A ROLE FOR THE HYDROXYBENZOIC ACID METABOLITES OF FLAVONOIDS

AND ASPIRIN IN THE PREVENTION OF COLORECTAL CANCER

BY

RANJINI SANKARANARAYANAN

A dissertation submitted in partial fulfillment of the requirements for the

Doctor of Philosophy

Major in Pharmaceutical Sciences

South Dakota State University

2021

ii

DISSERTATION ACCEPTANCE PAGE Ranjini Sankaranarayanan

This dissertation is approved as a creditable and independent investigation by a candidate

for the Doctor of Philosophy degree and is acceptable for meeting the dissertation

requirements for this degree. Acceptance of this does not imply that the conclusions

reached by the candidate are necessarily the conclusions of the major department.

Gunaje Jayarama, A00137123 Advisor Date

Omathanu Perumal Department Head Date

Nicole Lounsbery, PhD Director, Graduate School Date iii

ACKNOWLEDGEMENTS

The completion of this work would not have been possible without the help and contribution of various people who have in their own kind way supported its accomplishment.

I would like to express my heart-felt gratitude to my mentor

Dr. Jayarama Bhat Gunaje for having skillfully guided me through this project and for having generously extended his support throughout. Dr. Jay has taught me invaluable lessons about honesty, integrity, and listening to your data when it comes to scientific research. He has always been a pillar of support and has also been kind in sharing his many experiences to help me both professionally and personally. I thank him for this. These four years that I spent in the lab with him will be time I will fondly remember and cherish forever.

I would like to thank Dr. Joy Scaria (Department of Veterinary & Biomedical Sciences,

SDSU) and Dr. Hemachand Tummala for their valuable advice and for graciously allowing me to use their laboratory facilities and resources for the completion of this work.

I would like to thank my committee members Dr. Xingyou Gu and Dr. Suvobrata

Chakravarty for their critical inputs and valuable suggestions during the course of this investigation. I am also extremely thankful to Dr. Theresa Seefeldt, Dr. D. Ramesh Kumar

(University of Kentucky), and Dr. Severine Van slambrouck (Department of Chemistry and Biochemistry, SDSU) for all their encouragement, timely advice and help for the progression of my project. Each of them in their own kind way have been instrumental to for the completion of this work. This is especially true of Dr. Ramesh Kumar, who helped me out immensely with the bioinformatics section of the project and patiently guided me iv to better understand this field. I would also like to take this opportunity to thank Dr. Puttur

Prasad (Medical College of Georgia at Augusta University) for providing us with the transporter-expressing cancer cell line. A large part of my thesis would have been incomplete without the use of this critical cell line. I am extremely grateful to him for his generosity.

I am also grateful to Fraunhofer, Center for Molecular Biotechnology, USA, for providing me with an internship opportunity. My experience there helped me understand the nuances of drug delivery and allowed me to apply the theoretical knowledge I gained in my Ph.D. coursework into practice. It was an enriching experience and I thank them for this.

I would love to thank my colleagues, seniors and friends for providing an amazing environment to work in, these last 4 years. I would especially like to thank Dr. Rakesh

Dachineni, Maria and Linto Antony and their family, Shruti S. Menon, Prabhjot Kaur

Sekhon, Ekavali Sharma, Mawuli MacDonald, Chaitanya Valiveti, Dr. Mibin Joseph, Dr.

Somshuvra Bhattacharya, Emily Trias and Tana Lick, for their unwavering support and belief in me. My stay in Brookings was made pleasant because of these wonderful individuals who helped me make a home away from home.

I would sincerely like to thank Dr. Omathanu Perumal (the Head), Dr. Xiangming Guan

(Graduate Research Coordinator), and all the faculty and staff of the Pharmaceutical

Sciences Department for their constant support and encouragement during the course of my stay at SDSU. I would also like to thank them for offering me the opportunity to be the laboratory manager for the last three years. It taught me important lessons in people management and leadership. v

I would also like to take this opportunity to thank the department of Pharmaceutical

Sciences for their financial support and for providing me with all the facilities and resources required for the successful completion of this project.

Last but not the least, I would like to thank my family – my grandmother, parents, sister and brother-in-law without whose support and encouragement this journey would not have been complete.

I extend my heartfelt gratitude to them all.

______Ranjini Sankaranarayanan vi

CONTENTS

ABBREVIATIONS ...... xiii

LIST OF FIGURES ...... xvii

LIST OF TABLES ...... xx

ABSTRACT ...... xxi

CHAPTER 1: INTRODUCTION ...... 1

1.1 Colorectal cancer (CRC) ...... 3

1.1.1 Tumor suppressor genes and protooncogenes as drivers of CRC development . 4

1.1.1.1 APC and development of CRC ...... 5

1.1.1.2 Tumor protein P53 (P53) and development of CRC: ...... 5

1.1.1.3 SMAD4 and development of CRC ...... 6

1.1.1.4 RAS and BRAF and development of CRC ...... 7

1.1.1.5 PI3K signaling and development of CRC ...... 7

1.2 Cell cycle regulation and cancer development ...... 8

1.2.1 Dysregulation of the cell cycle in cancer: ...... 11

1.2.2 CDKs ...... 12

1.2.3 Cyclins ...... 12

1.2.4 CDK activating enzymes ...... 13

1.2.5 CKIs ...... 13

1.2.6 CDK Substrates ...... 14 vii

1.2.7 Checkpoint proteins ...... 14

1.3 CDK inhibitors for cancer treatment ...... 14

1.3.1 Targeted inhibition of CDK4 and CDK6 ...... 15

1.4 Cancer chemoprevention ...... 17

1.4.1 Drugs as agents of cancer prevention ...... 17

1.4.2 Phytochemicals as agents of cancer prevention ...... 19

1.5 Flavonoids in cancer prevention ...... 20

1.5.1 Flavonoid effects on signal transduction ...... 21

1.5.2 Antioxidant effects of flavonoids ...... 23

1.6 Aspirin as a chemopreventive agent...... 23

1.6.1 Pharmacological effects of Aspirin ...... 24

1.6.2 Mechanisms proposed for aspirin’s chemo-preventive effects...... 25

1.6.2.1 Inhibition of COX enzymes: the platelet hypothesis ...... 25

1.6.2.2 Inhibition of Nuclear Factor (NF)-κB signaling ...... 29

1.6.2.3 Activation of AMP-kinase and inhibition of mTOR signaling ...... 29

1.6.2.4 Inhibition of Wnt signaling and β-catenin phosphorylation ...... 30

1.6.2.5 Downregulation of c-myc, cyclin A2 and CDK2 ...... 30

1.6.2.6 Induction of polyamine catabolism ...... 31

1.6.2.7 Induction of DNA mismatch repair proteins ...... 31 viii

1.6.2.8 Acetylation of P53, glucose-6-phosphate dehydrogenase and other proteins

...... 32

1.6.2.9 Other mechanisms ...... 33

1.7 Metabolism of flavonoids ...... 34

1.8 Metabolism of aspirin...... 37

1.9 Biotransformation of flavonoids and aspirin by the gut microbiota ...... 39

1.10 Rationale for studies on flavonoid and aspirin metabolites as chemopreventive

agents and the hypothesis ...... 40

1.10.1 Hypothesis ...... 42

1.10.2 Approach ...... 42

CHAPTER 2: MATERIALS AND METHODS ...... 44

2.1 Materials ...... 44

2.2 Buffers ...... 48

2.3 Cell lines, culture media and other reagents ...... 48

2.4 Recombinant proteins and antibodies ...... 49

2.5 Cell culture ...... 49

2.6 Cell lysate preparation and Western Blotting ...... 49

2.7 In vitro CDK assay ...... 50

2.8 Molecular docking studies ...... 50

2.9 HPLC analysis for detection of metabolites from cancer cell lysates ...... 51 ix

2.10. RNA isolation and qRT-PCR ...... 51

2.11 Flow cytometric analysis ...... 52

2.12 Clonogenic assay ...... 52

2.13 Cell proliferation analysis through cell counting ...... 53

2.14 Bacterial culture medium ...... 53

2.15 Screening for bacterial species that were capable of metabolizing quercetin ...... 54

2.16 HPLC analysis for the detection of quercetin/ metabolites in bacterial culture

supernatant ...... 54

2.17 Lysis of bacteria and assay for determination of quercetin degrading enzyme

activity ...... 55

2.18 Statistical analysis ...... 56

CHAPTER 3: THE FLAVONOID METABOLITE 2,4,6-TRIHYDROXYBENZOIC

ACID IS A CDK INHIBITOR AND AN ANTI-PROLIFERATIVE AGENT ...... 57

3.1 Abstract ...... 57

3.2 Background and hypothesis ...... 58

3.3 Results: ...... 59

3.3.1 Dose dependent effect of 2,4,6-THBA on CDK 1, 2 and 4 enzyme activity ... 59

3.3.2 Comparison of the effect of 2,4,6-THBA, phloroglucinol and 3,4,5-THBA on

CDK enzyme activity ...... 60

3.3.3 Molecular docking studies show potential interactions of 2,4,6-THBA,

phloroglucinol and 3,4,5-THBA with CDKs...... 62 x

3.3.4. 2,4,6-THBA inhibits cell proliferation in cell lines expressing functional

SLC5A8 ...... 64

3.3.5 2,4,6-THBA is taken up by cells expressing functional SLC5A8 ...... 68

3.3.6. Clonogenic assay ...... 70

3.3.7 Cell cycle analysis and apoptosis assay ...... 71

3.3.8. Effect of other flavonoid metabolites (4-HBA; 3,4-DHBA; and 3,4,5-THBA)

on clonal formation ...... 72

3.4 Discussion ...... 77

CHAPTER 4: ASPIRIN METABOLITES 2,3‑DHBA AND 2,5‑DHBA INHIBIT

CANCER CELL GROWTH ...... 86

4.1 Abstract ...... 86

4.2 Background and hypothesis ...... 87

4.3 Results ...... 88

4.3.1 Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK enzyme

activity ...... 88

4.3.2 Docking studies reveal binding pockets for aspirin, salicylic, 2,3-DHBA, and

2,5-DHBA...... 93

4.3.3 Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on colony

formation ...... 96

4.4 Discussion ...... 101 xi

CHAPTER 5: IDENTIFICATION OF BACTERIAL SPECIES THAT CAN

METABOLIZE QUERCETIN ...... 111

5.1 Abstract ...... 111

5.2 Background and hypothesis ...... 112

5.3 Results ...... 115

5.3.1 Screening the human gut bacterial library for quercetin degrading strains .... 115

5.3.2 HPLC analysis of Bacillus glycinifermentans and Flavonifractor plautii reveal

different metabolites produced following quercetin degradation ...... 117

5.3.3 Degradation kinetics and different end products suggest different mechanisms

of quercetin degradation by B. glycinifermentans and F. plautii ...... 122

5.3.4 Clonogenic assays with DOPAC ...... 123

5.3.5 Clonogenic assay using HCT-116 with spent bacterial culture supernatant .. 124

5.4 Discussion ...... 128

CHAPTER 6: SUMMARY, PERSPECTIVES, AND FUTURE STUDIES ...... 134

6.1 Summary from Chapter 3 ...... 135

6.2 Summary from Chapter 4 ...... 136

6.3 Summary from Chapter 5 ...... 137

6.4 The metabolite hypothesis – a common mechanism for cancer prevention ...... 138

6.5 Perspective on the role of flavonoid degrading bacteria and their metabolites in

decreasing CRC ...... 141

6.6 Perspective on aspirin metabolites in decreasing CRC ...... 143 xii

6.7 Conclusions ...... 146

LITERATURE CITED ...... 148

LIST OF PUBLICATIONS DIRECTLY RELATED TO THIS DISSERTATION ...... 179

LIST OF OTHER PUBLICATIONS ...... 180

xiii

ABBREVIATIONS

2,3-DHBA 2,3-dihydroxybenzoic acid

2,4,6-THBA 2,4,6-trihydroxybenzoic acid

2,4-DHBA 2,4-dihydroxybenzoic aicd

2,5-DHBA 2,5-dihydroxybenzoic acid

2,6-DHBA 2,6-dihydroxybenzoic acid

3,4,5-THBA 3,4,5-trihydroxybenzoic acid

3,4-DHBA 3,4-dihydroxybenzoic acid

3-HBA 3-hydroxybenzoic acid

4-HBA 4-hydroxybenzoic acid

ALL Acute lymphoblastic leukemia

AML Acute myleoid leukemia

AMP Adenosine monophosphate

AMPK Adenosine monophosphate activated kinase

APC Adenomatous polyposis coli

ATP Adenosine triphosphate

BSA Bovine serum albumin

CAK CDK activating kinase

CDK Cyclin Dependent Kinase

CKI CDK inhibitor

CLL Chronic lymphocytic leukemia

COX Cyclooxygenase

CRC colorectal cancer xiv

CYP450 Cytochrome P450

DMSO Dimethysulfoxide

DNA Deoxyribonucleic acid

DOPAC 3,4-dihydroxyphenyl acetic acid

DR Death receptor

DTT Dithiothretiol

EDTA Ethylenediaminetetraacetic acid

EGCG Epigallocatechin gallate

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

EMT Epithelial Mesenchymal transition

FAP Familial adenomatous polyposis

FBS Fetal Bovine Serum

FDA Food and drug administration

G6PD Glucose-6-phosphate dehydrogenase

GAP GTPase activating protein

GI Gastrointestine

HBA Hydroxybenzoic acid

HDAC Histone deacetylase

HPLC High performance liquid chromatography

HPV Human Papilloma Virus

IKK IκB Kinase

IRS2 Insulin receptor substrate 2 xv

LCIS Lobular carcinoma in situ

MAPK Mitogen activated protein kinase

MCT Monocarboxylic acid Transporter miRNA micro RNA

MMP Matrix metalloprotease

MOPS 3-(N-morpholino)propanesulfonic acid mTOR mechanistic target of rapamycin

NF-κB Nuclear factor - κB

NSAID Non-steroidal anti-inflammatory drug

ODC Ornithine decarboxylase

P53 Tumor protein P53

PBS Phosphate buffered saline

PDGF Platelet derived growth factors

PG Prostaglandin

PGI2 prostacyclin

PI3K Phosphatidyl inositol 3 kinase

PTEN Phosphatase and Tensin homolog

Rb protein Retinoblastoma

RNA Ribonucleic acid

ROS Reactive oxygen species

SCFA Short chain fatty acid

SDS Sodium dodecyl sulphate

SERM Selective estrogen receptor modulator xvi

SSAT Spermidine/spermine N1-acetyl transferase

TFA Trifluoroacetic acid

TGF-β Transforming growth factor -β

TPA 12-O-tetradecanoylphorbol-13-acetate

TSG Tumor suppressor gene

TXA2 Thromoxane A2

USPSTF United States Preventive Services Task Force

VEGF Vascular endothelial growth factor

xvii

LIST OF FIGURES

Figure 1. 1: Stages of cancer ...... 3

Figure 1. 2: The Cell cycle ...... 11

Figure 1. 3: Basic backbone (center) and different classes of flavonoids ...... 21

Figure 1. 4: The Platelet hypothesis ...... 28

Figure 1. 5: Other suggested pathways for aspirin’s chemopreventive actions ...... 34

Figure 1. 6: Postulated degradation pathways of selected flavonoid compounds to generate

2,4,6-THBA, 4-HBA, 3,4-DHBA and 3,4,5-THBA ...... 36

Figure 1. 7: Metabolism of aspirin and flavonoids to generate hydroxybenzoic acids .... 38

Figure 3. 1: Dose dependent effect of 2,4,6-THBA on different CDKs ...... 60

Figure 3. 2: Comparison of the effect of phloroglucinol, 2,4,6-THBA and 3,4,5-THBA on

CDK1 (A), CDK2 (B) and CDK4 (C) activity ...... 61

Figure 3. 3: Molecular docking revealed potential interactions of different compounds with

CDKs...... 64

Figure 3. 4: Effect of 2,4,6-THBA on HCT-116 cell proliferation ...... 66

Figure 3. 5: Effect of 2,4,6-THBA on MDA-MB-231 and SLC5A8-pLVX cells ...... 67

Figure 3. 6: Western Blot analysis showing the levels of various CDKs and cyclins in

SLC5A8-pLVX cells in response to 2,4,6-THBA ...... 68

Figure 3. 7: Uptake studies of 2,4,6-THBA and its effects on colony formation in different cell lines ...... 69

Figure 3. 8: Western Blot demonstrating the expression levels of SLC5A8 protein in HCT-

116, HT-29, MDA-MB-231 and SLC5A8-pLVX cell lines ...... 70

Figure 3. 9: Effect of different HBAs on colony formation in HT-29 cells ...... 71 xviii

Figure 3. 10: Cell cycle and apoptosis assays ...... 72

Figure 3. 11: Colony formation assay following treatment with 3,4-DHBA ...... 74

Figure 3. 12: Colony formation assay following treatment with 3,4,5-THBA ...... 75

Figure 3. 13: Colony formation assay following treatment with 4-HBA ...... 76

Figure 3. 14: HPLC analysis showing the cellular uptake of different HBAs ...... 77

Figure 3. 15: A model depicting how flavonoid metabolites may exert its chemo-preventive effects on colonic tissue to protect against CRC ...... 84

Figure 4. 1: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK1 enzyme activity...... 89

Figure 4. 2: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK2 enzyme activity...... 90

Figure 4. 3: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK4 enzyme activity...... 91

Figure 4. 4: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK6 enzyme activity...... 92

Figure 4. 5: Line model showing the interactions of aspirin, salicylic acid, 2,3-DHBA and

2,5-DHBA with CDK1, CDK2, CDK4 and CDK6 ...... 94

Figure 4. 6: Effect of aspirin, salicylic acid and its metabolites on HCT-116 colony formation ...... 97

Figure 4. 7: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on HT-29 colony formation ...... 98

Figure 4. 8: Effect of aspirin, salicylic acid and its metabolites on MDA-MB-231 colony formation ...... 99 xix

Figure 4. 9: Effect of 2,4-DHBA and 2,6-DHBA on colony formation in MDA-MB-231 cells ...... 100

Figure 4. 10: Effect of 2,3-DHBA (A) and 2,5-DHBA (B) on cell number in MDA-MB-

231 cells ...... 101

Figure 4. 11: A model depicting how aspirin metabolites may exert its chemo-preventive effects on colonic tissue to protect against CRC ...... 108

Figure 5. 1: Catabolic pathways proposed for quercetin degradation ...... 114

Figure 5. 2: Quercetin degradation by the human gut bacterial library ...... 116

Figure 5. 3: Growth pattern of B. glycinifermentans (A) and F. plautii (B) in 7N minimal media ...... 118

Figure 5. 4: Quercetin degradation pattern of different bacterial species ...... 119

Figure 5. 5: Detection of metabolites generated by B. glycinifermentans (A) and F. plautii

(B) following quercetin degradation ...... 121

Figure 5. 6: Quercetin degrading enzyme as determined in the culture supernatant and cell lysates of B. glycinifermentans (A) and F. plautii (B) ...... 123

Figure 5. 7: Effect of DOPAC on SLC5A8-PLVX and HCT-116 colony formation .... 124

Figure 5. 8: Clonogenic assay using HCT-116 cells treated with spent bacterial culture supernatant ...... 126

Figure 5. 9: Clonogenic assay using SLC5A8-pLVX cells treated with spent bacterial culture supernatant ...... 128

Figure 6. 1: Metabolite hypothesis: Model depicting convergence of the pathways generating HBAs from parent compounds through host/microbial enzymes ...... 140 xx

LIST OF TABLES

Table 3. 1: Free energy binding values and hydrogen bond lengths for the interaction of

2,4,6-THBA, phloroglucinol, 3,4-DHBA, 4-HBA and 3,4,5-THBA with CDK1, CDK2 and

CDK4...... 63

Table 4. 1: Free energy binding values and hydrogen bond lengths for the interaction of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA with CDK1, CDK2, CDK4 and CDK6

...... 95

Table 6. 1: Table showing the source of the HBA metabolites, their potential to inhibit

CDKs and their ability to retard cancer cell growth ...... 139

xxi

ABSTRACT

A ROLE FOR THE HYDROXYBENZOIC ACID METABOLITES OF FLAVONOIDS

AND ASPIRIN IN THE PREVENTION OF COLORECTAL CANCER

RANJINI SANKARANARAYANAN

2021

Flavonoids are a group of polyphenolic compounds, with established antioxidant properties, that are present ubiquitously in plants, fruits, and vegetables. Their universal presence in nature is evident by the fact that more than 4000 types of flavonoids have been identified till date. Additionally, they have also become part of the daily diet through consumption of fruits such as berries, grapes, pomegranate, and cherries, and vegetables such as onions, kale, and eggplant. High levels of flavonoids are also consumed through beverages such as tea and red wine. Numerous studies have demonstrated that a diet rich in flavonoids decreases the incidences of colorectal cancer (CRC). Interestingly, the cancer prevention properties of flavonoids are also shared with a structurally unrelated compound

– aspirin. Aspirin is a drug commonly used since 1897 for its anti-inflammatory, anti- pyretic and pain-relieving properties; however, it is synthesized from salicylic acid, which is commonly found in plant sources. In the last two decades, it has been recommended as an anti-thrombotic drug to prevent heart attacks in the general population. The interest in aspirin research piqued following the initial report in 1988, suggesting that regular consumption of aspirin decreases CRC incidences. In view of the increased incidence of cancer worldwide, the reports of the ability of flavonoids and aspirin to decrease cancer was highly significant. This led to extensive research of these compound to elucidate and understand the mechanisms of their cancer preventive actions. However, despite decades xxii of efforts, a consensus has not been reached on their specific modes of action. This research investigation was thus initiated to address this lacuna and to identify novel pathways of cancer prevention by these compounds.

An important investigative line of research is to address the question – why are both aspirin and flavonoids preferentially protecting against CRC as opposed to cancers of the other tissues? Reports in literature suggested that a significant amount of both these compounds are left unabsorbed in the intestine, and therefore we considered the possibility that both flavonoids and aspirin may directly target colorectal tissues to exert their anticancer effects before they are absorbed into circulation. Apart from liver metabolism, reports also exist on the biotransformation of flavonoids and aspirin in the intestinal lumen, by the actions of the resident microflora. Hence, if one were to consider this line of investigation, it was also important to consider the metabolites that are generated from these compounds through microbial degradation in the intestine. Currently, there are minimal investigations carried out to determine the role of these metabolites in the prevention of CRC, and therefore represents an important area for further research. The studies described in this dissertation, therefore, focuses on metabolites that are known to be generated from flavonoids and aspirin, their cellular targets, and their ability to inhibit

CRC cell growth. We hypothesize that metabolites of flavonoids and aspirin may inhibit cancer cell growth through the inhibition of cyclin dependent kinases (CDKs). We focused on CDKs as the primary targets of these metabolites because cancer is marked by the dysregulation of the cell cycle. Since the gut microbiota are the major sources of these metabolites in the intestine, an initial investigation was also carried out to identify the bacterial species capable of degrading the flavonoid quercetin to generate bioactive xxiii metabolites. Owing to experimental constraints, the identification of bacterial species capable of biotransforming aspirin and salicylic acids have not been performed.

Flavonoids metabolism has been demonstrated to generate 3,4-dihydroxybenzoic acid

(3,4-DHBA), 3,4,5-trihydroxybenzoic acid (3,4,5-THBA), 2,4,6-trihydroxybenzoic acid

(2,4,6-THBA), 4-hydroxybenzoic acid (4-HBA), and 3,4-dihydroxyphenyl acetic acid

(DOPAC) to name a few. Among these, some studies exist on the ability of 3,4-DHBA,

3,4,5-THBA and DOPAC to prevent cancer cell proliferation. However, no studies exist on the anti-proliferative ability of 2,4,6-THBA against cancer cells. Chapter 3 focuses on this metabolite with regards to identification of cellular targets, as well as its effect on cancer cell growth. Our studies have demonstrated that 2,4,6-THBA inhibited CDK enzyme activity and cancer cell proliferation in vitro. 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4, and in silico studies identified key amino acids involved in these interactions. Interestingly, the structurally related compounds 3,4-DHBA and 3,4,5-

THBA did not inhibit CDK enzyme activity. We also showed that cellular uptake of 2,4,6-

THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter

(MCT), which is often silenced either through mutations or hypermethylations during tumor development. Consistent with this, in cells expressing the functional SLC5A8, significant uptake of 2,4,6-THBA was observed. Additionally, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation. We also observed that 3,4-DHBA and 3,4,5-THBA were not taken up by the cancer cells, however, they were very effective in inhibiting cancer cell growth independent of SLC5A8 expression, suggesting that their targets are most likely extracellular. These findings, for the first time, demonstrated that the flavonoid metabolite xxiv

2,4,6-THBA may exert its anti-cancer effects through a CDK- and SLC5A8-dependent pathway, while 3,4-DHBA and 3,4,5-DHBA may exert its anti-cancer effects through a

CDK- and SLC5A8-independent pathway.

Aspirin metabolism has been shown to generate 2,3-dihydroxybenzoic acid (2,3-

DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Chapter 4 examines the ability of these metabolites to inhibit CDKs and cancer cell growth. We demonstrated that

2,3‑DHBA and 2,5‑DHBA inhibits CDK1, CDK2, CDK4 and CDK6 enzyme activity although the concentrations required significantly varied among different CDK members.

In silico studies were performed to determine the potential sites of interactions of

2,3‑DHBA and 2,5‑DHBA with CDKs. Colony formation assays also showed that

2,5‑DHBA was highly effective in inhibiting clonal formation in HCT‑116 and HT‑29

CRC cell lines and in the MDA‑MB‑231 breast cancer cell line. In contrast, 2,3‑DHBA was effective only in MDA‑MB‑231 cells. Both aspirin and salicylic acid failed to inhibit all four CDKs and colony formation in these experiments. Based on the present results, it is suggested that 2,3‑DHBA and 2,5‑DHBA may contribute to the chemopreventive properties of aspirin, possibly through the inhibition of CDKs.

Having demonstrated the ability of flavonoid and aspirin metabolites to inhibit CDKs and cancer cell growth, we attempted to identify the bacterial species involved in its biotransformation. In chapter 5, we screened 91 human gut bacterial isolates for their ability to biotransform the flavonoid quercetin into different metabolites under anaerobic conditions, mimicking the human gut environment. We demonstrated that of the 65 strains screened, 6 were able to degrade quercetin including Bacillus glycinifermentans,

Flavonifractor plautii, Bacteroides eggerthi, Olsonella scatoligenes, Eubacterium eligens, xxv and Lactobacillus spp. Additional studies showed that B. glycinifermentans could generate

2,4,6-THBA and 3,4-DHBA from quercetin, while F. plautii generated DOPAC. In addition to the differences in the metabolites produced, the kinetics of quercetin degradation was also different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Investigations into the bacterial species capable of degrading aspirin was not carried out owing to difficulties with the detection of the metabolites (2,3-DHBA and 2,5-DHBA) through HPLC.

Summary

The results presented in this dissertation has demonstrated that 2,4,6-THBA generated from flavonoid biotransformation, and 2,3-DHBA and 2,5-DHBA generated from aspirin biotransformation are capable of inhibiting CDK enzyme activity and cancer cell growth.

