Effect of inhibitors of cysteine and serine proteases in anticancer drug-induced apoptosis in gastric cancer cells.

Activation of proteases can play an important role in apoptotic cell death induced by anticancer drugs. To assess involvement of activation of cysteine and serine proteases in anticancer drug-induced apoptosis, we tested effect of inhibitors of cysteine and serine proteases on sensitivity to anticancer drugs in MKN45 gastric cancer cells. Cytotoxic effect by adriamycin (ADM), SN-38 (active form of irrinotecan) and cisplatin (CDDP) was significantly prevented by cotreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) (p<0.01), a pancaspase inhibitor compared with drug alone using MTT assay. In contrast, cotreatment with N-acetyl-Tyr-Val-Ala-Asp aldehyde (AC-YVAD-CHO), a caspase 1 inhibitor did not prevent any cytotoxic effect of these drugs. Cotreatment of N-acetyl-Asp-Glu-Val-Asp aldehyde (AC-DEVD-CHO), a caspase 3 inhibitor prevented cytotoxic effect of VP-16 and SN-38 (p<0.01). Prevention of these cytotoxic effects by caspase inhibitors was not dose-dependent. Cotreatment of N-tosyl-L-lysyl chloromethylketone (TLCK), a serine protease inhibitor significantly prevented cytotoxic effect of ADM, SN-38, 5-fluorouracil (5-FU) and CDDP in a slight dose-dependent manner (p<0.01) except for etoposide (VP-16) and docetaxel (TXT), while an other serine protease inhibitor, N-tosyl-L-phenylalanyl chloromethylketone (TPCK) did not prevent any anticancer drug-induced cytotoxic effect. These effects were associated with prevention of internucleosomal DNA ladder formation in apoptosis. Further, protease inhibitors did not block induction of cytochrome c, that can explain the partial effect of prevention by anticancer-induced cell death. These results suggest that anticancer drug-induced cytotoxic effect is mediated by activation of serine protease (caspase-independent) as well as caspase-dependent pathway leading to apoptotic cell death, and that protease-independent pathway may also be involved in apoptotic pathways. The involvement of protease in signal transduction pathways may differ in cytotoxic action of drugs in gastric cancer cells.