Lloyd F. Alfonso

Molecular targets of aspirin and cancer prevention

Salicylates from plant sources have been used for centuries by different cultures to treat a variety of ailments such as inflammation, fever and pain. A chemical derivative of salicylic acid, aspirin, was synthesised and mass produced by the end of the 19th century and is one of the most widely used drugs in the world. Its cardioprotective properties are well established; however, recent evidence shows that it can also act as a chemopreventive agent. Its antithrombotic and anti-inflammatory actions occur through the inhibition of cyclooxygenases. The precise mechanisms leading to its anticancer effects are not clearly established, although multiple mechanisms affecting enzyme activity, transcription factors, cellular signalling and mitochondrial functions have been proposed. This review presents a brief account of the major COX-dependent and independent pathways described in connection with aspirin’s anticancer effects. Aspirin’s unique ability to acetylate biomolecules besides COX has not been thoroughly investigated nor have all the targets of its primary metabolite, salicylic acid been identified. Recent reports on the ability of aspirin to acetylate multiple cellular proteins warrant a comprehensive study to investigate the role of this posttranslational modification in its anticancer effects. In this review, we also raise the intriguing possibility that aspirin may interact and acetylate cellular molecules such as RNA, and metabolites such as CoA, leading to a change in their function. Research in this area will provide a greater understanding of the mechanisms of action of this drug.

Does aspirin acetylate multiple cellular proteins? (Review)

Aspirin is a salicylate drug that is extensively used for its anti-inflammatory, antipyretic, analgesic and anti-thrombotic effects. More recently, it has been shown to decrease the incidence of cancers of epithelial origin. In most cases, aspirin is relatively safe. However, it does cause a host of adverse effects and toxicities, including gastrointestinal bleeding, ulcerations, nephrotoxicity and hypersensitivity reactions. Although the inhibition of cyclooxygenases by aspirin, which leads to its anti-inflammatory/analgesic properties, has been well studied, the mechanisms involved in its chemopreventive effects as well as some of its adverse effects are as yet ill-defined. Studies over the past decades suggest that, besides cyclooxygenases, aspirin acetylates other cellular proteins. These studies used radiolabeled 3H or 14C aspirin, the only approach used to date for the detection of proteins acetylated by aspirin. In a recent study using protein-specific anti-acetyl lysine antibodies and immunological methods, we demonstrated the ability of aspirin to acetylate the tumor suppressor protein p53. In this review, we present current research from the literature on the aspirin-induced acetylation of proteins. We also describe an immunological approach to detecting acetylated proteins in aspirin-treated cells, and demonstrate that multiple proteins are acetylated. Since post-translational modification of proteins, such as acetylation, may lead to the alteration of their function, it is possible that some of the hitherto unexplained beneficial or adverse effects of aspirin could occur as a result of these modifications. The identification of these novel acetylation targets of aspirin represents a new area for investigation.

Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53

Aspirin’s ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21CIP1. Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin’s anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

Aspirin inhibits glucose‑6‑phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

Abstract 1638: Aspirin acetylates multiple cellular proteins in HCT-116 cells: Identification of novel targets

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Aspirin, a non-steroidal anti-inflammatory drug has been consistently associated with a reduced risk of colon cancer; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate, and post-transnationally modify cellular proteins in HCT-116 human cancer cells to understand the potential mechanisms by which it may exert anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immune-affinity purification, mass spectrometry and immunoblotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeletal proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde 3 phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiments showed that aspirin also acetylated glucose 6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6-phosphate dehydrogenase activity. Selective inhibition of glucose 6-phosphate dehydrogenase and other key proteins through acetylation may represent important mechanisms by which aspirin may exert its anti-cancer effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1638. doi:1538-7445.AM2012-1638

Abstract 4038: Aspirin acetylates p53 in colon cancer cells and induces the expression of p21 and Bax: Identification of aspirin acetylated sites on rp53

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Several studies over the past decade have shown that regular use of aspirin reduces the risk of cancers of various organs such as colon, breast, lung and prostate tissue. The mechanism by which aspirin exerts its anti-cancer effects is not clearly understood. Previous studies from our laboratory showed that aspirin acetylates the tumor suppressor protein p53 and modulates target gene expression in MDA-MB-231 breast cancer cells. We now have extended these studies to include HCT116 (wt-type p53) and HT-29 (mutant p53) colon cancer cells. Using anti-acetyl p53 antibody, we show in the present study that aspirin acetylated p53 at lysine 382 in both cell types. It also detected two other proteins of size 63 kDA and 35 kDa, which are likely to represent other isoforms of p53. Aspirin also acetylated recombinant p53 (rp53) in vitro, suggesting that the mechanism of cellular acetylation may be through a direct non-enzymatic chemical reaction. Mass spectrometry analysis of the aspirin-treated rp53 identified 7 acetylate lysine residues. In HCT116 and HT-29 cells, aspirin-induced acetylation of p53 was observed in a concentration dependent manner. The acetylated p53 was localized to the nucleus. Consistent with these observations, aspirin transiently increased p21, a protein involved in cell cycle arrest; however, the expression of the proapoptotic protein, Bax, was sustained. Treatment of cells with the anti-cancer drug, camptothecin, induced sustained induction of p21. Co-treatment with both camptothecin and aspirin decreased the expression of p21. Aspirin also potentiated camptothecin-induced cell death as measured by MTT assay, clonogenic cell survival assay and annexin V binding assay. Our results suggest that p53 pathway may contribute to the anti-cancer properties of aspirin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4038. doi:10.1158/1538-7445.AM2011-4038

Abstract 5041: Aspirin acetylates and activates p53 in HT-29 and HCT-116 colon cancer cells

Increasing evidences from human epidemiological studies, animal models and in vitro experiments suggest that regular use of aspirin may reduce the risk of cancer of the colon; however, the mechanisms are not well understood. Since aspirin9s protective effect is mainly observed in epithelial cell types which are more resistant to chemotherapy, an urgent need exists to identify the primary targets and cancer preventive pathways affected by aspirin. We hypothesized that aspirin9s anti-cancer effect may involve acetylation of the tumor suppressor protein p53 and modulation of target gene expression. In the present study, we demonstrate that exposure of HT-29 and HCT-116 colon cancer cells to aspirin caused acetylation of p53 at lysine 382 (K382). HT-29 cells harbor a mutant form of p53 (R273H), whereas HCT-116 contain wild type p53. Acetylated p53 was mainly localized to the nucleus. In both cell types aspirin induced acetylation of p53 in a concentration dependent manner, and this was associated with increased p53 DNA binding activity. Consistent with this, we also observed induction of p21, a protein involved in cell cycle arrest as well as Bax, a mitochondrial proapoptotic protein. Aspirin also caused acetylation of bacterially expressed recombinant p53 in vitro, suggesting that it is a non-enzymatic reaction. Our observation that aspirin acetylates p53 leading to induction of target gene expression suggest that its anti-cancer effect may involve activation of the p53 pathway. Since p53 is mutated in nearly 50% of all tumors which leads to loss of its function, our observation also suggests that aspirin may find use in chemotherapy to reactivate mutant p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5041.