UNIVERSITI PUTRA MALAYSIA

PROPERTIES OF VANILLIN AND ITS EFFECTS ON COLORECTAL CANCER IN VITRO AND IN VIVO

HO KET LI

IB 2011 27

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PROPERTIES OF VANILLIN AND ITS EFFECTS ON COLORECTAL CANCER

IN VITRO AND IN VIVO

HO KET LI

DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2011

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PROPERTIES OF VANILLIN AND ITS EFFECTS ON COLORECTAL CANCER

IN VITRO AND IN VIVO

By

HO KET LI

Thesis Submitted to the School of Graduate Studies,

Universiti Putra Malaysia,

In Fulfillment of the Requirements for the Degree of Doctor of Philosophy

August 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 Doctor of Philosophy

PROPERTIES OF VANILLIN AND ITS EFFECTS ON COLORECTAL CANCER

IN VITRO AND IN VIVO

By

HO KET LI

August 2011

Chairman: Professor Maznah Ismail, PhD

Faculty : Institute of Bioscience

Vanillin is responsible for the flavor and smell of vanilla, a widely used flavoring

agent. Previous studies showed that vanillin was a good red blood sickle cell inhibitor,

anti-microbial agent and anti-mutagen. However, vanillin must be administered at

high concentrations and prevented from being oxidized by the upper gastrointestinal

tract to be medically effective. Hence, the objectives of this study were: i. to

investigate the cytotoxic properties of vanillin on HT-29 colorectal cancer cell line; ii.

to assess the negative effect of vanillin when administered at high concentrations in

vivo; iii. to investigate the chemopreventive properties of vanillin on colorectal cancer

and iv. to study the effects of vanillin on expression of selected genes in vivo.

Methods used to study the cytotoxic effects include cytotoxicity assay, double

staining cell morphological analysis, cell cycle analysis, apoptosis test and cell

proliferation assay. The negative effect of oral and intra-peritoneal administration of

vanillin to Sprague-Dawley rats in unoxidized form at high concentrations (150 mg/kg

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and 300 mg/kg) was also investigated. Following the administration, animal behavior

was observed and recorded. After 14 weeks of vanillin administration, the effects of

vanillin on blood cells, kidney, liver and brain were studied. For the chemopreventive

properties of vanillin, rats were injected with azoxymethane (AOM) and subsequently

treated with vanillin. Aberrant crypt foci (ACF) counts and multiplicity were recorded.

RNA was extracted from colon for DNA repair, apoptosis, cell cycle, anti-

inflammation, proto-oncogene, colorectal cancer biomarker and tumor suppressor

gene expressions study. Findings from the in vitro study showed that vanillin was

cytotoxic towards HT-29 and 3T3 cells with the IC50 value of 400 µg/ml and 1000

µg/ml respectively. Vanillin also induced apoptosis and cell cycle arrest. Different

concentrations of vanillin showed arrest of cell cycle at different checkpoints. G0/G1

arrest was noted at lower concentration of vanillin (200 µg/ml) while G2/M arrest

occurred at higher concentration of vanillin (1000 µg/ml). From the in vivo study,

results showed that treatment of 300 mg/kg of vanillin by intra-peritoneal injection

caused the rats to be unconscious without exerting any negative effect on blood cells,

kidney and liver. Further analysis with GenomeLab GeXP genetic system on brain

tissues showed that the expression of most xenobiotic metabolism, cell progression,

tumour suppressor, DNA damage and inflammation genes was maintained at normal

level. However, the expressions of a few xenobiotic metabolism, cell cycle arrest and

apoptosis genes were up-regulated by 5 % ethanol injection. This shows that 5%

ethanol could pose negative effects onto the brain cells. Nevertheless, when 5 %

ethanol was injected together with vanillin, the expression of genes was comparable

to normal level. Hence, it is postulated that vanillin might have neuro-protective

property. For the chemopreventive effect, AOM-injected rats treated with vanillin

have significantly higher (p<0.05) ACF counts and multiplicity compared to the

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control group. The colon gene expression analysis showed that vanillin could

enhance recombinational repair and mismatch repair, arrest cell at cell cycle

checkpoints, increase the expression of tumour suppressor gene, colorectal cancer

biomarker and proto-oncogene. However, vanillin did not induce apoptosis and

inflammation in ACF-bearing colon. In conclusion, vanillin could induce cytotoxic

effects on colorectal cancer cells. It was not showing negative effects when

administered at high concentrations through oral and intra-peritoneal injections.

