikan tikus yang
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dirawat dengan asid fitik yang diekstrak daripada dedak beras ini telah
mengurangkan jumlah kejadian ACF. Tambahan pula, IP6 juga turut mengurangkan
jumlah pembentukan ACF ‘dysplasia’ dan bukan ‘dysplasia’ bergantung kepada dos
(p<0.05).
Bagi penamatan kedua pula, 6 ekor tikus yang berbaki dibunuh selepas 16 minggu
dirawat dengan asid fitik. Kolon atau usus tikus tersebut diambil dan dikaji untuk
analisis kejadian pembentukan tumor. Dapat dibuktikan bahawa terdapat
pengurangan terhadap kejadian pembentukan kanser setelah rawatan asid fitik ke atas
tikus yang diaruh dengan kolon kanser berbanding dengan tikus yang tidak dirawat
(p<0.05). Perubahan kawalan terhadap tranduksi isyarat Wnt/β-catenin telah
diimplifikasikan dalam proses karsinogenesis kolon menyebabkan pengumpulan β-
catenin adalah mekanisme terbesar di dalam haiwan yang diaruh dengan AOM.
Selain itu juga, ekspresi enzim yang melibatkan inflamasi contohnya COX-2
dibuktikan memainkan peranan penting dalam proses karsinogenesis. Oleh itu,
potensi asid fitik terhadap kunci komponen tranduksi isyarat Wnt/β-catenin dan
COX-2 sebagai terapi kanser dijalankan. Kolon tikus yang diambil pada masa
penamatan kedua dikaji untuk ekspresi β-catenin and COX-2 pada peringkat mRNA
dan protein oleh qRT-PCR dan ‘Western blot’. Kajian ini membuktikan rawatan
tikus dengan asid fitik telah mengurangkan atau ‘down’-regulasi ekspresi β-catenin
and COX-2 di dalam kanser kolon berbanding dengan kawalan. Oleh itu, telah
dibuktikan bahawa β-catenin dan COX-2 mungkin berpotensi sebagai sasaran dalam
pencegahan kanser kolon. Secara kolektif, keputusan yang dibentang dalam kajian
ini membuktikan asid fitik yang diekstrak daripada dedak beras telah menghalang
proliferasi kanser kolon secara in vitro, melalui perencatan pada kitaran sel dan
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seterusnya membawa kepada kematian sel yang diprogramkan, yang mana
kemudiannya disahkan melalui regulasi antara molekul pro- dan anti- apoptosis dan
laluan ‘caspase’. Tambahan pula, kajian in vivo pada model tikus yang diaruh dengan
kanser kolon menggunakan AOM menunjukkan IP6 telah menghalang pembentukan
kanser kolon melalui penyekatan terhadap ACF dan seterusnya mengurangkan
pembentukan tumor, iaitu melalui ‘down’-regulasi β-catenin dan COX-2.
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ACKNOWLEDGEMENTS
This has been a learning journey for me with new experiences and obstacles that
come with undertaking a PhD degree. I am forever thankful to my supervisor, Assoc.
Prof. Dr. Norhaizan Mohd Esa for all the opportunities that she has given me to excel
in my field of research. Dr Norhaizan has been a wonderful source of encouragement
and guidance. Her frankness plus friendly and enthusiastic approach has made the
journey that much more pleasant, so I sincerely thank her.
To Prof. Dr. Hairuszah Ithnin, thank you for being there, whenever I had questions or
was in need. Your expert guidance has been most helpful and appreciated. I also
want to thank to Dr Abdah Md Akim for all her guidance and assistance, particularly
in the molecular studies.
Special thanks to Miss Norazalina Saad for your time and assistance in many aspects.
I would like to thank the entire staffs of the Laboratory of Cancer Research UPM-
MAKNA including Mrs Noraini Mohd Ain, Mrs Tommini Salleh, Mrs Norlela
Ahmad and Mrs Noor Haizi Ruslan for their help as well as MOSTI and MOHE for
scholarship and financial assistance.
