Fig. 5

Combined rhein and EGFR inhibitors induce apoptosis in pancreatic cancer cell. a The indicated PANC-1 and Patu8988T cells were treated with DMSO (Control), 100 μM rhein, 5 μM erlotinib or the combination of both inhibitors. Whole-cell protein extracts were analyzed after 36 h of treatment by Western blotting with the indicated antibodies. GAPDH antibody was used as loading control. Results are representative of three independent experiments. b Pancreatic cancer cells PANC-1 and Patu8988T were treated with rhein and erlotinib 36 h and assayed for caspase-3 activity. c PANC-1 and Patu8988T cells were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination 36 h. The viable cells showed normal-shaped nuclei that were faintly stained with Hoechst 33258. The apoptotic cells exhibited shrunken nuclei with evidence of chromatin condensation. The results are representative of three replicate experiments (magnification, × 200). d Representative images for cell apoptosis stained with Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI). PANC-1 cells were treated with rhein, erlotinib or the combination of both inhibitors for 24 h, then cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry as described in the methods section. The statistical data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001