One common property among these metabolites is that all are derivatives of hydroxybenzoic acids (HBAs). The results obtained from this study suggested that HBAs may be common mediators of cancer prevention by both flavonoids and aspirin.

Interestingly, these HBAs are also abundantly present in fruits and vegetables as secondary metabolites produced through the shikimate pathway. Based on the findings obtained from these studies and previous reports from literature, we propose a novel hypothesis – “the metabolite hypothesis”, to explain the cancer preventive actions of flavonoids, aspirin, and a diet rich in HBAs. The metabolite hypothesis proposes that the HBAs produced through microbial degradation of flavonoids and aspirin or those consumed through the diet may be common mediators of CRC prevention.

xxvi

Significance

The studies described in this dissertation has identified a novel mechanism of cancer prevention by flavonoids and aspirin through the generation of HBAs. This pathway is paradoxically different from any of the previously proposed pathways of cancer prevetion by other investigators. Importantly, the discovery of 2,4,6-THBA, 2,5-DHBA, and 2,3-

DHBA as inhibitors of CDKS has important implications towards the development of a novel class of CDK inhibitors which can be used for the prevention of CRC. Additionally, the identification of the gut bacterial species, B. glycinifermentans and F. plautii, capable of generating 2,4,6-THBA, 3,4-DHBA and DOPAC, suggests the potential use of these bacteria as probiotics for the prevention of CRC and also for the general maintenance of gut health. We believe that these findings are important step forward in decrypting the complex mechanisms involved in cancer prevention. 1

CHAPTER 1: INTRODUCTION

Cancer is an ancient disease or a set of diseases that predates human civilization; its earliest description can be found in the ancient Egyptian text – the Edwin Smith Papyrus, dated 3000 BC [1]. This complex disorder evolves in stages that comprise of numerous transitions within the cell that eventually disrupts the hierarchical organization in multicellular organisms [2]. Hanahan and Weinberg, in 2000, first described the six hallmarks of cancer as self-sufficiency in growth signals, insensitivity to anti-growth signals, limitless replicative potential, evading apoptosis, sustained angiogenesis and tissue invasion and metastasis [3]. Later in 2011, with increased understanding of the disease, two more hallmarks were added to this list– deregulating cellular energetics and avoidance of immune destruction [4]. Our current knowledge of cancer identifies it fundamentally as a genetic disease whose development can be attributed largely to mutations in protooncogenes and tumor suppressor genes. Protooncogenes help in the regulation of cell growth and cellular differentiation. Once mutated, they convert to oncogenes, whose abnormal expression leads to the transition of a normal cell to a cancerous cell due to uncontrolled proliferation. Tumor suppressor genes on the other hand prevent the development of cancer by regulating the over-expression of proto-oncogenes and down- regulating aberrantly expressed genes. The inactivation/silencing of tumor suppressor genes leads to the dysregulation of an important set of genes/proteins that regulate the cell growth and differentiation across various stages [5]. Genetic alterations in cells can be attributed to both internal and external factors. Internal factors involve inherited genetic mutations, epigenetic factors, immune disorders and hormonal imbalance whereas common external causative agents include exposure to ultraviolet (UV) rays, industrial 2 waste, air pollutants, chemical carcinogens, and pathogens like human papilloma virus

(HPV) and Helicobacter pyrolii. In addition, life-style factors such as diet, physical inactivity, smoking, heavy alcohol use, and obesity also contributes to cancer development.

There has also been a link suggested between the age of an individual and increased cancer risk owing to epigenetic changes, particularly those that affect the methylation patterns in tumor suppressor genes and telomerase activity [6].

The process of transformation of a normal cell into a cancer cell (tumorigenesis) is classified into four stages – (a) Initiation (b) Promotion (c) Progression and (d) Metastasis.

Initiation comprises of genetic alterations or mutation of genes that occur spontaneously or due to continuous exposure to carcinogens. These mutations are believed to initially arise in genes affecting biochemical pathways pertaining to cell proliferation, differentiation, and survival. Promotion comprises of many reversible processes where actively proliferating, preneoplastic cells accumulate. Progression marks the final stage of neoplastic transformation, comprising of an increase in tumor size, and further mutations that increase the invasive potential of these cells. The final stage, metastasis, is characterized by the spread of cancer cells to other parts of the body through the circulatory and lymphatic systems (Fig. 1.1) [2, 7]. Cancer has affected millions of people around the world and the American Cancer Society estimated in 2018 that nearly 17 million people would be diagnosed globally with this deadly disorder, and it was also predicted that 1.8 million people would be diagnosed with cancer in 2020 in the United States of America alone; the global cancer burden is expected to reach to 27.5 million new cases by 2040 [8].

Cancer does not discriminate gender and affects men and women of all age groups equally.

It can also arise in any part/organ in the body, however, the three major organs affected by 3 cancer include the lungs, breast, and colon [9]. The work presented in this thesis will focus on colorectal cancer research.

Figure 1. 1: Stages of cancer

Carcinogenesis can be divided into 4 stages – (a) Initiation where a single cell gets genetically altered either spontaneously or due to exposure to carcinogenic agents; (b)

Promotion where neoplastic cells accumulate, and genetic instability increases; (c)

Progression is marked by increased tumor sized and invasive potential of the cells; and (d)

Metastasis that involves the spread of the cancer cells. Cancer prevention is possible in the initiation and promotion stages of carcinogenesis.

1.1 Colorectal cancer (CRC)

Colorectal cancer (CRC) is the second most common cause of cancer related deaths

[10, 11] and the third most common cancer diagnosis [12] in the United States of America.

Every year in the United States, around 150,000 cases of CRC is predicted and 1/3rd of these patients die from this disease. CRC usually begins as a benign adenomatous polyp that develops into an advanced form of adenoma with high-grade dysplasia, which 4 eventually leads to the development of invasive cancer [7]. Formation of benign precancerous polyps that is characterized by the aggregation of abnormally dividing cells in the intestinal lumen marks the initiation of CRC. Over time, these polyps gain numerous mutations and acquire the ability to invade the bowel wall and eventually spread to the adjacent lymph nodes, and distant metastatic sites. The risk of a polyp developing into CRC increases with increasing size of the polyps owing to increased accumulation of mutations and other genetic changes along with epigenetic modifications. As genetic damage increases over time, features of high-grade dysplasia start to present, and if left untreated, the polyps will develop the ability to invade adjacent tissues and grow beyond the confinement of the colon and rectum (Fig. 1.1). Stages 1 and 2 of CRC are usually operable, through excision of the region containing the tumor(s), while stage 3 has a 73% survival rate through surgery coupled with adjuvant chemotherapy. Stage 4 CRC is normally incurable, though life-expectancy can be improved with chemotherapy [7].

1.1.1 Tumor suppressor genes and protooncogenes as drivers of CRC development

Tumor suppressor genes (TSGs) and protooncogenes, following genetic alterations, are characterized through loss-of-function and gain-of-function effects respectively. These genetic alterations influence key biochemical pathways involved in growth and development, contributing to the malignant nature of cancers. More specific to CRC is the chromosomal instability that results in changes in the chromosomal copy number and structure. The most commonly encountered chromosomal instability in CRC include the physical loss of wild type tumor suppressor genes like Adenomatous polyposis coli (APC),

P53 and SMAD4 [13, 14]. CRCs are also characterized by the over-expression of oncogenes including RAS, BRAF and Phosphatidyl inositol 3 Kinase (PI3K) [15, 16]. Other 5 genetic factors that result in the formation of CRC include aberrant regulation of prostaglandin signaling, and over-expression of epidermal growth factor (EGF) receptors and vascular endothelial growth factor (VEGF) receptors in the diseased state [17-19].

Recent studies have also indicated a role for epigenetic regulation including global hypo- methylation, allele specific hypermethylation, miRNA based genetic regulation and histone modifications in the pathogenesis of CRC [20, 21]. Additionally, mutations in

DNA-repair genes like MLH1 and MLH2 have also been identified to contribute to CRC development [22]. All these factors individually or in combination lead to the development of CRC, and some of the key players in CRC progression are detailed below.

1.1.1.1 APC and development of CRC

Wnt signaling is characterized by the binding of β-catenin (an onco-protein) to the nuclear transcription factors TCF/LEF, resulting in the expression of growth promoting genes like c-myc, cyclin D1, matrix metalloproteinase (MMP)-7, and VEGF. Wnt- signaling is constitutively activated in progenitor cells of the colorectal epithelium, whereas in mature/maturing cells this pathway is inactivated by the expression of APC.

APC acts by binding to the excess β-catenin in the cytoplasm, leading to the subsequent ubiquitination and proteolysis of this onco-protein. Initiation of CRC development is marked by aberrant activation of the Wnt-signaling pathway that is characterized by the mutation of the APC gene, rendering it non-functional. This mutation in the TSG tips the balance to favor uncontrolled proliferation and differentiation of the colonic epithelia [13].

1.1.1.2 Tumor protein P53 (P53) and development of CRC:

The P53 TSG regulates the expression of nearly 150 genes in human cells.

Interestingly, it is mutated in nearly 50% of all cancers, and its inactivation is the second 6 key genetic step in CRC development. The mutations include a combination of missense mutations leading to protein inactivation, and a 17p chromosomal deletion that eliminates the second P53 allele. In normal cells, P53 is considered as the guardian of the genome owing to its ability to mediate cell cycle arrest by inducing the CDK-inhibitory protein p21Cip1 upon exposure of the cells to genotoxic agents. Arresting cell cycle permits the cells to repair the damaged DNA and prevents accumulation of further mutations. P53 is also capable of inducing few DNA repair proteins like p48 and P53R2. In cells that contain extensive DNA damage, P53 can induce pro-apoptotic proteins like Bax, resulting in cell apoptosis. It has been reported that inactivation of P53 negatively affects its tumor suppressor functions, and often coincides with the transition of an adenoma to invasive carcinoma [23, 24].

1.1.1.3 SMAD4 and development of CRC

SMAD4, SMAD2 and SMAD3 are transcription factors that mediates the transforming growth factor-β (TGF-β) signaling pathway. Once induced by the TGF-β receptor, the SMAD2/3-SMAD4 complex translocates to the nucleus. In the nucleus, this heterodimer is responsible for the transcription of numerous proteins involved in cell cycle arrest and apoptosis. These transcription factors are implicated in the expression of CDK inhibitors like p15, p21Cip1 and p27Kip1, and are also responsible for the expression of pro- apoptotic proteins like TIEG, DAPK, GADD45β and Bim, thus contributing to the regulation of cell growth. Mutations and deletions in SMAD proteins have been shown to inactivate the TGF-β signaling pathways, contributing to CRC. This mutational inactivation has been identified as the third step in the progression of CRC and is associated with the transition from adenoma to high-grade dysplasia or carcinoma [25, 26]. 7

1.1.1.4 RAS and BRAF and development of CRC

The protooncogenes RAS and BRAF are commonly mutated in CRC patients.

In normal cells, following growth factor stimulation, GTPase activity is elevated through

KRAS leading to the constitutive activation of the serine threonine kinase BRAF. BRAF activation leads to the downstream stimulation of the Mitogen Activated Protein Kinase

(MAPK) pathway, leading to cellular proliferation. In normal cells, this process is tightly regulated whereas in cancer cells it is dysregulated due to mutations in RAS and BRAF.

Mutational activation of RAS and BRAF have been shown to occur in 37% and 17% of

CRC cases respectively, which makes these oncogenes important drivers of cancer cell proliferation. It has been reported that BRAF mutations are detectable in small polyps, hyperplastic polyps, serrated adenomas and proximal colon cancers as compared to RAS mutations [27, 28].

1.1.1.5 PI3K signaling and development of CRC

Phosphatidylinositol 3-kinase (PI3K) is an important serine threonine kinase that feeds into the p42-44 MAPK pathway in cells stimulated with growth factors. Its temporal activation is critical for cell proliferation. PI3K is a heterodimer that consists of a regulatory and catalytic subunit – PI3KR and PI3KC respectively. Nearly 1/3rd of CRC patients have mutations in the catalytic subunit that leads to the constitutive activation of the enzyme. This in turn results in uncontrolled proliferation, resistance to apoptosis, angiogenesis and metastasis. Additionally, it is also observed that in some CRC patients

Phosphatase and TENsin homolog (PTEN), a protein involved in PI3K regulation has been genetically altered either through mutations or deletions, leading to the continuous 8 stimulation of this cascade. Interestingly, abnormal activation of PI3K signaling has also been observed in some CRC patients due to the amplification of the insulin receptor substrate 2 (IRS2) gene that is upstream of PI3K along with co-amplification of Akt and

PAK4 that are downstream mediators of PI3K signaling [29, 30].

1.2 Cell cycle regulation and cancer development

Cell cycle refers to the process involving the duplication of cellular contents followed by cell division. One of the hallmarks of cancer is the dysregulation of the cell cycle leading to uncontrolled cell proliferation [3, 4]. In the human body, cells will not divide unless they receive a prompt, in the form of a growth signal, from their environment to proliferate.

These signals result in a complex growth cycle, normally referred to as the cell cycle, that consists of four distinct stages namely G1, S, G2 and M phases, the end of which is marked by one parent cell dividing into two daughter cells. When cells are in the resting phase

(quiescent phase) and do not divide, it is known as the G0 phase. Each stage of the cell cycle is tightly regulated by a set of distinct proteins and check points to ensure proper functioning of the system. The G1 phase is the gap phase between the M phase and the S phase of the cell cycle, which is marked by doubling in cell size. The restriction check point at the end of the G1 phase ensures that the environment is favorable for proceeding into the S-phase. The S-phase or the synthesis phase involves the duplication of the genetic material and following histone synthesis the cells move into the G2 phase. In the G2-phase, protein synthesis continues (importantly α- and β-tubulins), microtubules are assembled and any damage to DNA is rectified and the cells enter mitotic or M-phase. The G2/M checkpoint blocks the entry of cells into the M phase if DNA replication is incomplete. In the M phase, the cells finally divide into two daughter cells. The metaphase-anaphase 9 transition checkpoint, before the anaphase of the mitotic cycle, blocks the progression of the anaphase if the chromatids are improperly aligned on the mitotic spindle in the middle of the cell. Progression of the cell through these stages lead to segregation of the duplicated chromatids followed by cytokinesis, resulting in the production of two daughter cells [31,

32].

The cell cycle is thus a tightly regulated system, and the progression of this system is controlled by a subfamily of cyclin dependent kinases (CDKs) that depend on cyclins for their activation. Each type of cyclin pairs with a specific CDK or a set of CDKs. The D type cyclins bind to CDK4/CDK6 and regulates the entry and progression of the cell through the G1 phase of the cell cycle. The E-type cyclins and the A-type cyclins bind to

CDK2 and are involved in part of G1 phase S, and G2 phases. The B-type cyclin binds to

CDK1 and is involved in the M phase of the cell cycle [32].

The activation of CDKs occurs when they bind to their partner cyclins. As the name suggests, cyclin levels fluctuate throughout the various stages of the cell cycle, resulting in the periodic activation of CDKs at the necessary phases. CDK levels on the other hand are constant throughout the entire process. CDKs 1, 2, 4 and 6 are directly involved in the cell cycle, whereas CDK7 contributes indirectly by acting as a CDK activating kinase (CAK).

Structurally, CDKs are bilobed proteins containing a large flexible loop known as the T- loop or the activation loop that rises from the C-terminal lobe to block the active site and prevent ATP binding. Hence, CDK activation requires a myriad of structural alterations to become active [33].

Once the CDK binds to its partner cyclin, it becomes partially activated. This enables the phosphorylation of the CDK by CAK, on the T-loop at a conserved threonine site. This 10 phosphorylation results in complete activation of the CDK and opens the ATP binding pocket of the enzyme. Once active, the enzyme can phosphorylate its substrate (such as condensins and lammins for CDK2 and retinoblastoma protein for CDK4 and CDK6) and bring about the necessary changes for progression of the cell cycle. The activity of these

CDK enzymes are also regulated by Wee1 and Myt1 kinases that inactivate CDKs by phosphorylating Tyr-15 and/or Thr-16 residues in the CDKs. Dephosphorylation of these sites by Cdc25 phosphatases can restore kinase activity. Apart from Wee-1 and Myt-1, a set of CDK inhibitory proteins can bind to either CDK alone or the CDK-cyclin complex and inhibit CDK activity. These inhibitors fall under the INK4 family or Cip/Kip family and includes p15, p16, p18, p19 (INK4) that specifically inactivate CDK4 and CDK6 and p21Cip1, p27Kip1 (Cip/Kip) that can inactivate all CDK-cyclin complexes [34-36]. The complex regulation of the cell cycle is depicted in Figure 1.2.

11

Figure 1. 2: The Cell cycle

The cell cycle is a tightly regulated process in normal cells. The different phases of the cell cycle (G1, S, G2 and M) are indicated along with their checkpoints (restriction checkpoint, G2/M checkpoint and Metaphase-Anaphase transition checkpoint). The sites of actions of CDKs and their binding partners cyclins in each stage of the cell cycle are also indicated.

1.2.1 Dysregulation of the cell cycle in cancer:

In cancer cells, the process of cell cycle is highly dysregulated due to fundamental alterations in the genetic control of cell division. This results in uncontrolled cell proliferation, making it an important hallmark of cancer [3]. As mentioned previously, genetic alterations occur mainly in protooncogenes and TSGs. While mutations in 12 protooncogenes involved in cell cycle regulation (such as EGFR, Src Kinase) promote cell growth, mutations in TSGs (such as pRB and P53) result in the loss of their ability to inhibit cell growth, both leading to uncontrolled cell division. Importantly, genetic mutations or overexpression of proteins have also been observed in other genes that regulate the cell cycle, contributing to their growth promoting effects. This includes mutations in numerous

CDKs, cyclins, CAKs, CDK substrates, CDK inhibitors (CKI), and checkpoint proteins.

An account of such dysregulated proteins are detailed below:

1.2.2 CDKs

CDKs 1 and 2 are reported to be overexpressed in colon adenomas, and focal carcinomas in adenomatous tissues [37]. Mutations in CDKs 4 and 6 or their amplification have been observed in numerous cancers [38]. A study with neuroblastoma cell line reported that mutations in CDK6 caused a loss of CKI binding, thereby bypassing the cell cycle block [39].

1.2.3 Cyclins

D-type cyclins are implicated in the regulation of retinoblastoma protein (Rb). In normal cells, biding of cyclin D to CDK4/6 leads to activation of kinase that phosphorylates the protein retinoblastoma (Rb) in the Rb-E2F complex. Phosphorylation of Rb results in the release E2F from this complex. Free E2F, a transcription factor, then helps in the progression of the cell cycle through G1 phase by upregulating cyclin A1, Cyclin E and

CDK2. Aberrant cyclin D1, D2 and D3 expression has been reported in various human cancers. Of these, cyclin D1 mutations are more prevalent in cancers of the breast, esophagus, bladder, lung and also in squamous cell carcinomas [40]. Cyclins E was reported to be over-expressed in breast and colon cancers and also in acute lymphoblastic 13

(ALL) and acute myeloid leukemias (AML). Additionally, cyclins A and E have also been found to be overexpressed in lung carcinoma [37].

1.2.4 CDK activating enzymes

Cdc25B is a CDK phosphatase that activates CDK by dephosphorylating the CDK at a site phosphorylated by Wee1 kinase. Over expression of this phosphatase thus leads to constitutive activation of CDKs, resulting in unrestricted cell proliferation. Over- expression of Cdc25B has been reported in nearly 32% of primary breast cancers. c-myc, an oncogene associated with numerous human cancers has also been shown to activate transcription of Cdc25A and Cdc25B. Interestingly, the activation and deregulation of

Cdc25 has also been demonstrated through Raf kinase that is a downstream product of the

RAS protooncogene which is often mutated in cancer [41, 42].

1.2.5 CKIs

p16, p21Cip1 and p27Kip1 proteins are established CKIs that act by clamping on to either CDKs or CDK-cyclin complexes. The p16 gene is genetically altered in many human tumors with common alterations including deletions, point mutations and hyper- methylations. Deletions of p16 gene has been found in 50% of gliomas and mesotheliomas,

40-60% of nasopharyngeal, pancreatic and biliary tract tumors and 20-30% of ALLs [40,

43]. As p16 targets CDK4/6, inhibition of this protein leads to unhindered progression through G1 phase of the cell cycle. Loss of p27Kip1 expression has been observed in colorectal, lung, breast and bladder cancers [37]. Genetic alterations have also been identified in p21Cip1 protein in some leukemia patients [44, 45].

14

1.2.6 CDK Substrates

Rb which is a substrate for CDK4/6 is often mutated in human retinoblastoma and lung cancers [46, 47]. Common genetic alterations of this protein include deletions and missense mutations leading a truncated or non-functional form of Rb. Additionally, Rb is also inactivated in some cells through HPV encoded E7 protein, adenovirus encoded E1A protein and Simian virus encoded large T antigen. Irrespective of the mechanism, loss of function of Rb would lead to unrestrained cell cycle progression [48].

1.2.7 Checkpoint proteins

As mentioned above, the TSG P53 is mutated in nearly 50% of all cancers. Point and missense mutations are frequently observed, leading to changes in the conformation and inactivation of this protein. Interestingly, HPV encoded E6 protein and adenovirus encoded E1B-55K protein can also alter or block P53 function [24, 49].

1.3 CDK inhibitors for cancer treatment

Numerous reports have documented the discovery of CDK inhibitors over the past two decades, owing to the importance of CDKs in cell cycle progression and its dysregulation in cancers. The very first generation of CDK inhibitory molecules developed were non- specific in their actions, targeting all CDKs. Hence, they were referred to as pan-CDK inhibitors. Examples of this class included flavopiridol, olomucine, and roscovitine [50].

Among these, flavopiridol (a semi-synthethic flavonoid) is known to inhibit CDKs 1, 2, 4, and 6 with IC50 < 200 nM. This compound was capable of inducing cell cycle arrest in G1 and G2 phases and certain studies also showed that it induced cytotoxic responses through inhibition of CDKs 7 and 9 [51]. Other studies have also shown flavopiridol has diverse distal cellular effects including transcriptional suppression, apoptosis, autophagy and 15 endoplasmic reticulum stress. While flavopiridol had a substantial in vitro inhibitory effect on CDK activity, it showed considerably diminished activity in vivo against solid tumors.

However, it had potent clinical effects against certain hematological malignancies such as chronic lymphocytic leukemia (CLL) and mantel cell leukemia and was the first CDK inhibitor to enter clinical trials. Roscovitine is a purine based CDK inhibitor that was also tested in clinic. However, the desired therapeutic efficacy was not observed, and all trials were discontinued [50].

The second generation of CDK inhibitors were designed to be more selective to individual CDKs and focused on improving the potency of tumor inhibition. Among these,

Dinaciclib is the most extensively studied CDK inhibitor. It had high inhibitory activity against CDKs 1, 2, 5 and 9 with IC50 values ranging from 1- 4 nM. It also showed inhibitory effect against CDK 4, 6 and 7 with IC50 values ranging from 60-100 nM. Importantly,

Dinaciclib was effective in inhibiting cell cycle in cancer cell lines isolated from various tumor types and it caused regression of solid tumors in mouse models [52, 53]. Compared to flavopiridol, dinaciclib was more effective in tumor suppression. However, it showed poor or no efficacy in clinical trials for the treatment of solid tumors [50]. The potential use of Dinaciclib in hematological malignancies is currently being tested in clinical trials.

1.3.1 Targeted inhibition of CDK4 and CDK6

To increase the specificity and efficacy that was not observed in previous CDK inhibitors, additional inhibitors were developed that targeted unique CDKs. This was important because the first and second generation of CDK inhibitors were broadly specific and therefore failed to meet expectations in clinical trials. The failure of these compounds was attributed to lack of clear understanding of their mechanism of action and low 16 specificity that was compounded by their inability to differentiate between cancerous and normal tissues [50]. As CDK4/6 plays a central role in the first phase of the cell cycle

(G0/G1), efforts were made to identify compounds that targeted these CDKs. The reasoning was that inhibiting CDK4/6 would arrest cell cycle at G0/G1 phase by preventing the phosphorylation of Rb and keeping Rb complexed with E2F. Screening of compounds identified pyrido[2,3-d]pyrimidin-7-one compounds with 2-aminopyridine side chains at

C2 position had high degree of selectivity to CDK4/6. This backbone was utilized to optimize the drug PD-0332991 (Palbociclib), which could selectively arrest the cells at the

G0/G1 phase in cell culture systems and animal models. The high degree of selectivity offered by Palbociclib was due to its unique ability to bind to the specialized ATP-binding pocket of CDK4/6, thereby preventing Rb phosphorylation. This compound is capable of inhibiting CDK4/6 at nanomolar concentrations and is highly selective compared to a range of other protein kinases [54]. The food and drug administration (FDA) approved this drug in the year 2015 for use against metastatic breast cancer. Following this, another CDK4/6 inhibitor was developed – LEE011 (Ribociclib) and this drug has also shown selective inhibition against CDK4/6 in nanomolar concentrations [55]. Ribociclib was approved by

FDA in 2017 for use against advanced metastatic breast cancer. A third inhibitor –

LY2835219 (Abemaciclib) was also developed to specifically target CDK4/6 and was FDA approved for use against advanced and metastatic breast cancer in 2017 [56].

The discovery of Palbociclib, Ribociclib and Abemaciclib has ushered a new era of targeted cancer therapy. While these compounds are in clinical use, they have serious side effects including neutropenia, and extend the lifespan of the patient for only up to two and 17 a half years [57]. This calls for the discovery of additional CDK inhibitory molecules that are safer to use with lesser side-effects.

1.4 Cancer chemoprevention

In the last decade, due to the increase in occurrences of cancer, the field of chemoprevention has come to the forefront as the preferred approach to cancer management. Chemoprevention involves the use of drugs, or other agents to reduce the risk or delay the occurrence or recurrence of cancer. It is classified into primary, secondary and tertiary chemoprevention. Primary prevention involves intervening before the onset of cancer in the general “healthy” population or those at risk for cancer development. This can be achieved through behavioral changes such as dietary intervention, physical exercise, smoking cessation, preventing overexposure to the sun, or regular screening and physical examination, and use of chemical compounds known to reduce the risk of cancer.

Secondary prevention is the intervention before the onset of precancerous processes; this includes screening individuals with premalignant lesions and administration of anti- cancerous agents to prevent cancer progression. Tertiary prevention refers to management of the cancer to slow or stop progression post diagnosis [58, 59].

1.4.1 Drugs as agents of cancer prevention

The FDA approved tamoxifen in 1998 for the prevention of invasive breast cancer based on the data obtained from the breast cancer prevention trial, which showed a 49% reduction in invasive breast cancer in more than 13,000 pre- and post-menopausal women with high risk of developing this disease [60]. Tamoxifen, a selective estrogen receptor modulator (SERM), is a synthetic drug that has been shown to be effective in preventing estrogen receptor positive breast cancers. This drug was found most effective in women 18 with a known genetic predisposition for BRCA1 and BRCA2 mutations, a history of lobular carcinoma in situ (LCIS) or atypical ductal hyperplasia [60]. However, the use of this drug also caused higher incidents of thromboembolic events, and endometrial cancer.

