Document Type

Dissertation - Open Access

Award Date

2021

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Jayarama Gunaje

Abstract

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, antipyretic 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 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 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 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, 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.
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.

Number of Pages

206

Publisher

South Dakota State University

Rights

Copyright © 2021 Ranjini Sankaranarayanan

Share

COinS