A thesis submitted in fulfilment of the requirement for the degree of Master of Biobehavioural Health Sciences

THE EFFECTS OF PROBIOTICS AND ACTINOMYCES NAESLUNDII ON ORAL CARCINOGENESIS

BY

WAN NUR FATIHAH BINTI WAN MOHD KAMALUDDIN

A thesis submitted in fulfilment of the requirement for the degree of Master of Biobehavioural Health Sciences

Kulliyyah of Nursing

International Islamic University Malaysia

DECEMBER 2020

ii

ABSTRACT

Oral cancer is one of the subclassification of head and neck cancer, which is one of the most prevalent cancers globally with high incidence and mortality rate. 90% of oral cancer constitutes of oral squamous cell carcinoma (OSCC). The prognosis status of the patients after treatment is determined by the relapse of the secondary oral tumour. The relapse of the secondary oral tumour leads to poor prognosis status of patients, hence reducing its five-year survival rate. Recently, probiotics have been heavily studied due to its health benefits and its contribution to cancer prevention. This study aimed to identify the anti-cancer properties of probiotics in oral carcinogenesis. A systematic review and meta-analysis were conducted to identify the list of probiotics that have a prominent effect on oral carcinogenesis. The identified probiotics were Acetobacter syzygii, Lactobacillus plantarum, Lactobacillus salivarius REN and AJ2. These probiotics elicited several effects against oral cancer, including anti-proliferative effects, modulation of protein expression, and apoptosis. The meta-analysis of the present study showed that L. salivarius REN is associated with a lower risk of oral carcinogenesis by 95%, with (OR=0.05, P<0.05). Thus, the ability of L. salivarius REN as an inhibitory agent for oral carcinogenesis is identified. Actinomyces naeslundii is one of the early oral colonisers that is associated with oral biofilm development.

However, the previous study has discovered the presence of A. naeslundii in polymicrobial biofilms was able to reduce the biofilm biomass and activity of the oral pathogens. Hence, this study also aimed to identify the protein similarities between L.

salivarius REN and A. naeslundii using in silico analysis. From the analysis, elongation factor Tu (EF-Tu) protein was identified with percent similarities of 79.5% in the present study. EF-Tu is a translation factor protein that is responsible for the modulation of the immune response. In addition, C. albicans is an opportunistic yeast that frequently isolated from the oral cavity. The previous study has shown that C. albicans is associated with oral carcinogenesis. The present study aims to determine the potential of A. naeslundii as an oral probiotic against oral carcinogenesis. The aggregation assay of the bacterium with C. albicans was conducted. The polymicrobial interactions of clinical strain C. albicans (ALC2) with A. naeslundii in nutrient broth exhibited lower co-aggregation compared to the auto-aggregation indicating the ability of A. naeslundii to reduce colonisation of C. albicans by inhibiting candidal dimorphism. In conclusion, L. salivarius REN could be a potential inhibitory agent against oral carcinogenesis. The high percentage protein similarities between L. salivarius REN with A. naeslundii shown the potential of A. naeslundii as an oral probiotic which able to inhibit oral carcinogenesis.

iii

ثحبلا ةصلاخ

ABSTRACT IN ARABIC

ىلع اراشتنا ناطرسلا عاونأ رثكأ دحأ وهو ،قنعلاو سأرلا ناطرسل ةيعرفلا تافينصتلا دحأ مفلا ناطرس دعي ىوتسم

لياوح .تايفولاو تبااصلإل عفترم لدعبم لماعلا 90

ةيفشرلحا يالالخا ناطرس نم ةنوكم مفلا ناطرس تلااح نم ٪

ساكتنا يدؤي .ةيوناثلا ةيومفلا مارولأا ساكتنا ةبسن للاخ نم جلاعلا دعب ضيرملل ةيصيخشتلا ةلالحا ديدتح متي .يومفلا ةلالحا ءوس لىإ ةيوناثلا ةيومفلا مارولأا سمبخ ةردقلما ةايلحا ديق ىلع ءاقبلا لدعم صيلقت لياتلباو ،ضيرملل ةيصيخشتلا

نم ةياقولا في اهتهماسمو ةيحصلا اهدئاوف ببسب فثكم لكشب كيتويبوبرلا ةسارد تتم ةيرخلأا ةنولآا في .تاونس برلا في ةدوجولما ناطرسلل ةداضلما صئاصلخا ىلع فرعتلا لىإ ةساردلا هذه تفده .ناطرسلا نطرستلا ىلع كيتويبو