Due to these serious side effects, its use is highly personalized. Raloxifene, another synthetic and second generation SERM, has similar effects in reducing invasive breast cancer in high-risk, post-menopausal women. Interestingly, this drug had lower incidences of thromboembolic events, and endometrial cancers when compared to tamoxifen [61].

Aspirin and other non-steroidal anti-inflammatory drugs (NSAID) have shown promising anti-cancer effects upon regular consumption for 5 or more years. Aspirin’s efficacy to reduce CRC is reported to be between 20-40%, and the evidence for this effect comes from multiple epidemiological and clinical studies which showed that its intake for

5 or more years reduces the risk associated with colorectal adenomas and carcinomas [62-

65]. This is also supported by animal studies where aspirin has been shown to decrease chemically induced carcinogenesis in colorectal tissues [66, 67]. These observations and evidences prompted the United States Preventive Services Task Force (USPSTF), to recommend “initiating low dose aspirin use for the primary prevention of cardiovascular disease and colorectal cancer in adults aged 50-59 years” in 2016 [68]. In view of these compelling evidences, numerous clinical trials have also been launched to address its efficacy against CRC [63, 69, 70]. Though aspirin has been recommended for the primary prevention of CRC by the USPSTF, its role in secondary and tertiary prevention has not been clearly established. Celecoxib, another NSAID that selectively inhibits cyclooxygenase-2 (COX-2) has also been approved by the FDA for the prevention of adenomatous polyps in patients with familial adenomatous polyposis (FAP), which is 19 associated with the development of colon polyps and a 100% risk for CRC [71]. While aspirin is recommended by the USPSTF for the prevention of CRC, celecoxib has been approved only for FAP patients and not the general public by the FDA.

HPV is one of the common causes of uterine cancer, and the vaccines Gardasil and

Cervarix are approved by the FDA as a preventive measure against cervical cancer, pre- cancerous or dysplastic cervical and vaginal lesions, and genital warts associated with HPV

[72]. As mentioned previously, HPV infection in uterine epithelial cells cause inactivation of Rb and P53 by viral encoded E7 and E6 proteins respectively, leading to uterine cancer.

These vaccines, however, are not approved for the treatment of cervical cancers.

1.4.2 Phytochemicals as agents of cancer prevention

Vitamin A was the first phytochemical compound discovered (in 1976) to possess cancer chemopreventive properties [73]. Since then, numerous other phytochemicals with chemopreventive properties have been identified and these include fibers, carotenoids such as lycopene, other vitamins (C, D, and E), and folate [74]. In addition, studies carried out in the past two decades have demonstrated that phytochemicals such as flavonoids, isothiocyanates, glucosinolates, and saponins have anti-tumor properties [75, 76]. The most prominent among these are the polyphenolic flavonoid compounds present in green and black tea, fruits, and vegetables such as berries, onions, kale, and grapes to name a few

[77]. Isothiocyanates present in cruciferous vegetables are another class of compounds that are extensively studied for its anti-cancer properties [78].

The focus of this thesis is to delineate the molecular mechanisms by which flavonoids and the common household drug aspirin prevent the occurrence of CRC. An 20 account of flavonoid’s and aspirin’s anti-cancer properties and known pathways leading to cancer prevention are detailed below.

1.5 Flavonoids in cancer prevention

Flavonoids are plant secondary metabolites found largely distributed among fruits and vegetables. They are a major component of the human diet and recent studies have shown that these compounds could potentially prevent the occurrences of numerous cancers including those of the colon, breast, and pancreas. The backbone of flavonoids comprises of an aromatic A-ring bound to a heterocyclic C-ring, which in turn is attached to a third aromatic B-ring through a carbon-carbon bond (Fig 1.3). More than 4000 types of flavonoids exist in nature and they are broadly categorized as flavones, flavonols, flavan-

3-ols (catechins), isoflavones, flavanones, anthocyanins and chalcones depending on the number and positions of the different functional groups appended or attached to this backbone (Fig. 1.3). Commonly studied flavonoids include Epigallocatechin gallate

(EGCG), Catechin, Quercetin, Anthocyanins, Genistein, and Kaempferol [79-83]. 21

Figure 1. 3: Basic backbone (center) and different classes of flavonoids

1.5.1 Flavonoid effects on signal transduction

Clinical and epidemiological studies have largely focused on the ability of flavan-

3-ols to prevent cancer. Most flavonoids are predicted to prevent the occurrences of cancer through various mechanisms including antioxidant activity, enzyme/receptor inhibition properties, regulation of apoptosis and the modification of signal transduction pathways.

EGCG is one of the most well studied flavonoids for anti-cancer effects both in vitro and in in vivo tumor models. Its interaction with laminin receptor in the extracellular matrix and insulin like growth factor receptors in the plasma membrane have been implicated in its role in cancer prevention [84]. It was also demonstrated in human immortalized cervical cells and cervical tumor cells that ECGC inhibits EGF dependent activation of EGFR, and subsequent activation of p42/p44 MAPKs, whose activation favors cell proliferation. The 22 reduction in p42/p44 MAPK was associated with reduced phosphorylation of downstream substrates such as p90RSK, FKHR, and Bad as well as increased expression of p21Cip1 and p27Kip1. This resulted in cell cycle arrest at G0/G1 phases. Importantly, sustained exposure of cells to EGCG caused cellular apoptosis [85]. In other studies, using LNCaP (androgen sensitive) and DU145 (androgen insensitive) prostate cancer cells, EGCG caused a significant dose and time dependent upregulation of p21Cip1, p27Kip1, p16 and p18 CKIs and downregulation of cyclin D1, cyclin E, CDK2, CDK4 and CDK6 [86]. Another study demonstrated the ability of EGCG to bind to several target proteins including PI3K, Ras-

GTPase activating protein (GAP) SH-domain-binding protein-1, Bcl/xl and Bcl-2, vimentin and Fyn, resulting in inhibitory activity against various breast cancer cell lines and animal models [87].

Another well studied flavonoid compound known for its anti-cancer effects is

Quercetin. It was shown to trigger DNA fragmentation, cleave poly (ADP-ribose) polymerase, up-regulate Bax and induce post-translational modification of Bcl-2 in human leukemia cell lines [88, 89]. It has also been recently identified that quercetin enhanced

TRAIL-induced apoptosis by causing the redistribution of Death receptor-4 (DR4) and

DR5 into lipid rafts, demonstrating their synergistic effects on cancer cell apoptosis [90].

Quercetin is also suggested to interact with the androgen receptor LNCaP and/or LAPC-4 to act against prostate cancer [91]. Additionally, it was also shown to be effective against azoxymethanol induce colonic neoplasia in mice models [92] and also reduced tumor volume in mice bearing MCF-7 and CT-26 tumors [93]. 23

There are numerous reports on the effects of flavonols on signal transduction pathways that include effects on cell cycle regulation, apoptosis, pro-inflammatory protein induction, and angiogenesis. These effects have been well documented in literature [94].

1.5.2 Antioxidant effects of flavonoids

The antioxidant effects of flavonoids are believed to occur due to the presence of multiple phenol groups in the ring structure that can scavenge reactive oxygen species

(ROS) including superoxides, singlet oxygen, peroxides and hydroxyl radicals. The antioxidant properties of anthocyanins have been demonstrated in various cell culture systems that include colonic, endothelial, leukemic, breast and liver cancer cells. These studies demonstrated the ability of flavonoids to directly scavenge reactive oxygen species, increase the oxygen-radical absorbing capacity of cells, reduce the formation of oxidative adducts in DNA, decrease lipid peroxidation, stimulate the expression of Phase 2 enzymes, inhibit carcinogen induced mutagenesis and reduce cell proliferation [95, 96].

1.6 Aspirin as a chemopreventive agent

As previously mentioned, aspirin and other NSAIDs have shown promising anti-cancer effects upon regular consumption for 5 or more years. The first report linking aspirin use and reduction in CRC was published in 1988 by Kune et al. that showed a statistically significant reduction in cases taking aspirin-containing medication in both men and women

[97]. An intriguing aspect of aspirin against CRC is its dose independent effect; it is reported that low-dose aspirin (75-81mg) is as effective in preventing CRC as higher doses

(≥ 325mg) [98]. Multiple mechanisms in cells have been proposed for aspirin’s anticancer effects and this includes inhibition of enzymes, modulation of signaling pathways and 24 transcription factors, and post translational modifications of proteins, although a consensus has not been reached till date.

1.6.1 Pharmacological effects of Aspirin

Following oral consumption, aspirin is absorbed mainly in the stomach and upper small intestine [99]; in addition, lymphatic uptake of aspirin has also been recently reported

[100]. Aspirin concentration maximally achieved in the plasma varies depending on the aspirin dose with anti-platelet (75 mg/day), analgesic (325-600 mg/ 4-6h) and anti- inflammatory (1.2 g/4-6h) doses reaching 7.31 μM, 25-80 μM, and 144 μM respectively

[69]. The half-life of aspirin is ~20 min. and it undergoes hydrolysis by intestinal, plasma and liver esterases to generate salicylic acid [69, 99]. The concentration of salicylic acid in the plasma has be estimated to be around 15 μM for anti-platelet doses, 500 μM for analgesic doses and 1.5-2.5 mM for anti-inflammatory doses. The half-life of salicylic acid is estimated to be 4-6 h in the plasma [69]. It is reported that the oral bioavailability of aspirin tablets is ~40-50%, while this is significantly reduced (~15 fold) in enteric coated and sustained release tablets [101]. However, some reports have estimated that aspirin’s oral bioavailability is ~68% [102].

Aspirin like all other NSAIDs works through the inhibition of COX (COX-1 and

COX-2) enzymes in the body. COX-1 is the only isoform present in mature platelets and is responsible for platelet aggregation. COX-2 is expressed in many tissues and its expression is induced in tissues during inflammation, wound healing, and neoplasia [98,

103]. The molecular mechanism of aspirin involves its ability to inhibit arachidonic acid metabolism through prostaglandin (PG) H synthase or COX pathway. Aspirin inhibits

COX-1 by acetylating Ser530, and acetylates COX-2 at Ser516, thereby inactivating them 25

[104-106]. Depending on the cell type, inhibition of COX activity affects synthesis of different PGs including PGD2, PGE2 and PGF2α, prostacyclin (PGI2) and thromboxane

A2 (TXA2). TXA2 is the major metabolite that promotes the activation and aggregation of platelets [107]. It is to be noted that aspirin is more selective to COX-1 as compared to

COX-2, with IC50 for COX-1 inhibition observed at 1.7 μM as compared to the >100 μM required for COX-2 inhibition [108, 109].

1.6.2 Mechanisms proposed for aspirin’s chemo-preventive effects.

As mentioned previously, daily use of aspirin has been shown to reduce the risk of

CRC and recurrence of adenomatous polyps. This observation sparked the interest of scientists, physicians and public alike for its potential use as a prophylactic drug against

CRC and studies conducted based on this interest has led to numerous hypotheses on its mechanism of action. Some of the major pathways proposed for its anti-cancer effects are discussed below.

1.6.2.1 Inhibition of COX enzymes: the platelet hypothesis

Cyclooxygenases are a group of enzymes that are involved in the synthesis of prostanoids including PGs and TXA2 from arachidonic acid. While TXA2 is majorly involved in platelet aggregation, PGs such as PGE2 have a role in inflammatory reactions.

Of the two COX enzymes, COX-2 overexpression is associated with cancer progression.

Unlike COX-1 that is constitutively present in all cell types including platelets, COX-2 is normally absent in many cell types. However, as mentioned above, COX-2 expression is elevated under inflammatory and other pathological conditions such as cancer [98, 103].

Owing to the involvement of COX-2 in cancer [110] it is suggested that aspirins’ anti- cancer effects may occur through the inhibition of this enzyme. Additionally, studies 26 conducted using COX-1 and COX-2 deletions have also demonstrated reduced intestinal tumorigenesis [111]. However, a direct inhibition of COX-2 by low-dose aspirin is thought to be unlikely due to the observed low plasma concentration (~7 μM) and a requirement for higher amounts for enzyme inhibition (IC50 278 μM) [109]. Thus, the fact that low-dose aspirin is as effective as higher doses in preventing CRC led to the suggestion that COX-1 inhibition in platelets could indirectly affect COX-2 expression levels in the colorectal tissues through sequential steps [65, 69, 98].

According to this theory (popularly referred to as the platelet hypothesis) the initiating event for tumorigenesis in the colorectal tissues is the aggregation and activation of platelets in the intestinal mucosa upon injury. Persistent activation of platelets, if left unchecked, will result in the local recruitment of immune cells, leading to inflammation.

Aggregation of platelets at these sites release platelet-derived growth factors (PDGF), cytokines (interleukin-1β) and lipid mediators (PGE2 and TXA2) that then promote growth/proliferation of adjacent nucleated cells in the colonic mucosa. It is further hypothesized that these agents may increase the levels of COX-2 (further increasing PGE2 levels) and result in epithelial–mesenchymal transition (EMT) of the intestinal epithelial cells, characteristic of early events in tumorigenesis. EMT activation in turn results in the generation of tumor-initiating cells and triggers tumor cell invasion and metastasis to distant organs [65, 69, 98]. It is to be noted that, PGE2 binding to EP1-4 receptors (G- protein coupled receptors) activates signal transduction pathways that promote adhesive, migratory and invasive behavior of cells during the development and progression of cancer

[18]. Therefore, according to the platelet hypothesis, platelet aggregation and aberrant activation at site of mucosal injury is the driving factor for the genesis of adenomatous 27 polyps and eventual progression to CRC. Aspirin’s chemopreventive effect against CRC is attributed to its ability inhibit COX-1 in platelets, prevent the release of lipid or protein mediators of inflammation and subsequently decrease COX-2 expression in colorectal tissues (Fig. 1.4).

Figure 1.4: Figure legend continued in next page

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Figure 1. 4: The Platelet hypothesis

According to this model, platelet activation and aggregation at the site of intestinal mucosal injury is fundamental to tumorigenesis in the colorectal tissues.

Activation of platelets can lead to the release of lipid mediators (e.g., PGE2) and growth factors (e.g., PDGF) from platelets that in turn may promote stromal cell growth and induce COX-2 expression in intestinal cells of the colonic mucosa. Such events can further lead to increased PGE2 production resulting in hyperplasia, EMT and transformation to a neoplastic phenotype [65, 98]. It is hypothesized that central to the prevention of CRC by aspirin is its ability to prevent platelet aggregation through inhibition of COX-1 leading to subsequent inhibition of thromboxane A2. Prevention of platelet aggregation blocks the release of lipid mediators and growth factors from platelets that induces the expression of COX-2 (in epithelial cells), which is implicated in tumorigenesis. This hypothesis thus states that sequential inhibition of COX-1 in platelets followed by inhibition of COX-2 expression in adjacent nucleated cells of the colorectal mucosa are important steps in aspirin’s cancer preventive actions.

29

1.6.2.2 Inhibition of Nuclear Factor (NF)-κB signaling

NF-κB is a transcription factor that is involved in many immune and inflammatory responses [112]. NF-κB is present as a heterodimer along with IκB in the cytoplasm. Upon receiving appropriate signals, IκB is phosphorylated by IκB Kinase

(IKK) that eventually leads to the ubiquitin mediated degradation of IκB. The free NF-κB then translocates to the nucleus where it acts as a transcription factor for many cellular processes including those that control cellular growth. Aspirin and salicylic acid have been shown to inhibit IKK, through direct interactions, thus preventing the translocation of NF-

κB to the nucleus. As aspirin is also an NSAID, it is additionally believed to prevent inflammation mediated carcinogenesis by affecting this signaling pathway. The effect exerted by aspirin on NF-κB requires concentrations ranging from 0.05-5 mM [113, 114].

1.6.2.3 Activation of AMP-kinase and inhibition of mTOR signaling

Adenosine monophosphate activated protein kinase (AMPK) is a critical cellular energy sensor that gets activated in response to stress. AMPK is involved in processes that synthesize ATP to meet the energy demands of the body. AMPK regulates numerous pathways including that of P53, fatty acid synthase, and mechanistic target of rapamycin (mTOR). As these pathways are crucial for cell survival and metabolism,

AMPK has also been implicated in cancer progression in numerous cancers including CRC

[112]. It has been shown that aspirin, at a concentration of 5 mM is capable of decreasing mTOR signaling by inhibiting the effectors S6K1 and 4E-BP1, that are involved in translation and protein synthesis, eventually leading to cell death. It was also demonstrated that aspirin could alter the AMP:ATP ratios in cells, leading to the activation of AMPK, and subsequent inactivation of mTOR signaling. Aspirin also inhibited mTOR signaling in 30

CRC patients treated with analgesic doses (600 mg/day for one week). In these patients, there was a decreased phosphorylation of S6K1 and S6 effectors, suggesting that their inhibition helps in the regulation of intracellular energy homeostasis and metabolism, contributing to the protective effect of aspirin against CRC [115].

1.6.2.4 Inhibition of Wnt signaling and β-catenin phosphorylation

Wnt-signaling is constitutively activated in progenitor cells of the colorectal epithelium, whereas in mature/maturing cells this pathway is inactivated by the expression of APC. APC acts by binding to the excess β-catenin in the cytoplasm, leading to the subsequent ubiquitination and proteolysis of this onco-protein. As mentioned previously, initiation of CRC development is marked by aberrant activation of the Wnt-signaling pathway that is characterized by the mutation of the APC gene, rendering it non-functional

[13]. This mutation in the tumor suppressor gene tips the balance to favor uncontrolled proliferation and differentiation of the colonic epithelia. It has been demonstrated that aspirin, tested at concentrations between 0.05-5 mM, stimulates the phosphorylation

(through inhibition of protein phosphatase 2A) and subsequent ubiquitination of β-catenin, leading to the inhibition of the Wnt/ β-catenin pathway [116].

1.6.2.5 Downregulation of c-myc, cyclin A2 and CDK2

c-myc is a nuclear transcription factor that controls 15% of the expression of all genes. It regulates multiple cellular functions such as cell proliferation, metabolism, apoptosis, growth and differentiation. It is frequently overexpressed in nearly 20% of all cancers, and this is associated with poor clinical outcome and an aggressive metastatic phenotype. Aspirin/Salicylic acid has been shown to downregulate the levels of c-myc mRNA and protein, between 0.25 and 2.5 mM, in a variety of cancer cells [117], and 31 similar results have been reported in another study [118]. Aspirin and salicylic acid have also been shown to downregulate cell cycle regulatory proteins cyclin A2 and CDK2 through proteasomal mediated pathways at concentrations ranging from 0.5-2.5 mM [119].

It is suggested that this may lead to cell cycle arrest and inhibition of cell proliferation.

1.6.2.6 Induction of polyamine catabolism

Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine synthesis. The transcriptional regulator c-myc activates the transcription of ODC which results in increased levels of mucosal polyamines that is implicated in tumorigenesis.

Aspirin use, between 20 and 100 μM, in Caco-2 cells has been associated with the transcriptional activation of SSAT (spermidine/spermine N1-acetyltransferase), a driver for polyamine catabolism. Reduced levels of polyamines are thus believed to result in reduced tumorigenesis [120].

1.6.2.7 Induction of DNA mismatch repair proteins

DNA mismatch repair is a mechanism that is evolutionarily conserved, to repair mutations that arise during DNA replication or damage. Epigenetic or somatic changes to mismatch repair genes have been associated with CRC development [121]. It was demonstrated that in colon cancer cells, exposure of aspirin between concentrations of 1-

10 mM induced DNA mismatch repair proteins by 2-7-fold, depending on the cell line studied. This in turn was associated with G0/G1 cell cycle arrest and apoptosis. The authors suggested that, this COX-independent pathway may contribute to aspirin’s anticancer effects [22]. 32

1.6.2.8 Acetylation of P53, glucose-6-phosphate dehydrogenase and other proteins

P53 is a tumor suppressor protein that regulates cell proliferation. In normal cells, it is activated through acetylation and phosphorylation following which it translocates to the nucleus and acts as a transcription factor. Nearly 50% of all tumors contain a mutated P53 and such mutations inactivate its tumor suppressor functions leading to enhanced cell proliferation. Aspirin has been shown to directly acetylate both wild-type and mutant P53, at concentrations between 0.05 and 2.5 mM, and this was associated with induction of p21 and Bax, suggestive of a role in restoration of P53 function and tumor suppression [122-124]. Aspirin was also shown to acetylate glucose-6-phosphate dehydrogenase (G6PD; at ≥ 100 μM), an enzyme in the hexose monophosphate shunt pathway and cause decreased enzyme activity. It was suggested that selective inhibition of

G6PD may represent an important mechanism by which aspirin may exert its anticancer effects through inhibition of ribonucleotide synthesis [125, 126]. Aspirin’s ability to decrease G6PD, potentially contributing to prevention of cancer cell growth, is an interesting finding because there are reports that have documented a direct correlation between G6PD deficiency and decreased cancer incidences [127].

Aspirin also appears to acetylate multiple proteins, over a range of concentrations starting from 100 μM, in cells grown in culture as well as in vivo [123, 125, 128, 129]. Two independent reports have documented aspirin’s ability to acetylate multiple proteins which include histones, cytoskeletal and heat-shock proteins, glycolytic and pentose pathway enzymes, proteins involved in translation, proteasomal subunits and mitochondrial proteins. The significance of these post-translational modifications has not been thoroughly investigated [125, 128]. Interestingly, Tatham et al., reported that although exposure of 33 cells to aspirin amplifies the acetylation status of proteins by several orders of magnitude, protein function is not altered as long as deacetylases are not inhibited. They suggested that aspirin-mediated acetylations do not possibly accumulate to levels capable of eliciting biological effects in most cases [129].

1.6.2.9 Other mechanisms

In addition to the pathways mentioned above, aspirin is also known to affect other targets. For example, it is suggested that aspirin (between 1-4 mM) might exert its chemopreventive effect against CRC by normalizing EGFR expression in gastrointestinal precancerous lesions [19]. It is also reported that at concentrations between 0.1-5 mM, aspirin and salicylic acid could inhibit TPA (12-O-tetradecanoylphorbol-13-acetate) induced activator protein 1 activity, preventing neoplastic transformation [130]. Another study showed that aspirin’s ability to inhibit colon cancer cell growth (between 5-10 mM) may involve downregulation of specificity protein transcription factors [131]. Both aspirin and salicylate are also reported to induce apoptosis through caspase activation in B-cell chronic lymphocytic leukemia cells at a concentration of 10 mM [132]. In addition, aspirin has been shown to exert cytotoxic and anti-cancer effects through generation of reactive oxygen species in melanoma cells at concentrations between 0.1-1 mM [133]. A recent study has also demonstrated that low-dose aspirin (100 mg/day for 7 days) causes acetylation of COX-1, inhibiting PGE2 synthesis, in human intestinal mucosal cells leading to decreased levels of S6 kinase, suggesting that this pathway may play a role in the prevention of colorectal carcinogenesis [134]. A compilation of some of these pathways is diagrammatically represented in Fig. 1.5.

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Figure 1. 5: Other suggested pathways for aspirin’s chemopreventive actions

Aspirin/Salicylic has been shown to modulate a variety of signaling pathways which may also contribute to its anticancer effects. It is known to inhibit NF-κB pathway, activate AMPK pathway, inhibit Wnt/β-catenin signaling pathway, cause degradation of c- myc mRNA and proteins, and acetylate P53 leading to induction of p21 and Bax protein expression, to name a few. See text for details; additional pathways described in the text are not depicted in this figure. -P, phosphorylated protein; -Ac, acetylated protein.

1.7 Metabolism of flavonoids

The degradation of flavonoids can occur through changes in physical parameters (like pH) [135], through microbial action in the gut before absorption [136, 137], and through host metabolism in the liver [138]. It is reported that the absorption of flavonoids is 1%– 35

15% in the intestine, and that it is extensively metabolized in the liver through conjugation reactions for subsequent elimination in the body [139-141]. It is also reported that these conjugated intermediates may be returned to the intestine through the bile, where it further undergoes deconjugation, subjecting them to further degradation through microbial metabolism [137, 142]. As mentioned previously, the basic backbone of flavonoids is highly conserved and comprises of an aromatic A-ring bound to a heterocyclic C-ring, which in turn is attached to a second aromatic B-ring (Figure 1.3). The functional groups, their number and position on this backbone will determine their stability and the metabolite(s) they generate [143]. Flavonoids are generally stable under acidic conditions but undergo rapid degradation to simpler phenolic compounds under alkaline conditions

(like in the intestine). Additionally, multiple studies have documented the ability of gut microbes to degrade flavonoids into simpler phenolic acids, many of which are HBAs. The most commonly observed HBAs include, 3,4-dihydroxybenzoic acid (3,4-DHBA; protocatechuic acid), 3,4,5-trihydroxybenzoic acid (3,4,5-THBA; gallic acid), 4- hydroxybenzoic acid (4-HBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), 2,4- dihydroxybenzoic acid (2,4-DHBA) and 2,4,6-trihydroxybenzoic acid (2,4,6-THBA; phloroglucinol carboxylic acid) [135-137, 139, 141, 144, 145]. A schematic of the HBAs generated from flavonoids is shown in Figure 1.6 and 1.7B. Flavonoid degradation also generates phenolic acids like vanillic acid, caffeic acid and 3,4-dihydroxyphenyl acetic acid

(DOPAC) [145-147].

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Figure 1. 6: Postulated degradation pathways of selected flavonoid compounds to generate 2,4,6-THBA, 4-HBA, 3,4-DHBA and 3,4,5-THBA

The parent flavonoid compounds (labelled with roman numerals) undergo autodegradation or microbial degradation to generate unstable intermediates such as chalcones or depsides which then further degrade to more stable phenolic acids such as 4-

HBA, 3,4-DHBA, 3,4,5-THBA and 2,4,6-THBA. The chemical structure of the flavonoid

A-ring (labelled within each flavonoid structure) is conserved and generates 2,4,6-THBA.

The flavonoid B-ring (labelled within each flavonoid structure) can generate 4-HBA, 3,4-

DHBA and 3,4,5-THBA depending on the number of hydroxyl groups. R1, R2 and R3 represent either hydrogen or hydroxyl groups and based on the position and number of –

OH groups in the B-rings flavonoids generate monohydroxy-, dihydroxy- or trihydroxybenzoic acids [136, 141, 148]. 37

1.8 Metabolism of aspirin

The reported half-life of aspirin is about 20 min [149] and its metabolism either through cytochrome P450 (CYP450) catalyzed reactions [150] or direct conjugation of salicylic acid by phase 2 enzymes [151] in the liver has been well documented. Once absorbed, intact aspirin is partially hydrolyzed to salicylic acid (half-life is 4–6 h) by esterases in the blood and liver [152]. Salicylic acid can then be directly excreted (1%–31%) or can be metabolized in a number of different ways for elimination through kidney or bile. It undergoes conjugation with glycine to form salicyluric acid which accounts for 20%–65% of the metabolites generated, whereas ether and ester glucuronides of salicylic acid constitute 1%–42% of metabolites following conjugation with glucuronic acid [153].

