Document Type

Dissertation - Open Access

Award Date

2017

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Jayarama B Gunaje

Keywords

Aspirin, CDKs and Cyclins, Cell Cycle, Colon Cancer, Polyphenols, Salicylic acid

Abstract

Background:
The pursuit of drugs that inhibit cyclin-dependent kinases (CDKs) has been an intense area of research for more than 15 years. Till date, although multiple CDK inhibitors have been identified and few are undergoing clinical trials, only two synthetic drugs have been approved by Food and Drug Administration (FDA) for use in the treatment of cancer. These two drugs are mainly used for the treatment of metastatic breast cancer in combination with other drugs; however they have toxicity associated with their use and extends patients life not more than 24 months. Therefore, there is an urgent need for developing newer drugs that are more safe and efficacious. Uncontrolled cell proliferation is a hallmark of cancer. In mammalian cells, cell cycle is controlled by the sequential activation of cyclin dependent kinases (CDKs). Four CDKs (CDKs 1, 2, 4 and 6) and their activating cyclins (A, B, D and E), play key roles in cell cycle progression. It has been established that CDK4,6/cyclin D and CDK2/cyclin E/A promote the passage through G1 and S phases, whereas CDK1/cyclin B regulates the transition through the late G2 and mitosis. In addition, specific proteins classified as CDK inhibitors capable of binding to cyclin/CDK complexes to inhibit their enzyme activity also play a significant role in regulating cell cycle. While, expression and activity of cyclins and CDKs are tightly regulated in normal cells, they are often deregulated in cancer cells through frequent overexpression and frequent inactivation. Studies carried in the past 2 decades have clearly established that regular aspirin use for 5 – 10 years decreases the cancers of the epithelial tissues particularly the cancers of the colon. This evidence came from numerous epidemiological studies, clinical trials, in-vitro cell culture experiments as well as experiments in animal models. Despite its potential role in cancer prevention, it is not clear precisely how aspirin exerts its chemopreventative effects in epithelial tissues. In this context, multiple targets and signaling pathways have been identified; however a unifying mechanism has not been identified till date. The objective of this dissertation is to investigate the novel mechanisms by which aspirin prevents the occurrences of cancer and discover newer protein targets that maybe responsible for mediating its chemopreventative actions. Understanding aspirin-mediated chemopreventive mechanism and pinpointing its direct cellular targets is of high value, if it is to be used as a prophylactic drug. We hypothesized that aspirin and/or its primary metabolite salicylic acid may target cell cycle regulatory proteins modulating their level as well as functions. To address this, numerous biochemical, molecular biological studies were carried out in multiple cancer cell lines along with molecular docking studies to determine the interactions between aspirin/salicylic acid with CDKs and cyclins. The studies carried out during the course of this dissertation work have established that aspirin, salicylic acid and salicylic acid metabolites and derivatives target all more 4 members of CDK family namely CDKs 1, 2, 4 and 6, the major findings of which are detailed below.
Results:
Major finding 1: Our studies demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2, B1, D3, CDKs 1, 2, 4 and 6 protein levels in a diverse panel of cancer cell lines. The decrease in cyclin A2 and cyclin B1 levels as well as CDK1 and CDK2 protein levels were associated with a corresponding decrease in the levels of messenger RNAs, suggesting that both aspirin salicylic acid regulate their expression at both transcriptional and post translational levels. Aspirin and salicylic acid also increased the levels of CDK inhibitors namely p21 and p27. The decrease in cyclin A2 and cyclin B1 protein levels appears to be mediated through 26S proteasomes.
Major Finding 2: Through biochemical and molecular modeling studies we showed that salicylic acid directly binds to CDK2. Molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. Extension of these studies showed that salicylic acid also binds to CDK1 using Asp146 and Lys33. Despite salicylic acid interacting with CDK 1 and 2 via interactions using amino acids in the active site of the enzyme, inhibition of the enzyme activity was not observed.
Major Finding 3: We investigated the ability of salicylic acid metabolites 2,3- dihydroxy benzoic acid (2,3-DHBA) and 2,5-dihydroxy benzoic acid (2,5-DHBA) known to be generated by cytochrome p450 metabolism to CDK enzyme activity. In-vitro CDK assays showed that both metabolites inhibited CDK1 enzyme activity. Interestingly several derivatives 2,4-dihydroxybenzoic acid (2,4, DHBA), 2,6-dihydroxybenzoic acid (2,6- DHBA) and 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) also inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, 2,4,6-THBA was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and preincubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supports the data obtained from molecular docking studies.
Conclusion:
We identified CDK1 and 2 as salicylic acid binding proteins. In addition, we have demonstrated the interactions of salicylic acid metabolites and derivatives with CDKs. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells may be responsible to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin’s chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of a novel CDK inhibitors in cancer prevention and treatment.

Library of Congress Subject Headings

Cancer -- Prevention.
Colon (Anatomy) -- Cancer -- Prevention.
Cyclin-dependent kinases.
Aspirin.
Salicylic acid.

Description

Includes bibliographical references (page 134-152)

Format

application/pdf

Number of Pages

176

Publisher

South Dakota State University

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Rights Statement

In Copyright