DNA was labeled with 100 mL/L propidium iodide. Cells were sorted by FACScan analysis, and cell cycle profiles were determined using ModFitLT V2.0 software (Becton Dickinson, San Diego, USA). Each experiment was performed in triplicate. Animal studies Tumors were induced by injecting 5 �� 106 HPAF-2 or L3.6pl cells in 200 ��L PBS sc into the flank region of NMRI Crizotinib side effects nude mice. Treatment was started when an average tumor volume of 150 mm3 was reached (usually after 2 wk). The verum groups received either NVP-LBH589 (25 mg/kg, 5 �� weekly) or gemcitabine (5 mg/kg, 1 �� weekly) or a combination of both (NVP-LBH589 at 25 mg/kg, 5 �� weekly plus gemcitabine at 5 mg/kg, 1 �� weekly) ip, whereas the control group received placebo (carrier solution 50 mL/L DMSO in D5W) only.
Treatment was continued for 28 consecutive days, tumors were measured daily with a Vernier caliper and tumor volumes were calculated using the formula tumor volume = 0.5 �� L �� W2, where L represents the length and W the width of the tumor. When treatment was finished, animals were sacrificed and tumors excised and weighed. TUNEL POD test Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (in situ cell death detection kit, POD) was used to detect apoptosis in paraffin sections from mouse tumor tissue. TUNEL was carried out following the manufacturer��s instructions (Roche, Penzberg, Germany) as previously described[28]. Apoptotic cells (red) were counted under a light microscope after fluorescence signal conversion using peroxidase-conjugated antibody and peroxidase substrate (DAB, Roche, Penzberg, Germany).
The number of positive cells was counted by an experienced pathologist (M.N.) in a total of 8 high power fields (HPFs) and expressed as mean percentage of total cells in these fields of the tumor. Necrotic tumor cells were excluded from the cell count. Immunohistochemical staining For MIB-1 staining, we used paraffin sections following a protocol that has been described elsewhere[29]. The number of positive cells was counted by an experienced pathologist (M.N.) in a total of 4 HPFs and expressed as mean percentage of total cells in these fields of the tumor. Statistical analysis Statistical calculations were performed using SPSS, version 10.0 (SPSS Inc., Chicago, USA). Numeric data were presented as mean value with SD or SEM. Inter-group comparisons were performed with the Student t-test and ANOVA. P < 0.05 was considered significant. RESULTS Inhibition of cell growth After 3 d of incubation, 7 of 8 tested cell lines were sensitive to NVP-LAQ824 (mean IC50 (3 d) = 0.18 �� 0.24 ��mol/L) and even more to NVP-LBH589 (mean IC50 (3 d) Cilengitide = 0.09 �� 0.14 ��mol/L). Only cell line Capan-2 demonstrated an IC50 (3 d) value > 1 ��mol/L for both compounds.