Experimental Study On The Effects Of Tea Polyphenols In Prostate Cancer PC-3M Cells

Objective:Tea,as a traditional health drink in China, has lasted for over three thousand years,and it is one of the three world's favourest beverages for it's speciality on health care.Today it's still popular.It has became more and more scientists' concerns that the habit of drinking tea regularly has advantages of keeping human health.Prostate cancer is the second largest cancer that result of the death,only second to lung cancer in men in western countries.The prostate tumor incidence in China,Japan and other asiatic countries in which peoples have the long-term habits of drinking tea is far lower than the west.Whether this difference is relative with the habits of drinking tea in above-mentioned zones?Many findings of experiments in vivo and in vitro have indicated that the main ingredient of tea:tea polyphenols(TPs) is anticarcinogenic,so it is reasonable that more and more investigators try to cure the tumor with the TPs.Our experiment also try to investigate the regulatory effect of tea polyphenol on proliferation inhibition apoptosis and invasion force of human androgen-independent prostate cancer PC-3M cells.Methods:The androgen-independent prostate cancer cells PC-3M is cultured in Iscove's Modified Dulbecco's Medium(IMDM) containing 10% of fetal bovine serum,and is incubatored in incubation at 37℃, 5% CO2 and constant humidity.When cell fusion reachs about 80%,intervention treatment is conducted.The PC-3M cells are divided into treatment group and control group.At 24 hour and 48 hour after intervention,we observe the change about cell number,morphology and state by phase contrast microscope.Final concentrations of TPs are 0,20,40,60,80,100ug/ml.The inhibitions of cell proliferation at 24 hour,48 hour and 72 hour after intervention are detected by MTT assay.Final concentrations of TPs are same with above-mentioned.The change of cell cycle phase at 24 hour after intervention is analyzed by flow cytometry.Final concentrations of TPs are 0,40,60,80ug/ml.The changes of the apoptosis at 24 hour and 48 hour after intervention is inspected by acridine orange/ethidium bromide(AO/EB) fluorescent staining method.Final concentrations of TPs are also 0,40,60,80ug/ml.By scratche assay,we observe the change of cell migration at 0h,4h,8h,12h and 24h after intervention with different concentrations of TPs.Final concentrations of TPs are also 0,40,60,80ug/ml.We conduct the transwell assay.We know the change of cell invision force when PC-3M cell is intervented for 24h and 48h with different concentrations of TPs.Final concentrations of TPs are 0,40,60,80ug/ml.Besides,the transcription and translation of the genes of cyclin D1,caspase-3, survivin,MMP-2,TIMP-2 in carcinoma cells are observed by RT-PCR and western blot.Final concentrations of TPs are 0,40,60,80ug/ml.Data shown here are from representative experiments repeated at least three times with similar results.The data are shown asχ±s.With SPSS 17.0 statistical software,the data are analysised as t-test for independent samples and analysis of variance for the single factor and multiple sets(a=0.05).Results:After being treated with tea polyphenols solution,the number,morphology and state of human androgen-independent prostate cancer PC-3M cell are all changed appropriately.Compared with control group,dosing groups cell proliferation is slower,and cell is weak.Under the microscope,the cell number decreases and cell is slender and collapse.In some local visual fields,fine filamentous "pseudopodia" is visiable.Cell transmittance is weakened,and grey-black particles increase in cytoplasm.The results of MTT assay about cell inhibition incidence are shown in Talbe 1.1,from which we can see that as TPs's concentrations being elevated and time being extended,proliferation inhibitory effect of TPs on the PC-3M cell has gradually enhanced.AO/EB staining reflects the apoptosis rate after PC-3M cell is treated with TPs at different concentrations as shown in Talbe 1.2,from which we can see that partial cells present the morphological change of apoptosis under the fluorescent microscope, and the apoptotic rates of the treatment groups increase respectively.Flow cytometry reflects cell cycle changes after prostate cancer PC-3M cell is treated with TPs for 24h,as shown in Talbe 1.3,from which we can see that the cells in S and G2/M phase increase, the cells in G0/G1 phase decrease.It demonstrates that the cell cycle of experimental group is blocked in the G1 phase,and proliferation index decrease.As shown in the scratch assay,as TPs concentration increasing, the lateral transfer distances of PC-3M cells decrease.And there is also change about cell morphology.Especially when the TPs concentrations is up to 80ug/ml,the cell on the edge of scratch creases and become round.It indicates that the TPs can attenuate the ablity of migration of prostate cancer PC-3M cell.It is shown in the photo of transwell assay and Talbe 1.4 that PC-3M cells which penetrated through the matrix and migrated to under-surface decrease after treatment with TPs.It explains that PC-3M cell invasion forces weaken.In part 1 of experiment,RT-PCR and Western blot result shows the transcription and translation of caspase-3 gene is up-rugulated,and the transcription and translation of cyclin D1 and survivin gene are down-rugulated.The above results in a certain range are time-dependent and dose-dependent,which to a certain extent explain and validate the phenomenons that TPs can cause prostate cancer PC-3M cell cycle arrest,proliferation depreciation and promotion of cell apoptosis.In part 2 of experiment,RT-PCR and Western blot result shows the transcription and translation of MMP-2 genes is down-rugulated,and the transcription and translation of TIMP-2 gene is up-rugulated.The above results in a certain range is time-dependent and(or) dose-dependent,which to a certain extent explain and validate the phenomenons that TPs can weaken prostate cancer PC-3M invasion.Conclusion:TPs can inhibit the proliferation of human androgen-independent prostate cancer PC-3M cell,induce the apoptosis and weaken PC-3M cell migration and invasion force.The transcription and translation of up-regulation of caspase-3 and TIMP-2 gene,and of down-regulation of cyclin D1,survivin and MMP-2 gene may one of its molecular mechanisms.