Each bar represents means SD from three independent experiments. after treated with 100 M Rhein or DMSO for 24 h. Representative images are shown. Each bar represents means SD from three independent experiments. ***< 0.001. (D) Cell cycle distribution were analyzed in Panc-1, MIAPaCa-2 and HPDE cells after 0-200 M Rhein treatment. Representative results are shown in the left panel. Statistical comparisons were performed. Each bar represents means SD from three independent experiments. *< 0.05; **< 0.01; ***< 0.001. (E) Western blot analysis of protein levels of cdk4, cdk6, cyclinD1, cyclinE, p21 and p27 in Panc-1, MIAPaCa-2 and HPDE cells after indicated concentration of Rhein treatment for 24 h. GAPDH was used as a loading control. Representative results of two independent experiments are shown. In the present study, we investigated the effects of Rhein treatment alone or in combination with oxaliplatin on PC cells. We demonstrated for the first time that Rhein can inhibit the PI3K/AKT pathway and display a synergistic effect with oxaliplatin by inducing apoptosis of PC cells both and values were evaluated by Student's two-tailed < 0.05 was considered as statistically significant. Results Rhein inhibits PC cell growth by inducing a G1 phase cell cycle arrest To investigate the effects of Rhein on cell growth, human PC cells (Panc-1 and MIAPaca-2) and the immortalized normal human pancreatic duct epithelial (HPDE) Z-DEVD-FMK cell line were treated with varying concentrations (0-200 M) of Rhein for 24 h and 48 h. CCK-8 assay results showed that Rhein inhibited cell viability in a dose- and time-dependent manner in Panc-1 and MIAPaca-2 cells, while HPDE cells were less sensitive to Rhein (Figure ?(Figure1B).1B). The respective inhibitory concentration (IC) 50 values at 24 and 48 h of Rhein treatment were approximately 89.9 M and 76.3 M in Panc-1 cells, 83.4 M and 72.6 M in MIAPaca-2 cells, and 157.1 M and 150.4 M in HPDE cells. Colony formation assays indicated that Rhein also inhibited the long-term survival ability of PC cells (Figure ?(Figure11C). To explore the mechanism of Rhein-induced growth inhibition in PC cells, cell cycle distribution was analyzed by flow cytometry. As shown in Figure ?Figure1D,1D, Rhein caused G1 phase arrest of Panc-1 and MIAPaca-2 cells in a dose-dependent manner. Conversely, the percentage of HPDE cells in G1 phase was only slightly elevated under a high concentration of Rhein treatment. To understand the mechanism underlying the G1 phase arrest in Rhein-treated PC cells, we next used western blots to investigate the effect of Rhein on the expression levels of proteins that regulate the G1 phase transition. As shown in Figure ?Figure1E,1E, Rhein treatment resulted in a dose-dependent decrease in CDK4, CDK6, cyclin Z-DEVD-FMK D1, and cyclin E protein levels and an increase in p21 and p27 protein levels in PC cells. However, expression levels of these proteins only slightly changed in Rhein-treated HPDE cells. Taken together, Rabbit polyclonal to PKC zeta.Protein kinase C (PKC) zeta is a member of the PKC family of serine/threonine kinases which are involved in a variety of cellular processes such as proliferation, differentiation and secretion. these results indicate that Rhein can inhibit PC cell growth by inducing a G1 phase cell cycle arrest. Rhein induces PC cell apoptosis by activation of caspase cascades and the mitochondrial apoptosis pathway To explore whether Rhein has an effect on Z-DEVD-FMK PC cell apoptosis, Panc-1.