DOX And STS Induce Human Hepatocellular Carcinoma Cell Line SMMC-7721 Apoptosis And Its Mechanism

One of main mechanisms of treating tumor with chemotherapy medicine is to induce cell apoptosis.The concrete mechanisms about how to start cancer cells' apoptosis and its signal transduction are also important problems in anti-tumor therapy of chemotherapy medicine.Doxorubicin hydrochloride(DOX) is a clinical anti-tumor antibiotic,and Staurosporine(STS) is a potent PKC inhibitor.DOX and STS are all common apoptosis inducer.Although DOX or STS can induce apoptosis clearly,the concrete mechanism in inducing apoptosis,and the expression and regulation of related signal molecules such as oncogene and antioncogene are also worth to study.We mainly analyzed the inductive effect of DOX or STS in SMMC-7721 cells and the possible apoptotic mechanism.Then we analyze the changes of mRNA and protein about Bax,Bcl-2,p53,PTEN,FAK,p-FAK and ILK.We try to make an approach to the changes of molecules relating to tumor in SMMC-7721 treated with DOX or STS in order to make sure the molecular mechanism of DOX or STS in tumor therapy.In all of this study,we used human hepatocellular carcinoma cell line SMMC-7721. Firstly,we detected cellar growth activity treated with DOX or STS in different concentration using MTT assay,and found that DOX or STS could obviously inhibit cellar growth.Secondly,we observe cells treated with DOX or STS using light microscope or fluorescence microscope,and found that cells in light microscope and nucleolus in fluorescence microscope change to that is similar to apoptotic changes. Thirdly,we detected mRNA and protein about caspase-3 in treating with DOX and STS using RT-PCR and Western blot assay,and found that caspase-3 mRNA was up-regulated and its protein was cut and activated.In order to analyze the concrete mechanism about the activation of capase-3,we detected mRNA of caspase-9 and caspase-8 in treating with DOX and STS,and found the up-regulation of capase-9, which implied that caspase-3 was activated through intrinsic pathway.Fourthly,we detected protein expression of PARP in treating with DOX and STS that is a main substrate of activatory caspase-3,and found that PARP was cut.Therefore,we demonstrated that DOX or STS induced SMMC-7721 cells apoptosis by activating caspase-3,and capase-3 may be activated through intrinsic pathway.In order to analyze the effect of molecules relating to tumor induced by DOX or STS,we detected mRNA and/or protein about Bax,Bcl-2,p53,PTEN,FAK,p-FAK (Tyr 397),ILK in treating with DOX and STS,and found that:1)The transcription of Bax was up-regulated and the transcription of Bcl-2 was down-regulated;2)DOX or STS could induce up-regulation of transcription and protein expression of p53 and PTEN to enter apoptotic process,and once cells started apoptosis,transcription and protein expression of p53 and PTEN were down-regulated;3) The transcription and protein expression of FAK were down-regulated;4) FAK phosphorylation was down-regulated after a brief up-regulation;5) DOX or STS had no effect on ILK protein expression.Taken together,our work demonstrated that DOX or STS induced SMMC-7721 cells to apoptosis by activating caspase-3,and capase-3 may be activated through intrinsic pathway in the first place.And apoptosis induced by DOX or STS had obvious effect on Bax,Bcl-2,p53,PTEN,FAK,p-FAK,but had no effect on ILK in the second place.Our study will help to investigate the apoptotic mechanism of chemotherapy medicine and the relationship between apoptosis and molecules relating to tumor.