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Purpose by in vitro cell culture methods, confirmed tanshinone Ⅱ A (Tan Ⅱ A) In vitro inhibition of human prostate cancer cell lines PC3 and DU145 growth and induction of apoptosis, and further analysis of cell cycle and apoptosis related gene protein expression. Explore the possible mechanism of Tanshinone Ⅱ A in PC3 and DU145, and eventually hopes to provide a new choice for the clinical treatment of advanced prostate cancer. Method of in vitro cultured human prostate cancer cell lines PC3 and DU145, respectively with different concentrations of tanshinone Ⅱ A (2.5,5.0,10.0,20.0 mg.L -1 ) cells were treated for 24, 48 and 72 hours after detection of the following indicators: light microscopy, electron microscopy of cell morphology changed; cell count, cytotoxicity assay (CCK-8 method), colony formation assay inhibition of cell growth and proliferation; 3. agarose gel electrophoresis analysis the cell DNA ladder fracture; 4. changes in the cell cycle by flow cytometry PI staining before and after treatment; 5.Western-blot to detect cell apoptosis gene proteins p53, p21, bcl-2, expression of Caspase3 in change, explore drugs mechanism. Results. Morphological observation: after 10.0mg.L -1 tanshinone light the microscope control group cells adherent growth, round or fusiform, volume, closely arranged smooth edge; Ⅱ A treated cells arranged in sparse cell shrinkage, membrane shrinkage, into a small round or irregular shape, and see more cells floating in the medium. With the prolonged duration (24h-48h-72h), the extent of its morphological changes gradually elevated; transmission electron microscope visible Tan Ⅱ A group of cells characteristic changes of apoptosis such as nuclear condensation and fragmentation, formation of apoptotic bodies. Cytotoxicity assay: various concentrations of tanshinone II A (2.5,5.0,10.0,20.0 mg.L -1 ) PC3 and DU145 growth was inhibited significantly stronger than in the control group, the The difference was statistically significant (P <0.05). In addition, it was found that, with the role of time and tanshinone Ⅱ A concentration gradually increased growth inhibition. The two-way ANOVA test significant difference (P <0.05) between the different times of cell viability 10.1mg.L -1 Tanshinone Ⅱ A treatment, different concentrations of tanshinone Ⅱ A treatment after 72h the cells significant difference (P <0.05), suggesting that drug effects between the survival time and concentration-dependent manner. Colony formation assay: Tanshinone Ⅱ A treatment PC3 and DU145 cell colony formation rate was significantly lower compared with the control group, a statistically significant difference (P <0.05). And colony formation rate decreased with the increase in drug concentration. Agarose gel electrophoresis: by 2.5,5.0,10.0 mg.L -1 Tanshinone Ⅱ A treated DU145 cells and 5.0,10.0 mg.L --1 handled the PC3 cells DNA ladder fracture, while the control group did not occur to this phenomenon. Flow cytometry cell cycle: of tanshinone II A (5.0, 10.0, 20.0 mg.L -1 ) PC3 and DU145 cells after, G 0 / G 1 of proportion significantly higher S-phase fraction decreased significantly higher rate of apoptosis. 6 apoptosis-related protein expression detected: Western-blot detection, compared with the control group, Tanshinone Ⅱ A significantly increased the expression of p21 protein, lowered the bcl-2 protein expression, and to varying degrees upward of p53 and Caspase3 the expression level. Conclusion Tanshinone Ⅱ A significantly inhibit the growth of prostate cancer cell lines PC3 and DU145, induction of apoptosis in vitro, the role of time and concentration-dependent. Tanshinone Ⅱ A possible mechanism of action of the cells were arrested in G 0 / G 1 of significantly reduced cell proliferation index, and affect the protein expression of apoptosis-related genes, such as raised the expression of p53, p21, Caspase3 down bcl-2 expression, thereby inducing tumor cell apoptosis. The tests showed that tanshinone Ⅱ A potential value in the clinical treatment of prostate cancer, and worthy of further research and exploration.
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