salicylates and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Focal adhesion kinase (FAK) is a signaling molecule associated with cell survival. Previously, we showed that thimerosal, a reactive oxygen species (ROS) generator, can acutely induce FAK tyrosine phosphorylation (within minutes) and chronically induce apoptosis (within days) by redox modulation in HeLa S cells. In the present study, we report that a prolonged oxidative stress by thimerosal induces a remarkable cleavage of FAK, which is accompanied with apoptosis. In fact, the kinetics of FAK cleavage has a good correlation with and actually preceding the apoptosis that was independent of anoikis. The effects were almost completely blocked by the pretreatment with either N-acetyl-l-cysteine (ROS scavenger) or Z-VAD-FMK (pan-caspase inhibitor), suggesting ROS-induced caspase activation as a key mechanism. They could be also reproduced by hydrogen peroxide alone, which appeared to be responsible for thimerosal-mediated oxidative stress-induced apoptosis. Additionally, the down regulation of FAK with antisense oligonucleotide dramatically augmented thimerosal-induced apoptosis. We could observe similar results using human corneal epithelial cells. Taken together, our results show that FAK is a critical cellular target of caspases during oxidative stress (particularly by hydrogen peroxide), resulting in the acceleration of subsequent apoptosis regardless of the anchorage status of cells. From the present results, it is more likely that not cell detachment but the proteolytic cleavage (or inhibition) of FAK is a key modulator as well as a promising indicator of apoptosis in epithelial cells under oxidative stress.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Benzoquinones; Caspase 3; Caspase Inhibitors; Cell Line, Transformed; Chelating Agents; Cornea; Cysteine Proteinase Inhibitors; Egtazic Acid; Epithelial Cells; Focal Adhesion Kinase 1; Focal Adhesion Kinase 2; HeLa Cells; Humans; Hydrogen Peroxide; Lactams, Macrocyclic; Oxidative Stress; Protein Kinase Inhibitors; Reactive Oxygen Species; Rifabutin; RNA, Small Interfering; Salicylates; Sulfhydryl Compounds; Thimerosal

We analyzed the effect of aspirin, salicylate, and other nonsteroidal antiinflammatory drugs (NSAIDs) on the viability of B-chronic lymphocytic leukemia (B-CLL) cells. Aspirin induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 for cells from 5 patients was 5.9 +/- 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In some cases, 2.5 mmol/L aspirin produced an important cytotoxic effect after 4 days of incubation. No effect was observed with other NSAIDs, at concentrations that inhibit cyclooxygenase, such as ketorolac (10 micromol/mL), NS-398 (100 micromol/mL), or indomethacin (20 micromol/mL), thus suggesting the involvement of cyclooxygenase-independent mechanisms in aspirin-induced cytotoxicity. Salicylate also produced dose-dependent cytotoxic effects on B-CLL cells and the mean IC50 for cells from 5 patients was 6.96 +/- 1.13 mmol/L (range, 5 to 7.8 mmol/L). Both aspirin and salicylate induced DNA fragmentation and the proteolytic cleavage of poly(ADP(adenosine 5'-diphosphate)-ribose) polymerase (PARP), demonstrating that both compounds induce apoptosis of B-CLL cells. Finally, inhibition of caspases by Z-VAD.fmk blocked proteolytic cleavage of PARP, DNA fragmentation, and cytotoxicity induced by aspirin. Mononuclear cells from normal donors showed a lower sensitivity than cells from B-CLL patients to aspirin as determined by analysis of cell viability. B and T lymphocytes from normal donors and T lymphocytes from CLL patients are more resistant to aspirin-induced apoptosis, as determined by analysis of phosphatidylserine exposure. These results indicate that aspirin and salicylate induce apoptosis of B-CLL cells by activation of caspases and that this activation involves cyclooxygenase-independent mechanisms.

Topics: Aged; Amino Acid Chloromethyl Ketones; Annexins; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; B-Lymphocytes; Cell Survival; Cyclooxygenase Inhibitors; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Enzyme Activation; Female; Humans; Indomethacin; Ketorolac; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Neoplasm Proteins; Nitrobenzenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Salicylates; Salicylic Acid; Sulfonamides; Tolmetin; Tumor Cells, Cultured