However, the ACF count and multiplicity indicate that vanillin was a co-mutagen

instead of chemopreventive agent in AOM-injected rats. Nevertheless, it should be

noted that only intraperitoneally injected vanillin would become co-mutagen, orally

administered vanillin is not a co-mutagen.

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Abstrak tesis dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi keperluan Ijazah Doktor Falsafah

FUNGSI DAN KESAN VANILLIN ATAS KANSER KOLON SECARA IN VITRO DAN IN VIVO

Oleh

HO KET LI

August 2011

Pengerusi: Profesor Maznah Ismail, PhD

Fakulti: Institut Biosains

Vanilin bertanggungjawab untuk memberi rasa dan bau vanila, satu agen perisa

yang diguna dengan meluas. Kajian lepas menunjukkan bahawa vanilin merupakan

perencat sel darah merah sabit, agen anti-mikrob dan anti-mutagen yang baik.

Namun demikian, vanilin perlu diberikan pada kepekatan yang tinggi dan tidak boleh

dioksidakan di bahagian perut dan usus untuk kesan perubatannya yang berkesan.

Oleh yang demikian, tujuan kajian ini adalah untuk 1. menyelidik kesan sitotoksik

vanilin ke atas sel kanser kolon HT-29; ii. menyelidik kesan negatif vanilin apabila

diberikan pada kepekatan yang tinggi secara in vivo; iii. menyelidik kesan

pencegahan vanilin terhadap kanser kolon dan iv. menyelidik kesan vanilin atas

penzahiran gen kolon. Kaedah yang digunakan untuk mengkaji kesan sitotoksik

vanilin termasuk: Ujian sitotosik, analisis morfologi sel dengan dua-pewarnaan,

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analisis kitaran sel, ujian apoptosis) dan ujian pertumbuhan sel. Kesan negatif vanilin

apabila diberi kepada tikus Sprague-Dawley dengan cara oral dan suntikan intra-

peritoneal pada kepekatan yang tinggi (150 mg/kg dan 300 mg/kg) juga dikaji.

Selepas vanilin diberikan, tingkah-laku tikus diperhatikan dan dicatatkan. Selepas

diberi vanilin untuk 14 minggu, kesan negative vanillin atas sel darah, ginjal, hati dan

otak juga dikaji. Bagi kesan pencegahan kanser kolon, tikus disuntik dengan

azoksimetana (AOM) dan kemudiannya dirawat dengan vanilin. Bilangan “aberrant

crypt foci” (ACF) dan keserbaragamannya direkodkan. RNA diekstrak dari kolon

untuk kajian penzahiran gen termasuk: gen pembaikian DNA, gen apoptosis, gen

kitaran sel, gen perencat keradangan, proto-onkogen, biomarker kanser kolon dan

gen penindas kanser. Keputusan kajian in vitro menunjukkan bahawa vanilin adalah

sitotosik terhadap sel HT-29 dan 3T3 dengan IC50 400 µg/ml dan 1000 µg/ml

masing-masing. Vanilin juga boleh menyebabkan apoptosis dan perhentian kitaran

sel. Vanilin dalam kepekatan yang berbeza akan menghentikan kitaran sel pada fasa

yang berbeza. Perhentian sel pada fasa G0/G1 boleh dicapai dengan menggunakan

vanilla pada kepekatan yang rendah (200 µg/ml) dan perhentian pada fasa G2/M

boleh dicapai dengan menggunakan vanilin pada kepekatan yang tinggi (1000

µg/ml). Daripada kajian kesan negative vanillin, rawatan 300 mg/kg secara suntikan

menyebabkan tikus mengalami keadaan tidak sedar tanpa menunjukkan kesan

negatif terhadap sel darah, ginjal dan hati. Analisis selanjutnya melalui system

genetic GenomeLab GeXP ke atas tisu otak tikus menunjukkan bahawa penzahiran

kebanyakan gen metabolime xenobiotik, perkembangan sel, penindas kanser,

kerosakan DNA dan keradangan adalah pada paras normal. Walau bagaimanapun,

penzahiran beberapa gen metabolime xenobiotik, penahanan kitaran sel dan

apoptosis telah ditingkatkan dengan suntikan 5 % etanol. Ini menunjukkan 5%

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ethanol boleh menyebabkan kesan negative atas sel otak. Namun demikian, apabila