Additionally, CYP450 enzymes in the liver can also metabolize salicylic acid to 2,5- dihydroxybenzoic acid (2,5-DHBA; gentisic acid) and 2,3-dihydroxybenzoic acid (2,3-

DHBA; pyrocatechuic acid) that accounts for 1%–8% of the dose. 2,5-DHBA can further undergo conjugation with glycine to form gentisuric acid [150]. Aspirin has also been reported to be metabolized in the gut by the resident microflora. In this regard, Kim et al. demonstrated the importance of human fecal microbiota to degrade aspirin to salicylic acid and hydroxylated salicylic acids [154]. They showed that when rats were administered aspirin along with ampicillin, the bioavailability of aspirin increased when compared to rats administered with aspirin alone. Supporting this observation, a very recent study by

Zhang et al., in 2019, also showed that administration of aspirin to rats following amoxicillin treatment decreased aspirin metabolism in the intestine, as compared to rats treated with aspirin alone [155]. The authors of both studies have suggested an important role for the intestinal microflora in the biotransformation of aspirin before its absorption 38 into circulation. It is also important to emphasize that intestinal epithelial cells also express

CYP450 enzymes [156], although their capability to generate these HBAs is not well studied. A schematic of the HBAs generated from aspirin is shown in Figure 1.7.

Figure 1. 7: Metabolism of aspirin and flavonoids to generate hydroxybenzoic acids

(A) Aspirin metabolism generates 2,3-DHBA and 2,5-DHBA through CYP450 reactions in the liver [157]. DHBAs have also been shown to be generated through microbial metabolism of aspirin/salicylic acid [154]. (B) Flavonoid metabolism generates

2,4,6-THBA, 3,4-DHBA, 3,4,5-THBA, 4-HBA, 2,4-DHBA, 2,6-DHBA through microbial degradation in the intestine [137, 142, 143, 158]. R-R5 represent various functional groups

(example -hydroxy, -ketone, -hydrogen, -methoxy etc.) that are appended/attached to the flavonoid backbone to generate different groups of flavonoids. 39

1.9 Biotransformation of flavonoids and aspirin by the gut microbiota

Following consumption, glycosylated flavonoids can be converted to their aglycone forms by the host intestinal enzymes such as lactase-phlorizin hydrolase following which the aglycone is subjected to further metabolism by the intestinal CYP enzymes [159].

Alternatively, other flavonoid-glycosides that cannot be deconjugated by the intestinal enzymes pass on to the colon where they are biotransformed by the microbial enzymes

[141, 160, 161]. The sugar moieties of these flavonoid-glycones usually serve as the sole carbon and energy source to the gut microbes following preferential fermentation. Owing to the anaerobic nature of the intestine, most of the reactions catalyzed by the gut microbiota rely on reduction and hydrolysis reactions. For flavonoids, these reactions include ring fission, O- and C-glycosylation, demethylation, dehydroxylation, reduction of carbon-carbon double bonds, ester cleavage, isomerization, and decarboxylation, to name a few. Most of these reactions, individually or in combination, lead to the generation of simpler phenolic acids from the A- and B- rings following the cleavage of the C-ring including 2,4,6-THBA, 3,4,5-THBA, 3,4-DHBA, 2,5-DHBA, 2,4-DHBA, 2,6-DHBA, 3-

HBA, 4-HBA, and phloroglucinol [136]. 3,4-DHBA and 3,4,5-THBA are the most abundant phenols generated from B-ring cleavage and they have been extensively studied for their therapeutic potential [144, 162-165]. While the biotransformation of flavonoids has been studied to a certain extent, limited studies exist on the metabolism of aspirin by intestinal microbiota and this has been mentioned on page 37.

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1.10 Rationale for studies on flavonoid and aspirin metabolites as chemopreventive agents and the hypothesis

Eradication of cancers in the world requires a three-pronged approach involving educating the public towards adapting a healthier lifestyle, discovery of drugs to prevent cancer, and discovery of drugs to treat cancer. Through decades of effort, we have achieved only one of these milestones as we have many drugs to treat cancer. However, educating the public to adapt to a healthier lifestyle has only been partially successful, while the efforts to discover drugs to prevent cancer are lacking. Drugs currently recommended to prevent cancer, such as tamoxifen and aspirin, were originally meant for the treatment of other ailments and were repurposed for cancer prevention. The alarming rate at which cancer is diagnosed every year in the world (which range in millions) suggests that it is going to be the leading cause of death, surpassing heart disease. So much has been understood about the risk factors for cancers, molecular mechanisms of cancer initiation and progression and the mechanisms of drugs that are used in the treatment of cancer. It is a genetic disease that arises due to mutations in protooncogenes and tumor suppressor genes and can occur due to sporadic mutations or through hereditary means. Based on scientific evidence, it is argued that cancer is a preventable disease. Thus, aside from the treatment options available for cancer, there is an urgent need to raise public awareness along with discovering chemopreventive agents for the eradication of this deadly disorder.

Synthetic drugs against cancer target different proteins and enzymes within the cells and often have serious side effects and associated toxicity. One of the reasons for this is that the human body has not been exposed to these drugs for their efficient metabolism and elimination, causing collateral damage in normal cells. Therefore, identification of natural 41 products or their metabolites for chemoprevention are more appealing as they would be expected to have less toxicity and side effects. In this context, the demonstration of the ability of flavonoids and aspirin to prevent CRC interested us to pursue this line of investigation.

Studies previously performed by Dr. Dachineni in our lab provided the lead for investigation into the possibility that the metabolites and derivatives of aspirin and salicylic acid might be important contributors for the prevention of tumorigenesis. As a part of his research investigation, he demonstrated the ability of aspirin metabolites 2,3-DHBA and

2,5-DHBA, and salicylic acid derivative 2,4,6-THBA to inhibit CDK enzyme activity in vitro. Interestingly, 2,4,6-THBA is also known to be generated as a metabolite of flavonoid degradation in the intestine by host microbes. However, the previous studies were limited in nature and examined the effect of these compounds superficially on CDK enzyme activity, and a detailed study was not conducted [119, 166]. The unexplored areas include the effect of these metabolites on cancer cell growth and identification of the bacterial species that generate these metabolites from flavonoid degradation. The investigation described in this thesis are therefore an extension of the previous studies on the effect of these natural compounds on CDKs and how they may contribute to cancer prevention through inhibition of cell growth. CDKs are often dysregulated and overexpressed in cancers and therefore are attractive targets for cancer therapy [4]. In this regard, as mentioned earlier, there are only three FDA approved drugs which act as CDK inhibitors

(Palbociclib, Ribociclib, and Abemaciclib). Therefore, further characterization of these compounds as CDK inhibitors for potential use in cancer prevention appeared significant. 42

This thesis contains four sections – one investigating the effect of 2,4,6-THBA on

CDKs 1, 2, 4 and 6 and its growth inhibitory effects in cancer cells, along with other flavonoid metabolites, another investigating the ability of aspirin metabolites 2,3-DHBA and 2,5-DHBA to inhibit these CDKs and cancer cell growth. The third section investigates the ability of human gut bacterial isolates to biotransform flavonoids and the final section describes a novel hypothesis (the metabolite hypothesis), based on the results obtained from this study.

1.10.1 Hypothesis

We hypothesize that flavonoids and aspirin may mediate their anti-cancer effects through their respective hydroxybenzoic acid (HBA) metabolites, known to be generated in the gut by bacterial action. Additionally, we hypothesize that the anti-cancer effects of these metabolites may occur through CDK inhibition.

1.10.2 Approach

The inhibitory effect of the metabolites of flavonoids and aspirin on CDKs were determined using in vitro CDK assays. The potential interactions of these metabolites with

CDKs were determined by molecular docking studies. The growth inhibitory potential of these metabolites was tested using various cancer cell lines in vitro, utilizing cell count and clonogenic assays. Approaches such as qRT-PCR and western blotting was used to measure the levels of CDK inhibitors upon treatment of cells with HBAs. Uptake of metabolites into cells were studied using high performance liquid chromatography

(HPLC). Effects of these metabolites on cell cycle progression was determined by flow cytometry. Identification of bacterial species capable of degrading the flavonoid quercetin 43 was performed by screening 94 human gut bacterial strains grown strictly under anaerobic conditions and the metabolites generated were determined using HPLC.

The investigations carried out in this thesis is the first attempt to understand the role of HBAs, known to be generated from flavonoids and aspirin, on cancer cell growth.

Our goal in this project was to delineate the mechanistic aspects of CDK inhibition by

HBAs, characterize potential transporters involved in the uptake of HBAs into cells, identify other molecular events that lead to inhibition of cancer cell growth, and identify bacterial species involved in the generation of these metabolites.

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CHAPTER 2: MATERIALS AND METHODS

2.1 Materials

100bp DNA ladder [Invitrogen (Carlsbad, CA, USA)] 2,3-DHBA [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] 2,4,6-THBA [Sigma Aldrich (St. Louis, MO, USA)] 2,5-DHBA [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

3,4,5-THBA [Sigma Aldrich (St. Louis, MO, USA)] 3,4-DHBA [Sigma Aldrich (St. Louis, MO, USA)] 32P γ-ATP [MP Biochemicals (Solon, OH, USA)] 32P γ-ATP [PerkinElmer (Waltham, MA, USA)] 4-HBA [Sigma Aldrich (St. Louis, MO, USA)] 6X DNA loading dye [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Acetonitrile [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Agarose [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Aluminum potassium sluphate (AlK(SO4)2 (anhydrous)) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Ammonium acetate [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Ammonium chloride (NH4Cl) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Ammonium Persulfate [Biorad laboratories (Hercules, CA, USA)] Annexin V/7-AAD kit [Beckman Coulter (Miami, FL, USA)] Aspirin [Sigma Aldrich (St. Louis, MO, USA)] β-mercaptoethanol [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Biotin [Sigma Aldrich (St. Louis, MO, USA)]

Boric acid (H3BO3) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Bovine Serum Albumin (BSA) Heat shock fraction [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Bradford’s reagent [Biorad laboratories (Hercules, CA, USA)] Brain Heart Infusion (BHI) Broth [Sigma Aldrich (St. Louis, MO, USA)] 45

Bromophenol blue [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Calcium chloride (CaCl2 (anhydrous)) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Calcium chloride dihydrate (CaCl2.2H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Calcium Pantothenate [Sigma Aldrich (St. Louis, MO, USA)] Chloroform [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Cobalt nitrate hexahydrate (Co(NO3)2.6H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Coomassie stain [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Copper (II) sulphate pentahydrate (CuSO4.5H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Crystal violet [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] D-glucose [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Dimethylsulfoxide (DMSO) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Dithiothretiol (DTT) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] DNase I [Invitrogen (Carlsbad, CA, USA)] Doxycycline [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Ethanol [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Ethyl acetate [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Ethylenediaminetetraacetic acid [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Folic acid [Sigma Aldrich (St. Louis, MO, USA)] Glycerol [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Glycine [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

H1 Histones [EMD Millipore (Billerica, MA, USA)] Hemin [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Histidine [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Immobilon membranes [EMD Millipore (Billerica, MA, USA)]

Iron (II) sulphate heptahydrate (FeSO4.7H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] 46

Isopropyl alcohol [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] L-cystein [Sigma Aldrich (St. Louis, MO, USA)] Magnesium Chloride [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Magnesium chloride hexahydrate (MgCl2.6H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Magnesium sulphate heptahydrate (MgSO4.7H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Manganese sulphate monohydrate (MnSO4.H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Menadione [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Methanol [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Monopotassium phosphate [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] MOPS (3-(N-morpholino) propanesulfonic acid) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] n-butanol [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Nickel chloride hexahydrate (NiCl2.6H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Nicotinic acid [Sigma Aldrich (St. Louis, MO, USA)] p-Aminobenzoic acid [Sigma Aldrich (St. Louis, MO, USA)] Phosphate buffered saline (PBS) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Potassium dihydrogen phosphate (K2HPO4) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Potassium Sulphate (K2SO4) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Protease inhibitor tablets [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Pyridoxine hydrochloride [Sigma Aldrich (St. Louis, MO, USA)] qPCR reagents [Applied Biosystems (Foster City, CA, USA)] Quercetin [Sigma Aldrich (St. Louis, MO, USA)] Resazurine [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Riboflavin [Sigma Aldrich (St. Louis, MO, USA)] RT-PCR reagents [New England Biolabs (Ipswich, MA, USA)] 47

Salicylic acid [Sigma Aldrich (St. Louis, MO, USA)]

Sodium Acetate (CH3COOH) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Sodium bicarbonate (NaHCO3) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Sodium Chloride (NaCl) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Sodium dodecyl sulphate (SDS) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Sodium hydroxide (NaOH) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Sodium molybdate dihydrate (Na2MoO4.2H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Sodium selenite (Na2SeO3) (anhydrous) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Sodium tungstate dihydrate (Na2WO4.2H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Super Signal™ West Pico Chemiluminescent Substrate [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Thiamine [Sigma Aldrich (St. Louis, MO, USA)] Thioctic acid [Sigma Aldrich (St. Louis, MO, USA)] Tricine [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Tris-base [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] TRIzol reagent [Invitrogen (Carlsbad, CA, USA)] Trypan blue [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] trypsin-EDTA [Sigma Aldrich (St. Louis, MO, USA)]

Tween 20 [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] Tween 80 [Sigma Aldrich (St. Louis, MO, USA)] Vitamin B12 [Sigma Aldrich (St. Louis, MO, USA)] Yeast extract [Sigma Aldrich (St. Louis, MO, USA)]

Zinc sulphate heptahydrate (ZnSO4.7H2O) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)] ZORBAX C-18 column [Agilent technologies (Santa Clara, CA, USA)]

48

2.2 Buffers

4X Protein dye: 20% SDS (4 ml); 2M Tris.Cl (pH 6.8); Glycerol (8 ml); Bromophenol blue

(40 mg) and β-mercaptoethanol (200 μl/ml).

10X Running buffer: Tris base (30 g); Glycine (144); SDS (10 g); make up to 1 L with

water.

Lysis buffer: 1M Tris.Cl (pH 7.4) – 2ml; 4M NaCl (3.75 ml); Glycerol (15 ml); Triton X-

100 (1 ml); make up to 100 ml with water.

Protein transfer buffer: Tris base (11.6 g)l Glycine (5.85 g); Methanol (500 ml); make up

to 2 L with water.

Washing buffer: 1M Tris.Cl (pH 7.4) – 20 ml; NaCl (11.5 g); Make up to 2 L with water;

Tween 20 – 1 ml.

2.3 Cell lines, culture media and other reagents

HCT-116, HT-29 and Caco-2 (Human colon cancer cells) from ATCC (Manassas, VA,

USA)

MDA-MB-231 (Human breast cancer cells) from ATCC (Manassas, VA, USA)

MDA-MB-231 cells expressing functional SLC5A8 (SLC5A8-pLVX cell line) was kindly provided by Dr. Puttur Prasad, Medical College of Georgia.

RPMI 1640 media (for both the MDA-MB-231 cell lines) [Thermo Fisher Scientific Inc.,

(Waltham, MA, USA)]

McCoy’s 5A media (for all other cell lines) [Thermo Fisher Scientific Inc., (Waltham, MA,

USA)] 49

Fetal Bovine Serum (FBS) [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

Penicillin-streptomycin antibiotics [Thermo Fisher Scientific Inc., (Waltham, MA, USA)]

2.4 Recombinant proteins and antibodies

anti-p21Cip1, anti-p27Kip1, anti-CDK1, anti-CDK2, anti-CDK4, anti-cyclinA2, anti- cyclinB1, anti-cyclinD1, and anti-β tubulin antibodies were purchased from Cell Signaling

Technology (Danvers, MA, USA); anti-SLC5A8 from Invitrogen (Carlsbad, CA, USA); goat anti-rabbit and goat anti-mouse antibodies were obtained from Bio-Rad (Hercules,

CA, USA). CDK1/Cyclin B1, CDK2/Cyclin A2, CDK4/Cyclin D1, Retinoblastoma (C- term) and kinase buffer were purchased from SignalChem (Richmond, BC, Canada).

2.5 Cell culture

MDA-MB-231 cells were grown in RPMI media while HCT-116 and HT-29 cells were cultured in McCoy’s 5A media, both supplemented with 10% FBS and antibiotics for 24h before treatment with specified compounds for indicated times; all cell lines were maintained at 37 °C and 5% CO2. SLC5A8-pLVX cells were grown in the presence of doxycycline (5μg/ml) for the induction of SLC5A8. Authentication of cell lines was done by

ATCC through their DNA-STR profile.

2.6 Cell lysate preparation and Western Blotting

Following treatment with compounds at the specified concentrations, cells were washed with 1X PBS and lysates were prepared as previously described [117]. Fifty micrograms of total protein was separated on an 8 or 12% polyacrylamide gel, transferred to immobilon membrane and immunoblotted with indicated antibodies.

50

2.7 In vitro CDK assay

In vitro CDK assays, to measure enzyme activity, were performed as described by the manufacturer [167-170] and previously published protocols [119, 166, 171]. Briefly, purified enzyme was aliquoted into the reaction buffer and incubated with indicated compounds (aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA) at various concentrations for 10 min. at room temperature. The reaction mixture was incubated with kinase buffer containing 15 µM ATP, 2 µCi of [32P] γ-ATP, 5 µg of H1 Histone (7.5 μg/reaction added as substrate for CDKs 1 and 2) or retinoblastoma (1.5 μg/reaction added as substrate for

CDKs 4 and 6), at 30°C for 20 min in a final volume of 50 μl. The reactions were halted by adding EDTA to a final concentration of 20 mM and addition of 4X loading buffer. The samples were boiled for 10 min, analyzed by 8 or 10% SDS-PAGE, stained using coomassie brilliant blue (R250) to confirm equal loading of the H1 histones and

Retinoblastoma protein. The molecular weight of the proteins was confirmed by molecular weight markers. The gel was dried and exposed to X-ray film. NIH ImageJ software was used to quantify the intensities of the bands. The normalization of the band intensities of the phosphorylated H1 histones/ Retinoblastoma proteins were determined by comparing to the control, which is the phosphorylation in the absence of these compounds.

2.8 Molecular docking studies

The crystallographic three-dimensional structures of CDK1 (4Y72 A chain), CDK2

(1FIN A chain) and CDK4 (3G33 A chain) were retrieved from the Protein Data Bank

(PDB). Energy minimization for these proteins was performed using Gromacs 3.3.1 package utilizing GROMOS96 force field[172]. The energy-minimized molecules were used as the receptors for virtual small molecule docking with 2,4,6-THBA, 3,4-DHBA, 51

3,4,5-THBA, 4-HBA, phloroglucinol, aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA using AutoDockVina. The results were visualized by PYMOL molecular graphics system version 1.3.

2.9 HPLC analysis for detection of metabolites from cancer cell lysates

High Performance Liquid Chromatography (HPLC) was utilized to determine the uptake of flavonoid metabolites. Cells were exposed to different compounds at indicated concentrations and lysates were prepared as described previously[166]. Protein concentration was determined; 300μl of the lysates were taken and proteins were precipitated using 15μl of trifluoroacetic acid (TFA). Samples were centrifuged at

14,000rpm for 5 min, the supernatant was transferred to fresh tubes and the pH was adjusted to about 4-5. Finally, 300μl of acidified methanol was added to the samples. HPLC analysis was conducted using a Waters HPLC system equipped with a 1525 binary pump and a W2998 PDA detector. An isocratic method was used to elute the compound in reverse phase using an Agilent ZORBAX (SB-CN, 5µm, 4.6×250mm) column. The mobile phase contained 20mM ammonium acetate (adjusted to pH 4.0 with acetic acid) and methanol

(90:10 ratio), with a flow rate of 0.7 ml/min. The injection volume was 40l. Compounds were detected at a wavelength of 260nm. Quantification was done by generating a standard graph using known amounts of each compound.

2.10. RNA isolation and qRT-PCR

Total RNA was isolated from treated and untreated cells as previously described using TRIzol reagent[124]. RT-PCR was performed according to the manufacturer’s instructions (NEB). Briefly, total RNA from 2,4,6-THBA treated cells were isolated and reverse transcribed for 1h at 42°C using M-MuLV reverse transcriptase followed by 52 amplification of the cDNA product. The qPCR experiment was set by adding equal volumes of each template to the SYBR Fast Master mix along with ROX dye (Applied

Biosystems) and 5pmols of each primer (p21Cip1 Forward:

AGCTCAATGGACTGGAAGG Reverse: TGGATGAGGAAGGTCGCT; p27Kip1

Forward: GCTGAGGAACTGACGTGG Reverse: AGGGCAGTGAGGATAGGT). The final volume was adjusted to 20µl using nuclease free water (NEB) and the reaction mixture was amplified through one cycle of 50°C for 2 min, 95°C for 2 min; 40 cycles of

95°C for 15 sec, 60°C for 1 min and 72°C for 1 min; and one melt cycle of 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. GAPDH was used as internal control for the qPCR reaction (Forward: CCACTCCTCCACCTTTGAC Reverse:

ACCCTGTTGCTGTAGCCA).

2.11 Flow cytometric analysis

SLC5A8-pLVX cells were treated for 72h with varying concentrations of 2,4,6-

THBA. Adherent cells were collected by trypsinization and pooled with floating cells, washed with 1X PBS. Cell cycle analysis was performed by adding Vybrant® DyeCycle™

Green Stain at a final concentration of 250nM to 1ml cell suspension. The samples were analyzed by flow cytometry following incubation for 30 min at 37°C. To detect apoptosis, cells were stained with an Annexin V/7-AAD kit as previously described[173]. All experiments were carried out by the CytoFLEX flow cytometer (Beckman Coulter) using

CytExpert 2.0 software.

2.12 Clonogenic assay

Clonogenic assays were performed as previously described[174]. Cells were seeded at a density of 500 cells/100mm plate and grown for 48h following which specified 53 compounds were added at the concentrations indicated. The spent media was replaced with fresh media containing the respective compounds every 5-6 days. Cells were incubated for

14-21 days, fixed with 100% methanol for 20 min, and stained with 0.5% crystal violet prepared in 25% methanol. The colonies were then photographed and quantified using NIH

ImageJ software.

2.13 Cell proliferation analysis through cell counting

Approximately 250,000 cells were seeded per 100 mm plates containing 10% FBS and grown overnight. Cells were left untreated (control) or were treated with drugs at various concentrations and incubated for 48 hours. The floating cells in the spent media (if any) were collected, pooled with the trypsinized cells, and counted using the Nexcelom

Cellometer Auto T4 cell counter.

2.14 Bacterial culture medium

Modified Brain Heart Infusion (mBHI) broth was prepared as described by Ghimire et al [175] with minor modifications and this media was used as a base medium for growing the strains. Briefly, yeast extract (5.0 g/L), L-cysteine (0.3 g/L), 1 ml/L resazurine (0.25 mg/ml) and 1 ml/L menadione (5.8 mM) was added to the standard BHI base ingredients.

The prepared media was purged with nitrogen gas until it became completely anaerobic.

The medium was then autoclaved at 121°C and 15 psi for 30 min. The sterilized medium was then supplemented with 1 ml/L of hemin solution (0.5mg/ml), 10 ml/L of ATCC vitamin mixture (ATCC, USA), and 10 ml/L of ATCC mineral mixture (ATCC, USA).

For experiments studying quercetin degradation, 7N minimal media was used as described by Rodrigus-Castano et al. [176], with slight modifications. Briefly, 50 mM

MOPS⋅NaOH (pH 7.2), 0.02% resazurin, 2 mM tricine, 0.025% tween-80, 20 mM 54

CH3COONa, 20 mM NaCl, 14 mM NH4Cl, 0.25 mM K2SO4, 0.5 mM MgCl2⋅6H2O, 0.5 mM CaCl2⋅2H2O, 10 μM FeSO4⋅7H2O, 20 mM NaHCO3, 1 mM KH2PO4, 1 μg/ ml menadione, 1.9 μM hemin, 0.2 mM histidine, 8 mM L-cysteine, and 40 mM D-glucose were added, and the prepared media was purged with nitrogen until it became completely anaerobic. Following this, the media was supplemented with 1 × ATCC trace minerals and

1 × ATCC vitamin supplements. The media was then filter-sterilized (0.22 μm pore diameter). Quercetin was dissolved in ethanol (75mg/ml) and added to this media to obtain a final concentration of 75 µg/mL to give 7NQ media.

2.15 Screening for bacterial species that were capable of metabolizing quercetin

We first analyzed 57 human fecal isolates from the human gut bacterial library [175] for their ability to grow in 7NQ media. Briefly, the liquid culture of each strain was adjusted to an O.D.600 of 0.5 and 20µL of this culture was inoculated into 1ml of 7NQ media. The bacterial culture was then grown for 48h anaerobically. Following incubation, the O.D.600 of the strains were measured and compared to their growth in 7N media. HPLC was performed to analyze the ability of the tested bacteria to degrade quercetin.

2.16 HPLC analysis for the detection of quercetin/ metabolites in bacterial culture supernatant

High Performance Liquid Chromatography (HPLC) was utilized to determine the concentration of quercetin in the samples. 7NQ media and ethyl acetate was taken in a ratio of 1:2 and the samples were vortexed thoroughly following which phase separation was achieved by centrifuging the samples at 14000rpm for 5 mins. The organic phase was aspirated to a fresh tube and was dried under a steady stream of Argon gas. Following this,

400μL of the mobile phase was added to the dried samples and vortexed to mix the 55 contents. The samples were centrifuged at 14000 rpm for 5 min to pellet any debris and the supernatant was subjected to HPLC analysis. HPLC analysis was conducted using an

Agilent HPLC system equipped with PDA detector. An isocratic method was used to elute the compound in reverse phase using an Agilent ZORBAX (5µm, 4.6×250mm) column.

The mobile phase for quercetin detection consisted of acetonitrile and 2% acetic acid

(40:60 ratio), with a flow rate of 0.8 ml/min. The injection volume was 40µl. Quercetin was detected at a wavelength of 370nm. Quantification was done by generating a standard graph using known amounts of quercetin.

The metabolites of quercetin degradation were extracted with double the volume of n- butanol from the spent media. The n-butanol extract was dried under a steady stream of argon gas and the dried samples were dissolved in the mobile phase. The mobile phase for metabolite detection consisted of acetonitrile and 30mM ammonium acetate [pH 4.0;

89:11(v/v)]. This solution was added to methanol at a ratio of 80:20 ratio. The HPLC apparatus and column used was the same as that used for quercetin detection. The flow rate for the method was maintained at 0.8 ml/min. The injection volume was 40µl. 2,4,6-THBA,

3,4-DHBA and DOPAC was detected at a wavelength of 270nm. Quantification was done by generating a standard graph using known amounts of each metabolite.

2.17 Lysis of bacteria and assay for determination of quercetin degrading enzyme activity

Bacterial cell lysates were prepared as described previously with minor modifications

[177]. Briefly, cells were inoculated in 7N (control) and 7NQ media to an O.D.600 of 0.05 and were then cultivated anaerobically at 37 °C for 24 h. The cells were then harvested, washed twice with buffer A (50 mM Tris-Cl pH 7.5, 20 mM NH4Cl, 1% v/v glycerol, and 56

100 mM NaCl), and then lysed by suspension in buffer A containing 0.5 mg/ml of lysozyme and 2 units/ml of DNase I followed by incubation at 37 °C for 20 min with occasional vortexing. The cell suspension was then centrifuged at 17,000 xg for 30 min and the supernatant was recovered as the crude lysate.