To my fellow postgraduates students especially my housemates, Nursyuhada, Siti
Syazani, Salmiah and Munira Saira special thanks to you all for your continuous
support and friendship.
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I also would like to take this opportunity to acknowledge my best friends,
Nurhidayah and Nur Suzana and not to forget Siti Suriani for being there during my
critical preparation for thesis submission. Thank you girls!
Lastly, I want to give my most heartfelt thanks to my mother, Mrs Che Fadzilah Saad
and the rest of my family for all the endless encouragement, love and support.
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I certify that a Thesis Examination Committee has met on (22nd
May 2013) to
conduct the final examination of Nurul Husna Shafie on her thesis entitled "Anti-
cancer activity and mechanism of action of rice bran phytic acid on colon cancer in
vitro and in vivo" 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 Doctor of
Philosophy.
Members of the Thesis Examination Committee were as follows:
Asmah Rahmat, PhD
Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Patimah Ismail, PhD
Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)
Noorjahan Banu Binti Mohammed Alitheen, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Klaus W.J. Wahle, PhD
Professor
School of Medicine and Dentistry
Aberdeen University
United Kingdom
(External Examiner)
NORITAH OMAR, PhD
Assoc. Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 2 AUGUST 2013
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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 Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Norhaizan Mohd Esa, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Hairuszah Ithnin, PhD
Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
Abdah Md Akim, PhD
Senior Lecturer
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
BUJANG 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.
Nurul husna
NURUL HUSNA SHAFIE
Date: 30 AUGUST 2013
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TABLE OF CONTENTS
Page
ABSTRACT
ABSTRAK
ACKNOWLEDGEMENTS
APPROVAL
DECLARATION
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
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CHAPTER
1. INTRODUCTION
1.1 Research Background
1.2 Problem statement
1.3 Significance of study
1.4 Objectives
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2. LITERATURE REVIEW
2.1 Cancer
2.1.1 Definition of cancer
2.1.2 Mutation and carcinogenesis
2.2 Cell Cycle
2.2.1 Cell cycle and cancer
2.2.2 The cell cycle and its therapeutic targets
2.3 Terminology of cell death
2.3.1 Apoptosis
2.2.1.1 Morphological and biochemical features
2.2.1.2 Involvement of proteolysis
2.3.2 Apoptosis signaling pathways
2.3.2.1 Extrinsic pathway
2.3.2.2 Intrinsic pathway
2.3.2.2.1 The Bcl-2 family
2.3.3 Apoptosis and tumorigenesis
2.3.4 Apoptosis and cancer therapy
2.4 Treatment of cancer
2.3.1 Surgery
2.3.2 Radiation therapy
2.3.3 Chemotherapy
2.5 Colorectal cancer
2.5.1 Molecular pathway of colon carcinogenesis
2.5.1.1 The canonical or suppressor pathway
2.5.1.2 The microsatellite instability pathway
2.5.1.3 Dietary fibre and protection against
colorectal cancer
2.6 Rice bran
2.6.1 Phytic acid
2.6.1.1 Phytic acid as anticancer agents
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2.6.1.2 Potential mechanism of action and
molecular targets for phytic acid
2.6.1.2.1 Induction of apoptosis and cell
cycle arrest
2.6.1.2.2 Gene alteration
2.6.1.2.3 Enhanced immunity
2.6.1.2.4 Repression of angiogenesis
and metastasis
2.6.1.2.5 Antioxidant properties
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3. MATERIALS AND METHODS