5 % etanol disuntik bersama-sama dengan vanilin, penzahiran gen kembali ke paras

normal. Vanilin dijangka mempunyai kesan perlindungan sel neuro. Untuk kesan

pencegahan, tikus yang disuntik dengan AOM dan dirawat dengan vanilin

mempunyai kiraan ACF dan keserbaragaman ACF yang lebih tinggi (p<0.05)

berbanding dengan kumpulan kawalan. Analisis penzahiran gen menunjukkan

bahawa vanilin boleh meningkatkan mekanime pembaikian DNA rekombinasi,

pembaikian DNA mismatch repair (MMR), menahan kitaran sel, meningkatkan

penzahiran gen penindas kanser, biomarker kanser kolon dan proto-onkogen. Walau

bagaimanapun, vanilla tidak mengaruh apoptosis atau keradangan dalam kolon

yang mengandungi ACF. Kesimpulannya, vanilin boleh mengarah kesan sitotoksik

ke atas sel kanser kolon dan tidak memberi kesan negatif apabila diberi pada

kepekatan yang tinggi secara oral dan suntikan intra-peritoneal. Walau

bagaimanapun, vanilin merupakan co-mutagen dan bukan agen perlindungan kimia

dalam tikus yang disuntik dengan AOM. Namun demikian, hanya vanillin yang diberi

dengan cara suntikan akan menjadi co-mutagen. Vanillin yang diberikan secara oral

bukan co-mutagen.

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ACKNOWLEDGEMENT

First and foremost, I would like to thank my supervisor, Professor. Maznah Ismail for

her supervision, support and guidance. Without her full support, I would not be able

to finish my research.

Besides, special thanks to all my co-supervisors, Associate Professor Dr. Chong Pei

Pei and Dr. Latifah Saiful Yazan. They have given me support and precious

comments when I was facing problems in my research

A big thank you also dedicated to all the staff and friends in the Laboratory of

Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia. Kak Siti

Muskinah had been a great help and her contributions to my work will always be

remembered.

Last but not least, I am greatly thankful to my parents, siblings, grandmother, uncles

and aunties for their full support of my studies. They make me feel proud of my

achievements.

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I certify that a Thesis Examination Committee has met on 18th August, 2011 to conduct the final examination of Ho Ket Li on his thesis entitled “Properties of Vanillin on Colorectal 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:

Suhaila binti Mohamed, Ph.D. Professor Institut of Biosains Universiti Putra Malaysia (Chairman)

Fauziah bt Othman, Ph.D.

Professor Faculty of Medicine and Health Science Universiti Putra Malaysia (Member) Sharida Binti Fakurazi, Ph.D.

Professor Madya Faculty of Medicine and Health Science Universiti Putra Malaysia (Member) Ranjana Prasad Bird, Ph.D.

Professor Vice President Research Sunset Avenue Windsor Canada (External examiner)

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This thesis submitted to the Senate of the Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the supervisory Committee were as followed:

Maznah Ismail, PhD

Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Chong Pei Pei, PhD

Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) Latifah Saiful Yazan, PhD

Senior Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)

_________________________________

HASANAH MOHD. GHAZALI, 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 other institutions.