Quercetin degrading enzyme activity was analyzed by measuring the decrease in the absorbance of the reaction mixture at 367nm as previously described [177]. Each reaction was set up as follows: 30 µl of 45mM quercetin dissolved in DMSO was added to 870 µl of 50 mM Tris-Cl buffer (pH 7.5) and preincubated for 5 min. The enzymatic reaction was started by adding 100 µl of either the spent culture supernatant or the cell lysate and the absorbance was recorded at 367 nm following 2 min incubation at 25°C. The molar extinction coefficient was calculated using the blank samples under the same conditions as the reaction mixture.

2.18 Statistical analysis

All experiments were repeated 3-6 times independently of each other. One-way

ANOVA followed by Tukey’s post-hoc tests were adopted to compare group differences to control and significance was defined as p<0.05.

57

CHAPTER 3: THE FLAVONOID METABOLITE 2,4,6-TRIHYDROXYBENZOIC

ACID IS A CDK INHIBITOR AND AN ANTI-PROLIFERATIVE AGENT

3.1 Abstract

Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their antioxidant and anti-inflammatory properties. It is hypothesized that the metabolites of flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6- trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-

THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-

THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter (MCT). Consistent with this, in cells expressing functional SLC5A8, 2,4,6-

THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation.

These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC.

58

3.2 Background and hypothesis

The goal of this study was to determine the ability of 2,4,6-THBA, generated from the flavonoid A-ring, to inhibit CDK activity and cancer cell growth. Two important observations previously reported by us on 2,4,6-THBA led to the present study. First, while determining the ability of salicylic acid derivatives (2,3-dihydroxybenzoic acid and 2,5- dihydroxybenzoic acid) to inhibit CDK enzyme activity, we observed that 2,4,6-THBA also inhibited its activity in vitro [166]. Secondly, among all the hydroxybenzoic acids

(HBAs) tested in that study, 2,4,6-THBA exhibited the highest level of CDK inhibition which prompted us to further examine its potential to inhibit cancer cell growth. CDKs are often dysregulated and overexpressed in cancers and therefore are attractive targets for cancer therapy [178]. The present investigation was carried out to study 1) the effect of

2,4,6-THBA on CDKs 1, 2 and 4; 2) the interactions of 2,4,6-THBA with CDKs though in silico analysis; 3) to identify a transporter for its cellular uptake; and 4) its ability to inhibit cell proliferation in colon cancer cell lines. In this study we demonstrate that 2,4,6-THBA dose-dependently inhibits activities of CDKs 1, 2 and 4, and identify the specific amino acids involved in these interactions. We show that SLC5A8, a member of the monocarboxylic acid transporter (MCT) family, is required for the cellular uptake of 2,4,6-

THBA. In cell lines ectopically expressing SLC5A8, 2,4,6-THBA induced the expression of CDK inhibitors p21Cip1 and p27Kip1 (mRNA and protein) and this was associated with decreased cell proliferation. Interestingly, the other two metabolites, 3,4-DHBA and 3,4,5-

THBA, which failed to inhibit CDK activity, potently inhibited cancer cell proliferation independent of a functional SLC5A8. These results suggest that the flavonoid metabolite

2,4,6-THBA may mediate its anti-cancer effects through a CDK- and SLC5A8-dependent 59 pathway, whereas 3,4-DHBA and 3,4,5-THBA are likely to exert their effect through a

CDK- and SLC5A8-independent pathway.

3.3 Results:

3.3.1 Dose dependent effect of 2,4,6-THBA on CDK 1, 2 and 4 enzyme activity

In a previous report, we showed that 2,4,6-THBA inhibits CDK1 activity in vitro; however, a detailed study of its effects on other CDKs or its biological implications was not investigated [166]. We, therefore, initiated this study by analyzing the dose-dependent inhibitory effect of 2,4,6-THBA on recombinant CDKs 1, 2 and 4. Figure 3.1A-C (upper panel) shows the effect of 2,4,6-THBA on CDK1, 2 and 4 enzyme activity over a range of concentrations (62.5µM to 1mM for CDK1 and 2; and 125µM to 1mM for CDK4). The quantified data representing the degree of inhibition is shown in the lower panel. The IC50 of 2,4,6-THBA was 580±57µM for CDK1, 262±29µM for CDK2 and 403±63µM for

CDK4. These experiments also revealed that 2,4,6-THBA was most effective against

CDK2 as compared to CDKs 1 and 4. The middle panels of Fig. 3.1A-C show that each of the lanes contain similar levels of the substrate. In a separate experiment, we also tested the inhibitory effect of 2,4,6-THBA on CDK6 activity; however, owing to the inconsistency in the obtained results, these data are not reported. 60

Figure 3. 1: Dose dependent effect of 2,4,6-THBA on different CDKs

A, B, and C show the dose dependent effects of 2,4,6-THBA on CDK1, CDK2 and

CDK4 enzyme activity respectively. H1 histones were used as the substrate for CDK1 and

CDK2, while retinoblastoma protein was used as the substrate for CDK4. Upper panel shows phosphorylation of H1 Histones (CDK1 and CDK2) and Retinoblastoma protein

(CDK4) in the presence of different concentrations of 2,4,6-THBA. The middle panel shows the Coomassie blue stained proteins. Lower panel represents the quantification of the bands in upper panel.

3.3.2 Comparison of the effect of 2,4,6-THBA, phloroglucinol and 3,4,5-THBA on

CDK enzyme activity

To investigate the importance of the carboxyl (-COOH) and hydroxyl (–OH) functional groups and their positions within the aromatic ring, two structurally related compounds, phloroglucinol and 3,4,5-THBA (gallic acid), were tested for their effect on

CDK activity and compared against 2,4,6-THBA at a concentration of 500µM. As shown in Fig. 3.2, 2,4,6-THBA inhibited the activity of all three CDKs confirming the results 61 obtained in Fig. 3.1A-C. Interestingly, 3,4,5-THBA had a marginal effect, if any, on CDK1 and CDK4 and failed to inhibit CDK2, whereas phloroglucinol which lacks a –COOH group had no effect on CDK4 but slightly inhibited CDK1 and 2 (Fig. 3.2 lower panel).

These results suggest that the presence of a –COOH group and the relative positions of the

–OH groups on the benzene ring are important for exhibiting effective CDK inhibition.

Figure 3. 2: Comparison of the effect of phloroglucinol, 2,4,6-THBA and 3,4,5-THBA on CDK1 (A), CDK2 (B) and CDK4 (C) activity

Upper panel shows phosphorylation pattern of H1 Histones (CDK1 and CDK2) and

Retinoblastoma protein (CDK4), in the presence of different compounds. The middle panel shows Coomassie blue stained proteins. The lower panel shows quantification of the blots in the upper panel.

62

3.3.3 Molecular docking studies show potential interactions of 2,4,6-THBA,

phloroglucinol and 3,4,5-THBA with CDKs

AutoDock Vina was used to predict the potential interactions of 2,4,6-THBA, 3,4-

DHBA, 4-HBA, phloroglucinol and 3,4,5-THBA with CDKs 1, 2 and 4. The binding free energy and hydrogen bond lengths were also determined. The results of the docking studies are shown in Table-3.1 and Fig. 3.3A-C (space-filling model). The active site of the widely studied CDK2 enzyme has a total of 16 amino acids (Ile10, Gly11, Glu12, Val13, Val64,

Glu81, Phe82, Leu83, Asp86, Asp127, Lys129, Gln131, Asn132, Leu134, Asp145,

Thr165) [179]. All the compounds tested interacted with CDKs (except 4-HBA and CDK1) and utilized amino acids either residing in the active site or at alternate binding pockets.

Docking studies revealed that 2,4,6-THBA potentially interacts with CDK1 through

Arg123, Arg151, and Gly154; with CDK2 it utilizes Lys33, Gln81and Leu83; and with

CDK4, it interacts through Cys73 and His158. Phloroglucinol interacts with CDK1 using

Leu125 and Arg123; with CDK2 it utilizes Asp145 and Lys33; and with CDK4 through

Phe78 and Glu74. Interactions of 3,4,5-THBA with CDK1 occurs through Asp146; through

Lys33 for CDK2; and with CDK4 utilizing Glu74, Cys73 and Cys68. Interactions of 2,4,6-

THBA and phloroglucinol with CDK1 appear to be occurring in a pocket other than the active site; in contrast 3,4,5-THBA interacts with the amino acids in the active site. In

CDK2 and CDK4, all three compounds seem to interact with amino acids in the active site.

These results also suggest that 2,4,6-THBA can potentially interact with residues involved in CDK2-ATP-interaction.

63

Table 3. 1: Free energy binding values and hydrogen bond lengths for the interaction of 2,4,6-THBA, phloroglucinol, 3,4-DHBA, 4-HBA and 3,4,5-THBA with CDK1,

CDK2 and CDK4.

S.No Receptor Ligand Interacting Measurement No of Energy amino (A°) H value acids bonds (kCal/mol) 2,4,6-THBA Arg123, 2.5, 2.4, 2.0 3 -6.0 Gly154, Arg151 3,4,5-THBA Asp 146 2.1 1 -6.1 1. CDK1 Phloroglucinol Leu125, 2.4, 2.6 2 -4.9 Arg123 3,4-DHBA Asp146 2.8 1 -6.3 4-HBA - - 0 -6.4 2,4,6-THBA Leu83, 2.2, 2.3, 2.4 3 -5.6 Gln81, Lys33 3,4,5-THBA Lys33 2.5 1 -5.8 2 CDK2 Phloroglucinol Asp145, 2.2,2.4 2 -5.2 Lys33 3,4-DHBA Leu83(2), 3.2, 3.1, 2.0 3 -5.8 Glu81 4-HBA Lys33 2.9 1 -5.7 2,4,6-THBA His158, 21, 2.8 2 -5.8 Cys73 3,4,5-THBA Glu74, 2.2, 2.8, 1.9 3 -5.8 Cys73, Cys68 3. CDK4 Phloroglucinol Phe78, 2.1, 2.7 2 -5.0 Glu74 3,4-DHBA Cys68, 2.5, 2.7 2 -5.7 Glu75 4-HBA His158 3.2 1 -5.7 64

Figure 3. 3: Molecular docking revealed potential interactions of different compounds with CDKs

Space-filling model showing the orientation of the compounds and the potential binding pockets in CDK1 (A), CDK2 (B) and CDK4 (C) for 2,4,6-THBA (upper panel), phloroglucinol (middle panel) and 3,4,5-THBA (lower panel).

3.3.4. 2,4,6-THBA inhibits cell proliferation in cell lines expressing functional

SLC5A8

Since 2,4,6-THBA effectively inhibited CDK activity in vitro, the possibility that it could inhibit cell proliferation of human CRC cells was explored. Surprisingly, when

HCT-116 cells were exposed to 2,4,6-THBA (125- 1000 μM) for 72 h, no decline in cell number was observed (Fig. 3.4). Additionally, the levels of cell cycle inhibitory proteins 65 p21Cip1 and p27Kip1 also remained unaltered, and similar results were obtained in HT-29 and Caco-2 cell lines (data not shown). This prompted us to speculate that the unresponsiveness of these cells may be due to the lack of endogenous functional transporters for 2,4,6-THBA. We reasoned that 2,4,6-THBA being a monocarboxylic acid may be transported through the MCT SLC5A8 [180], which is expressed in the GI and proximal tubule for the transport of monocarboxylic acids [181, 182]. Interestingly, in various cancers this protein is either inactivated through mutations (F251V) [182] or its expression is suppressed through promoter methylation[183]; this possibly explains the lack of an inhibitory effect of 2,4,6-THBA in CRC cell lines. Since a CRC cell line expressing a functional SLC5A8 was unavailable, we sought to determine the effect of

2,4,6-THBA on a breast cancer cell line (MDA-MB-231) previously characterized and reported to express this protein under the control of the tet-promoter (SLC5A8-pLVX)

[184]. In contrast, parental MDA-MB-231 cells have been reported to express low levels of SLC5A8 [185].

66

Figure 3. 4: Effect of 2,4,6-THBA on HCT-116 cell proliferation

Cells were treated with 2,4,6-THBA for 72h. Floating cells were collected, the adherent cells were washed, trypsinized and pooled with the floating cells and counted.

To investigate if the effect of 2,4,6-THBA required SLC5A8 transporter function,

MDA-MB-231 and SLC5A8-pLVX cells were treated for 72h at different concentrations from 125µM to 1000µM. Cells were counted for the inhibition of proliferation or analyzed for the induction of various cell cycle regulatory proteins namely p21Cip1, p27Kip1, CDK1,

CDK2, CDK4, cyclin A2, cyclin B1 and cyclin D1. Figure 3.5A shows that no significant inhibition of cell proliferation was observed in MDA-MB-231 cells at concentrations from

125-750μM; however, a reduction was observed at 1000μM. Consistent with this, 2,4,6-

THBA upregulated p21Cip1 and p27Kip1 protein levels at 1000μM (data not shown). In contrast, in SLC5A8-pLVX cells, there was a clear decrease in cell density and number at all concentrations tested, indicative of an inhibitory effect on cell proliferation (Fig. 3.5B and C). The decreased cell number correlated with increased levels of p21Cip1 and p27Kip1

(Fig. 3.5D). In these lysates, protein levels of CDK1, CDK4, cyclin A2 and cyclin D1 67 remained unaltered while levels of CDK2 and cyclin B1 were reduced at higher concentrations (≥750μM; Fig. 3.6). Further examination of mRNA levels revealed an increase of p21Cip1 at all concentrations whereas p27Kip1 levels were upregulated beginning at 500µM (Fig. 3.5E and F), suggesting that 2,4,6-THBA is an effective anti-proliferative agent. Collectively, these results indicate that SLC5A8 is a likely transporter for 2,4,6-

THBA, and that its uptake contributes to the observed increase in the levels of p21Cip1 and p27Kip1, leading to inhibition of cell proliferation.

Figure 3. 5: Effect of 2,4,6-THBA on MDA-MB-231 and SLC5A8-pLVX cells

A, Graphic representation of the lack of an effect of 2,4,6-THBA on MDA-MB-

231 cells. B, Microscopy images showing the effect of 2,4,6-THBA in SLC5A8-pLVX cells. C, Graphic representation showing the progressive decrease in cell number upon treatment with 2,4,6-THBA in SLC5A8-pLVX cells. D, Western Blot analysis showing the increase in p21Cip1 and p27Kip1 protein levels in SLC5A8-pLVX cells in response to 2,4,6-

THBA. E and F, qPCR analysis showing fold-increase in p21Cip1 and p27Kip1 mRNA levels respectively in SLC5A8-pLVX cells in response to 2,4,6-THBA. 68

Figure 3. 6: Western Blot analysis showing the levels of various CDKs and cyclins in

SLC5A8-pLVX cells in response to 2,4,6-THBA

3.3.5 2,4,6-THBA is taken up by cells expressing functional SLC5A8

In independent experiments, using HPLC, cellular uptake of 2,4,6-THBA by

SLC5A8-pLVX cells, MDA-MB-231 and HCT-116 cells was investigated to further confirm that SLC5A8 is a transporter for 2,4,6-THBA. Figure 3.7A demonstrates the dose- dependent uptake of 2,4,6-THBA by various cancer cell-lines. In SLC5A8-pLVX cells, uptake was observed at 125µM and increased linearly up to 1000µM. As anticipated, reduced uptake was observed in the parental MDA-MB-231 cells with lower SLC5A8 expression (Fig. 3.8). Importantly, in HCT-116 cells, there was no uptake of 2,4,6-THBA despite the detection of SLC5A8 in the cell lysate (Fig. 3.8), which may be due to the presence of a dysfunctional transporter as previously reported [185]; this is also consistent with the lack of inhibition of proliferation in these cells (Fig. 3.7D). Together, these results implicate SLC5A8 in the transport of 2,4,6-THBA. 69

Figure 3. 7: Uptake studies of 2,4,6-THBA and its effects on colony formation in different cell lines

A, HPLC analysis showing uptake of 2,4,6-THBA after 72h in SLC5A8-pLVX, MDA-

MB-231 and HCT-116 cells. B, C and D, effect of 2,4,6-THBA (14-21 days) on colony formation in SLC5A8-pLVX, MDA-MB-231 and HCT-116 cells respectively.

Quantification of the data is shown beside the crystal violet stained image of the colonies.

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Figure 3. 8: Western Blot demonstrating the expression levels of SLC5A8 protein in

HCT-116, HT-29, MDA-MB-231 and SLC5A8-pLVX cell lines

Despite the expression of SLC5A8 in HCT-116 cells, uptake of 2,4,6-THBA was not observed (Fig. 3.7A). In contrast, uptake was observed in SLC5A8-pLVX cells expressing the functional transporter. Consistent with the low expression of SLC5A8 in

MDA-MB-231cells, low levels of 2,4,6-THBA was observed in these cells.

3.3.6. Clonogenic assay

Clonogenic assay was performed to demonstrate the effectiveness of 2,4,6-THBA against colony formation in SLC5A8-pLVX, MDA-MB-231, HCT-116 and HT-29 cells.

Figure 3.7B demonstrates that in SLC5A8-pLVX cells, 2,4,6-THBA dose dependently decreased the colony formation beginning at 125µM with a 50% reduction observed around

400µM. In addition to the decrease in the number of colonies, 2,4,6-THBA caused a reduction in the size of the colonies. In the MDA-MB-231 cells, 2,4,6-THBA also reduced the number of colonies after 250μM with 50% reduction observed at about 446μM (Fig.

3.7C). In contrast, the colony formation in HCT-116 (Fig. 3.7D) and HT-29 cells (Fig.

3.9A) remained unaffected at all concentrations. This further demonstrates that SLC5A8 is a transporter for 2,4,6-THBA and its uptake leads to inhibition of cell proliferation. 71

Figure 3. 9: Effect of different HBAs on colony formation in HT-29 cells

Figure shows the effect of 2,4,6-THBA (A), 3,4,5-THBA (B), 3,4-DHBA (C) and

4-HBA (D) on colony formation in HT-29 cells.

3.3.7 Cell cycle analysis and apoptosis assay

We next determined the effect of 2,4,6-THBA on cell cycle arrest and apoptosis in

SLC5A8-pLVX cells. Figure 3.10A demonstrates that no significant shift nor accumulation of a specific phase was observed in different stages of the cell cycle under treated conditions as compared to the untreated control. Interestingly, there was also no change in the number of cells undergoing apoptosis following 2,4,6-THBA treatment (Fig. 3.10B).

These results when combined with the cell count data obtained in Fig. 3.5C suggests that

2,4,6-THBA is likely to slow down the rate of proliferation without causing an accumulation at any specific stage of the cell cycle, implying that the inhibition may be equal across all phases of the cell cycle. This is further supported by the earlier observation in Fig. 3.1 that 2,4,6-THBA is an effective inhibitor of CDKs 1, 2 and 4. Lack of apoptosis 72 observed also indicates that 2,4,6-THBA is likely to be a cytostatic agent rather than a cytotoxic agent at the concentrations tested.

Figure 3. 10: Cell cycle and apoptosis assays

Graphic representation of A, the cell cycle analysis and B, apoptosis assay following treatment with 2,4,6-THBA in SLC5A8-pLVX cells. The percentage of cells in each stage was determined through flow cytometry.

3.3.8. Effect of other flavonoid metabolites (4-HBA; 3,4-DHBA; and 3,4,5-THBA)

on clonal formation

Next, the effect of metabolites generated from the flavonoid B-ring namely 4-HBA,

3,4-DHBA and 3,4,5-THBA on colony formation was investigated for comparison with

2,4,6-THBA. The data in Fig. 3.2 showed that 3,4,5-THBA did not inhibit CDK activity, and our previous observations indicated that 3,4-DHBA is also incapable of inhibiting

CDK1 [166]. Despite the lack of CDK inhibition by these compounds, the possibility that they may exhibit their known anti-proliferative activity [144, 162, 164, 165] following uptake through SLC5A8 was considered. Consistent with previous reports in other cell 73 lines, 3,4-DHBA and 3,4,5-THBA efficiently inhibited clonal formation in SLC5A8- pLVX, MDA-MB-231, and HCT-116 cells (Figs. 3.11 and 3.12). Inhibition of cell proliferation with 3,4-DHBA was observed beginning at a concentration of 15.62μM for

SLC5A8-pLVX, 7.3μM for MDA-MB-231 and 125μM for HCT-116 cells. Inhibition with

3,4,5-THBA was observed starting at 7.3μM in all cell-lines. The lack of CDK-inhibition by 3,4-DHBA [166] and 3,4,5-THBA (Fig. 3.2) and the results obtained in clonogenic assay clearly indicated that their mechanism of action is CDK-independent. Interestingly,

4-HBA failed to inhibit clonal formation at all concentrations tested (Figs. 3.9 and 3.13) which suggests that the flavonoid metabolites are highly selective in their actions.

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Figure 3. 11: Colony formation assay following treatment with 3,4-DHBA

Effect of different concentrations of 3,4-DHBA on colony formation in SLC5A8- pLVX (A), MDA-MB-231 (B), and HCT-116 (C) cells. 75

Figure 3. 12: Colony formation assay following treatment with 3,4,5-THBA

Effect of different concentrations of 3,4,5-THBA on colony formation in

SLC5A8-PLVX (A), MDA-MB-231 (B), and HCT-116 (C) cells. 76

Figure 3. 13: Colony formation assay following treatment with 4-HBA

Effect of different concentrations of 4-HBA on colony formation in SLC5A8- pLVX (A), MDA-MB-231 (B), and HCT-116 (C) cells.

The ability of 3,4-DHBA and 3,4,5-THBA to inhibit proliferation in HCT-116 cells

(Figs. 3.11 and 3.12) was surprising as in these cells the structurally related 2,4,6-THBA failed to inhibit cell proliferation. These contrasting results prompted the study of their 77 uptake in SLC5A8-pLVX, MDA-MB-231 and HCT-116 cells. The data obtained from

HPLC analysis showed that 3,4-DHBA and 3,4,5-THBA were not taken up whereas cellular uptake of 4-HBA was consistently observed in all three cell lines tested (Fig. 3.14).

Inhibition of cell proliferation by 3,4-DHBA and 3,4,5-THBA in all three cell lines without their cellular uptake suggests that the biological targets for these compounds are extracellular. The uptake of 4-HBA in all cell lines might be mediated either through an alternative transporter or it passively diffuses across the cell membrane.

Figure 3. 14: HPLC analysis showing the cellular uptake of different HBAs

HPLC analysis showing the cellular uptake in SLC5A8-pLVX, MDA-MB-231 and

HCT-116 cells following incubation with 3,4-DHBA (A), 3,4,5-THBA (B) and 4-HBA (C) in the cytosol.

3.4 Discussion

In this chapter, we report several novel observations including a mechanism by which flavonoid metabolites known to be generated in the GI tract may exert their anti-cancer effects against CRC through CDK-dependent and -independent pathways. This study investigated 2,4,6-THBA generated from flavonoid A-ring and 4-HBA, 3,4-DHBA and

3,4,5-THBA from the flavonoid B-ring (Fig. 1.6) for their potential to inhibit cancer cell 78 growth. Our results demonstrated that the inhibitory effect of 2,4,6-THBA on purified

CDKs in vitro was dose dependent with IC50 observed between 250-600µM, while molecular docking studies revealed the key amino acid interactions. We also identified

SLC5A8 as a transporter for 2,4,6-THBA, and using SLC5A8-pLVX cells, we demonstrated that its cellular uptake for 72h was sufficient to cause induction of p21Cip1 and p27Kip1, leading to inhibition of cell proliferation. Although endogenous SLC5A8 was detected in HCT-116 cells, they were insensitive to 2,4,6-THBA treatment. Additionally, using clonogenic assays, we showed that 2,4,6-THBA was highly effective in inhibiting clonal formation in the cell lines expressing functional SLC5A8. The IC50 of clonal inhibition corresponded to the IC50 of CDK inhibition, suggesting that 2,4,6-THBA may mediate its effect through a CDK-dependent pathway. We believe the inhibition of proliferation can occur through at least two different mechanisms – one by direct binding of 2,4,6-THBA to CDKs leading to their inhibition and the other through upregulation of

CDK inhibitory proteins p21Cip1 and p27Kip1. Although 3,4-DHBA and 3,4,5-THBA were incapable of inhibiting CDKs, they efficiently inhibited cell proliferation in all cell lines tested including HCT-116 and HT-29 (Figs. 3.9, 3.11 and 3.12). This suggests that the mechanism of inhibition by 3,4-DHBA and 3,4,5-THBA is both an SLC5A8- and CDK- independent phenomenon. Not all metabolites exhibited anti-proliferative effects as 4-

HBA although taken up by all cell lines tested was ineffective in inhibiting cell proliferation which suggests that the metabolites are selective in their actions. Our results, for the first time demonstrate a role for flavonoid metabolites in the inhibition of cancer cell growth occurring through both CDK-dependent and -independent mechanisms, and also establishes a functional role for SLC5A8 in the transport of 2,4,6-THBA. 79

Molecular docking studies indicated that 2,4,6-THBA binds to CDKs 1, 2 and 4 at specific sites within these enzymes. The CDK structure is highly conserved among the different family members with a bi-lobe fold that harbors a conserved ATP-binding domain

[186, 187]. The interactions of 2,4,6-THBA with CDKs 2 and 4 at the ATP-binding site may explain the enhanced inhibition of enzyme activity in comparison to the lesser inhibition of CDK1 by 2,4,6-THBA which interacts at an allosteric site. Interestingly, the structurally related compound 3,4,5-THBA failed to inhibit CDK activity although docking studies provided evidence of its binding to all CDKs (Table 3.1). This suggests that the presence of hydroxyl groups at specific positions on the benzene ring in these compounds confers the ability to bind and cause enzyme inhibition. The –COOH group seems to be important in increasing the potency of this inhibition as phloroglucinol which lacks the –

COOH group was only marginally effective in inhibiting CDK1 and CDK2. Therefore, we suggest that both the presence of –COOH and the position of the -OH groups are important for efficient CDK inhibition. Further studies involving kinetics of inhibition are required to fully understand the nature of these interactions and its implications towards inhibition of enzyme activity.

Detection of 2,4,6-THBA in SLC5A8-pLVX cells through HPLC suggests that

SLC5A8 is a natural transporter for this monocarboxylic acid. The attributed physiological functions of SLC5A8 includes transport of short chain fatty acids (SCFAs), salicylates, propionate, butyrate, lactate, pyruvate, and benzoates to name a few [182]. Numerous studies have shown that SLC5A8 is likely to be a tumor suppressor protein[183, 184] and the mechanism is predicted to involve inhibition of histone deacetylases (HDACs) through the uptake of SCFAs by these transporters [182, 185]. It is a sodium-coupled MCT and is 80 expressed in the apical membrane of the colonic and intestinal epithelium [181]. It is silenced in many cancers including colon, thyroid, kidney, stomach, brain, breast, pancreas and prostate. Studies suggest that silencing of SLC5A8 contributes to tumorigenesis [188].

We speculated that the lack of response observed following 2,4,6-THBA treatment of

HCT-116 and other CRC cell lines is due to the absence of a wild-type functional SLC5A8.

In support of this view, no cellular uptake of 2,4,6-THBA was observed in these cells.

