Cells were grown, treated, lysed and centrifuged, and supernatants were used. Based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide by caspase-3, resulting pNA was measured photometrically at 405 nm after incubation at 37��C and 5% CO2. A pNA calibration curve was used to calculate results. Statistical analysis Statistical calculations were performed using selleck chemicals Vismodegib SPSS, version 10.0 (SPSS Inc., Chicago, USA). Numeric data were presented as mean value with SD. Inter-group comparisons were performed with the Student t test. P values less than 0.05 were considered significant. RESULTS Inhibition of cell growth After 3 d of incubation, tested cell lines were sensitive to NVP-AEW541 (mean IC50 = 0.51 �� 0.

44 ��mol/L) with CC-LP-1 being the most sensitive and Mz-ChA-1 the least sensitive cell line (IC50 values were calculated with linear regression model). Response differed markedly between the group of extrahepatic CC cell lines (mean IC50 = 0.29 �� 0.15 ��mol/L) and the group of the two GBC cell lines (mean IC50 = 1.06 �� 0.47 ��mol/L) (P < 0.05). Inhibition of cell growth was more pronounced if incubation time was extended to 6 d (treated twice, on days 0 and 4) with a mean IC50 value of 0.22 �� 0.15 ��mol/L. Although not statistically significant, there was again a difference in response for the 6 d experiment between the group of extrahepatic CC cell lines (mean IC50 = 0.18 �� 0.06 ��mol/L) and the group of the two GBC cell lines (mean IC50 = 0.3 �� 0.32 ��mol/L) (Figure (Figure1A1A and andB,B, Table Table11).

Table 1 Inhibition of cell growth by in vitro treatment with NVP-AEW541 Figure 1 In vitro cell growth inhibition. A: Treatment of seven human biliary tract cancer cell lines with NVP-AEW541 for 3 d (n = 3); B: 6 d incubation (n = 3); C: Incubation of selected cell lines EGI-1 and Mz-ChA-1 with calculated IC50 for 24-96 h (n = 3). … Cell lines Mz-ChA-1, showing a weak, and EGI-1, showing an intermediate response to NVP-AEW541, were selected for further studies of drug mechanism, because each of these lines represented one entity of BTC (GBC and extrahepatic CC). Firstly, the in vitro cell growth inhibition experiment was repeated with a broader spectrum of drug doses to determine the correct 3 d IC50 concentration.

To verify calculated 3 d IC50 concentrations and to determine the most effective Anacetrapib length of incubation, additional experiments with drug incubation times ranging from 1 to 4 d, with 3 d IC50 concentration, were carried out (Figure (Figure1C),1C), which showed best response after 3 d of incubation. Using data from this experiment, replication times for Mz-ChA-1 (68.16 h) and EGI-1 (30.7 h) were calculated. This difference could be one factor influencing response to NVP-AEW541. DMSO, the solvent of NVP-AEW541, had no influence on cell growth when administered alone (data not shown).