لكشب يومفلا نطرستلا ىلع يرثأتلا ىلع ةرداقلا كيتويبوبرلا ةمئاق ديدحتل يولت ليلتحو ةيجهنم ةعجارم ءارجإ تم .يومفلا ( ةيلفنرقلا ةيصعلا ةيللخا :نم لاك اهديدتح تم تيلا ةيرهلمجا ةيلحا تانئاكلا تنمضت .زربا Acetobacter syzygii

،)

ا ةينبللا ةيصعلاو ( ةيتابنل

Lactobacillus plantarum ( ةيباعللا ةينبللا ةيصعلاو ،)

Lactobacillus

salivarius REN يايرتكبو ،)

AJ2 كلذ في ابم ،مفلا ناطرس دض تايرثأتلا نم ديدعلا كيتويبوبرلا هذه ترثاأ .

لا رهظأ .جمبرلما يالالخا توم طيشنتو ،ينتوبرلا يربعت ليدعتو ،رثاكتلل داضلما يرثأتلا ةيصعلا نأ ةساردلا هذله يولتلا ليلحت

ةبسنب مفلا ناطرسب ةباصلإا رطخ ضافنخبا ةطبترم ةيباعللا ةينبللا 95

( ٪ OR=0.05 ،

P<0.05 ديدتح تم اذلهو .)

( ةيدنولسيانلا ةيرطفلا ةيعشلا .يومفلا نطرستلل طبثم لماعك ةيباعللا ةينبللا ةيصعلا ةردق Actinomyces

naeslundii ئاوأ دحأ يه )

تفشتكا دقف كلذ عمو .ةيومفلا ةيويلحا ةيشغلأا ءوشنب ةطبترلما ةيومفلا تارمعتسلما ل

ليلقت ىلع اًرداق ناك تباوركيلما ةددعتم ةيويلحا ةيشغلأا في ةيدنولسيانلا ةيرطفلا ةيعشلا دوجو نأ ةقباسلا تاساردلا لباو .ةيومفلا ضارملأا تاببسم طاشنو ةيويلحا ةيشغلأل ةيويلحا ةلتكلا هجوأ ديدتح لىإ اًضيأ ةساردلا هذه تفده دقف ليات

ليلحتلا دعب تم .بيوسالحا ليلحتلا مادختسبا ةيدنولسيانلا ةيرطفلا ةيعشلاو ةيباعللا ةينبللا ةيصعلا في تانيتوبرلا ينب هباشتلا ةلاطتسلاا لماع ينتورب ديدتح Tu

(EF-Tu) يانلا ةيرطفلا ةيعشلاو ةيباعللا ةينبللا ةيصعلا ينب

هباشت ةبسنب ةيدنولس

تغلب 79.5 .٪

EF-Tu نأ اضيأ ليلحتلا فشكو ،ةيعانلما ةباجتسلاا ليدعت نع لوؤسم يجمرت لماع ينتورب وه

( ءاضيبلا ةضيبلما Candida albicans

ترهظأو ،يومفلا فيوجتلا نم لكشب الهزع امئاد متي ةيزاهتنا ةيرخم يه )

طبترم ءاضيبلا ةضيبلما نأ ةقباسلا تاساردلا ةيعشلا تنااكمإ ديدتح لىإ ةيلالحا ةساردلا تفده .يومفلا ناطرسلبا ة

ترهظأو ،ءاضيبلا ةضيبلما عم يايرتكبلا مكارت رابتخا ءارجإ تم .يومفلا نطرستلا دض يومف كيتويبوبرك ةيدنولسيانلا ةيرطفلا ( ءاضيبلا ةضيبملل ةيكينيلكلإا ةللاسلل تباوركيلما ةددعتلما تلاعافتلا ALC2

عم ) في ةيدنولسيانلا ةيرطفلا ةيعشلا

ليلقت ىلع ةيدنولسيانلا ةيرطفلا ةيعشلا ةردق لىإ يرشي امم تياذلا عيمجتلبا ةنراقم اضفخنم اًكترشم اًمكارت يذغم طيسو ةيرطفلا ةيعشلا ناكمبإ هنأ ينبتي ،اماتخ .تاضيبملل يلكشلا جاودزلاا طيبثت قيرط نع ءاضيبلا ةضيبلما رامعتسا دنولسيانلا ةيصعلا ينب نييتوبرلا هباشتلل ةيلاعلا ةبسنلا ترهظأ .يومفلا نطرستلا دض دعاو طبثم لماع ةباثبم نوكت نأ ةي

.يومفلا نطرستلل طبثم يومف كيتويبوبرك لمعلا ىلع يرخلأا ةردق ةيدنولسيانلا ةيرطفلا ةيعشلاو ةيباعللا ةينبللا

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Biobehavioural Health Sciences

………

Asst. Prof. Dr. Noratikah Othman Supervisor

………..