Importantly, in SLC5A8-pLVX cells expressing the functional SLC5A8, 2,4,6-THBA not only induced p21Cip1 and p27Kip1 RNA and protein expression, but also reduced cell proliferation. Our demonstration that 2,4,6-THBA inhibits cell growth in cancer cells expressing functional SLC5A8 suggests that such a phenomenon may indeed exist in vivo and may contribute to its anti-cancer effects against CRC.

The mechanism by which 2,4,6-THBA inhibits CDK activity requires additional investigations. Unlike the conventional drugs Palbociclib and Ribociclib that target

CDK4/6 and cause G0/G1 cell cycle arrest [189], 2,4,6-THBA appears to target CDKs 1,

2, and 4. The lack of inhibition at any particular stage of the cell-cycle observed in our study (Fig. 3.10A) may be attributed to the effect of 2,4,6-THBA on multiple CDKs (Fig.

3.1A-C), leading to a decrease in the overall growth rate without causing accumulation of cells at any given stage. Exposure of SLC5A8-pLVX cells to 2,4,6-THBA at concentrations from 125µM-1000µM slowed the proliferation (Fig. 3.5C) without causing significant apoptosis (Fig. 3.10B) suggesting that it is more likely to be a cytostatic rather than a cytotoxic agent.

The exposure of SLC5A8-pLVX cells as well as MDA-MB-231 cells to 2,4,6-THBA for 14-21 days resulted in a decrease in colony formation. The ability of 2,4,6-THBA to 81 inhibit cell proliferation in MDA-MB-231 cells was surprising considering the low expression levels of SLC5A8 and the fact that acute treatment (72h) with 2,4,6-THBA did not cause any significant decrease in cell proliferation. This discrepancy between acute and chronic treatment in MDA-MB-231 cells can be explained by the limited uptake of 2,4,6-

THBA during acute exposure, whereas chronic exposure of these cells to 2,4,6-THBA could result in significant accumulation sufficient to cause inhibition of cell proliferation.

This was consistent with the HPLC data that showed lower uptake of 2,4,6-THBA in

MDA-MB-231 cells when compared to SLC5A8-pLVX cells (Fig. 3.7A). Consistent with the previously reported lack of functional SLC5A8 in HCT-116 cells [185], 2,4,6-THBA had no effect on the colony formation (Fig. 3.7D), and HPLC also showed no uptake of

2,4,6-THBA in these cells (Fig. 3.7A). These results suggest that the transporter activity of

SLC5A8 is required for 2,4,6-THBA-mediated inhibition of cell proliferation.

Tumor progression involves distinct stages – initiation, promotion, progression and metastasis. Of these stages, it is suggested that chemo-preventive agents preferentially act within the initiation and promotion stages to reverse the process of carcinogenesis [190].

They are further classified into blocking agents and suppressing agents depending on their ability to prevent carcinogenesis and suppress neoplastic cell growth respectively [191].

Based on the properties reported for phenolic compounds [95, 192] and the results presented in this study, we propose that 2,4,6-THBA can act as both – a blocking and a suppressing agent. Since it is a phenolic compound with multiple hydroxyl groups, it can act as a blocking agent and exert its effect through its antioxidant properties [96]. It can also be viewed as a suppressing agent through its direct inhibitory effect on CDKs 1, 2 and

4 leading to reduced rate of cell proliferation. Its antioxidant and anti-proliferative actions 82 could collectively contribute to cancer prevention by slowing down the rate of cell proliferation and providing an opportunity to repair the DNA damage or for immune surveillance by Natural Killer- and cytotoxic T-cells for cancer cell destruction [193, 194].

Although 3,4-DHBA and 3,4,5-THBA failed to inhibit CDK activity in vitro, their ability to inhibit colony formation in all cell lines tested irrespective of the SLC5A8 expression is also an important observation. It suggests that these compounds exert their anti-proliferative effect through an SLC5A8- and CDK-independent mechanism. These flavonoid metabolites have previously been shown to inhibit cancer cell growth through multiple signaling pathways [144, 162]; however, no direct primary target(s) have been identified. It appears that depending upon the cell line tested, 3,4-DHBA and 3,4,5-THBA can inhibit colony formation within a range of 7.3–250 μM and 7.3-31.25 μM respectively.

These concentrations are significantly lower compared to those of 2,4,6-THBA. The lack of detection of these compounds in HPLC studies suggests that they are not taken up by the cells, and hence we suggest that their primary target is likely to be extracellular.

The discovery of the flavonoid metabolite 2,4,6-THBA as an inhibitor of CDKs and the demonstration of its ability to inhibit cell proliferation is a significant finding. Although the IC50 of 2,4,6-THBA and other compounds tested in this study are in micromolar concentrations, it could be argued that it is physiologically relevant in view of their abundance in the diet [141, 145]. CDKs and other potential cellular targets probably have evolved to be less sensitive to these compounds to avoid complete inhibition or perturbance of cell cycle at lower concentrations. We suggest that the reported chemo-preventive actions of flavonoids could be explained at least in part through the ability of its metabolite

2,4,6-THBA to target CDKs by direct inhibition and through upregulation of CDK 83 inhibitory proteins; this would tip the balance strongly towards cell-cycle arrest, leading to reduced cell proliferation. A model depicting how flavonoid metabolites may prevent the occurrences of CRC is shown in Fig. 3.15. Since the metabolites are also generated through microbial degradation, it highlights the importance of GI microflora in the prevention of

CRC. Effective cancer prevention may therefore require both the chemo-preventive agents and the responsible microbial partners for their degradation. Identification of the microbial species contributing to this process as well as the mechanism of action of the metabolites on GI tissue is an important area for future study.

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Figure 3. 15: A model depicting how flavonoid metabolites may exert its chemo- preventive effects on colonic tissue to protect against CRC

We suggest that 2,4,6-THBA generated by auto-degradation of flavonoids or those through the action of microflora is taken up by the colonic epithelial cells through SLC5A8.

Since SLC5A8 is expressed only in the apical surface (and not in the basolateral surface)

[182], 2,4,6-THBA may accumulate to pharmacologically relevant concentrations within cells, sufficient to inhibit CDKs. This would lead to a reduced rate of cell proliferation through 2 distinct mechanisms – one by direct inhibition of CDKs by 2,4,6-THBA and the other through the upregulation of p21Cip1 and p27Kip1, possibly through an indirect pathway.

The reduced rate of proliferation will provide an opportunity for DNA repair to occur or destruction of cancer cells through immune surveillance. The metabolites such as 3,4-

DHBA and 3,4,5-THBA that are generated from flavonoids may also contribute to chemoprevention by targeting extracellular proteins.

In this chapter, using a variety of biochemical, molecular biology and computational approaches, we report that the flavonoid metabolite 2,4,6-THBA inhibits CDK enzyme 85 activity and exhibits potent anti-proliferative effects. We showed that cellular uptake of

2,4,6-THBA required the expression of SLC5A8, an MCT. Investigations were also carried out to determine the effectiveness of three other metabolites– 4-HBA, 3,4-DHBA and

3,4,5-THBA. Of these only 3,4-DHBA and 3,4,5-THBA inhibited cancer cell proliferation and this was independent of both SLC5A8 transport and CDK inhibition. These findings for the first time, suggests that the flavonoid metabolite 2,4,6-THBA along with 3,4-DHBA and 3,4,5-THBA may contribute to the chemo-preventive effects of flavonoids against

CRC. In addition, our studies also highlight the need of further investigations directed towards the role(s) played by flavonoid metabolites in the prevention of cancer. Our finding that 2,4,6-THBA is an effective inhibitor of CDKs that acts as an anti-proliferative agent suggests that it has the potential to be developed into a novel class of CDK inhibitors.

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CHAPTER 4: ASPIRIN METABOLITES 2,3‑DHBA AND 2,5‑DHBA INHIBIT

CANCER CELL GROWTH

4.1 Abstract

Although compelling evidence exists on the ability of aspirin to treat colorectal cancer

(CRC), and numerous theories and targets have been proposed, a consensus has not been reached regarding its mechanism of action. In this regard, a relatively unexplored area is the role played by aspirin metabolites 2,3‑dihydroxybenzoic acid (2,3‑DHBA) and

2,5‑dihydroxybenzoic acid (2,5‑DHBA) in its chemopreventive actions. In a previous study, we demonstrated that 2,3‑DHBA and 2,5‑DHBA inhibited CDK1 enzyme activity in vitro. The aim of the present study was to understand the effect of these metabolites on the enzyme activity of all CDKs involved in cell cycle regulation (CDKs 1, 2, 4 and 6) as well as their effect on clonal formation in three different cancer cell lines. Additionally, in silico studies were performed to determine the potential sites of interactions of 2,3‑DHBA and 2,5‑DHBA with CDKs. We demonstrated that 2,3‑DHBA and 2,5‑DHBA inhibits

CDK‑1 enzyme activity beginning at 500 μM, while CDK2 and CDK4 activity was inhibited only at higher concentrations (>750 μM). 2,3‑DHBA inhibited CDK6 enzyme activity from 250 μM, while 2,5‑DHBA inhibited its activity >750 μM. Colony formation assays showed that 2,5‑DHBA was highly effective in inhibiting clonal formation in

HCT‑116 and HT‑29 CRC cell lines (250‑500 μM), and in the MDA‑MB‑231 breast cancer cell line (~100 μM). In contrast 2,3‑DHBA was effective only in MDA‑MB‑231 cells

(~500 μM). Both aspirin and salicylic acid failed to inhibit all four CDKs and colony formation. Based on the present results, it is suggested that 2,3‑DHBA and 2,5‑DHBA may 87 contribute to the chemopreventive properties of aspirin, possibly through the inhibition of

CDKs.

4.2 Background and hypothesis

The goal of this chapter was to understand if aspirin metabolites 2,3-DHBA and 2,5-

DHBA can prevent cancer cell growth. Our laboratory has been focusing on determining a role for aspirin and salicylic acid metabolites 2,3-dihydroxybenzoic acid (2,3-DHBA) and

2,5-dihydroxybenzoic acid (2,5-DHBA), known to be generated in the body by the cytochrome P-450 (CYP450) catalyzed reactions [150], in the prevention of CRC.

Interestingly, similar dihydroxybenzoic acids have also been reported to be generated from aspirin by the gut microflora [154]. In a previous study we demonstrated the ability of 2,3-

DHBA and 2,5-DHBA to inhibit CDK1 activity in vitro [166]. However, an extended study on the effect of these metabolites on other CDKs involved in cell-cycle regulation (CDK2,

CDK4 and CDK6) as well as the demonstration of their ability to inhibit cancer cell growth were not reported. As a dysregulated cell cycle is one of the hallmarks of cancer, it was important to determine if 2,3-DHBA and 2,5-DHBA affected cancer cell growth by inhibiting these CDKs to gain a comprehensive understanding of their mechanism of action. In the present study, we investigated the effect of 2,3-DHBA and 2,5-DHBA on

CDKs 1, 2, 4 and 6 enzyme activities and determined their potential sites of interaction within these enzymes. In addition, we also performed studies to determine the effect of these metabolites on cancer cell proliferation.

88

4.3 Results

4.3.1 Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK enzyme

activity

We initiated this study by determining the dose-dependent effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on recombinant CDKs 1, 2, 4 and 6 by measuring the phosphorylation pattern through in vitro kinase assays. Figure 4.1 shows that CDK1 activity remained unaffected following treatment with aspirin and salicylic acid at all concentrations tested (125 µM to 1 mM); however, 2,3-DHBA and 2,5-DHBA inhibited

CDK1 activity, confirming our previously published results [166]. In both cases, inhibition was around 20% at 500 μM, 40% at 750 μM and 60% at 1000 μM. CDK2 activity remained unchanged upon exposure to aspirin and salicylic acid while inhibition was observed with

2,3-DHBA and 2,5-DHBA, especially at higher concentrations (>750 μM; Fig. 4.2).

Similar to CDK1 and CDK2, aspirin and salicylic acid both failed to inhibit CDK4 and

CDK6 enzyme activity (Figs. 4.3 and 4.4). 2,3-DHBA and 2,5-DHBA inhibited CDK4 activity at 1000 µM. 2,3-DHBA was able to inhibit CDK6 enzyme activity beginning at

250 µM, while 2,5-DHBA inhibited CDK6 enzyme activity at concentrations >750 μM.

These results show 2,3-DHBA and 2,5-DHBA were able to inhibit CDKs 1, 2, 4, and 6 activities to different degrees. It is also important to note that the inhibition observed with

CDK1 by 2,3-DHBA and 2,5-DHBA, and CDK-6 by 2,3-DHBA was significantly greater compared to inhibition of other CDKs by these compounds. 89

Figure 4. 1: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK1 enzyme activity

A, B, C and D show the effect of different concentrations of aspirin, salicylic acid,

2,3-DHBA and 2,5-DHBA respectively on CDK1 enzyme activity. H1 histones were used as the substrate for CDK1. Upper panel shows phosphorylation of H1 Histones in the presence of different concentrations of each compound. The middle panel shows the coomassie blue stained proteins. Lower panel represents the quantification of the bands in upper panel. The concentration of the drugs used are indicated in micromolar (μM). 90

Figure 4. 2: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK2 enzyme activity

A, B, C and D show the effect of different concentrations of aspirin, salicylic acid,

2,3-DHBA and 2,5-DHBA respectively on CDK2 enzyme activity. H1 histones were used as the substrate for CDK2. Upper panel shows phosphorylation of H1 Histones in the presence of different concentrations of each compound. The middle panel shows the coomassie blue stained proteins. Lower panel represents the quantification of the bands in upper panel. The concentration of the drugs used are indicated in micromolar (μM). 91

Figure 4. 3: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK4 enzyme activity

A, B, C and D show the effect of different concentrations of aspirin, salicylic acid,

2,3-DHBA and 2,5-DHBA respectively on CDK4 enzyme activity. Retinoblastoma protein was used as the substrate for CDK4. Upper panel shows phosphorylation of retinoblastoma protein in the presence of different concentrations of each compound. The middle panel shows the coomassie blue stained proteins. Lower panel represents the quantification of the bands in upper panel. The concentration of the drugs used are indicated as micromolar

(μM). 92

Figure 4. 4: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on CDK6 enzyme activity

A, B, C and D show the effect of different concentrations of aspirin, salicylic acid,

2,3-DHBA and 2,5-DHBA respectively on CDK6 enzyme activity. Retinoblastoma protein was used as the substrate for CDK6. Upper panel shows phosphorylation of retinoblastoma protein in the presence of different concentrations of each compound. The middle panel shows the coomassie blue stained proteins. Lower panel represents the quantification of the bands in upper panel. The concentration of the drugs used are indicated as micromolar

(μM). 93

4.3.2 Docking studies reveal binding pockets for aspirin, salicylic, 2,3-DHBA, and

2,5-DHBA

AutoDock Vina was used to predict the potential interactions of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA with CDKs 1, 2, 4 and 6. The binding free energy and hydrogen bond lengths were also determined. The results of the docking studies are shown in Table 4.1 and Figure 4.5 (line model). All the compounds tested interacted with CDKs and utilized amino acids either residing in the active site or at alternate binding pockets.

Docking studies revealed that 2,3-DHBA potentially interacts with CDK1 through

Asp146; with CDK2 it utilizes Lys33, and Asp145; with CDK4, it interacts through Cys68 and His158 and with CDK6 it binds to Asp134, Tyr292, Ala291 and Thr198. 2,5-DHBA interacts with CDK1 using Asp146; with CDK2 it utilizes Asp145 and Lys33; with CDK4 through His158; and with CDK6 it uses Lys43, Tyr24 and Gly25. Salicylic acid and aspirin also interacted with all 4 CDKs (Table 4.1), although CDK inhibition was not observed in vitro (Figs. 4.1-4.4). Notable interactions are that between the drug molecules and Asp146 of CDK1, as this amino acid is involved in CDK-ATP interactions [179]. Although salicylic acid interacted with CDK1 through Asp146 and Lys33 and CDK2 through

Asp145 and Lys33, it is not clear at this stage why it failed to cause inhibition of the enzyme activity. This may be due to the formation of an intramolecular hydrogen bond between its carboxyl and hydroxyl groups which may hinder its interactions with CDKs. 94

Figure 4. 5: Line model showing the interactions of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA with CDK1, CDK2, CDK4 and CDK6

95

Table 4. 1: Free energy binding values and hydrogen bond lengths for the interaction of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA with CDK1, CDK2, CDK4 and

CDK6

S. Receptor Ligand Interacting Measurement No of Energy No amino acids (A°) H value bonds (kCal/mol)

Aspirin Asn133, Tyr15 2.5, 3.0 2 -6.6 Salicylic Asp146, Lys33 2.8, 3.5 2 -6.4 1 CDK1 acid 2,3-DHBA Asp146 2.9 1 -5.9 2,5-DHBA Asp146 2.9 1 -5.9 Aspirin Asp145, 3.2, 2.6, 3.1, 4 -6.2 Glu51, 3.0 Lys33(2) Salicylic Asp145, 3.3, 3.0, 3.3 3 -5.7 2 CDK2 acid Lys33(2) 2,3-DHBA Lys33, Asp145 3.2, 3.2 2 -5.6 2,5-DHBA Asp145, 3.2, 2.9, 2.9 3 -5.9 Lys33(2) Aspirin His158, 2.9, 2.0 2 -5.8 Gln183 Salicylic His158, 2.8, 1.8 2 -5.4 3. CDK4 acid Gln183 2,3-DHBA His158, Cys68 3.1, 2.2 2 -5.8 2,5-DHBA His158 3.1 1 -5.4 Aspirin Lys43 2.9 1 -5.4 Salicylic Gly165, 3.1, 2.8, 3.0 3 -5.1 acid Leu166, Arg144 4. CDK6 2,3-DHBA Asp134, 2.0, 3.0, 3.2, 5 -5.4 Tyr292, 2.9, 2.9 Ala291(2), Thr198 2,5-DHBA Lys43(2), 3.2, 3.2, 3.2, 4 -5.3 Tyr24, Gly25 3.2 96

4.3.3 Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on colony formation

Clonogenic assay was performed to determine the effectiveness of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on colony formation in HCT-116 and HT-29 colon cancer cells as well as MDA-MB-231 breast cancer cells. HCT-116 and HT-29 cells were chosen to study the effect of these compounds as these cell line have been extensively used in studies pertaining to aspirin’s biological effects [195, 196]; MDA-MB-231 cells were chosen to determine if this effect was mimicked in another cancer cell line. We observed that aspirin and salicylic acid both failed to inhibit colony formation in HCT-116 (Fig. 4.6A and B), HT-29 (Fig. 4.7A and B) and MDA-MB-231 cells (Fig. 4.8A and B). 2,3-DHBA was effective in MDA-MB-231 cells at higher concentrations (>500 μM) (Fig. 4.8C), while it was ineffective in HCT-116 (Fig. 4.6C) and HT-29 cells (Fig. 4.7C). Interestingly, 2,5-

DHBA exhibited a dose dependent effect on colony formation in all cell lines examined

(Figs. 4.6D, 4.7D, 4.8D). In HCT-116 cells, decreased colony formation was observed with

2,5-DHBA at concentrations >250 μM, in HT-29 cells at concentrations >200 μM, and in

MDA-MB-231 cells, beginning at 50 μM. In contrast, two other salicylic acid derivatives

2,4-DHBA and 2,6-DHBA were unable to inhibit colony formation in MDA-MB-231 cells

(Fig. 4.9). These results clearly show that although the sensitivity varies among different cell lines, both 2,3-DHBA and 2,5-DHBA exhibit the ability to inhibit cancer cell proliferation in contrast to aspirin and salicylic acid.

Interestingly, 2,3-DHBA and 2,5-DHBA were ineffective in inhibiting cell proliferation in MDA-MB-231 cells over a range of concentrations (125-1000 μM) for 24-

48h (Fig. 4.10). We suggest that the lack of inhibition in acute treatment observed may be related to the poor uptake of these compounds. 97

Figure 4. 6: Effect of aspirin, salicylic acid and its metabolites on HCT-116 colony formation

Effect of aspirin (A), salicylic acid (B), 2,3-DHBA (C) and 2,5-DHBA (D) on colony formation in HCT-116 cells. Quantification of the data is shown beside the crystal violet stained image of the colonies. The graph is represented as mean ± standard deviation.

The concentration of the drugs used are indicated as micromolar (μM). 98

Figure 4. 7: Effect of aspirin, salicylic acid, 2,3-DHBA and 2,5-DHBA on HT-29 colony formation

Effect of aspirin (A), salicylic acid (B), 2,3-DHBA (C) and 2,5-DHBA (D) on colony formation in HT-29 cells. Quantification of the data is shown beside the crystal violet stained image of the colonies. The graph is represented as mean ± standard deviation.

The concentration of the drugs used are indicated as micromolar (μM). 99

Figure 4. 8: Effect of aspirin, salicylic acid and its metabolites on MDA-MB-231 colony formation

Effect of aspirin (A), salicylic acid (B), 2,3-DHBA (C) and 2,5-DHBA (D) on colony formation in MDA-MB-231 cells. Quantification of the data is shown beside the crystal violet stained image of the colonies. The graph is represented as mean ± standard deviation. The concentration of the drugs used are indicated as micromolar (μM). 100

Figure 4. 9: Effect of 2,4-DHBA and 2,6-DHBA on colony formation in MDA-MB-231 cells

Quantification of the data is shown beside the crystal violet stained image of the colonies. The concentration of the drugs used are indicated as micromolar (μM).

101

Figure 4. 10: Effect of 2,3-DHBA (A) and 2,5-DHBA (B) on cell number in MDA-MB-

231 cells

Cells were seeded at a density of 250,000 cells/ 100mm plate overnight and treated with the indicated compounds for 48h. Cells were then trypsinized and counted using an automated cell counter. More than 96% of the cells were viable. The concentration of the drugs used are indicated as micromolar (μM).

4.4 Discussion

Despite the growing evidence for aspirin’s ability to decrease CRC, the mechanisms involved remain poorly defined. Numerous hypotheses commencing from the COX inhibition in platelets [65, 98, 197] to increased expression of DNA repair proteins, transcription factors and interconnected signaling pathways have been proposed [198-201]; however, a consensus has not been reached regarding the target/pathway(s) primarily responsible for its anti-cancer effects [202]. In this research paper, we tested the hypothesis that aspirin metabolites 2,3-DHBA and 2,5-DHBA may mediate its chemopreventive actions by inhibiting CDKs and affecting cell growth. We provide evidence that 2,3-DHBA 102 and 2,5-DHBA inhibit CDKs involved in cell-cycle regulation (CDKs 1, 2, 4 and 6), although to different degrees. Inhibition of CDK1 by 2,3-DHBA and 2,5-DHBA was observed at 500 μM, and CDK-6 by 2,3-DHBA was observed starting at 250 µM, however, inhibition of other CDKs by these compounds required much higher concentrations (750

µM), suggesting that CDK1 and CDK6 are better targets for 2,3-DHBA and/or 2,5-DHBA.

In contrast, aspirin and salicylic acid both failed to inhibit CDK-1, 2, 4 and 6 enzyme activity. Through molecular docking studies we identified potential sites of these interactions; most of the interacting amino acids appear to be localized in the active site of these enzymes. Importantly, we also demonstrated the ability of aspirin/salicylic acid metabolites to inhibit clonal formation in cancer cell lines. 2,5-DHBA inhibited colony formation in a dose-dependent manner in three different cancer cell lines (HCT-116, HT-

29 and MDA-MB-231). In contrast, 2,3-DHBA exhibited effective inhibition in MDA-

MB-231 cells alone. These results suggest that 2,3-DHBA and 2,5-DHBA may be involved in aspirin’s chemopreventive actions. Our findings raise two important questions: 1) What are the sources of the aspirin metabolites 2,3-DHBA and 2,5-DHBA?; 2) Are there unique scenarios that gives an anatomical advantage to 2,3-DHBA and 2,5-DHBA to act against

CRC?

Plain aspirin (non-enteric coated) is absorbed in the stomach where the acidic environment protects it from deacetylation and ionization, resulting in its faster absorption; however, aspirin may also be absorbed in the upper small intestine and enter into the hepatic portal vein [203-205]. In addition, lymphatic uptake of some aspirin and salicylic acid has been reported [205]. We hypothesize that aspirin’s preferential effect against CRC

(over other tissues) may be related to the generation of salicylic acid that occurs both 103 through hydrolysis and the action of gut esterases [206] in the intestine or colon. Intact aspirin’s bioavailability is between 50-68% [101, 102], and it is estimated that nearly 29-

39% of aspirin may be hydrolyzed in the GI tract, particularly in the duodenum and ileum

[102]. Therefore, it can be argued that epithelial cells of the intestine and colon are likely to be exposed to substantially higher concentrations of salicylic acid compared to other tissues in the body. With enteric coated aspirin, the concentration of aspirin/salicylic acid exposed to the GI cells may be even greater than non-enteric coated tablets as the bioavailability of intact aspirin from enteric coated tablets is significantly lower compared to plain aspirin due to slow release and absorption in the intestine [203, 207].

Aspirin in the systemic circulation is known to be metabolized in the liver to 2,3-DHBA and 2,5-DHBA through the action of cytochrome P450 (CYP450s) enzymes. However, it is unlikely that these liver generated metabolites contribute to the anti-cancer effect in colorectal tissues due to their low concentrations in the plasma [150]. We suggest that there are two routes through which intestinal epithelial cells may get exposed to 2,3-DHBA and

2,5-DHBA. As intestinal epithelial cells also express CYP450s similar to the isoforms expressed in the liver involved in aspirin metabolism [208], one would expect aspirin metabolites to be generated in these cells; however, it is not clear at this stage to what extent these metabolites are produced within the GI cells. Since exposure of HCT-116, HT-29 and MDA-MB-231 cells to aspirin or salicylic acid (up to 1 mM) failed to inhibit clonal formation, it is likely that the contributions of cellular CYP450s to the formation of 2,3-

DHBA and 2,5-DHBA is negligible, although a small amount may still be produced through this route. We have not determined the effect of aspirin and salicylic acid on clonal formation at concentrations greater than 1 mM, therefore we are not sure if higher 104 concentrations will have an effect on clonal formation. An alternative possibility and an attractive hypothesis is that the significant amount of aspirin / salicylic acid left unabsorbed

(32-50%) in the lumen of the intestine and colon maybe metabolized by the intestinal microflora to generate hydroxyl derivatives of salicylic acid, which may include both 2,3-

DHBA and 2,5-DHBA. Bacteria are capable of metabolizing drugs through hydroxylations

[209], and one report in 2016 showed that incubation of aspirin with human fecal suspensions containing microbiota resulted in the degradation of aspirin to salicylic acid and hydroxylated salicylic acids [154]. The authors also showed that in rats orally administered with aspirin and ampicillin, the bioavailability of aspirin in the plasma was much higher compared to rats administered with aspirin alone, suggesting that microbiota contributes to the degradation of aspirin in the GI tract. An estimation of the salicylic acid present in the lumen of human gut has not been made, nor has the characterization of the hydroxyl derivatives of salicylic acid produced in the lumen been reported till date. The absence of experimental evidence on the potential formation of 2,3-DHBA and 2,5-DHBA, beyond what is reported in literature by Kim et al. [154], is a limitation of this study. Further work is also required to determine how these metabolites are taken up by the normal, non- cancerous epithelial cells of the intestine and colon and their effect on colony formation, which is also a limitation of this study. Regarding the uptake mechanism, one possibility is that 2,3-DHBA and 2,5-DHBA may be taken up by the monocarboxylate transporter

(MCT) SLC5A8 as it is implicated in the transport of other derivatives of salicylic acid

[171]. The differential sensitivity of HCT-116 and HT-29 vs. MDA-MB-231 could be attributed to the differential expression of this transporter due to mutational/ methylation- driven inactivation of the SLC5A8 gene as explained in the previous chapter. 105

We observed that 2,5-DHBA was universally effective in inhibiting colony formation as compared to 2,3-DHBA. Significant inhibition of clonal formation was observed with

2,5-DHBA at 500 μM in HCT-116 cells, at 250 μM in HT-29 cells and at 100 μM in MDA-

MB-231 cells, whereas 2,3-DHBA failed to inhibit colony formation in HT-29 and HCT-

116 cells (125-1000 μM). However, it inhibited the colony formation at ~500 μM in MDA-

MB-231 cells. It appears that 2,3-DHBA and 2,5-DHBA show differential inhibitory effects in different cancer cell lines and it is unclear whether this is due to the differences in uptake of these compounds by the cell lines tested. Two other salicylic acid derivatives namely 2,4-DHBA and 2,6-DHBA failed to inhibit colony formation in MDA-MB-231 cells (Fig. 4.9). This suggests that among the different salicylic acid derivatives tested, 2,5-

DHBA is universally effective whereas 2,3-DHBA is more selective as an anti-proliferative agent.

