The analysis of apoptosis was performed selleck chemicals Vorinostat after exposure to 50��M of ZOL by flow cytometric detection of Annexin-V immunostaining. Cells were pulse exposed … Caspase- and PARP-dependency of ZOL-induced apoptosis in PC cells We investigated the molecular mechanisms of ZOL-induced apoptosis in these cells. We found that exposure of PC cells to ZOL increased PARP cleavage, a key enzyme in the apoptotic cascade, as demonstrated by Western blot analysis, which showed a higher expression of the 116kDa proenzyme and the appearance of an 89kDa cleavage product in treated cells. The maximal effect was detected after 72h of ZOL exposure (Figure 3A). We also found that caspase-9 was cleaved/activated after cell exposure to the drug, while no effect was detected on caspase-3.

These data were confirmed by the use of specific caspase inhibitors. We demonstrated that the caspase-9 inhibitor Z-VAD and the specific caspase-6 inhibitor VEID almost completely prevented apoptotic death of PC cells, while the DEVD caspase-3 inhibitor only slightly affected apoptosis induced by ZOL, as detected by flow cytometric analysis of Annexin-V staining (Figure 3B). These findings suggest that apoptosis induced by ZOL on human PC cells is caspase-9-, caspase-6- and PARP-dependent, but caspase-3-independent. Figure 3 Role of caspase-9/-3 and PARP in apoptotic cell death induced by ZOL. (A) Detection of caspase-9/-3 and PARP in PC cells after ZOL treatment. Western blotting analysis was performed on protein extracts from solubilized whole cell pellets …

Zoledronic acid inhibits the p21ras/Raf1/MEK/ERK1-2 mitogenic and pKB/Akt antiapoptotic pathways in PC cells In order to investigate the molecular mechanism of ZOL-induced growth inhibitory activity, we analysed whether the drug would affect the function of the ras/raf1/MEK/ERK1-2 pathway. We observed a strong decrease of the intracellular content of p21ras and of its substrate Raf-1 as evaluated by Western blotting (Figure 4A and B). In fact, 48h treatment with 50��M ZOL induced a two-to-three-fold reduction of the expression of the two signalling components (Figure 4A and B). Subsequently, we studied the effects of ZOL on the activity of ERK1-2, the terminal enzyme of the ras/raf-1 antiapoptotic and survival pathway. We found a strong decrease of the activity of the two enzymes, as demonstrated by the immunodetection of the phosphorylated forms of ERK1-2 (Figure 4E).

On the other hand, no changes in the intracellular Carfilzomib levels of ERK1-2 were found in the ZOL-treated PC cells (Figure 4D). These data suggest a significant inhibition of the signalling activity of this mitogenic and survival pathway. Another important and ras-dependent survival pathway of human cancer cells is mediated by the activation of the intracellular kinase pKB/Akt. Therefore, we also studied the effects of ZOL on Akt activity.