(G) Effect of NAC on PEITC-induced GSH depletion. indicates an enhanced cytotoxicity for the combination. To determine whether PEITC in combination with CDDP decreases cell viability via an increase in apoptosis, annexin V-fluorescein isothiocyarate (Annexin V-FITC)/propidium iodide (PI) double labeling flow cytometry was used to determine the percentage of cells entering apoptosis. Flow cytometry analysis showed that CDDP caused about 10% of the cells to enter apoptosis, but co-treatment with PEITC dramatically enhanced CDDP-induced apoptosis to 40% (Figure 1CC1D). A similar pro-apoptotic effect of the combined treatment PFK-158 with PEITC and CDDP was also observed in human cholangiocarcinoma RBE cells (Figure 1EC1H). Together, these data demonstrated that PEITC can enhance CDDP-induced apoptosis in BTC cells. PEITC enhances the sensitivity of SP cells and xenograft tumors to CDDP Recent studies have shown that SP cells isolated from various cancer cell lines and primary tumors possess cancer stem-like properties [13C16]. SP cells can effectively avoid the effects of chemotherapeutic drugs, and are considered to be the root cause of tumor recurrence and metastasis. Therefore, we tested the effect of PEITC-CDDP co-treatment on SP cells from GBC-SD cells. The proportions of SP cells was 5.2% (Figure ?(Figure2A).2A). PFK-158 As shown in Figure ?Figure2B,2B, flow cytometry analysis of SP cells treated with PEITC, CDDP, or a PEITC-CDDP combination showed that CDDP alone caused little cell apoptosis, but when combined with PEITC, markedly enhanced apoptosis at 24 hrs. These results demonstrate that PEITC significantly enhances the sensitivity of SP cells to CDDP. Open in a separate window Figure 2 PEITC-CDDP co-treatment sensitizes SP cells and inhibits xenograft tumor growth without obvious toxic effects(A) FACS analysis on single cell suspension of GBC-SD cells stained with Hoechst 33342 dye showing SP cells. SP cells are enclosed within the area demarcated in black. Verapamil inhibited the efflux of the dye and caused the disappearance of SP cells. A representative plot of the frequency of SP cells is provided. (B) SP cells from GBC-SD cells were treated with PEITC, CDDP or PEITC-CDDP combination for 24 hrs and apoptosis detected by Annexin V/PI assay. Data shown PFK-158 is average of three independent experiments. *< 0.05. (C) GBC-SD cells were transplanted into nude mice. When tumor size reached approximately 50 mm3, mice were randomly sorted into four equal groups. The tumor-bearing mice were intra-peritoneally injected with physiological saline as a control, PEITC, CDDP or PEITC-CDDP combination for 10 days. Xenografts were excised and weighed. Each dot represents weight of one tumor, and the mean tumor weights of each PFK-158 group is indicated by solid lines (right panel; 7). *< 0.05. (D) Volume of the tumors was measured twice a week, and a tumor growth curve created for each group (7). *< DC42 0.05,. (E) Mice were weighed twice a week, and a weight curve created for each group (7). To further examine the synergistic effect of PEITC and CDDP anticancer effect in the PEITC-CDDP combined group was further evident PFK-158 in the tumor growth curve data (Figure ?(Figure2D).2D). Systemic toxic effects of the treatments in these mice were evaluated by measuring the loss in body weight. No notable differences were observed between the treated groups (Figure ?(Figure2E).2E). Collectively, these results demonstrate that PEITC-CDDP co-treatment can effectively inhibit tumor growth without obvious toxic effects siRNA (SiMcl1) for 48 hrs and reduction in Mcl-1 was analysed by western blot (C) Apoptosis analysis using Annexin V/PI flow cytometry in GBC-SD cells transfected with siRNA after treatment with CDDP for 24 hrs. Data shown is average of three independent experiments. *< 0.05. (D) Immunoblot analysis of Mcl-1 protein level in SP and MP cells from GBC-SD cells. PEITC enhances the cytotoxicity of CDDP through proteasomal degradation of Mcl-1 and and and plasmid for.