__________________

HO KET LI

Date: 18th August 2011

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TABLE OF CONTENT

PAGE

ABSTRACT II

ABSTRAK V

ACKNOWLEDGEMENT VIII

APPROVAL X

DECLARATION XI

LIST OF TABLES XV

LIST OF FIGURES XVI

LIST OF ABBREVIATIONS XIX

CHAPTER

1. INTRODUCTION 1

1.1 Significance of Study 5

1.2 General Objective 6

1.3 Specific Aims 6

2. LITERATURE REVIEW 2.1 Vanillin 7

2.1.1 Traditional Uses of Vanillin 9

2.1.2 Modern Uses of Vanillin 10

2.1.3 Bioavailability of Vanillin 13

2.1.4 Negative Effect of Vanillin 15

2.2 Colon 21

2.3 Cancer 22

2.3.1 Colorectal Cancer 25

2.3.2 Risk Factor of Colorectal Cancer 28

2.3.3 Aberrant Crypt Foci (ACF) 28

2.4 Characteristics of Anti-Cancer Drugs 29

2.5 Apoptosis 30

2.6 Cell Cycle 33

2.6.1 Cell Cycle Checkpoints 34

2.6.2 Cyclin-Dependent Kinase and Cyclin 35

2.7 Liver and Kidney Functions 36

2.8 Xenobiotic Metabolisms 37

2.9 Brain and Unconsciousness 38

2.10 Blood Cells 39

2.11 DNA-Repair Mechanism 42

2.12 Azoxymethane (AOM) 50

2.13 GeXP Analysis 50

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3. APOPTOSIS AND CELL CYCLE ARREST OF HUMAN COLORECTAL CANCER CELL LINE HT-29 INDUCED BY VANILLIN

3.1 Introduction 58

3.2 Materials and Methods 59

3.2.1 Materials 59

3.2.2 Preparation of Cell Culture Medium 59

3.2.3 Culture of HT-29 Cells and NIH/3T3 Cells 60

3.2.4 Cytotoxicity Test 60

3.2.5 Double Staining Cell Morphological Analysis 61

3.2.6 Cell Cycle Analysis 62

3.2.7 Apoptosis Test 63

3.2.8 Cell Proliferation Assay 63

3.2.9 Statistical Analysis 64

3.3 Results 64

3.3.1 Effects of Vanillin and Vanillic Acid on Cell Viability 64

3.3.2 Effects of Vanillin on Cell Morphology 66

3.3.3 Effects of Vanillin on the Cell Cycle 71

3.3.4 Incidence of Apoptosis in HT-29 Cells Following the Treatment with Vanillin 75

3.3.5 Effects of Vanillin on HT-29 Cells Proliferation 80

3.4 Discussion 83

4. ANTI-COLORECTAL CANCER PROPERTIES OF VANILLIN ON RATS

VIA ORAL AND INTRA-PERITONEAL ADMINISTRATION AND ITS RELATIONSHIP WITH DNA-REPAIR MECHANISMS 87

4.1 Introduction 87

4.2 Materials and Methods 88

4.2.1 Materials 88

4.2.2 Solubility of Vanillin in Ethanol 89

4.2.3 Animals and Treatments 89

4.2.4 Body Weight 92

4.2.5 Aberrant Crypt Foci (ACF) Counts 92

4.2.6 Aberrant Crypt Foci (ACF) Multiplicity 93

4.2.7 Tissue Samples and RNA Extraction 93

4.2.8 GeXP Analysis of Rat’s Colon Genes Expression 95

4.2.9 Statistical Analysis 100

4.3 Results 101

4.3.1 Solubility of Vanillin in Ethanol 101

4.3.2 Aberrant Crypt Foci (ACF) Counts 101

4.3.3 Aberrant Crypt Foci (ACF) Multiplicity 102

4.3.4 GeXP Analysis of Rat’s Colon Genes Expression 104

4.4 Discussion 112

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5. NEGATIVE EFFECTS OF VANILLIN ON RATS VIA ORAL AND INTRA-PERITONEAL ADMINISTRATION

5.1 Introduction 117

5.2 Materials and Methods 118

5.2.1 Materials 118

5.2.2 Animals and Treatments 118

5.2.3 Body Weights and Blood Sampling 120

5.2.4 Organ Weight 120

5.2.5 Whole Blood Cell Count 120

5.2.6 Liver and Kidney Functions Test 121

5.2.7 Tissue Samples and RNA Extraction 123

5.2.8 GeXP Analysis of Rat’s Brain Genes Expression 124

5.2.9 Statistical Analysis 129

5.3 Results 130

5.3.1 Animal Behavior 130

5.3.2 Organ Weight 135

5.3.3 Whole Blood Cell Count 137

5.3.4 Liver and Kidney Functions Test 137

5.3.5 Rat’s Brain Genes Expression 139

5.4 Discussion 144

6. GENERAL DISCUSSION, CONCLUSION AND FUTHER WORK

6.1 General Discussion and Conclusion 150

6.2 Further Work 152

REFERENCES 153

APPENDICES 167

BIODATA OF STUDENT 182