3.1 Materials
3.1.1 Rice bran
3.1.2 General reagents and chemicals
3.1.3 Instruments and tools
3.2 Methods
3.2.1 Preparation of phytic acid
3.2.1.1 Stabilization of rice bran
3.2.1.2 Production of defatted rice bran
3.2.1.3 Extraction of phytic acid from
defatted rice bran
3.2.1.4 Neutralization of phytic acid
3.2.2 Identification of phytic acid
3.2.2.1 Anion-Exchange Purification
3.2.2.2 High Performance Liquid
Chromatography (HPLC)
analysis for determination of IP6
3.2.3 Study in vitro
3.2.3.1 Cell lines
3.2.3.2 Growth of cell cultures
3.2.3.3 Harvesting and splitting of cell cultures
3.2.3.4 Cryopreservation and thawing of
cell cultures
3.2.3.5 MTT Cell proliferation assay
3.2.3.6 Cell cycle distribution analysis
3.2.3.7 Detection of apoptotic cell death
3.2.3.8 Genes expression
3.2.3.8.1 Extraction of total RNA
3.2.3.8.2 Quantification of RNA
3.2.3.8.3 Agarose gel electrophoresis
3.2.3.8.4 cDNA synthesis for
Real-Time PCR analysis
3.2.3.8.5 Quantitative Real-Time
Polymerase Chain Reaction
3.2.3.8.5.1 Specific primer
for qRT PCR
3.2.3.8.5.2 Real-Time PCR
reactions
3.2.3.8.5.3 qPCR Reaction
setup
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3.2.3.8.5.4 Plate Setup and
qRT-PCR
reaction
3.2.3.8.5.5 Real-time PCR
data analysis
3.2.3.9 Protein expression
3.2.3.9.1 Extraction of total protein
3.2.3.9.2 Quantification of protein
sample
3.2.3.9.3 SDS-PAGE
3.2.3.9.4 Western Blotting
3.2.3.9.4 Densitometry
3.2.4 Study in vivo
3.2.4.1 Animals
3.2.4.2 Ethical approval
3.2.4.3 Carcinogen treatment
3.2.4.4 Experimental design
3.2.4.5 Detection of aberrant crypt foci (ACF)
3.2.4.6 Histological classification of ACF
3.2.4.7 Tumor assessment
3.2.4.8 Gene expression
3.2.4.9 Protein expression
3.2.5 Statistical analysis
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4. RESULTS AND DISCUSSION
4.1 Extraction and determination of phytic acid 4.2 Bioassay in vitro
4.2.1 Growth inhibitory effect of IP6 on human colorectal
cancer cells (HT-29)
4.2.2 Effect of IP6 on cell cycle kinetics 87
4.2.3 Apoptosis induction analysis of IP6- treated HT-29 92
4.2.4 Genes expression
4.2.4.1 Extraction of total RNA 98
4.2.4.2 Concentration of extracted RNA 98
4.2.4.3 mRNA expression of Bcl-2 and caspase
family genes in IP6- treated HT-29 cells
4.2.5 Protein expression
4.2.5.1 Protein concentration
4.2.5.2 Bcl-2 and caspase family protein
expression in IP6- treated HT-29 cells
4.2.6 Changes in mRNA and protein expression level of
Bax, Bcl-xl, Caspase-3 and Caspase-8 contribute to
IP6 induces apoptotic cell death in HT-29 cell
4.3 Bioassay in vivo 4.3.1 Body weight
4.3.2 Effect of phytic acid on the incidence of ACF in rat
colon cancer
4.3.3 Histological classification of ACF
4.3.4 Tumor assessment
4.3.5 Tumor- associated gene expression
4.3.5.1 Extraction of total RNA
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4.3.5.2 Concentration of extracted RNA
4.3.5.3 IP6 down-regulated mRNA level of β-
catenin expression in IP6- treated AOM-
induced rat colon cancer
4.3.5.4 Cyclooxygenase-2 expression in IP6-
treated AOM induced rat colon cancer
4.3.6 Protein expression
4.3.6.1 Protein quantification
4.3.6.2 Reduction of β-catenin and COX-2
protein expression in IP6- treated
AOM-induced rat colon cancer
4.3.6.3 Down-modulatory effects of IP6 on β-
catenin and COX-2 in AOM- induced rat
colon cancer
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5. CONCLUSION 5.1 Summary
5.2 Conclusion
5.3 Future research and recommendation
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REFERENCES
APPENDICES 182
BIOGRAPHY OF AUTHOR
LIST OF PUBLICATIONS
LIST OF ORAL AND POSTER PRESENTATION
LIST OF AWARDS
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