The Anti-cancer Effects And Mechanisms Of Oleanolic Acid In Human Prostate Cancer Cells

Part 1 The in vitro anti-cancer effects of oleanolic acid in human prostate cancer cellsObjective:To explore the in vitro anti-cancer effects of oleanolic acid (OA) on human prostate cancer (PCa) and its mechanisms.Methods:Human PCa PC-3, DU145 and LNCaP cells were treated with different concentrations of OA. The effects of OA on cell survival and proliferation of PC-3, DU145 and LNCaP cells were assessed by CCK-8 assay. The effect of OA on colony-forming ability of PC-3, DU145 and LNCaP cells was analyzed by colony formation assay. Flow cytometry analysis was used to assess the effect of OA on apoptosis and cell cycle distribution in PC-3, DU145 and LNCaP cells. Transwell assay was employed to analyze the effects of OA on cell migration and invasion of PC-3, DU145 and LNCaP cell. Caspase activity assay kits were adopted to detect the effect of OA on the activities of caspase-8, caspase-9 and caspase-3 in PC-3, DU145 and LNCaP cells. Mitochondrial membrane potential assay kit with JC-1 was used to examine the effect of OA on the loss of mitochondrial membrane potential in PC-3, DU145 and LNCaP cells. Western blotting analysis was adopted to assess the effects of OA on the expression levels of cleaved PARP, Bax, Bcl-2, Bcl-xL, CDK4, cyclin Dl, cytochrome c, PI3K, Akt, p-AktSer473, p-BadSer136, p-GSK-3βSer9, FOXO1 and p-FOXO1 proteins in PC-3, DU145 and LNCaP cells.Results:CCK-8 assay showed that OA remarkably inhibited cell survival and proliferation of PC-3, DU145 and LNCaP cells in a dose-dependent manner. Flow cytometry analysis revealed that OA obviously induced apoptosis and cell cycle G0/G1 phase arrest of PC-3, DU145 and LNCaP cells in a dose-dependent manner. Colony formation assay showed that OA evidently repressed colony-forming ability of PC-3, DU145 and LNCaP cells in a dose-dependent manner. Transwell assay revealed that OA obviously suppressed migration and invasion of PC-3, DU145 and LNCaP cells in a dose-dependent manner. In addition, OA remarkably increased the activity of caspase-8, caspase-9 and caspase-3 and the expression level of cleaved PARP in PC-3, DU145 and LNCaP cells in a dose-dependent manner. Western blotting analysis showed that OA dose-dependently increased the expression level of pro-apoptotic Bax protein, and decreased the expression levels of anti-apoptotic Bcl-2 and Bcl-xL proteins as well as CDK4 and cyclin D1 proteins. OA dose-dependently increased the loss of mitochondrial membrane potential and the release of cytochrome c from mitochondrion to cytoplasm in PC-3, DU145 and LNCaP cells. Western blotting analysis showed that OA dose-dependently reduced the expression levels of PI3K, p-AktSer473, p-BadSer136 and p-GSK-3pSer9 proteins in PC-3, DU145 and LNCaP cells, but didn’t change the expression levels of FOXO1 and p-FOXO1 proteins.Conclusions:OA can dose-dependently inhibit human PCa cell survival, proliferation, migration and invasion in vitro; OA can activate mitochondrial pathway and inhibit the expressions of cell cycle-related proteins and the activity of the PI3K/Akt pathway.Part 2 Oleanolic acid inhibits human prostate cancer cell survival and proliferation in vitro through the PI3K/Akt pathwayObjective:To explore the mechanisms of oleanolic acid (OA) in inhibiting human prostate cancer (PCa) cell survival and proliferation in vitro.Methods:Plasmid transfection was used to upregulate Akt expression in human PCa PC-3 and DU145 cells. qRT-PCR and western blotting analysis were employed to verify the transfection efficiency of Akt plasmid. The effect of upregulation of Akt expression on OA-inhibited cell survival and proliferation of PC-3 and DU145 cells was assessed by CCK-8 assay. The effect of upregulation of Akt expression in OA-inhibiting colony-forming ability of PC-3 and DU145 cells was analyzed by colony formation assay. Flow cytometry analysis was used to assess the effect of upregulation of Akt expression in O A-inducing cell apoptosis and cell cycle arrest of PC-3 and DU145 cells. Caspase activity assay kits were employed to detect the effect of upregulation of Akt expression in OA-activating caspase-9 and caspase-3 in PC-3 and DU145 cells. Mitochondrial membrane potential assay kit with JC-1 was used to assess the effect of upregulation of Akt expression on OA-induced loss of mitochondrial membrane potential in PC-3 and DU145 cells. Western blotting analysis was employed to evaluate the effect of upregulation of Akt