Asst. Prof. Dr. Mohd Hafiz Arzmi Co-Supervisor

………..

Asst. Prof. Dr. Edre Mohamad Aidid

Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Biobehavioural Health Sciences

………..

Asst. Prof. Dr. Mohd Arifin Kaderi

Internal Examiner

………..

Dr. Sarahani Harun External Examiner

This thesis was submitted to the Kulliyyah of Nursing and is accepted as a fulfilment of the requirement for the degree of Master of Biobehavioural Health Sciences

………..

Assoc. Prof. Dr. Salizar Mohamed Ludin

Dean, Kulliyyah of Nursing

v

DECLARATION

I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Wan Nur Fatihah Binti Wan Mohd Kamaluddin

Signature ... Date ... 30 November 2020

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

THE EFFECTS OF PROBIOTICS AND ACTINOMYCES NAESLUNDII ON ORAL CARCINOGENESIS

I declare that the copyright holders of this dissertation are jointly owned by the student and IIUM.

Copyright © 2020 Wan Nur Fatihah Binti Wan Mohd Kamaluddin and International Islamic University Malaysia. All rights reserved.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Wan Nur Fatihah Binti Wan Mohd Kamaluddin

……..……….. ………..

Signature Date 30 November 2020

vii

ACKNOWLEDGEMENTS

In the name of Allah, the Most Gracious, the Most Merciful. Alhamdulillah, finally I managed to finish my master’s thesis successfully with the help, encouragement, and supports from many people. I would like to take this opportunity to express my gratitude to the people who have contributed in the successful completion of this project.

Firstly, it is my utmost pleasure to dedicate this work to my dear parents and my family for their endless support, love, and best wishes. I am grateful to my family for the sacrifices they have made on my behalf. Their prayer for me was what sustained me this far and contributed a lot to the completion of this project. I thank you for your support and patience.

I wish to express my deepest appreciation to my beloved supervisor, Assistant Professor Dr. Noratikah Othman, for her caring, patience, non-stop encouragement, advice, tremendous help, and support in completing this project. And not to forget, my enthusiast co-supervisor, Assistant Professor Dr. Mohd Hafiz Arzmi, for his never- ending encouragement and constant support that contributed a lot to the completion of this project. I feel motivated and encouraged every time I meet with my supervisor and co-supervisor as they continuously gave useful remarks, comments, and ideas through the learning process of this project that I manage to coordinate, especially in writing this thesis. I also want to express my gratitude to Assistant Professor Dr. Edre Aidid and Assistant Professor Dr. Faisal for their constant help in completing the project.

Without their help, the completion of this is impossible to be achieved.

I wish to express my appreciation and thanks to those who provided their time, effort, and support for this project. To the members of my COCRII group, Nurul Alia Risma Rismayuddin, Munirah Mokhtar, Afifah Hanin, Sharmeen Nellisa, Hasna Ahmad, and Engku Anis Fariha, thank you for the support and constant motivation in helping me finish this project. And thank you for sticking with me through thick and thin.

Lastly, the guidance and support received from all the people who have contributed either directly or indirectly to this project were vital for the success of this project. I am so grateful for their constant and help. May Allah reward and bless all of your kindness and cooperation that you have done in this world and hereafter.

Once again, we glorify Allah for His endless mercy on us of which is enabling to successfully round off the efforts of writing this dissertation. Alhamdulillah.

