Document Type

Dissertation - Open Access

Award Date

2016

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Jayarama Bhat Gunaje

Keywords

aspirin, chemoprevention, c-myc, colorectal cancer, p53, salicylic acid

Abstract

Background

Aspirin has generated a significant interest in recent years as a potential chemopreventive agent supported by strong evidence from epidemiological data; however, the mechanisms are not well understood. The objective of this dissertation is to identify novel cyclooxygenase (COX)-independent pathways by which aspirin exerts its anticancer effects in epithelial cancer cell lines. We investigated the effect of aspirin on p53, glucose 6-phosphate dehydrogenase (G6PD), and c-Myc, all of which are known to play a major role in cancer development. p53 is a tumor suppressor protein, often mutated in cancers causing its inactivation. Expression of G6PD is elevated in many cancers, and this significantly contributes to increased synthesis of DNA and RNA through the production of pentose sugars. c-Myc is a transcription factor (TF), which is mutated and constitutively activated in 20% of all cancers. The studies on p53 and G6PD are an extension of the previous work carried out in our laboratory, in which we demonstrated the ability of aspirin to acetylate these proteins in MDA-MB-231 human breast and HCT 116 human colon cancer cells, leading to modulation of their functions. It was therefore important to extend these studies to other cancer cell lines to gain an insight into the mechanisms of chemoprevention by aspirin, and to more clearly establish the generality of the observations. The studies on c-Myc are novel, and few reports exist on aspirin’s ability to modulate c-Myc functions in the literature. Understanding these aspirin-mediated chemopreventive mechanisms is very important because they have translatable implications for chemoprevention and chemotherapy. This is because: 1) nearly 50% of all tumors contain mutated p53 with varying degree of inactivation, and therefore, it is desirable to identify molecules that reactivate mutant p53 to curtail the cancer cell growth; 2) G6PD catalyzes the first reaction in the pentose phosphate pathway (PPP), and involved in the synthesis of ribose sugars required for nucleic acid synthesis, important for tumor growth. The expression levels of G6PD are elevated in many cancers, and therefore, it is a potential target in cancer treatment. 3) Because of the established role of c-Myc in cancer, it has been considered as an important target in cancer therapy. Our central hypothesis was that a significant portion of aspirin’s anti-cancer effects occur through modulation of the functional activity of p53, G6PD and c-Myc, collectively contributing to suppression of cancer cell growth.

Results

Our studies demonstrate that aspirin exposure causes acetylation of wild-type p53 in HCT 116 cells and mutant p53 in HT-29 human colon cancer cells. Acetylated p53 was localized to the nucleus, and this was associated with an increased expression of p21CIP1, a protein involved in cell cycle arrest. Mass spectrometry (MS) analysis of aspirin acetylated recombinant wild-type p53 identified 10 lysines as targets of aspirin-mediated acetylation. This included 5 lysines in the DNA binding domain, 4 in the nuclear localization signal domain and 1 in the C-terminal regulatory domain. Five of these lysines acetylated by aspirin were common to the sites that are naturally acetylated by acetyltransferases. This included lysine 120 and 320, which were previously demonstrated to be important for the expression of p21CIP1 and Bax. This suggests that chemical acetylation of p53 by aspirin may lead to expression of p53-target genes in cancer cells harboring wild-type or mutant p53. Aspirin acetylated G6PD in both HCT 116 and HT-29 cells; however, the extent of acetylation was greater in HCT 116 cells than HT-29 cells. Increased acetylation of G6PD was associated with an increased inhibition of the enzyme activity. Mass spectrometry analysis revealed that aspirin targeted 14 lysines in G6PD; 4 in the NADP binding domain, and 1 in the active site of the enzyme. It is likely that the observed inhibition of G6PD enzyme activity by aspirin is due to acetylation of the essential lysine in the active site, or the NADP substrate binding sites, or both. These results suggest that aspirin may contribute to anti-cancer effect through inhibition of G6PD and reduced synthesis of pentose sugars. We demonstrate that aspirin and its primary metabolite salicylic acid caused the down-regulation of c-Myc protein and mRNAs in several human cancer cell lines. This suggests that c-Myc regulation by aspirin and salicylic acid is likely to occur at both transcriptional and post-translational levels. Down-regulation of the c-Myc protein was partially sensitive to treatment with lactacystin, an inhibitor of 26S proteasomal pathway, which suggests the involvement of cysteine proteases. This is the first detailed investigation demonstrating the down-regulation of c-Myc by aspirin and salicylic acid in cancer cells. Conclusion Our findings suggest that, aspirin or its metabolite salicylic acid can target p53, G6PD and c-Myc, leading to modulation of their functions within cells; collectively these contribute to their anti-cancer effects in epithelial tissues.

Library of Congress Subject Headings

Aspirin

Cancer -- Chemoprevention

Cancer cells

Cyclooxygenases

Description

Includes bibliographical references (page 42-45)

Format

application/pdf

Number of Pages

149

Publisher

South Dakota State University

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Rights Statement

In Copyright