Study Of The Effect And Mechanism Of Tanshinone �� A In Combination With Cisplatin On The Growth Of Hela Cells

Cervical carcinoma is one of the most common malignant tumor.In female carcinoma,the incidence of cervical carcinoma is the second only to mammary cancer, and it's the first leading cause of death from cancer in women.It's a serious suffering in women widely.In the past researches,cervical carcinoma was thought to be insensitive to chemotherapeutics.So chemotherapy was only a part in the combinedtherapy in advanced and recurring cervical carcinoma.But,recently researchers find combinedtherapy can improve 5-year survival rate and harvest many satisfactory results in preclinical and clinical studies of cervical carcinoma.However, chemotherapeutics,take cisplatin(DDP)for example,have many inevitable toxic and side effects.In other words,we can't use enough dose of the drugs to kill tumour cells. So,the urgent affairs are to develop effective anti-tumor drugs with less hypotoxicity. TanshinoneⅡA(TanⅡA)is an effective ingredient isolated from salvia miltiorrhiza BUNGE.Recent studies show that TanⅡA can inhibit proliferation and induce differentiation or apoptosis of many kinds of carcinoma cells.Those characters make TanⅡA possess good applying prospects as a new anti-tumor drug.DDP is a DNA damage drug,and it's the choice drug in cervical carcinoma chemotherapy,we want to study the effects of TanⅡA combined with DDP on the growth of Hela cells and the mechanism of action in vitro.The purpose of this study is to provide a new drug to cure the cervical cancer,and enrich the basic study on the anti-tumor effects of TanⅡA.Methods1.Cell culture:Hela cells freezed in liguid nitrogen were revived in routine method,and inoculated in RPMI-1640 blended with 10%new-born calf serum cultured in culture bottle.The bottle was put in the incubator with the condition of 37℃,5%CO2 for 72h with 0.01%DMSO,2ug/ml TanⅡA,5ug/ml DDP,and 2ug/ml TanⅡA combined with 5ug/ml DDP respectively. 2.The staining inhibitory effect of them on Hela cells' growth was analyzed by MTT assay.3.The cell apoptosis was measured by HE staining.4.The cell apoptotic rate was measured by Flow-cytometry.5.The cell apoptosis related protein P53,Bcl-2 and Bax were detected by Western Blot.Results1.The proliferation of Hela cells was inhibited by TanⅡA,DDP and the combination,respectively.Inhibition ratios were 40.01%,48.18%and 78.19%in TanⅡA group,DDP group and the combination group,respectively.There was a marked difference in the inhibition ratio among the TanⅡA group,DDP group and the combination group(p＜0.05).The coefficient of drug in interaction(CDI)was less than 1,so TanⅡA combined with DDP can generate synergistic effect.2.HE staining showed the apoptosis induced by TanⅡA,DDP and the combination,respectively,but the control group didn't.3.Apoptotic rates were 11.1%,11.4%and 17.6%in cells treated with TanⅡA, DDP and the combination,respectively.There was a significant difference in the percentage of apoptotic cells among the TanⅡA group,DDP group and the combination group(p＜0.05).4.The P53 protein expressed in each group,and it was increased in the combination group.The Bcl-2 protein also expressed in each group,but there was no much difference among them.Western Blot demonstrated Bax protein expression was also increased in the combination group,but it didn't express in the control group.Conclusion1.The proliferation of Hela cells could be inhibited in vitro by TanⅡA,DDP and the combination,respectively.And the combination could enhance the inhibitory effect.TanⅡA combined with DDP can generate synergistic effect.2.TanⅡA,DDP and the combination could induce cell apoptosis respectively. And the induction of apoptosis on Hela cells was enhanced by the combination. 3.The mechanisms of apoptosis-inducing may be associated with the restoration of the functions of P53 tumor suppressor and the up-regulation of Bax(apoptosis-promoting protein).Meanwhile,they expressed more in the combination group than in the single ones may result in the higher apoptotic rate.4.Inhibitting proliferation of Hela cells and inducing apoptosis may be the anti-tumor mechanisms of TanⅡA,DDP and the combination.