viii

TABLE OF CONTENTS

Abstract ... ii

Abstract in Arabic ... iii

Approval Page ... iv

Declaration ... v

Copyright Page ... vi

Acknowledgements ... vii

List of Tables ... xi

List of Figures ... xiii

List of Abbreviations ... xiv

CHAPTER ONE: INTRODUCTION ... 1

1.1 Oral Cancer ... 1

1.2 Probiotics and Cancer ... 4

1.3 Oral Diseases ... 5

1.4 Problem Statement ... 6

1.5 Research Objectives... 7

1.6 Research Questions ... 7

1.7 Hypotheses ... 8

1.8 Significance of the Study ... 8

CHAPTER TWO: LITERATURE REVIEW ... 9

2.1 Oral Microbiome ... 9

2.1.1 Composition of Oral Microbiota ... 9

2.1.2 Streptococcus Species ... 10

2.1.3 Actinomyces Species ... 11

2.1.4 Candida Species ... 12

2.2 Overview of Probiotics ... 13

2.3 The Role of Probiotics in Cancer Prevention ... 15

2.4 The Role of Probiotics in Oral Health ... 18

2.5 Bioinformatics and Computational Biology ... 19

CHAPTER THREE: PROBIOTIC INHIBITS ORAL CARCINOGENESIS: A SYSTEMATIC REVIEW AND META- ANALYSIS ... 21

3.1 Introduction... 21

3.2 Methodology ... 23

3.2.1 Formulation of the Review Question ... 23

3.2.2 Search Strategy ... 23

3.2.3 Eligibility Criteria ... 24

3.2.4 Data Extraction ... 24

3.2.5 Assessment of Risk of Bias In the Included Studies ... 25

3.2.6 Statistical Analysis ... 25

3.3 Result ... 27

3.3.1 Study Selection ... 27

ix

3.3.2 Study Characteristics ... 27

3.3.3 Assessment of the Included Studies ... 31

3.3.4 Effects of Lactobacillus salivarius REN on Oral Cancer ... 31

3.4 Discussion ... 32

3.4.1 Probiotics Elicited Anti-proliferative Effects on Oral Carcinogenesis ... 32

3.4.2 Modulation of Protein Expression Induced by Probiotics against Oral Cancer ... 34

3.4.3 Apoptosis Assessment of Probiotic against Oral Cancer Cells and Healthy Cells ... 35

3.4.4 Lactobacillus salivarius REN as Inhibitory Agent against Oral Carcinogenesis ... 40

3.5 The Properties of Lactobacillus salivarius REN ... 41

3.6 Effects of Probiotics Used on Cancer Patients Receiving Treatments ... 42

CHAPTER FOUR: CHARACTERIZATION OF POTENTIAL PROBIOTICS PROPERTIES IN ACTINOMYCES NAESLUNDII USING IN-SILICO APPROACH ... 43

4.1 Introduction... 43

4.2 Methodology ... 45

4.2.1 Data Mining ... 45

4.2.2 Multiple Alignment of Conserved Genomics Region ... 45

4.2.3 Sequence Alignment ... 46

4.3 Bioinformatics Analysis ... 46

4.3.1 Comparison of Actinomyces naeslundii Genome with Known Probiotics ... 46

4.3.2 The Protein Similarities between Lactobacillus salivarius REN and Actinomyces naeslundii ... 55

4.3.3 Domain Analysis of Elongation Factor Tu (EF-Tu) ... 57

4.3.3.1 Transcription Factor, GTP-binding domain ... 58

4.3.3.2 Elongation Factor Tu, domain 2 ... 59

4.3.3.3 Elongation Factor (EF-Tu), GTP-binding domain ... 59

4.3.3.4 Translation Elongation Factor, EFTu/EF1A, C-terminal ... 60

4.3.3.5 Translation Elongation Factor, EFTu-like, domain 2 ... 60

4.3.3.6 Small GTP-binding Protein Domain ... 60

4.4 Discussion ... 61

4.4.1 The Role of Elongation Factor (EF-Tu) in Inducing Immune Responses ... 61

CHAPTER FIVE: COAGGREGATION ASSAY OF ACTINOMYCES NAESLUNDII WITH CANDIDA ALBICANS ATCC AND CLINICAL ISOLATE (ALC2) ... 63

5.1 Introduction... 63

5.2 Methodology ... 65

5.2.1 Growth of Microorganisms ... 65

5.2.2 Aggregation Assay ... 65

5.2.3 Statistical Analysis ... 67

5.3 Results ... 67

5.3.1 Morphology of Candida albicans and Actinomyces naeslundii .... 67

x

5.3.2 Auto-aggregation and co-aggregation of Candida albicans and

Actinomyces naeslundii ... 69

5.4 Discussion ... 73

CHAPTER SIX: CONCLUSION ... 75

6.1 Conclusion ... 75

6.2 Limitation of the Study ... 77

6.3 Future Directions ... 77

REFERENCES ... 78

APPENDIX A: CONFERENCE AND PUBLICATIONS ... 93

APPENDIX B: PUBLICATION EVIDENCE: PROBIOTIC INHIBITS ORAL CARCINOGENESIS: A SYSTEMATIC REVIEW AND META-ANALYSIS ... 94

APPENDIX C: PUBLICATION EVIDENCE: MEDICAL RESEARCH SYMPOSIUM ... 95

APPENDIX D: PUBLICATION EVIDENDE: INTERNATIONAL JOURNAL OF OROFACIAL AND HEALTH SCIENCES .... 96

APPENDIX E: RISK OF BIAS ASSESSMENT OF THE INCLUDED STUDIES BY USING JOANNA BRIGGS INSTITUTE (JBI) CRITICAL APPRAISAL TOOLS ... 97

xi

LIST OF TABLES

Table No. Page No.

