Mediation of curcumin-induced apoptosis of human multiple myeloma cells through caspase activation.
(A-C) U266 cells (2 × 106/mL) were incubated in the absence or presence of curcumin (50 μM) for the indicated times. The cells were washed, and total proteins were extracted by lysing the cells. Then, 60 μg extracts were resolved on 10% SDS-PAGE gel, electrotransferred to a nitrocellulose membrane, and probed with anti–caspase-9 (panel A); anti–caspase-7 (panel B); anti-PARP (panel Ci); and anti–cleaved PARP (panel Cii) antibodies as described in “Materials and methods.” (D) Detection of caspase activation by fluorescence microscopy. Untreated or curcumin-treated U266 cells (12 hours) were examined for caspase activation by Apo Logix carboxyfluorescein caspase detection kit. Cells were analyzed under light microscopy (LM) and by fluorescence microscopy (FM). Green fluorescence indicates activated caspases. Original magnification, × 200. (E) Suppression of curcumin-induced PARP cleavage by caspase-3 inhibitor. U266 cells (2 × 106/mL) were preincubated with caspase inhibitors Ac-DEVD-CHO (10 μM) or Ac-YVAD-CHO (10 μM) for 2 hours and then treated with curcumin (50 μM) for 24 hours. Thereafter, cell extracts were prepared and analyzed for PARP cleavage by using either anti-PARP antibody (i) or antibodies that recognize only cleaved PARP (ii) as described in “Materials and methods.” (F) Caspase-3 inhibitor protects cells from curcumin-induced cytotoxicity. U266 cells (5000/0.1 mL) were incubated with different concentrations of caspase inhibitors Ac-DEVD-CHO or Ac-YVAD-CHO for 2 hours and then treated with curcumin. After 24 hours, cell viability was determined by the MTT method, as described in “Materials and methods.” The results are shown as the means (± SDs) percentage viability from triplicate cultures.
