Studies Of Tanshinone IIA On Growth Inhibition And Apoptosis Induction In Prostate Cancer Cell Lines

ObjectiveTo evaluate the effect of tanshinoneâ…¡A on growth inhibition and apoptosis induction in human prostate cancer cell lines(PC3 and DU145) in vitro.To investigate the cell cycle and apoptosis-related genes protein expression changes of PC3 and DU145 cells treated with tanshinoneâ…¡A.Explore the possible mechanism of tanshinoneâ…¡A and provide a gist for the treatment of advanced prostate cancer.MethodsThe PC3 and DU145 cells were cultured in vitro and treated with tanshinoneâ…¡A in different dose(2.5,5.0,10.0,20.0mg.L^-1) and different time(24h,48h,72h) for following measurement.1.Observation of morphologic changes under the light microscope(LM) and transmission electron microscope(TEM).2.CCK-8 assay and colony-forming assay were used to investigate the inhibition of cells' growth.3.Agarose gel electrophoresis was used to examine the DNA fragments.4.Changes of cell cycle were analyzed with PI staining,flow cytometry(FCM).5.Western-blot assay was used to detect the apoptosis-related genes p53,p21,bcl-2, Caspase3 protein expression changes.Results1.LM and TEM observation:Under light microscope,the PC3 and DU145 cells in control group were large,roundish and serried.After treated by 10.0mg.L^-1 tanshinoneâ…¡A,the cells became small,detached or sparse and membranous frothed or wizened.The characteristic morphologic changes of apoptosis such as chromatin condensation and apoptotic body were observed under transmission electron microscope.2.Cytotoxicity test:In CCK-8 assay,all the tanshinoneâ…¡A dose levels(2.5,5.0,10.0, 20.0mg.L^-1) induced growth inhibition in both of the 2 cell lines.The inhibitory effect became stronger with the passage of time after treatment with tanshinoneâ…¡A,as well as the increase of doses.The differences between different dose and treatment time groups were statistically significant,which demonstrated the growth and proliferation of PC3 and DU145 cells were inhibited in a dose- and time- dependent manner.3.Colony-forming assay:The colony-forming rates in both of PC3 and DU145 cells were obviously suppressed by every dose level oftanshinoneâ…¡A.4.Agarose gel electrophoresis:DNA "ladder" was presented in 2.5,5.0 and 10.0 mg.L^-1 tanshinoneâ…¡A groups in DU145 cells,and in 5.0,10.0 mg.L^-1 groups in PC3 cells,but not observed in control groups.5.Cell cycle analysis:Compared with control groups,the cells treated with tanshinoneâ…¡A were arrested in G₀/G₁ phase and decreased in S phase.The apoptosis index was increased in flow cytometry.6.Western-blot assay:Compared with control groups,the protein expression of apoptosis-related genes p53,p21,Caspase3 in treated cells were up-regulated and bcl-2 down-regulated.ConclusionThe findings of the study suggested that tanshinoneâ…¡A could inhibit the growth and induce apoptosis in PC3 and DU145 cells in vitro.The possible mechanisms of tanshinoneâ…¡A were arresting the cell cycle in G₀/G₁ phase,up-regulating p53,p21, Caspase3 expression of protein and down-regulating bcl-2 expression.Tanshinoneâ…¡A could be a potential effective candidate for the clinical treatment of advanced prostate cancer patients.