3.3.2 Overview of the included study including the references, probiotic strain involved, type of sample used, mechanism exerted, and the

outcome exerted by the probiotics 29

3.4.3.1 Late apoptosis, early apoptosis and necrosis value for treated/untreated cell line with Acetobacter syzygii. KB cell is human oral cancer cell line meanwhile KDR cell is a normal epithelial cell

line 38

3.4.3.2 Necrosis, late apoptosis, and early apoptosis for cell lines treated with or without Lactobacillus plantarum. KB cell line is a human oral

cancer cell line 39

4.3.1 Summary of the protein similarities of probiotics identified from the

multiple genome alignment (MAUVE) 48

4.3.2 The protein similarities between Lactobacillus salivarius REN and

Actinomyces naeslundii 56

5.3.2 Percentage of auto and co-aggregation of two Candida albicans strains, ATCC MYA-4901 and ALC2 with Actinomyces naeslundii

(An) in RPMI-1640 (hyphal form) and NB (yeast form) 71

xii

LIST OF FIGURES

Figure No. Page No.

2.1.4 The yeast form (left) and the hyphal form (right) of C. albicans. The

arrow shows the yeast and hyphal form, respectively. 13 3.2 The flow diagram of the protocol based on the Preferred Reporting

Items for Systematic Review and Meta-analysis (PRISMA) (Moher et

al., 2015) 26

3.4.4 Forest plot on the effect of Lactobacillus salivarius REN on oral

cancer 40

4.3.1.1 Multiple genome alignment analysis of protein similarities between four probiotics and A. naeslundii. The red box indicates similarities of protein

RecA 49

4.3.1.2 Multiple genome alignment analysis of protein similarities between four probiotics and A. naeslundii. The red box indicates similarities of protein

Elongation Factor Tu 50

4.3.1.3 Multiple genome alignment analysis of protein similarities between four probiotics and A. naeslundii. The red box indicates similarities of protein 30s

ribosomal S7 51

4.3.1.4 Multiple genome alignment analysis of protein similarities between four probiotics and A. naeslundii. The red box indicates similarities of protein 30s

ribosomal S12 52

4.3.1.5 Multiple genome alignment analysis of protein similarities between four probiotics and A. naeslundii. The red box indicates similarities of protein

DNA-directed RNA polymerase subunit beta 53

4.3.1.6 Multiple genome alignment analysis of protein similarities between four probiotics and A. naeslundii. The red box indicates similarities of protein

DNA-directed RNA polymerase subunit beta' 54

4.3.2 EF-Tu protein similarities between Lactobacillus salivarius REN (accession id: AKI04200) and Actinomyces naeslundii (accession id:

WP_076067072) by using EMBOSE Watcher Application 56 4.3.3 InterPro database for Elongation Factor Tu (EF-Tu) 57 5.3.1 Gram-stained of the Candida albicans and Actinomyces

naeslundii after 24-h incubation when observed under light

microscope at 1000x magnification 68

xiii

5.3.2.1 Percentage of auto-aggregation of Candida albicans strain (ATCC and ALC2) in (A) RPMI-1640 and (B) NB after 1h of

incubation period 70

5.3.2.2Gram-stained of aggregation assay between mono and dual-culture of Candida albicans strain and Actinomyces naeslundii (An) after 1h of incubation, when observed with light microscope with 1000x

magnification 72

xiv

LIST OF ABBREVIATIONS

HNC Head and Neck Cancer

OSCC Oral Squamous Cell Carcinoma

PMD Potentially Malignant Disorder

HPV Human Papillomavirus

WHO World Health Organization

TNM Tumor, nodes and metastasis classification of malignant tumors

IBS Irritable bowel syndrome

IBD Irritable bowel diseases

PANC-1 Pancreas cancer cell line

HT-29 Colon cancer cell line

ALC2 Clinical isolate of Candida albicans

SDGs Sustainable Development Goals

HMP Human microbiome project

ISAPP International Scientific Association for Probiotics and Prebiotics

HCA Heterocyclic aromatic amines

AFB 1 Aflatoxin B1

TCGA The Cancer Genome Atlas

IAP Inhibitor of Apoptosis protein

QS Quorum Sensing

QSI Quorum Sensing Inhibitory

PRISMA Preferred Reporting Items for Systematic Review and Meta- analysis

RCT Randomized Controlled Trial

OR Odds Ratio

WNF Wan Nur Fatihah

EMA Edre Mohamad Aidid

AFI Ahmad Faisal Ismail

NAO Noratikah Othman

MHA Mohd Hafiz Arzmi

NAR Nurul Alia Risma

NAH Noor Afifah Hanin

JBI Joanna Briggs Institute

I² Heterogeneity

TCA-8113 Human tongues squamous cell carcinoma

4NQO 4-nitroquioline-1-oxide

KB Human oral cancer cell line

KDR Normal epithelial cell line hu-BLT mice Humanised BLT mice OSCSC Oral squamous cell stem cell

