| Part1: In vivo Suppression of androgen independent prostate cancergrowth by PC-SPESⅡPurpose To evaluated efficacy of POSPES Ⅱ against DU145 and PC-3 xenografts growth .Methods 128 male Balb/c nu/nu nude mice were randomly divided into two groups, DU145 or PC-3 cells were injected subcutaneously, and each group were randomly divided into four groups of different dose and 16 mice each:control, PC-SPESⅡ125,250 and 500mg/kg.d .On the day after the incubation mice were fed the 0.9% saline solution or PC-SPESII every day for 8 weeks. At the end of 2,4,8 weeks, viscera were removed for side effects and toxicity analysis. 80 mice complete 8 weeks observe and 10 in each dose groups. The latent of growth were monitored , and tumor sizes were measured every week with vernier calipers and the volume calculated by the formula:length×widthXheight×π/6. At the end of experiments, all tumors were removed for histological study, and group of 250mg/kg. d were analyzed by electron microscopy. Statistical analysis was done with Oneway ANOVA and SAS repeated measures analysis of variance.Result PC-SPES II feeding resulted in suppression of androgen independent prostate cancer growth without any adverse effect. The growth of DU145 tumor were significant delayed (P=0.021) , and tumor growth inhibition in a significant dose-dependent manner(P=0.0002). At the end of study , DU145 tumor volume inhibition by PC-SPES II 125,250,500mg/kg. d was 29.41%, 44. 2%, and 66.15% .No statistical significance was noted in either the latent of growth or volume of the PC-3 tumors (P=0. 252 or 0. 0787) . Electron microscopy DU145 tumor showed typical apoptosis morphological changes, and PC-3 showed significant reduce of mitochondrial.Conclutions PC-SPESII inhibits proliferation of androgen independent prostate cancer in vivo, and apoptosis maybe one of the mechanisms.Part 2:The effect of PC-SPESII on serum levels of IL-6 of nude mice withDU145 or PC-3 xenograftsPurpose To analysis the serum levers of IL-6 after treatment with PC-SPES II and compare to controls.Methods 93 male Balb/c nu/nu nude mice were divided into three groups, 9 without injected tumor cells named non-tumor group,the DU145 or PC-3 groups were divided into four groups of different dose and 10-11 mice each-.control, PC-SPESII 125, 250 and 500mg/kg. d . On the day after the incubation , non-tumor and control groups were fed with 0.9% saline solution and others with PC-SPESII every day for 8 weeks. At the end of 2, 4, 8 weeks, 3-5 mice serum were collected and IL-6 were measured using commercially available enzyme linked immunosorbent assays (ELISA) . We also measured the lever of IL-6 mRNA of DU145 and PC-3 tumors using RT-PCR after PC-SPES II 250mg/kg. d were fed for 8 weeks. Statistical significance was determined by Oneway ANOVA and difference between two group using t test.Results Serum levels of IL-6 were significantly elevated in DU145 and PC~3 nude mice (P<0. 0001) when comparing to those without tumors. Serum IL-6 of DU145 mice were significantly elevated with growth of tumor at the end of 8 weeks (P=0. 016), but no statistical significance was noted in PC-3 mice(P=0.778). Serum levels of IL-6 could be significant inhibited by PC-SPESII in a dose dependent manner both in DU145 and PC-3 mice (P^0. 002 or P<0. 0001), and that was the case of IL-6 mRNA. Conclutions Tumor-secreted IL~6 and serum IL-6 of androgen independent prostate cancer could be decreased by PC-SPESII. Part 3 :The effects of PC-SPES II on Gl/S regulatory factors and apoptosis related proteins in androgen independent prostate cancer xenograftsPurpose To elucidate the molecular mechanism of growth arrest and apoptosis induction of PC-SPES II in androgen independent prostatecancer xnografts.Methods 80 male Balb/c nu/nu nude mice were divided into two groups, DU145 or PC-3 cells were injected subcutaneously, and each group were randomly divided into four groups of different dose and 10 mice each:control, PC-SPESII125, 250 and 500mg/kg.d . On the day after the incubation mice were fed the 0.9% saline solution or PC-SPESII every day for 8 weeks. At the end of At the end of experiments, all tumors were removed , the expression of Gl/S regulatory factors P16, P21, P-Rb and apoptosis related protein Bax, Bcl-2 and cleaved-PARP were analyzed by Western blot and Immunohistochemistry. Statistical significance was determined by Oneway ANOVA and difference between two group using t test. Results Results of Western blot:The expressions of P16 was elevated significantly (P=0. 001) and P21 and phosphorylated Rb(P-Rb)were not alterd (P=0. 188 or P=0. 778) in the DU145 group treated with PC-SPES II as compared with the control. While the expression of P16, P21 were elevated significant in a dose-dependent manner (P<0. 0001) , and P-Rb was decreased in a dose-dependent manner (P=0. 005)in the PC-3 group. In addition, the expression of proapoptotic protein Bax was increased( P=0.003 ) Bcl-2 and antiapoptotic protein Bcl-2 was decreased significantly (P=0. 001) in the DU145 group. While Bcl-2 was decreased only at 500mg /kg. d(P=O. 021), Bax decreased at 125mg/kg. d and increased to the level of control at 500mg/kg. d in the PC-3 group. The expressions of cleaved-PARP in tumor of both group were increased significantly(P=0. 002 or P=0. 02) and finally leading to the promotion of apoptosis after treatment of PC-SPES II . Results of Immunohistochemistry: Expression of each of protein in PC-3 group and P16 , P~Rb and Bax in DU145 group were consist with Western blot . While the P21 was elevated significant (P=0. 001) and the Cleaved-PARP increased or Bcl-2 decreased became more significantly in a dose-dependent manner in the DU145 group. Conclutions The possible molecular mechanism of PC-SPES II is speculated to regulate some gene expressions related to Gl/S arrest and apoptosis.
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