Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets.

Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts 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 immuno-affinity purification, mass spectrometry and immuno-blotting. 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, cytoskeleton 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 experiment 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. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

[1]  M. Mann,et al.  Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions , 2009, Science.

[2]  P. Brooks,et al.  Clinical Pharmacokinetics of the Salicylates , 1985, Clinical pharmacokinetics.

[3]  K. Srivenugopal,et al.  Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells. , 2009, International journal of oncology.

[4]  M. Cascante,et al.  Oxythiamine and dehydroepiandrosterone induce a G1 phase cycle arrest in Ehrlich's tumor cells through inhibition of the pentose cycle , 1999, FEBS letters.

[5]  Stephanie Spange,et al.  Acetylation of non-histone proteins modulates cellular signalling at multiple levels. , 2009, The international journal of biochemistry & cell biology.

[6]  R. Gaynor,et al.  The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. , 1998, Nature.

[7]  S. Mazurek.  Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumor cells. , 2011, The international journal of biochemistry & cell biology.

[8]  G. Duggin,et al.  ASPIRIN, PROTEIN TRANSACETYLATION AND INHIBITION OF PROSTAGLANDIN SYNTHETASE IN THE KIDNEY , 1978, British journal of pharmacology.

[9]  Weidong Zhou,et al.  Mass spectrometry analysis of the post-translational modifications of alpha-enolase from pancreatic ductal adenocarcinoma cells. , 2010, Journal of proteome research.

[10]  K. Stuhlmeier,et al.  Importance of Glucose-6-phosphate Dehydrogenase Activity for Cell Growth* , 1998, The Journal of Biological Chemistry.

[11]  A. Lane,et al.  Histone deacetylase inhibitors in cancer therapy. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[12]  J. Shen,et al.  Role of acetylation and extracellular location of heat shock protein 90alpha in tumor cell invasion. , 2008, Cancer research.

[13]  Eran Segal,et al.  Proteome-wide prediction of acetylation substrates , 2009, Proceedings of the National Academy of Sciences.

[14]  Marta Cascante,et al.  Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29 , 2009, International journal of cancer.

[15]  E. Gottlieb,et al.  Targeting metabolic transformation for cancer therapy , 2010, Nature Reviews Cancer.

[16]  G. Semenza,et al.  Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression , 2010, Proceedings of the National Academy of Sciences.

[17]  G. Roth,et al.  Isolation and covalent structure of the aspirin-modified, active-site region of prostaglandin synthetase. , 1983, Biochemistry.

[18]  Ru Wei,et al.  The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth , 2008, Nature.

[19]  Bruno C. Hancock,et al.  Suppression of Intestinal Polyposis in Apc Δ716 Knockout Mice by Inhibition of Cyclooxygenase 2 (COX-2) , 1996, Cell.

[20]  A. Mancuso,et al.  p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase , 2011, Nature Cell Biology.

[21]  J. Vane,et al.  The mechanism of action of aspirin. , 2003, Thrombosis research.

[22]  Tony Kouzarides,et al.  Acetylation: a regulatory modification to rival phosphorylation? , 2000, The EMBO journal.

[23]  J. Turnay,et al.  Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5 , 2005, Journal of cellular biochemistry.

[24]  M. Rowland,et al.  Absorption kinetics of aspirin in man following oral administration of an aqueous solution. , 1972, Journal of pharmaceutical sciences.

[25]  O. Lockridge,et al.  Reaction of human albumin with aspirin in vitro: mass spectrometric identification of acetylated lysines 199, 402, 519, and 545. , 2010, Biochemical pharmacology.

[26]  S. Ogino,et al.  Aspirin Use and Survival After Diagnosis of Colorectal Cancer , 2009 .

[27]  O. Warburg.  On the origin of cancer cells. , 1956, Science.

[28]  P. Rothwell,et al.  Eff ect of aspirin on long-term risk of colorectal cancer : consistent evidence from randomised and observational studies , 2007 .

[29]  D. Alberts,et al.  Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  K. Srivenugopal,et al.  Does aspirin acetylate multiple cellular proteins? (Review). , 2009, Molecular medicine reports.

[31]  R. Farr,et al.  In vitro Acetylation of Plasma Proteins, Enzymes and DNA by Aspirin , 1968, Nature.

[32]  E. Giovannucci,et al.  Primary prevention of colorectal cancer. , 2010, Gastroenterology.

[33]  Hani Doss,et al.  Aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs in cancer prevention: a critical review of non-selective COX-2 blockade (review). , 2005, Oncology reports.

[34]  E. Newsholme,et al.  Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells. , 1990, The Biochemical journal.

[35]  F. A. Green,et al.  Acetylation of erythrocytic membrane peptides by aspirin , 1981, Transfusion.

[36]  R. D. Williams,et al.  Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation. , 1997, Cancer research.

[37]  S. Ghosh,et al.  Inhibition of NF-kappa B by sodium salicylate and aspirin. , 1994, Science.

[38]  Guo-Ping Zhao,et al.  Acetylation of Metabolic Enzymes Coordinates Carbon Source Utilization and Metabolic Flux , 2010, Science.

[39]  Marta Cascante,et al.  Pentose phosphate cycle oxidative and nonoxidative balance: A new vulnerable target for overcoming drug resistance in cancer , 2006, International journal of cancer.

[40]  M. Luciani,et al.  Aspirin blocks proliferation in colon cells by inducing a G1 arrest and apoptosis through activation of the checkpoint kinase ATM. , 2007, Carcinogenesis.

[41]  M. Cascante,et al.  Nonoxidative pentose phosphate pathways and their direct role in ribose synthesis in tumors: is cancer a disease of cellular glucose metabolism? , 1998, Medical hypotheses.

[42]  J. Vilček,et al.  Sodium salicylate induces apoptosis via p38 mitogen-activated protein kinase but inhibits tumor necrosis factor-induced c-Jun N-terminal kinase/stress-activated protein kinase activation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Jing Chen,et al.  Tyrosine Phosphorylation Inhibits PKM2 to Promote the Warburg Effect and Tumor Growth , 2009, Science Signaling.

[44]  Yixue Li,et al.  Regulation of Cellular Metabolism by Protein Lysine Acetylation , 2010, Science.

[45]  B. Rigas,et al.  Effects of nonsteroidal anti-inflammatory drugs on proliferation and on induction of apoptosis in colon cancer cells by a prostaglandin-independent pathway. , 1996, Biochemical pharmacology.

[46]  T. Mak,et al.  Regulation of cancer cell metabolism , 2011, Nature Reviews Cancer.

[47]  P. Brooks,et al.  Pharmacokinetics of aspirin and salicylate in elderly subjects and in patients with alcoholic liver disease , 2004, European Journal of Clinical Pharmacology.

[48]  I. Weinstein,et al.  Role of COX-independent targets of NSAIDs and related compounds in cancer prevention and treatment. , 2003, Progress in experimental tumor research.

[49]  Saroj P. Mathupala,et al.  Hexokinase-2 bound to mitochondria: cancer's stygian link to the "Warburg Effect" and a pivotal target for effective therapy. , 2009, Seminars in cancer biology.

[50]  E. Abraham,et al.  Glycation of human lens crystallins: effect of age and aspirin treatment. , 1993, Ophthalmic research.

[51]  Y. Bang,et al.  Histone Deacetylase Inhibitors for Cancer Therapy , 2006, Epigenetics.

[52]  R. Gaynor,et al.  The anti-inflammatory agents aspirin and salicylate inhibit the activity of IκB kinase-β , 1998, Nature.