NK Natural Killer

MAPK Mitogen-activated protein kinase PTEN Phosphatase and tensin homolog

ACF Aberrant crypt foci

DMH 1,2-dimethylhydrazine

xv

MAUVE Multiple Alignment of Conserved Genomic Sequences with Rearrangements

DDBJ DNA Databank of Japan

ENA European Nucleotide Archive

EF-Tu Elongation factor Tu

DC Dendritic Cells

PAMP Pathogen associated molecular pattern

rEF-Tu Recombinant EF-Tu

NB Nutrient Broth

BHI Brain Heart Infusion

OD Optical Density

SD Standard deviation

SAPs Secreted aspartyl proteinases

HIV Human immunodeficiency virus

1

CHAPTER ONE INTRODUCTION

1.1 ORAL CANCER

The incidence rate of cancer has been shown to increase every year, regardless of age and gender (National Cancer Institute, 2015). Cancer is a genetic disease that occurs when there are changes to the genes (National Cancer Institute, 2017). It influences the function of the genes and may result in abnormal activities of the cells. Most people that were affected by cancer are mainly caused by carcinogen from external sources.

However, certain cancers are hereditary where individuals may inherit from their parents. A carcinogen is defined as any substance that may cause changes in the human body, which may alter the normal function of human physiology. These carcinogens may come in various types such as natural carcinogens, chemical carcinogens and radionuclide carcinogens (National Cancer Institute, 2015). As mentioned by Smith et al., (2016), according to the review done by International Agency for Research on Cancer (IARC), there are 10 key characteristics accompanying carcinogens mechanisms, which are: a) electrophilic or metabolically activated, b) genotoxic, c) alters DNA repair or causing genomic instability, d) induces epigenetic alterations, e) induces oxidative stress, f) induces chronic inflammation, g) immunosuppressive, h) modulates receptor-mediated effects, i) causes immortalization, and j) alters cell proliferation, cell death or nutrient supply. These characteristics were effective in ruling out the agents with regard to the carcinogenic hazard.

Oral cancer is one of the most prevalent diseases worldwide, with approximately 354,864 new cases being reported along with 177,384 deaths reported (Bray et al., 2018). Oral cancer is a subclassification within head and neck cancer (HNC), which it

2

is the sixth leading mortal cancer worldwide, with 630,000 new cases reported annually, followed by 350,000 deaths every year (Vigneswaran & Williams, 2014). Oral cancer sites include the buccal mucosa (cheek), tongue, the floor of the mouth, and lip (Tsantoulis et al., 2007). Ninety percent (90%) of oral cancer arises from oral squamous cell carcinoma (OSCC). OSCC is an aggressive and lethal type of oral cancer with high incidence and mortality rate (Chi et al., 2015). The main factors that contributed to oral carcinogenesis are high consumption of alcohol and tobacco-smoking (Migueláñez- Medrán et al., 2019). Additionally, other factors that contribute to oral carcinogenesis include genetic factors, poor oral hygiene, and human papillomavirus (HPV) infection (Candotto et al., 2017). In 2005, in an epidemiological study, it was reported that HPV DNA was detected in 35.6% of oropharyngeal cancers (Kreimer et al., 2010). HPV oncogenic factors HPV16 was accounted for most HPV-positive cases, with 87%

(Kreimer et al., 2010). The evaluation of the association of HPV with the risk of oropharyngeal cancer was determined through epidemiologic studies (Gröbe et al., 2013).

Besides, potentially malignant disorders (PMDs) of the oral cavity has also been proposed as the aetiological factor for the development of oral squamous cell carcinoma (Sankari et al., 2015). According to the review done by Bughsan and Farouq (2020), many PMDs can become OSCC under certain underlying factors. Contrasting to the term proposed by WHO Collaborating Centre for Oral Cancer in 2007, expressed that not all lesions and conditions described under the term of “potentially malignant disorder” may develop into cancer (Warnakulasuriya et al., 2007).

On average, the survival rate of patients with oral cancer are five years.

Currently, the preferred treatment option for treating oral cancer is mainly via surgery.

