Embelin partially inhibited caspase activation and did not induce caspase-dependent cell death in cholangiocarcinoma cells.

<p>(A) Mz-ChA-1 cells were treated with embelin for 24 hours and caspase 3/7 activity measured biochemically. Untreated cells were used for comparison and caspase activity in untreated cells normalized at 1.0. (B) BDEneu cells were treated with embelin (48 hours) and caspase 3/7 activity measured. (C) Caspase 3/7 activity was measured at an earlier time point (4 hours) in Mz-ChA-1, KMCH, and HuCCT cells to test for early caspase activation. Following 4 hours of vehicle, embelin (15 µM) or staurosporine (1 µg/mL), caspase 3/7 activity was measured biochemically. (D) BDEneu cells treated with 50 µM embelin for 48 hours were assayed for DAPI-positive nuclei with and without co-treatment with the caspase inhibitor Z-VAD-fmk (50 µM). DAPI-positive nuclei are presented as percent of total cells, n = 3, mean +/− SEM. Comparison of embelin versus embelin+Z-VAD-fmk was not significantly different. Panels A, B, C & D data are mean of 3 or 4 experiments +/− SEM; ** p<0.01, *** p<0.001 versus vehicle, ANOVA with Bonferroni correction. (E) Mz-ChA-1 cells were treated with embelin (5–15 µM) in DMSO or DMSO alone (Veh) for 24 hours. Whole cell lysates were blotted for PARP. Actin was included as a loading control. Apparent molecular weight for each protein is indicated to the right.