HSP90 Inhibitors, Geldanamycin and Radicicol, Enhance Fisetin-Induced Cytotoxicity via Induction of Apoptosis in Human Colonic Cancer Cells
Figure 2
Geldanamycin (GA) and radicicol (RAD) increased caspase-3 and PARP protein cleavage in fisetin- (FIS-) treated COLO205 cells. (a) GA and RAD induced cleavage of caspase-3 and PARP in FIS-treated COLOL205 cells. COLO205 cells were treated with GA (2 μM) or RAD (5 μM) with or without FIS (60 μM) for 24 h, and the expressions of caspase-3, PARP, and α-tubulin protein were detected by western blotting using specific antibodies. (b) Concentration-dependent investigation of GA on caspase-3 and PARP protein cleavage in FIS-treated COLO205 cells. (c) Effects of GA (2 μM), RAD (5 μM), FIS (60 μM), FIS (60 μM)+GA (2 μM), and FIS (60 μM)+RAD (5 μM) on caspase-3 enzyme activity were examined using Ac-DEVD-pNA as a caspase-3-specific peptidyl substrate. Cells were treated with the indicated components for 24 h, and caspase-3 activity was measured using Ac-DEVD-pNA as a substrate. (d) The addition of the caspase-3 peptidyl inhibitor, Ac-DEVD-FMK, inhibited FIS+GA-induced DNA ladder formation in COLO205 cells. Cells were incubated with the caspase-3 inhibitor, Ac-DEVD-FMK, for 2 h, followed by FIS+GA stimulation for additional 24 h, and DNA integrity was analyzed. Each data point was calculated from three triplicate groups, and data are displayed as the mean ± S.E. and denote a significant difference from the control (C) group.