However, the procedure has a low success rate in advanced stage of OSCC as this cancer

3

tend to have a recurrence. The prognosis status of the patients after treatment was determined through the remission of the secondary oral tumour. Patients with recurrence of the secondary oral tumour have poor prognosis status. As stated by Zini et al., (2010), the recurrence of the secondary oral tumour is the causal of reduced survival rate, approximately 50%-60% among patients. The recurrence affected the five-year survival rate and disease-free survival of patients with OSCC (Lindenblatt et al., 2012). It may be due to the aggressive local invasion and metastasis that gave the poor prognosis (Wang et al., 2013).

Patients' condition might be improved if they were diagnosed at an early stage.

However, two-third of the patients were diagnosed during the later stages, mainly in stage III and stage IV, thus, leading to the poor prognosis. According to Oral Cancer Foundation (2019), the mortality rate that is associated with oral cancer in 2019 is high because oral cancer is often diagnosed during their late stage. To date, there is no comprehensive program that can opportunistically screen oral cancer in the early stage.

The current diagnosis method that is used to estimate the prognosis and survival of oral cancer patients is the tumour, nodes, and metastasis classification of malignant tumours (TNM classification) (Jadhav & Gusta, 2013). It also provides detailed guidance on the treatment regimen to be followed in each case of OSCC. Thus, the healthcare practitioners must provide a preventive approach against the recurrence of the secondary oral tumour in order to improve the patients’ survival rate and prognosis.

Consequently, improving the patients’ quality of life.

4 1.2 PROBIOTICS AND CANCER

Probiotics is defined as ‘Live microorganisms which, when administered in adequate amounts, confer a health benefit to the host’ (WHO, 2001). Probiotics have sparked interest over the years due to its positive health benefits. It is widely used as therapy for the prevention and treatment of gastrointestinal disorders, including irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), pathogenic bacterial or viral infection, and antibiotic-associated bacteria. The mechanisms of probiotics were associated with health benefits, which emphasised the strengthening of the gut epithelial barrier and modulation of the immune system (Collado et al., 2010). However, the effectiveness of the probiotics in providing health benefits remains unclear. The most common bacteria strains that are used as probiotics are Bifidobacteria and Lactobacillus species. These bacteria have been added into many functional foods and dietary supplements (Fijan, 2014; Terpou et al., 2019).

Probiotics has been reported to possess anti-cancer properties. Lactobacillus strain, specifically L. acidophilus 606 and L. casei ATCC 393, was shown to inhibit the growth of pancreas (PANC-1) and colon (HT-29) cancer cell line (Choi et al., 2006). L.

acidophilus 606 was found to be the most effective at inhibiting the growth of these cancer cell lines (HT-29, HeLa, and PANC-1) at 21-28% survival rate compared with control at 10⁸ CFU ml^-1. Furthermore, L. acidophilus 606 was also found to be less cytotoxic to healthy cells compared to L. casei ATCC 393. It is also suggested that L.

acidophilus 606 may be used as natural cancer therapeutic agents due to its lower toxicity effects (Choi et al., 2006).

Interestingly, more strains of probiotics with anticancer properties have been discovered. Hence, this study will elucidate the potential proteins/genes in probiotics strains that may have the potential to prevent oral carcinogenesis.

5 1.3 ORAL DISEASES

In 2016, as reported by the Global Burden of Disease Study, it is estimated that half of the world population (3.58 billion people) were affected with oral diseases. Oral diseases may contribute to the health and economic burdens, thus affected people’s quality of life (Peres et al., 2019). There are a few examples of oral diseases that cause major public concern worldwide. They are dental caries, periodontal disease, oral mucosal lesions, oropharyngeal cancer, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS)-related oral disease and orodental trauma (Petersen et al., 2005). The main factors of oral diseases were strongly associated with behavioural risk factors such as taking unhealthy diets that are high in free sugars, tobacco usage, and harmful use of alcohol. Besides, dysbiosis of the oral microbiome was suggested to play a role in the occurrence of oral disease (Sharma et al., 2018). The development of periodontitis resulted from the colonisation of anaerobic bacteria in the periodontal pocket, which caused the loss of attachment between teeth and gingivae (Darveu, 2010). Meanwhile, gingivitis is another common periodontal disease which is caused by the colonisation of gram-negative bacteria (Zijnge et al., 2010).

Dental caries or tooth decay is one of the common oral diseases that affects people of all ages (Heng, 2016). Dental caries results from the accumulation of acid production due to the fermentation of carbohydrates. The excess uptake of carbohydrates leads to the demineralisation of the tooth, due to the formation of biofilm that contains acidogenic and aciduric species (Takahashi & Nyvad, 2011). Thus, this showed that the dysbiosis of oral microbiome also leads to the development of oral diseases.