In contrast to the clear inhibitory effect of 2,3-DHBA and 2,5-DHBA in clonal formation (21 days), both compounds failed to show any appreciable inhibitory effect on cell number when cells were treated for 24-48 hours (acute treatment) at various concentrations (125 to 1000 µM; see Fig. 4.10). Similarly, in acute treatment experiments, both compounds failed to show an effect on cell cycle progression as measured by flow cytometry, and owing to the lack of an effect, we have not included this data. This discrepancy in the results between acute and chronic treatment may be related to the time required for uptake and accumulation of these compounds to exert an inhibitory effect. We suggest that acute exposure for 24-48 h may result in limited uptake of the drugs resulting in milder effects which are difficult to discern in flow cytometry analysis and cell counting, whereas chronic exposure for 21 days may result in significant accumulation of the drugs 106 sufficient to cause inhibition of cell proliferation as measured by clonal formation assays.

As we have not performed flow cytometry analysis on clonal cells following chronic exposure, it is not clear what stages of the cell cycle are affected by these compounds, and this is another limitation of the study.

Although the IC50 of the compounds tested in this study are in micromolar concentrations for both CDK inhibition and clonal formation, it could be argued that it is physiologically relevant in view of their abundance in the intestine due to the hydrolysis of aspirin to salicylic acid in the gut [101, 102]. It is also important to note that 2,3-DHBA and 2,5-DHBA are abundantly available in fruits and vegetables [210] and one study demonstrated that the phenolic acid content generated from the diet in the gut can rise to micromolar concentrations [211]. CDKs and other potential cellular targets probably have evolved to be less sensitive to these compounds to avoid complete inhibition of cell cycle at lower concentrations. The importance of 2,5-DHBA in cancer prevention was also demonstrated in a recent study which showed that it is effective in inhibiting colony formation and DNA synthesis in C6 glioma cells in vitro, and enhanced survival of Ehrlich breast ascites carcinoma bearing mice [212]. In addition, 2,5-DHBA has been implicated in a previous report as a potential aspirin metabolite formed through the action of tyrosinase enzyme in melanoma cells. The study stated that p-quinone formation from 2,5-DHBA was responsible for aspirin’s anti-melanoma effects through ROS formation [133]. Thus, although both the studies implicate anti-cancer effects of 2,5-DHBA, the mechanisms described are starkly different; in melanocytes it occurs through depletion of GSH and generation of ROS leading to cell apoptosis [133], while in our study (in HT-29, HCT-116, and MDA-MB-231 cells) it involves inhibition of CDKs leading to suppression of cancer 107 cell growth. This suggest that 2,5-DHBA may contribute to anti-cancer effects through distinct mechanisms in different cell types.

The demonstration of 2,3-DHBA and 2,5-DHBA to inhibit cancer cell growth is a significant observation in view of aspirin’s reported ability to decrease CRC. An unanswered question is: what is/are the target(s) for 2,3-DHBA and 2,5-DHBA in cells?

Our previously published data [166] and the present study show that 2,3-DHBA and 2,5-

DHBA inhibited CDK1/CDK6 activity. However, at this stage it is not known if these two observations are related, or if the inhibition of proliferation by these compounds is due to an alternative mechanism not involving CDKs. In this context, a previous study reported that 2,5-DHBA can interfere with FGF function by disrupting the receptor-growth factor signaling complex in vitro and in cell culture studies [213]. At this stage, we believe that

2,5-DHBA does not target COX-2 to mediate its chemo-preventive effect in the GI epithelial cells as HCT-116 cells are reported to lack COX-2 expression while HT-29 cells express inactive COX-2 [214].Since both these cell lines were inhibited by 2,5-DHBA, it suggests that the aspirin metabolites can inhibit cancer cell proliferation through a COX-2 independent mechanism. Therefore, greater efforts need to be placed into these relatively unexplored areas to determine how 2,3-DHBA and 2,5-DHBA exert their chemopreventive actions. Whether microbiota generated salicylic acid metabolites becomes available for

CDK inhibition following their uptake into the GI cells is an interesting area for future study. If proven, this will highlight the contribution of the gut microflora to aspirin’s chemoprevention against CRC, through formation of 2,3-DHBA and 2,5-DHBA. A model depicting how 2,3-DHBA and 2,5-DHBA generated in the GI lumen through the microbial degradation of aspirin/salicylic acid, leading to inhibition of CDKs and cell proliferation is 108 shown in Fig. 4.11. We suggest that the ability of aspirin to inhibit tumor formation in the intestinal/colonic mucosa may be a local effect via salicylic acid metabolites generated in the gut acting on epithelial cells and may not require absorption into the blood.

Figure 4. 11: A model depicting how aspirin metabolites may exert its chemo- preventive effects on colonic tissue to protect against CRC

We hypothesize that aspirin and salicylic acid may impede events/pathways crucial to tumor formation through its metabolites 2,3-DHBA and 2,5-DHBA. We suggest that

2,3-DHBA and 2,5-DHBA generated from salicylic acid by the action of gut microflora is taken up by the colonic epithelial cells. The uptake of these metabolites may occur through

SLC5A8, a monocarboxylic acid transporter. Accumulation of these metabolites within the epithelial cells may result in CDK inhibition. This may cause reduced cell proliferation, allowing an opportunity for DNA repair and immune surveillance, thereby preventing tumorigenesis. Alternatively, it is also possible that 2,3-DHBA and 2,5-DHBA may have additional targets contributing to cancer prevention. 109

Nearly 35 years have passed since the initial report of aspirin’s ability to decrease incidences of CRC [97], however, debates still continue on its mode of action. Although the platelet hypothesis is widely discussed in literature, it has its own short-comings and appears to work indirectly through a set of sequential steps involving two COX isoforms

(COX-1 and COX-2) [65, 98, 103, 197, 215, 216]. COX-1 is the only form of COX in platelets and is constitutively expressed in all tissues, whereas COX-2 is inducible and expressed under inflammatory conditions. COX-2 levels are also elevated in many cancers

[204, 217-220]. The half-life of intact aspirin is about 15-20 min [149] in the systemic circulation and this period appears to be enough to cause inhibition of COX-1 in platelets

[107]. As chronic inflammation is linked to cancer, it is suggested that aspirin may mediate its effect through inhibition of these COX enzymes. However, aspirin is more effective in the inhibition COX-1 (IC50 1.67 μM) than COX-2 (IC50 278 μM) [109]. Since low dose aspirin (75-160 mg) is as effective as higher doses [64], the possibility of aspirin directly targeting COX-2 as a potential mechanism to decrease cancer is thought to be unlikely because lower doses administered per day is insufficient to cause COX-2 inhibition. It is suggested that aspirin’s effects may involve sequential inhibition of COX-1 and COX-2

[65, 98, 215]. This theory states that inhibition of COX-1 in platelets leads to prevention of platelet aggregation which subsequently prevents the release of lipid mediators (e.g.,

PGE-2; TXA2), cytokines and growth factors (e.g. PDGF) from the alpha-granules. These events could inhibit the induction of COX-2 in regions of GI mucosal lesions, preventing tumorigenesis.

If inhibition of COX-1 in platelets in the blood following its absorption is the primary mechanism as proposed in the platelet hypothesis [65, 197, 215], one would expect aspirin 110 to decrease the incidences of cancer to the same extent in all tissues in the body, unless one assumes that GI mucosal tissues are more prone to lesions which leads to platelet aggregation, creating an environment suitable for eliciting an inflammatory response. It is intriguing that aspirin that is only present for less than 20 min. in the circulation, while capable of preventing platelet activation and aggregation, could have such a profound effect against CRC development. While inhibition of COX-1 in platelets may play a role

(e.g. in the inhibition of metastasis), it is likely that other pathways may play a major role in aspirin’s cancer preventive actions.

Although the “platelet hypothesis” is an attractive theory, we suggest that alternative mechanisms as proposed in this study involving locally generated salicylic acid metabolites by the gut microflora from aspirin as potential contributors to chemoprevention should be considered. As ~50% of orally administered aspirin is left unabsorbed in the GI lumen

[101, 102], one would expect the concentration of aspirin from an 81 mg tablet to be in the range of 0.3 mM to 1.4 mM in the gut assuming that the total GI fluid volume under fed and fasting conditions are ~750 ml and ~160 ml respectively [221]. Presence of microflora in the GI lumen may thus be able to generate 2,3-DHBA and/or 2,5-DHBA from aspirin/salicylic acid, sufficient to cause inhibition of cell proliferation, allowing aspirin to act locally on colorectal tissues, thus adding merit to our hypothesis. It is likely that locally generated metabolites will provide a more direct effect on colorectal tissues against CRC as compared to the indirect mechanism involving sequential steps proposed in platelet hypothesis through COX-1 and COX-2 inhibition. In vivo studies utilizing germ-free mice should shed light on the role of aspirin/salicylic metabolites and the microflora in aspirin’s chemo-preventive properties. 111

CHAPTER 5: IDENTIFICATION OF BACTERIAL SPECIES THAT CAN

METABOLIZE QUERCETIN

5.1 Abstract

The studies presented in chapter 3 demonstrated that flavonoid metabolites inhibit cancer cell proliferation through both CDK -dependent and -independent mechanisms.

Literature evidence suggest that the gut microbiota are capable of flavonoid biotransformation to generate bio-active metabolites including 2,4,6-THBA, 3,4-DHBA,

3,4,5-THBA and DOPAC. In this chapter, we screened 94 human gut bacterial isolates for their ability to biotransform the flavonoid quercetin into different metabolites. We demonstrated that of the 94 strains screened, 6 were able to degrade quercetin including

Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthi, Olsonella scatoligenes, Eubacterium eligens, and Lactobacillus spp. Additional studies showed that

B. glycinifermentans could generate 2,4,6-THBA and 3,4-DHBA from quercetin while F. plautii generated DOPAC. In addition to the differences in the metabolites produced, we also demonstrated that the kinetics of quercetin degradation was different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Similar to the anti-proliferative effects of 2,4,6-THBA and

3,4-DHBA demonstrated previously, DOPAC also exhibited potent inhibitory effect against colony formation, in vitro. Thus, as B. glycinifermentans and F. plautii generate metabolites possessing anti-proliferative activity, we suggest these strains have the potential to be developed into probiotics to improve human gut health. 112

5.2 Background and hypothesis

In the past, clinical and epidemiological studies have largely focused on the ability of flavanols to prevent cancer. Of these flavanols, quercetin has been extensively studied not only for its anti-cancer effects but also for its anti-inflammatory, anti-thrombotic, anti- neurodegenerative, anti-infectious and immunomodulatory activities [222, 223]. Quercetin is one of the most abundant flavonoids in the diet and can be found in many foods including onions, apples, grapes, berries, citrus fruits, tea, cherries and broccoli. It has been estimated that humans consume 10–100 mg of quercetin every day on average through their diet

[222]. The reported health benefits are predicted to occur through quercetin’s (a) interaction with cellular receptors, (b) modification of signal transduction pathways, (c) antioxidant properties, and (d) apoptosis regulation, to name a few [224-226].

Quercetin however is not freely present as an aglycone in most of these food sources.

It is usually conjugated to a sugar moiety (forming glycosides) that confers water solubility and chemical stability to these compounds [227]. The sugar moiety attached to quercetin is usually glucose or rhamnose, but it could also be galactose, arabinose, xylose, or other sugars [228]. Quercetin glycosides are poorly absorbed from the intestine, and hence these linkages are cleaved by enzymes either present in the small intestine or the colon to facilitate absorption [141, 229]. As the enzymes produced by the human body is sometimes incapable of cleaving the glycosidic linkages, microbial enzymes produced have been implicated in these deconjugation reactions. For example, studies have suggested that the hydrolysis of rutin (quercetin-3-O-rutinoside; from onions, berries etc.) to quercetin occurs through the action of the gut microbiota [230, 231]. Studies highlighting the importance of 113 the gut microflora in human health are now emerging, wherein the microbiota is considered as a separate organ by itself [232-235].

Numerous studies have implicated the dysregulation of the microbial ecology in the intestine to be a cause for colorectal cancers (CRC). These studies have also indicated that diet is a major factor governing the dysbiosis wherein regular intake of fruits and vegetables seem to favorably shift the ecology of the gut microbes to a cohort that can prevent the occurrence of CRC [139, 236-240]. Increasing studies are also implicating a beneficial role for the microbial metabolites in human health, especially in cancer [136,

171, 241]. Taken together, these studies are indicative of the importance of the gut microbes and their catabolites following flavonoid degradation in influencing human health. Hence, identifying the mechanisms involved in flavonoid catabolism, and the various microbial enzymes involved in this process, would aid in understanding and establishing the key contributors of the beneficial metabolites.

Studies conducted this far have identified quercetin-2,3-dioxygenase (quercetinase) as the first enzyme in Bacillus spp. that is required for the metabolism of quercetin [242]. The reaction catalyzed by quercetinase is shown in Fig. 5.1A. As can be seen from this reaction, the ester product 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate can then be cleaved by the gut esterases to 2,4,6-THBA and 3,4-DHBA. Other studies have also demonstrated that alternative pathways may also exist for quercetin degradation, generating 3,4-dihydroxyphenylacetic acid (DOPAC) through biotransformation of this flavonoid by other bacteria (Fig 5.1B)[136, 243]. The generation of DOPAC from quercetin is predicted to occur through the involvement of phloretin hydrolase [244]. A recent study also suggested the involvement of ene-reductases in the biotransformation of 114 flavones and flavonols [245]. Apart from these enzymes, other enzymes known to be involved in flavonoid metabolism include chalcone isomerase and enoate reductase from

Eubacterium ramulus [246], and peroxidases, dehydrogenases, demethylases and tyrosinases from a variety of bacteria [247].

Figure 5. 1: Catabolic pathways proposed for quercetin degradation

(A) Reaction representing the microbial catabolism of quercetin to 2-(3,4- dihydroxybenzoyloxy)-4,6-dihydroxybenzoate and carbon monoxide by quercetinase found in Bacillus spp. [248] (B) Proposed mechanism of quercetin degradation to generate

DOPAC [136]

In the preceding chapters we demonstrated the ability of 2,4,6-THBA, one of the metabolites generated from flavonoid degradation by the gut microbiota, to inhibit cancer cell growth, possibly through inhibition of CDK enzymes [171]. This provided a basis for the hypothesis that the human gut microbiota maybe crucial for the generation of anti- cancerous metabolites from flavonoids. The goal of this chapter was to identify the bacteria capable of degrading quercetin, a flavonol, and identify the metabolites, with a focus on 115

2,4,6-THBA, 3,4-DHBA and 3,4-dihydroxyphenyl acetic acid (DOPAC). Our results identified 6 bacterial strains (from the human gut bacterial library) with flavonoid degrading capability. Among these strains, Bacillu glycinifermentans generated 2,4,6-

THBA and 3,4-DHBA, while Flavonifractor plautii generated DOPAC from quercetin, with different kinetics of degradation between the two bacterial strains. Additionally, we demonstrated that DOPAC has potent anti-proliferative properties against cancer cells, highlighting the importance of the contribution of bacteria in generating bio-active metabolites for the prevention of cancers.

5.3 Results

5.3.1 Screening the human gut bacterial library for quercetin degrading strains

Ninety-one bacterial strains obtained from the human gut library and 3 standard strains obtained from DSM, Germany were individually screened for their ability to degrade quercetin under anaerobic conditions in 7N minimal media containing 75μg/ml quercetin (7NQ media) as described in materials and methods. To ensure that the quercetin peak did not coincide with any other components present in the spent media, we also grew the cells in 7N media and analyzed the spent supernatant through HPLC. We also had a blank 7NQ media (without any bacterial inoculation; control) to account for the auto- degradation of quercetin. The results obtained from the bacterial spent media were compared to that of the 7NQ media control and the results are represented as percentage of control. As can be seen from figure 5.2, only 6 of the 94 strains tested had the ability to degrade >20% of the quercetin that was supplemented to the media (indicated in orange).

This includes Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthi,

Olsonella scatoligenes, Eubacterium eligens, and Lactobacillus spp. 116

Figure 5. 2: Quercetin degradation by the human gut bacterial library

Figure shows the degrading capability of each bacterial species as percentage degradation when compared to control. The results indicate that 6 strains (in orange) of the

94 strains tested exhibited >20% quercetin degradation. 117

5.3.2 HPLC analysis of Bacillus glycinifermentans and Flavonifractor plautii reveal

different metabolites produced following quercetin degradation

Among the 6 bacterial strains that could degrade quercetin (>20%), Bacillus glycinifermentans (B. glycinifermentans) and Flavonifractor plautii (F. plautii) exhibited maximum quercetin degradation under anaerobic incubation for 48 h (Fig. 5.2). Therefore, it was decided to focus on these two strains for their ability to biotransform quercetin under experimental conditions. It is important to note that while B. glycinifermentans is a newly identified bacteria [249, 250] present in the human gut, its abundance in the human gut is not fully characterized. On the other hand, F. plautii appears to be fairly distributed in individuals as reported in literature [251]. It is also important to highlight that under the culture conditions, the growth of B. glycinifermentans was profuse while that of F. plautii was sparce (Fig 5.3). As demonstrated in Fig 5.3, peak growth occurred at 21h and subsequently decreased after 36h for B. glycinifermentans. On the other hand, the growth pattern of F. plautii was slower but sustained over the time-period examined. This indicated that these two bacteria have different growth patterns in 7N minimal media. Despite the poor growth that F. plautii exhibited, it was able to degrade quercetin much faster than B. glycinifermentans. A time-course of the degradation pattern of quercetin by these two bacterial strains is shown in Fig. 5.4. As can be seen, the degradation of quercetin was almost complete at 48h for B. glycinifermentans, while this was achieved within 12 h by

F. plautii.

118

Figure 5. 3: Growth pattern of B. glycinifermentans (A) and F. plautii (B) in 7N minimal media

The bacterial strains were inoculated to an OD600 of 0.05 and allowed to grow for

48h under anaerobic conditions. Aliquots from the culture media was removed periodically and OD600 was measured at regular intervals. 119

Figure 5. 4: Quercetin degradation pattern of different bacterial species

(A) Kinetics of quercetin degradation by B. glyinifermentans (B) Kinetics of quercetin degradation by F. plautii 120

HPLC analysis of the spent media for B. glycinifermentans and F. plautii revealed that each bacterial species produced different metabolites upon incubation with quercetin for 24 h. 2,4,6-THBA and 3,4-DHBA was clearly detectable in the spent media isolated from B. glycinifermentans culture. The levels of 2,4,6-THBA was comparatively lower than that of 3,4-DHBA (Fig. 5.5A), while 4-HBA and DOPAC was not detected. In contrast, the spent media from F. plautii revealed the presence of DOPAC, however, 2,4,6-

THBA, 3,4-DHBA and 4-HBA were undetected (Fig. 5.5B). Consistent with the results obtained in Fig. 5.4, F. plautii completely degraded quercetin within 24 h of exposure while

B. glycinifermentans showed > 60% quercetin degradation (Fig. 5.5). 121

Figure 5. 5: Detection of metabolites generated by B. glycinifermentans (A) and F. plautii (B) following quercetin degradation

HPLC revealed that B. glycinifermentans generated 2,4,6-THBA and 3,4-DHBA

(A) while F. plautii generated DOPAC (B) upon exposure to Quercetin for 24h.

Concentration, estimated through standard curves using pure compounds, of each product is expressed in μg/ml. 122

5.3.3 Degradation kinetics and different end products suggest different mechanisms

of quercetin degradation by B. glycinifermentans and F. plautii

The differences in the metabolites produced between B. glycinifermentans and F. plautii possibly suggested that that these bacteria utilize different pathways for quercetin biotransformation. Therefore, an investigation was carried out to determine the presence of quercetin degrading enzyme activity in the culture supernatant and bacterial cell lysates.

Bacteria were grown under identical conditions as described in Fig. 5.5, and the supernatant and bacterial cell lysates were obtained. When assayed for the quercetin degradation, B. glycinifermentans exhibited higher units of enzyme activity in the supernatant when compared to cell lysate (Fig. 5.6A). One unit of enzyme activity is defined as the amount of enzyme required to convert 1 µmol of quercetin in one min at 25°C [177]. Interestingly, the supernatant of F. plautii did not exhibit any quercetin degrading activity; its cell lysate however showed minimal enzyme activity (Fig. 5.6B). Taken together, this indicates that

B. glycinifermentans mediated degradation of quercetin occurs possibly through a secreted enzyme while F. plautii requires the live culture for the biotransformation of quercetin that most likely occurs intracellularly or at the cell surface.

123

Figure 5. 6: Quercetin degrading enzyme activity as determined in the culture supernatant and cell lysates of B. glycinifermentans (A) and F. plautii (B)

As can be seen from the figure, higher enzyme activity was observed in the culture supernatant of B. glycinifermentans when compared to the cell lysate (A). In contrast, minimal enzyme activity was observed in the cell lysate of F. plautii, while there was no quercetin degradation observed in its culture supernatant (B).

5.3.4 Clonogenic assays with DOPAC

Clonogenic assay was performed to demonstrate the effectiveness of DOPAC against colony formation in SLC5A8-pLVX and HCT-116 cells. Figure 5.7A demonstrates 124 that in SLC5A8-pLVX cells, DOPAC decreased the colony formation beginning at 7.3

µM. In HCT-116 cells (Fig. 5.7B), higher concentrations of DOPAC were required to inhibit colony formation (≥ 15.62 μM). The anti-proliferative effect of DOPAC, observed in this study is similar to the observations reported previously in literature [145].

Figure 5. 7: Effect of DOPAC on SLC5A8-PLVX and HCT-116 colony formation

Effect of DOPAC on colony formation in (A) SLC5A8-pLVX cells and (B) HCT-

116 cells. Quantification of the data is shown beside the crystal violet stained image of the colonies. The graph is represented as mean ± standard deviation. The concentration of the drugs used are indicated as micromolar (μM).

5.3.5 Clonogenic assay using HCT-116 cells with spent bacterial culture supernatant

The results obtained in Fig 5.7 on the effect of pure DOPAC on HCT-116 and

SLC5A8-pLVX cells prompted us to examine the growth inhibitory properties of the spent 125

bacterial culture supernatant on both cell lines. Bacterial cells were inoculated at an OD600 of 0.05 in 25 mL of 7N minimal media, with and without quercetin (37.5 μg/ml for B. glycinifermentans and 75 μg/ml for F. plautii) and grown anaerobically for 24 h. These concentrations were selected to ensure that incubation with the corresponding bacteria for

24 h, will result in complete degradation of quercetin (based on previous data). The culture supernatant was then harvested through centrifugation, and any remaining bacterial cells/particles were removed through filtration (0.22μm filters). HPLC analysis showed that supernatants from both bacterial cultures contained only the metabolites and did not contain any quercetin (data not shown), suggesting that any anti-proliferative effect observed would most likely be linked to the metabolites. One milliliter of the sterile culture supernatant was used for colony formation assay as described in Fig. 5.7. Following incubation for 14-21 days, with three changes in medium and treatments, the colonies were stained using crystal violet and colonies were counted. As can be seen from Fig 5.8, the culture supernatant of B. glycinifermentans containing quercetin showed marginal inhibition (~ 3% inhibition compared to supernatant without quercetin) whereas significant inhibition was observed in the cells incubated with the culture supernatant of F. plautii grown in the presence of quercetin (~ 65% inhibition compared to supernatant without quercetin).

126

Figure 5. 8: Clonogenic assay using HCT-116 cells treated with spent bacterial culture supernatant

Effect of spent supernatants from B. glycinifermentans and F. plautii cultures grown in the absence (-Quercetin) and presence (+Quercetin) of quercetin on colony formation in HCT-116 cells. Quantification of the data is shown beside the crystal violet stained image of the colonies. The graph is represented as mean ± standard deviation. While in some plates the distribution of the 500 cells seeded were homogeneous, others had clusters of cells localized to certain regions of the plate, owing to their placement in the incubator. All plates were however seeded with 500 cells/plate and allowed to form colonies.

The observed marginal effect of the B. glycinifermentans supernatant on HCT-116 cells is likely to be due to the lower concentrations of 3,4-DHBA and 2,4,6-THBA as observed through HPLC (data not shown). Estimation of the metabolites in the culture supernatant showed that 1 ml of the B. glycinifermentans supernatant contained 68.93 μg and 16.17 μg of 3,4-DHBA and 2,4,6-THBA respectively. This roughly translates to 40.66 127

μM of 3,4-DHBA and 8.64 μM of 2,4,6-THBA in a volume of 10 ml used for colony formation assay. On the other hand, 1 ml of the culture supernatant from F. plautii contained 20.45 μg of DOPAC, which is equivalent to 11.05 μM of DOPAC in 10 ml of culture supernatant, and this concentration appears to be sufficient to cause inhibition of cell proliferation. However, when we tested for the anti-proliferative ability of the bacterial culture supernatants of B. glycinifermentans and F. plautii in SLC5A8-pLVX cells, inhibition of colony formation was observed irrespective of whether the supernatant was obtained from the bacterial cultures grown in the presence or absence of quercetin (Fig

5.9). This suggests that SLC5A8-pLVX cells are inherently sensitive to the components present in the bacterial culture supernatant. Further work is required to identify the nature of this inhibition.

128

Figure 5. 9: Clonogenic assay using SLC5A8-pLVX cells treated with spent bacterial culture supernatant

Effect of spent supernatants from B. glycinifermentans and F. plautii cultures grown in the absence (-Quercetin) and presence (+Quercetin) of quercetin on colony formation in SLC5A8-pLVX cells. As can be seen, there was inhibition of cell growth irrespective of whether the supernatant was obtained from the bacterial cultures grown in the presence or absence of quercetin, suggesting that some component of the bacterial culture supernatant was inhibiting cancer cell growth. Further work is required to identify the nature of this inhibition.

