Activation of p53 signalling in acetylsalicylic acid-induced apoptosis in OC2 human oral cancer cells.
BACKGROUND
Nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid (ASA, aspirin) are well known chemotherapeutic agents of cancers; however, the signalling molecules involved remain unclear. The aim of this study was to investigate the possible existence of a putative p53-dependent pathway underlying the ASA-induced apoptosis in OC2 cells, a human oral cancer cell line.


METHODS
The methyl tetrazolium (MTT) assay was employed to quantify differences in cell viability. DNA ladder formation on agarose electrophoresis was used as apoptosis assay. The expression levels of several master regulatory molecules controlling various signal pathways were monitored using the immunoblotting techniques. Flow cytometry was used to confirm the effect of ASA on cell cycle. Patterns of changes in expression were scanned and analyzed using the NIH image 1.56 software (NIH, Bethesda, MD, USA). All the data were analyzed by ANOVA.


RESULTS
Acetylsalicylic acid reduced cell viability and presence of internucleosomal DNA fragmentation. In the meanwhile, phosphorylation of p53 at serine 15, accumulation of p53 and increased the expression of its downstream target genes, p21 and Bax induced by ASA. The expression of cyclooxygenase-2 was suppressed. Disruption of p53-murine double minute-2 (MDM2) complex formation resulted in increasing the expression of MDM2 60-kDa cleavage fragment. Inhibited the activation of p42/p44 mitogen-activated protein kinase (MAPK) by PD98059, a specific inhibitor of extracellular regulatory kinase (ERK), significantly decreased cell viability and enhanced the expression of p53 induced by ASA. The result of the cell-cycle analysis showed that ASA and PD98059 induced the cell cycle arrested at the G0/G1 phase and resulted in apoptosis.


CONCLUSIONS
Nonsteroidal anti-inflammatory drug-inhibited cyclooxygenase is not the only or even the most important mechanism of inhibition. Our study presents evidences that activation of p53 signalling involved in apoptosis induced by ASA. Furthermore, the apoptotic effect was enhanced by blocking the activation of p42/p44 MAPK in response to treatment with ASA, thus indicating a negative role for p42/p44 MAPK.
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001241:Aspirin / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D051546:Cyclooxygenase 2; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009062:Mouth Neoplasms / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D011451:Prostaglandin-Endoperoxide Synthases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D015398:Signal Transduction / Q000187:drug effects; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D020928:Mitogen-Activated Protein Kinases; C022616:thiazolyl blue; D001241:Aspirin