HeLa cells exposed to apicularen A exhibited a 3 fold increase in caspase 3 activity compared to control cells. In addition, apicularen A increased the active form of caspase 3 selleck inhibitor and the cleaved form of PARP. Taken together, these results indicate that apicularen A induces apoptotic cell death in HeLa cells. PMA increases apicularen A induced cytotoxicity in HeLa cells The role of PKC in the mechanism of action of antitumor agents is controversial since PKC synergizes with some agents and Inhibitors,Modulators,Libraries antagonizes others. PMA was used to assess the effect of PKC on apicularen A induced cytotoxicity. PMA has a similar chemical structure to DAG and activates PKCs by interacting with the DAG binding site. HeLa cells were exposed to 100 nM api cularen A and 20 nM PMA, as shown in Figure 2A and Table 1, PMA synergistically increased Inhibitors,Modulators,Libraries the cytotoxicity of apicularen A in a time dependent manner.

This finding was supported by time lapse video microscopy, showing that combination of PMA and apicularen A strongly in duced cell death to a Inhibitors,Modulators,Libraries greater extent than apicularen A alone. Next, flow cytometry was used to assess the potential Inhibitors,Modulators,Libraries effect of PMA on the cell cycle. Forty percent of apicularen A treated cells were apoptotic at 48 hours, while no apoptosis was detected in control or PMA treated cells. In addition, 80% of the cells exposed to the combination of apicularen A and PMA were apoptotic at 48 hours, in dicating that PMA increased apicularen A induced apop totic cell death. Since apicularen A induced apoptotic cell death through caspase activation, we investi gated whether the cytotoxicity induced by the combin ation of PMA and apicularen A also depended on caspase activation.

The pan caspase inhibitor Z VAD fmk did not block the cytotoxicity induced Inhibitors,Modulators,Libraries by the drug combin ation , indicating that the synergy is caspase independent. Since PMA is involved in several PKC independent cellular processes associated with cell proliferation and differentiation, the role of PKC activation on the synergy with apicularen A was tested by exposing neverless cells to PKC inhibitor Ro31 8220 before adding PMA and apicularen A. Ro31 8220 showed no cytotoxicity in HeLa cells at 48 hours, and pretreatment with Ro31 8220 completely blocked the synergistic apoptotic activity of PMA and apicularen A. The PKC inhibitor Go6983 also suppressed the effect of PMA on apicularen A induced cytotoxicity. These results suggest that PKC activation increases apicularen A induced apoptotic cell death. PKC mediates the effect of PMA on apicularen A induced cytotoxicity To identify which PKC isotype is involved in the in crease in apicularen A induced cell death observed in the presence of PMA, cells were pretreated with siRNAs specific for individual PKCs and exposed to PMA and apicularen A.