Subsequently, the fixation buffer was removed and cells washed with 1ml of 1 �� washing buffer. For visualisation of filamentous actin, the cells were exposed to rhodamine�Cphalloidin (100��gml?1) (Sigma) for 10min at 4��C and washed with washing buffer. After final washes, coverslips were mounted on the dishes using ref 3 a 50% solution of glycerol in PBS. The cells were examined under a LEICA TCS SP2 confocal microscope. Statistical analysis Results are expressed as mean ��s.e. The statistical significance of differences between the experimental points was analysed using the t-test; differences were considered significant when P<0.05. RESULTS Zoledronic acid induces antiproliferative effects on PC cells The effect of ZOL on BxPC-3, CFPAC and PANC-1 PC cells was investigated in vitro using the MTT assay.

Treatment with ZOL (1�C100��M) produced a dose-dependent reduction of cell growth after 72h of treatment (Figure 1A) and the IC50 was calculated in a range of 10�C50��M (Figure 1B). Figure 1C and D show the morphological changes of cultured PC cells after 72h exposure to ZOL (50��M). Untreated cells (Figure 1C) were flat and well spread, but exposure to the drug (Figure 1D) resulted in significant antiproliferative effects, retraction of cells from the substratum, rounding up and loss of contact between neighbouring cells. Altogether, these findings indicate that ZOL exerts growth inhibitory activity on PC cells. Figure 1 Zoledronic acid inhibits the growth of PC cells. (A) The cell lines BxPC-3, CFPAC-1 and PANC-1 (3 �� 104ml?1) were seeded in 96-well plates and incubated for 24h.

After 24h, medium was removed and replaced with … Zoledronic acid induces apoptotic death of PC cells To clarify the mechanisms of ZOL-induced growth inhibition, we performed apoptotic assays on PC cells exposed to this compound. Activation of apoptosis was detected by Annexin-V staining, which is early expressed on the outer side of the cell membrane only when apoptosis is triggered and by PI which directly measures fragmented DNA. Figure 2 shows the apoptotic death of BxPC-3, CFPAC-1 and PANC-1 cells after 6, 12 and 24h exposure to 50��M ZOL. Apoptotic cell death was detected in 35�C90% of treated PC cells, suggesting a significant role of apoptotic death in the in vitro activity of ZOL. Notably, induction of the apoptosis was independent from the length of ZOL exposure.

After 6�C12h from the beginning of ZOL exposure, Annexin-V staining was equally detected in cells treated by a 30�C180min pulse as AV-951 compared with cells treated with continuous drug exposure, suggesting that induction of the apoptotic process is an early event in pancreatic cells exposed to ZOL. DNA fragmentation demonstrated by the PI experiment became evident after 24h and again was independent from the length of exposure (pulse vs continuous).