expression in OA-inhibiting the expressions of p-AktSer473, p-BadSer136, Bcl-2, p-GSK-3pSa9 and cyclin D1 proteins in PC-3 and DU145 cells.Results:qRT-PCR and western blotting analysis showed that Akt expression was successfully upregulated by plasmid transfection in PC-3 and DU145 cells, and then the activity of Akt protein was increased. CCK-8 assay showed that upregulation of Akt expression remarkably reversed the inhibitory effect of OA on cell survival of PC-3 and DU145 cells. Flow cytometry analysis revealed that upregulation of Akt expression obviously reversed the enhancing effect of OA on apoptosis of PC-3 and DU145 cells. CCK-8 assay displayed that upregulation of Akt expression clearly reversed the inhibitory effect of OA on cell proliferation of PC-3 and DU145 cells. Colony formation assay showed that upregulation of Akt expression evidently reversed the inhibitory effect of OA on colony-forming ability of PC-3 and DU145 cells. Flow cytometry analysis revealed that upregulation of Akt expression obviously reversed the inducing effect of OA on G0/G1 phase arrest in PC-3 and DU145 cells. In addition, Upregulation of Akt expression remarkably reversed the activating effect of OA on caspase-9 and caspase-3 in PC-3 and DU145 cells. Upregulation of Akt expression evidently reversed the promoting effect of OA on the loss of mitochondrial membrane potential in PC-3 and DU145 cells. Western blotting analysis showed that upregulation of Akt expression clearly reversed the inhibitory effect of OA on the expressions of p-BadSer136, Bcl-2, p-GSK-3pSer9 and cyclin D1 proteins in PC-3 and DU145 cells.Conclusions:OA inhibited human PCa cell survival and proliferation in vitro through the PI3K/Akt pathway.Part 3 Oleanolic acid inhibits the in vivo tumorigenesis of human prostate cancer PC-3 cells through the PI3K/Akt pathwayObjective:To explore the mechanisms of oleanolic acid (OA) in inhibiting the in vivo tumorigenesis of human prostate cancer (PCa) PC-3 cells.Methods:Four-week-old BALB/c nude male mice were randomized into four groups (five mice per group):NC, NC+OA, Akt and Akt+OA. PC-3 cells (3×106) transfected with or without Akt plasmid were subcutaneously inoculated into the right axilla of each mouse in the corresponding groups. Nude mice were intraperitoneally administered with OA (150 mg/kg/day) in the corresponding groups. Tumor volume was measured using a caliper every three days from the 6th day. On day 21, nude mice were sacrificed and then the tumors were isolated and weighed. The transcriptional level of Akt was detected by qRT-PCR analysis in the tumor tissues derived from PC-3 cells.the expression level of Akt protein was detected by western blotting analysis in the tumor tissues derived from PC-3 cells. The expression levels of Akt, p-AktSer473, Ki-67, p-BadScr136, Bcl-2, p-GSK-3pSer9 and cyclin D1 proteins were examined by immunohistochemistry analysis in the tumor tissues derived from PC-3 cells.Results:In vivo tumorigenesis of PC-3 cells in the NC+OA group was slower than that in the NC group, and the isolated tumors in the NC+OA group were lighter than those in the NC group, which indicated that OA could inhibit in vivo tumorigenesis of PC-3 cells. Whereas, in vivo tumorigenesis of PC-3 cells transfected with Akt in the Akt+OA group was faster than that in the NC+OA group, and the isolated tumors in the Akt+OA group were heavier than those in the NC+OA group, which implied that upregulation of Akt expression reversed the inhibitory effect of OA on in vivo tumorigenesis of PC-3 cells. qRT-PCR and western blotting analysis showed that the expression level of Akt protein was increased in the tumor tissues from the Akt and Akt+OA groups versus the NC and NC+OA groups, which confirmed that the expression of Akt protein was successfully upregulated. Immunohistochemistry analysis showed that the expression levels of Ki-67, p-BadSer136, Bcl-2, p-GSK-3βSer9 and cyclin Dl proteins in the tumor tissues from the NC+OA group were decreased, compared with the NC group, while the expression levels of Ki-67, p-BadSer136, Bcl-2, p-GSK-3βSer9 and cyclin D1 proteins in the tumor tissues from the Akt+OA group were higher than that from the NC+OA group, which implied that upregulation of Akt expression reversed the inhibitory effect of OA on the expression of Ki-67, p-BadSer136, Bcl-2, p-GSK-3βSer9 and cyclin D1 proteins in the tumor tissues from PC-3 cells.Conclusions:OA could inhibit the in vivo tumorigenesis of human PCa PC-3 cells through the PI3K/Akt pathway.