A vast amount of epidemiological, preclinical and clinical studies have revealed aspirin as a promising chemopreventive agent, particularly in epithelial carcinogenesis. Despite the wide attention inhibition of cyclooxygenases has received, it is clear that aspirin elicits a myriad of molecular effects that counteract the carcinogenic episodes. Since aspirin's protective effect was mainly observed in epithelial cell types which are more resistant to chemotherapeutic efforts, an urgent need exists to dissect and identify the primary targets and cancer preventive pathways affected by aspirin. In preliminary studies, we have obtained the first and strong evidence for a dose- and time-dependent acetylation of p53 tumor suppressor protein by aspirin in MDA-MB-231 human breast cancer cells, several cancer cells belonging to different tumor types and also in normal liver cells. In MDA-MB-231 cells, aspirin induced the levels of p53 target genes namely p21CIP1, a protein involved in cell cycle arrest, and Bax, a proapoptotic protein;however, p21 induction was transient (1-12h);where as, induction of Bax was sustained (24 h). Interestingly, in DNA damaged cells (induced by camptothecin), aspirin treatment (24 h) inhibited the p21 induction, while the Bax induction was unaffected. Built on these findings, the central hypothesis of this R03 pilot project is that aspirin-induced multi-site acetylation of p53 alters its transcription factor function by shifting the gene expression spectrum from those that elicit cell cycle arrest / prosurvival properties to those that promote and drive cell death. Since deletion of p21 gene has been previously shown to increase the sensitivity of cells towards apoptosis, our observation that aspirin inhibits p21 suggests a potential mechanism by which it may exert anti-cancer effects in DNA damaged cells. The studies proposed in this application will determine the mechanisms by which aspirin regulates apoptosis in DNA damaged cells via inhibition of p21. We will use MDA-MB-231 and MCF-7 breast cancer cells as well as normal human Peripheral Blood Mononuclear Cells in our study. The experiments in Aim 1 will investigate the molecular basis of aspirin-mediated inhibition of p21 using real time RT-PCR, electrophoretic mobility shift assays, and run on transcription assays. We will also identify aspirin-induced acetylation sites on p53.
In Aim II, we will determine the ability of aspirin to augment apoptosis in cells exposed to DNA damaging drugs by clonogenic cell survival assays and flow cytometry. In addition to camptothecin, all studies will be extended to include doxorubicin and cisplatin, to determine if aspirin also modulates p21 / Bax expression by these DNA damaging drugs. These studies will provide a novel mechanism by which aspirin may exert anticancer effects in DNA damaged cells via acetylation of p53, induction of Bax and inhibition of p21.
Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors. This proposal examines the mechanism by which aspirin exerts its chemopreventive / anti- cancer actions via acetylation of the tumor suppressor protein, p53 and modulation of its target genes.
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