5.4 Discussion

The flavonoid quercetin has been implicated for its health benefits against numerous disorders including inflammation, hypertension, obesity, and atherosclerosis [252].

Importantly, reports also indicate a role for quercetin in the prevention of many types of cancers [224, 237]. Quercetin is predicted to exert its anti-cancer effects through numerous 129 mechanisms including (a) interacting with growth receptors, (b) modifying signal transduction pathways including the MEK/Erk pathway and Nrf2/keap1 signaling, (c) through its antioxidant properties, and (d) apoptosis regulation [224-226]. Increasing evidence in literature suggests that the metabolites of quercetin may be responsible for its observed health benefits. Three observations that support a role for quercetin metabolites include: (1) pH dependent degradation of quercetin in the intestine (in the basic environment), (2) the low absorption of quercetin in the intestine resulting in low bioavailability, and (3) the biotransforming capability of the resident gut microflora. The results presented in this chapter has demonstrated the ability of select human gut bacteria to generate the metabolites 2,4,6-THBA, 3,4-DHBA and DOPAC. These results now tie in well with those presented in chapter 3 where we demonstrated the ability of some of these metabolites to inhibit cancer cell growth. It also gives credence to the metabolite hypothesis proposed in chapter 6 which suggests that: (1) bacteria are capable of biotransforming flavonoids and (2) the cancer preventive actions of flavonoids are likely mediated by its metabolites acting on specific cellular targets.

Our attempts to screen the human gut bacterial library yielded 6 bacteria capable of degrading quercetin. These include B. glycinifermentans, F. plautii, B. eggerthi, O. scatoligenes, E. eligens, and Lactobacillus spp. Of these strains, this is the first demonstration of quercetin degradation by B. glycinifermentans, B. eggerthi, O. scatoligenes, and E. eligens. All 6 species identified in this study have been demonstrated to be present in human fecal content, suggesting that several species in the gut are capable of biotransforming quercetin. Our demonstration of the ability of F. plautii and 130

Lactobacillus species degrading quercetin is also consistent with previously published reports [136, 243, 251, 253].

Although F. plautii has previously been reported to degrade quercetin to generate

DOPAC, studies described in this chapter, for the first time, has identified the ability of B. glycinifermentans to biotransform quercetin to generate 2,4,6-THBA and 3,4-DHBA. This is a novel finding. B. glycinifermentans, which was reported to be part of the human fecal content by Ghimre et al. [175], was initially characterized as being present in fermented soybean paste and hence the name B. glycinifermentans [249]. Interestingly, based on the analysis of its complete genome, this bacterium was suggested for use as a probiotic for livestock to enhance immune stimulation, enzyme production and pathogen inhibition

[250]. The link between our observation that it is capable of biotransforming quercetin to generate 2,4,6-THBA and 3,4-DHBA, and its suggested use as a probiotic in the previous report [250], makes this bacterial strain an interesting candidate for the maintenance of human gut health. The isolates investigated in this study involved three Bacillus spp, namely B. circulans, B. glycinifermentans and B. andreraoultii (Fig. 5.2). It is intriguing to note that among these 3 strains, only B. glycinifermentans was able to biotransform quercetin under experimental conditions, while the other 2 strains did not have any effect.

Further studies such as the comparison of the genome sequences or transcriptomic analyses are needed to understand the unique ability of B. glycinifermentans to biotransform quercetin when compared to the other two Bacillus strains.

The detection of DOPAC as a metabolite of quercetin generated by F. plautii in this study confirms the previous reports in literature [251]. In this study, we did not detect the presence of 2,4,6-THBA and 3,4-DHBA in the spent media from F. plautii. Although the 131 reason for this is not currently known, it is clearly not related to the sensitivity of the techniques employed, as both HBAs were detected in the spent media from B. glycinifermentans. An interesting possibility is that the quercetin degradation pathway utilized by F. plautii is radically different from that of B. glycinifermentans, and hence generates different metabolites. This is supported by the observation that the spent culture supernatant from B. glycinifermentans had quercetin degrading enzyme activity while this was absent in the culture supernatant of F. plautii (Fig. 5.6). This observation also suggests that the quercetin degrading enzyme in B. glycinifermentans and F. plautii is likely to be differentially localized (secreted Vs membrane bound/intracellular). Also, from the degradation kinetics, it is evident that F. plautii completely degrades quercetin within 12 h of incubation, whereas B. glycinifermentans requires around 48 h for complete quercetin degradation. This along with the minimal enzyme activity detected in the cell lysate (Fig.

5.6B) suggests that the degradation of quercetin by F. plautii may require the presence of live bacterial cells. A recent study by Yang et al., demonstrated the involvement of ene- reductase, chalcone isomerase, enoate reductase and phloretin hydrolase in the generation of metabolites from flavones and flavonols [245]. Hence, the lack of quercetin degradation in the culture supernatant and minimal activity in the pellet observed in our study (Fig

5.6B) may also be related to the requirement of four different enzymes for the generation of DOPAC from quercetin. Alternatively, it is also possible that the enzymes involved in

F. plautii is sensitive to the experimental conditions when assayed in vitro, which may not be the case for B. glycinifermentans. Hence, further investigation should shed light on the specific pathways utilized by these bacteria for quercetin degradation. 132

The importance of 2,4,6-THBA and 3,4-DHBA in the inhibition of cancer cell growth has been well established in the preceding chapters (Chapter 3), and in this chapter we have demonstrated the anti-proliferative effect of DOPAC on cancer cells. It is to be noted that

DOPAC has also been shown to inhibit cancer cell proliferation by other investigators in various cancer cell types [145, 243, 254]; this is believed to occur through its antioxidant properties. The ability of B. glycinifermentans to degrade quercetin to 2,4,6-THBA and

3,4-DHBA, and F. plautii to generate DOPAC from quercetin suggests that, in an in vivo scenario, the colorectal tissues are likely to be directly exposed to these anti-cancerous metabolites. Interestingly, one report also demonstrated that F. plautii along with 3 other bacterial species, was capable of inhibiting the growth of Clostridium perfringens, a pathogenic bacterium, highlighting its importance in gut health [255].

Human gut is known to harbor 300-500 species of bacteria [256, 257], this study has investigated the potential of only 94 bacterial strains to degrade quercetin. Therefore, additional screening is required to establish the contribution of other bacterial strains to

CRC prevention as well. However, it is interesting to note that of the 94 strains screened, only 6 of them exhibited the ability to degrade quercetin. This suggests that the flavonoid biotransforming ability may be narrowly restricted to only a few species of bacteria, highlighting the importance of these bacteria in the prevention of CRC. While the focus of our study was on quercetin, it is to be noted that the diet also contains other flavonoid members (such as anthocyanins, EGCG, Catechins, cyanidin-3-glucoside etc.) that may be biotransformed by the gut microflora [258]. For example, DOPAC, 2,4,6-THBA and 3,4-

DHBA has been reported to be metabolites produced upon green and black tea consumption [145, 259] while 2,4,6-THBA and 3,4-DHBA have been demonstrated to be 133 generated upon cyanidin-3-glucoside consumption [141]. Also, in this study, the bacterial strains were grown individually to screen for their ability to degrade quercetin, it is still unknown how quercetin may be degraded in the presence of other bacteria in co-cultures.

It is known that some bacterial species influence the growth of others, diet is also suggested to contribute to this selection [258, 260]. Therefore, additional studies are required to establish how diet influences the growth of these species and overall degradation of quercetin in vivo. It will be interesting to explore the metabolism of quercetin by other species of bacteria, enzymes involved in this process and characterize the metabolites generated individually or in a community setting. Additional studies are also required, to test the bacterial culture supernatants containing these metabolites, for their ability to inhibit cancer cell proliferation. It is also important to emphasize that besides bacteria, the microflora in the human gut also comprise of viruses, fungi and archaea [261] and hence their contributions to cancer prevention may also be significant.

The studies described in this chapter has identified 6 strains of bacteria capable of degrading quercetin to give different bio-active metabolites, some of which have been previously characterized to have anti-proliferative effects against cancer cells. This study also established clear differences between two bacterial species in terms of their ability to degrade quercetin, in addition, also showed the generation of structurally different metabolites. We believe that bacteria mediated biotransformation of flavonoids and generation of the metabolites are important contributors to cancer prevention observed in a diet rich in flavonoids.

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CHAPTER 6: SUMMARY, PERSPECTIVES, AND FUTURE STUDIES

Cancer is a genetic disease which arises due to mutations in protooncogenes and tumor suppressor genes whose imbalances in expression leads to uncontrolled cell proliferation.

Therefore, effective preventive measures for cancer requires identification of compounds that target proteins that regulate cell proliferation. In this regard, flavonoids and aspirin hold great promise for the prevention of cancer; however, despite considerable efforts in identifying their mechanism of action, a consensus has not been reached. It appears that flavonoids and aspirin and their metabolites affect many enzymes, proteins, transcription factors, and signaling pathways involved in cell proliferation and cancer development, making the identification of its primary target difficult. An effort has been made in this investigation to identify a common pathway that may explain the cancer prevention properties of flavonoids and aspirin.

The studies carried out in this dissertation explored the role of metabolites in CRC prevention by flavonoids and aspirin. The metabolites tested included 2,4,6-THBA, 3,4-

DHBA, 3,4,5-THBA, and 4-HBA known to be generated from flavonoid metabolism and

2,3-DHBA and 2,5-DHBA, known to be generated from aspirin metabolism. Studies were conducted on how these metabolites affect CDK enzyme activity using commercially obtained enzymes and the effect of these compounds on cancer cell growth were also investigated. Additionally, a human gut bacterial library was screened for identifying the bacterial strains capable of degrading the flavonoid quercetin, and the metabolites generated were identified. The major findings from this study are indicated below:

135

6.1 Summary from Chapter 3

• 2,4,6-THBA dose dependently inhibited CDKs 1, 2 and 4 enzyme activity,

however, 3,4-DHBA, 3,4,5-THBA and 4-HBA were ineffective in inhibiting these

enzymes. This suggests that the position and number of the hydroxyl groups in the

benzene ring may be key determinants for exerting inhibitory effects.

Phloroglucinol, that lacks the carboxyl group, failed to inhibit CDK enzyme activity

which demonstrated the importance of the carboxyl group in enzyme inhibition.

• Molecular docking studies revealed key interactions of 2,4,6-THBA with CDKs 1,

2 and 4. The interactions were found to be in the active site of the enzymes,

suggesting that 2,4,6-THBA may compete with the ATP-binding site. This suggests

that these metabolites may act as ATP-competitive inhibitors.

• Despite the potent inhibitory effect of 2,4,6-THBA on CDK enzyme activity, it

failed to inhibit cancer cell growth in HCT-116 and HT-29 colon cancer cells.

Further studies demonstrated that 2,4,6-THBA dose-dependently inhibited cancer

cell growth in a breast-cancer cell line expressing a functional MCT – SLC5A8

(SLC5A-pLVX cells). This demonstrated that a transporter function is necessary

for the growth inhibitory effect of 2,4,6-THBA.

• Consistent with the growth inhibition observed in transporter expressing cells,

HPLC studies demonstrated significant and dose-dependent uptake of 2,4,6-THBA

in cells having functional transporters (SLC5A8).

• In SLC5A8-pLVX cells, 2,4,6-THBA dose-dependently induced p21Cip1 and

p27Kip1 CDK inhibitory proteins as measured by qPCR and western blotting. 136

• Cell cycle analysis revealed that 2,4,6-THBA does not cause accumulation of cells

at any stage of the cell cycle; neither did it induce apoptosis. This suggests that

2,4,6-THBA possibly arrests cell cycle at all stages, without causing accumulation

at any particular stage, and without inducing apoptosis. These results also suggest

that 2,4,6-THBA is mostly a cytostatic agent than a cytotoxic agent.

• As expected, 2,4,6-THBA failed to inhibit colony formation in HT-29, HCT-116

cells; however, it dose-dependently inhibited colony formation in SLC5A8-pLVX

cells. In contrast, 3,4-DHBA and 3,4,5-THBA inhibited colony formation in all

three cell lines, while 4-HBA did not inhibit colony formation in any cell line.

HPLC analysis further revealed that 3,4-DHBA and 3,4,5-THBA were not taken up

into the cells, and 4-HBA was taken up by all cell lines tested.

• The results above show that 2,4,6-THBA causes cancer cell growth arrest through

a CDK and SLC5A8 -dependent pathway whereas 3,4-DHBA and 3,4,5-THBA

arrest cancer cell growth through a CDK and SLC5A8 -independent pathway. The

inability of 3,4-DHBA and 3,4,5-THBA to inhibit CDK, their lack of cellular

uptake and their ability to inhibit cell growth suggests that their targets are most

likely extracellular. These results suggest that although structurally related, the

targets and modes of actions for these HBAs are different in contributing to their

ability to prevent cancer cell growth.

6.2 Summary from Chapter 4

• Aspirin metabolites 2,3-DHBA and 2,5-DHBA inhibited commercially available

CDK 1 and 6 enzyme activity in vitro. 137

• Consistent with the enzyme inhibition, these compounds also inhibited cancer cell

colony growth. However, it is to be noted that 2,5-DHBA was universally effective

and inhibited HT-29, HCT-116 and MDA-MB-231 cell lines while 2,3-DHBA was

more effective in MDA-MB-231 cells.

• Molecular docking studies revealed key interactions of 2,3-DHBA and 2,5-DHBA

with specific amino acids in the active site of the CDK enzymes.

• Structurally related derivatives of salicylic acid, 2,4-DHBA and 2,6-DHBA, which

were previously reported to inhibit CDK enzyme activity [166], failed to inhibit

colony formation in cancer cell lines tested.

• Importantly, aspirin and salicylic acid themselves were not effective in inhibiting

both CDK enzyme activity and cancer cell colony formation even at 1 mM

concentration. This again highlights the importance of metabolites in aspirin’s anti-

cancer effects.

6.3 Summary from Chapter 5

• Ninety-four human gut bacteria were screened for their ability to degrade quercetin

under anaerobic conditions mimicking the human gut environment.

• Six bacterial species – B. glycinifermentans, F. plautii, B. eggerthi, O. scatilogenes,

E. eligens, and Lactobacillus spp. exhibited the ability to degrade >25% of the

supplemented quercetin.

• B. glycinifermentans degraded quercetin to yield 2,4,6-THBA and 3,4-DHBA.

• F. plautii degraded quercetin to yield DOPAC. 138

• Besides the different metabolites, the kinetics of quercetin degradation by B.

glycinifermentans and F. plautii appear very different, suggesting that different

pathways may be utilized by these species for quercetin biotranformation.

• This study also suggests the possibility that B. glycinifermentans and F. plautii may

be good probiotic candidates along with flavonoids to improve human gut health.

However, in vivo studies are required to explore this possibility.

6.4 The metabolite hypothesis – a common mechanism for cancer prevention

The results obtained in this dissertation has led to the proposal of a novel hypothesis that potentially explains HBA as common mediators of cancer prevention by flavonoids and aspirin. The unstable nature of flavonoids, the rapid hydrolysis of aspirin in the gut and the reports on the cancer-preventive potential of their degraded products through inhibition of cancer cell growth collectively suggest that the HBAs generated from flavonoids and aspirin may be key contributors to their cancer prevention properties. Unabsorbed flavonoids and aspirin/salicylic acid may act as substrates for the gut microbes to degrade them into simpler HBAs generating 2,3-DHBA, 2,5-DHBA, 3,4-DHBA, 3,4,5-THBA and

2,4,6-THBA. This generation of HBAs in the gut compel us to propose a central role for these molecules in cancer prevention by aspirin, flavonoids and the diet. As these HBAs are metabolites generated through biotransformation in the body and are also secondary metabolites in plants with the capacity to inhibit cancer cell growth, we would like to refer to this hypothesis as “metabolite hypothesis”. A model depicting the convergence of the pathways generating HBAs from aspirin, flavonoids and diet through microbial/host enzymes is shown in Fig. 6.1. Cancer prevention by HBAs is relatively underexplored, and further investigations are required to identify potential targets (intracellular vs. 139 extracellular), their uptake mechanisms by cells, signaling pathways and target gene expression. Such studies would have tremendous implications in developing new strategies for cancer prevention. Table 6.1 describes the various metabolites generated from aspirin and flavonoids and their varying abilities to inhibit CDKs and cancer cell growth.

Table 6. 1: Table showing the source of the HBA metabolites, their potential to inhibit

CDKs and their ability to retard cancer cell growth

Compound Aspirin Flavonoid CDK Inhibition Reference metabolite metabolite inhibition of cancer cell growth 2,3-DHBA + - + + [262] 2,5-DHBA + - + + [262] 2,4,6-THBA - + + + (in the [171] presence of a functional SLC5A8) 3,4,5-THBA - + - + [165, 171] 3,4-DHBA - + - + [162, 171] 2,4-DHBA - + + - [262] 2,6-DHBA - + + - [262] 4-HBA - + - - [171] 140

Figure 6. 1: Metabolite hypothesis: Model depicting convergence of the pathways generating HBAs from parent compounds through host/microbial enzymes

We propose that actions of HBAs, generated through biotransformation of aspirin and flavonoids, retard rate of cell proliferation. This would provide an opportunity for immune surveillance leading to either the destruction of cancer cells or DNA repair in cells containing damaged DNA, providing genetic stability, both of which are important steps in the prevention of tumorigenesis. While 2,4,6-THBA is likely to retard cell proliferation through CDK inhibition and upregulation of p21Cip1 and p27Kip1, the exact mechanisms of cell growth inhibition by other HBAs is still not understood [171, 262]. 141

6.5 Perspective on the role of flavonoid degrading bacteria and their metabolites in decreasing CRC

Flavonoids being unstable, undergo degradation to generate simpler phenolic acids and this process can occur through alterations in the pH, temperature and through microbial degradation to generate phenolic acids making it likely that the colonic epithelial cells are exposed to large amounts of these compounds. One of the major issues described in connection with flavonoids is their poor bioavailability; yet, they have been described as effective in reducing CRC. This raises the possibility that, along with the parent flavonoids, the phenolic acids may significantly contribute to cancer prevention. Evidence published from literature in the last 10 years are in favor of a role for the metabolites of flavonoids rather than the parent compounds in cancer prevention. The studies described in this dissertation further reiterates the importance of these metabolites in cancer prevention observed for flavonoid compounds.

Of the four major metabolites generated from flavonoid metabolism, the observation that 2,4,6-THBA inhibits CDK enzyme activity and cancer cell proliferation is highly significant. These findings have not been reported previously, although some studies have documented the ability of 3,4-DHBA and 3,4,5-THBA in inhibiting cancer cell growth in vitro and tumor formation in vivo. As the A-ring of flavonoids is highly conserved, it is expected that 2,4,6-THBA is likely to be a major phenolic acid metabolite, whereas the generation of 3,4,5-THBA and 3,4-DHBA will depend on the hydroxylation pattern of the

B-ring. A novel finding of this dissertation is the ability of 2,4,6-THBA to inhibit CDKs 1,

2 and 4 enzyme activity and cancer cell growth. This observation has immense clinical implications because 2,4,6-THBA has the potential to be used as a prophylactic compound 142 for chemoprevention. Alternatively, derivatives of 2,4,6-THBA can also be developed as novel inhibitors of CDKs, through modifications of their functional groups, which would be useful for cancer treatment. Therefore, efforts should be focused in future investigations on identifying the other potential targets for 2,4,6-THBA and their mechanism(s) of action in colorectal tissues. An understanding of how flavonoids are degraded by the human colonic bacteria may be useful to devise viable strategies to prevent the occurrences of cancer through the use of appropriate probiotics and flavonoids. In this dissertation we have identified 6 bacterial strains capable of degrading quercetin, suggesting their potential use as probiotic supplements.

Increasing evidence in literature suggest a role for microbiota in human health. Human gut has been shown to contain 300-500 species of microbes including bacteria, fungi, and protozoa [256]. It is considered a separate organ by itself because the lack of such a microbial community has serious effect on human physiology, biochemistry, and health.

Several studies have examined the effects of polyphenols against human gut microflora including their influence on the composition and activity of beneficial and pathogenic bacterial species. Polyphenolic compounds are abundant in green and black tea, in colored fruits and vegetables, and in chocolate and red wine [77, 88]. The bioactive components in the diet such as epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and catechins inhibit the growth of pathogens including

Helicobacter pyrolii, Staphylococcus aureus, E. coli O157:H7, Salmonella typhimurium

DT104, Listeria monocytogens, Pesudomonas aeruginosa, Hepatitis C virus, and fungi of the Candida genus [263]. On the other hand, polyphenolic compounds have also been shown to enhance the growth of commensals and beneficial bacteria, while suppressing the 143 pathogenic strains [264]. Tea derived phenolic compounds and their derivatives have been shown to inhibit pathogens such as Clostridium perfringens, and Clostridium difficile.

Interestingly, commensal anaerobic species such as Clostridium spp., Bifidobacterium spp., and probiotics such as Lactobacillus spp. were less severely affected [265]. Other studies also confirmed these findings and demonstrated that polyphenolic compounds alter the gut microbial ecology by increasing the bacteria that are beneficial and therefore exert a positive effect on gut health [266]. In view of reports suggesting that habitual consumption of a diet rich in flavonoids affect the microbial ecology, along with the fact that microbial dysbiosis in the gut can lead to the occurrence of CRC, the role played by flavonoids in CRC prevention should be considered for further studies.

6.6 Perspective on aspirin metabolites in decreasing CRC

Aspirin’s preferential effects against CRC as compared to cancers of other tissues raises the possibility that it may act directly on colorectal tissues before its absorption into circulation. Such an effect requires substantial concentrations of aspirin in the intestinal milieu which we believe is achievable. Since the bioavailability of aspirin is ~50% [101] and there is significant hydrolysis of aspirin occurring in the intestine [102], colorectal tissues are likely to be exposed to millimolar concentrations of aspirin and salicylic acid.

This is particularly true following consumption of enteric-coated tablets as its absorption is significantly lower than regular aspirin [203, 267] making it more available in the lumen of the intestine. In such an environment, both aspirin and salicylic acid can target specific proteins in the colonic cells to modulate their function and bring about changes required for effective chemoprevention. In this context, intact aspirin has the potential to target proteins through acetylation, and along with salicylic acid, may also target specific 144 transcription factors such as NF-κB or c-myc in colorectal tissues. Alternatively, other signaling pathways like Wnt/β-catenin or Akt/mTOR pathways may also be regulated as described above by aspirin and salicylic acid under these conditions.

Equally important are also the role of aspirin metabolites 2,3-DHBA and 2,5-DHBA in its chemopreventive effects. Both DHBAs have been demonstrated to inhibit CDK enzyme activity and colony formation in cancer cells, although 2,5-DHBA was more potent when compared to 2,3-DHBA [166, 268]. In view of the ability of intestinal bacteria, capable of degrading aspirin and salicylic acid to DHBAs, their contribution to the chemopreventive effect of aspirin should also be considered. Except for a few reports, a role for HBAs in cancer prevention has not been investigated in depth [166, 212, 268]. Exploring the role played by HBAs in aspirin’s cancer prevention should take precedence in future investigations as they are also generated following degradation of flavonoids [141, 269-

271], which are known to prevent cancer. In addition, there is plenty of evidence in literature that increased consumption of fruits and vegetables, that is rich in HBAs, is inversely correlated to the occurrences of cancer [272, 273]; an interesting target proposed for HBAs are CDKs present in colonic epithelial cells [166, 171, 274]. If one considers

HBAs as possible common mediators of CRC prevention, it provides the simplest explanation through which all three compounds (aspirin, flavonoids and a diet rich in

HBAs) can exert anti-cancer effects. Thus, the metabolite hypothesis borne out of the finding that HBAs inhibit cancer cell growth needs to be explored in future research with regards to its targets and mechanisms of action.

Interestingly, in one randomized clinical study, aspirin was shown to increase the abundance of Prevotella, Akkermansia, and Ruminococcaceae species while decreasing 145 the levels of Bacteroides, Parabacteroides and Dorea species, that have collectively been associated with reduced CRC risk [275]. Another in vivo experiment with germ-free and conventionalized mice also demonstrated that aspirin was able to increase the probiotic bacteria Bifidobacterium and Lactobacillus, suggestive of a role in CRC prevention.

However, this study also demonstrated that the aerobic microbe Lysinibacillus sphaericus was capable of degrading aspirin in the lumen; supplementation with antibiotics prevented such microbe-mediated aspirin degradation. Their study also pointed out the ability of aspirin to inhibit tumor formation in germ-free mice, raising the possibility that aspirin may prevent CRC through a microbiome-independent mechanism, and suggested that this may involve inhibition of COX-2 and PGE2 synthesis [276]. This raises the possibility that aspirin may exert anti-cancer effects independent of the formation of metabolites. A role for aspirin in increasing the beneficial bacteria which may contribute to human gut health and cancer prevention requires additional study. Under normal conditions, a portion of aspirin taken orally may be left unabsorbed, and therefore has the potential to act directly on colorectal tissues. However, aspirin may also be subjected to metabolism, generating

2,3-DHBA and 2,5-DHBA, that in turn act on the colorectal tissues to prevent cancer. As

2,5-DHBA and 2,3-DHBA have been shown to inhibit cancer cell growth [212, 268], it would be interesting to determine if these aspirin metabolites can potentiate the cancer preventive properties of aspirin when administered to germ-free mice. Such studies should validate the significance of both intact aspirin and its metabolites collectively contributing to CRC prevention.

146

6.7 Conclusions

It has been established over the course of time that increased consumption of fruits and vegetables has health benefits, making Hippocrates’ dictum “Let food be your medicine and medicine your food” all the more significant. Identification of polyphenols and phenolic acids commonly found as a component of the diet has now led to the generation of nutraceutical and/or pharmaceutical compounds that have revolutionized the health sector. Considering the increasing incidences of CRC worldwide, it is now more important than ever to come up with viable preventive strategies against this deadly disease. We believe that HBAs merit further studies to understand their role in cancer prevention as the proposed mechanism (metabolite hypothesis) involving HBAs is a simple and tenable explanation for CRC prevention by both aspirin and flavonoids. Since intestinal epithelial cells are the first to get exposed to HBAs following their consumption or metabolism (from aspirin/flavonoids), in our view CRC prevention is likely to be a local effect. The colorectal tissues may thus have an anatomical advantage as they are the first to get exposed to these

HBAs, making them preferred targets. We believe that effective cancer prevention requires the partnership between the parent compounds and the right microbial species responsible for HBA generation. We also propose that the variability previously observed in many epidemiological studies [64, 83, 277] could be attributed to the lack of an appropriate microbial ecology necessary for their degradation. Therefore, a strategy involving supplementation of the diet with appropriate probiotics and aspirin/flavonoids, or directly consuming HBAs may prove useful in CRC prevention. In this regard, the bacterial strains identified in this dissertation (B. glycinifermentans, F. plautii, B. eggerthi, O. scatoligenes, 147

E. eligens, and Lactobacillus spp.), merit further investigation for their use as probiotics to improve gut health.

148

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