Activation of caspase-3 in renal cell carcinoma cells by anthracyclines or5-fluorouracil

The caspase family of proteases is speculated to have a crucial role in apo ptosis. The effect of treatment with adriamycin (ADR), cisplatin (CDDP), 5- fluorouracil (5-FU), vinblastine (VLB), IFN-alpha, or IFN-gamma on the acti vation of caspase-3, -6, -8, and -9 in renal cell carcinoma (RCC) cells was investigated, to clarify the mechanisms of chemo- and immunotherapeutic ag ent-mediated apoptosis. Caspase activity was determined by a quantitative c olorimetric assay. Apoptosis was monitored by acridine-orange staining assa y. Treatment of ACHN cells with CDDP, VLB, IFN-alpha, or IFN-gamma did not activate caspase-3, but its activity was increased 7.2-fold (p=0.0001) with ADR and 2.8-fold (p=0.0385) with 5-FU in comparison with control. Furtherm ore, when the ADR treatment time was shortened from 24 to 8 or 2 h, the sam e caspase-3 activation occurred. Activation of caspase-3 was also observed in six freshly isolated human RCC cells after the treatment with ADR. Of th e six freshly derived RCC cells treated with 5-FU, caspase-? activity was i ncreased 3.1-fold (p=0.0051) and 2.4-fold (p=0.0346) in two of them, respec tively. Epirubicin and pirarubicin, compounds closely related to ADR, also respectively enhanced 4.2-fold (p=0.0052) and 2.8-fold (p=0.0147) caspase-3 activity in ACHN cells. The activation of caspase-3 observed with a colori metric assay was confirmed with immunocytochemical analysis using the anti- active caspase-3 mAb, which specifically recognizes the active form of casp ase-3. Furthermore, both active caspase-3 and apoptosis triggered by either ADR or 5-FU were inhibited significantly by the general caspase inhibitor Z-VAD-FMK, or a specific caspase-3 inhibitor DMQD-CHO. These findings provi de a mechanistic explanation for anthracyclines and 5-FU induced-apoptosis.