Streptococcus mutans is the primary factors that caused human dental caries as they are acidogenic and aciduric (Forssten et al., 2010). Besides, S. mutans,

6

Actinomyces naeslundii has also become the interest of researchers as it also plays a role in periodontal and caries infections. However, previous studies proved otherwise where A. naeslundii is also associated to good oral health where the studies found that the growth of A. naeslundii when co-cultured with S. mutans showed inhibition of S.

mutans growth (Arzmi et al., 2016). Thus, proved the ability of A. naeslundii in sustaining good oral health.

1.4 PROBLEM STATEMENT

Oral cancer is a part of head and neck cancer (HNC), which is one of the most prevalent diseases worldwide with 354,864 new cases being reported along with 177,384 deaths reported (Bray et al., 2018). Most common type of oral cancers is oral squamous cell carcinoma (OSCC). OSCC patient’s often get diagnosed during its late stage. The prognosis of the patient with OSCC was determined by the recurrence of the secondary oral tumour. The recurrence of the secondary oral tumour in OSCC patients indicates the poor prognosis status of the patient. It also reduces the survival rate and disease-free of the patients, thus affected the patient’s quality of life. Hence, the preventive approach against the recurrence of the secondary oral tumour is needed to improve the patient’s survival rate and quality of life.

7 1.5 RESEARCH OBJECTIVES

The study aims to achieve the following objectives:

1- To identify the probiotics that have effects on oral carcinogenesis based on the evidence collected through systematic review and meta-analysis.

2- To identify the similarities of proteins in probiotics with Actinomyces naeslundii by comparing genomic sequence.

3- To determine the interaction and coaggregation ability of Actinomyces naeslundii with clinical strain isolate of Candida albicans (ALC2) strain through coaggregation assay.

1.6 RESEARCH QUESTIONS

This study aims to answer the following research questions:

1. What are the probiotics that have effects on oral carcinogenesis?

2. How many proteins are similar and what are the percentage of protein similarities between genomic sequence of probiotics and Actinomyces naeslundii?

3. What is the percentage of coaggregation between Actinomyces naeslundii with the clinical strain isolate of Candida albicans (ALC2)?

8 1.7 HYPOTHESES

The hypotheses for this study are:

1. Probiotics exerts several mechanisms in inhibiting oral carcinogenesis.

2. The higher proteins similarity number and percentage between probiotics and Actinomyces naeslundii determined the higher possibility of Actinomyces naeslundii to act as potential oral probiotic against oral carcinogenesis.

3. The high coaggregation percentage between Actinomyces naeslundii and clinical isolate Candida albicans inhibits the interaction of Candida albicans.

1.8 SIGNIFICANCE OF THE STUDY

To our knowledge, there are still insufficient study to elucidate the role of probiotics in oral carcinogenesis. To date, the relapse of the secondary oral tumour affected the patient’s quality of life as it decreases their survival rate. The relapse of the secondary oral tumour also indicates poor prognosis status, hence a proper study that can identify the preventive approach against the relapse of the secondary oral tumour is needed. This study is the first study that mainly focuses on the effects of probiotics against oral carcinogenesis. This study will also elucidate the effect of A. naeslundii as a preventive approach against oral carcinogenesis, mainly in focusing its activity against the development of the pre-malignant lesion of oral cancer. The preventive approach against the relapse of the secondary oral tumour may reduce the incidence of the relapse of the secondary oral tumour, hence improving the patient’s survival rate. This study is aligned with the third sustainable development goals (SDGs), which aims to provide excellent health and well-being for all ages.

9

CHAPTER TWO LITERATURE REVIEW

2.1 ORAL MICROBIOME

Oral microbiota or oral microbiome is defined as the collective genome of microorganisms that reside in oral cavity, which is the second largest microbial community in human after the gut (Deo & Deshmukh, 2019). Initially, these oral microbiomes eventually played an essential role in maintaining the normal oral physiological environment and are associated with human health (Gholizadeh et al., 2016). However, oral microbiome dysbiosis always occurred due to the imbalance in the oral environment. The dysbiosis of oral microbiome is associated with various local and systemic human diseases, including dental caries, periodontal disease, obesity, and cardiovascular disease (Wade, 2013).

2.1.1 Composition of oral microbiota

The human mouth is heavily colonised by microorganisms, including viruses, protozoa, fungi, archaea and bacteria (Wade, 2013). Approximately, 700 prokaryotes species have been detected in the human oral cavity, which belongs to 185 genera and 12 phyla, namely Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Spirochaetes, SR1, Synergistetes, Saccharibacteria (TM7), and Gracilibacteria (GN02) (Perera et al., 2016; Zhao et al., 2017). Based on the data provided by the National Institute of Health Research common fund Human