Study On The Effect And The Mechanisms Of Sodium Valproate On The Growth, Differentiation And Apoptosis Of Breast Carcinoma Cell

Objective;The histone acetylizad level is dynamic accommodated by histone acetyltransferase(HAT)and histone deacetylase(HDAC).HATs lead to the relaxation of chromatin structures and genes transcriptional activation,while HDACs associate with chromatin condensation and transcriptional silence.Alteration in eukaryotic chromatin structures which caused by acetylizad modification to the N-terminal domains of core histone appeared to play a central role in the regulation of gene transcription,and recent studies have demonstrated the correlation of histone acetylizad level or histone deacetylase with the genesis and development of some kinds of tumors,thus a new approach of tumor chemotherapy emerged—to activate HATs and/or to suppress HDACs.Sodium Valproate(VPA-Na),which has been used widely as an anticonvulsant for over 20 years and been famous for its low noxious and prolonged effectiveness,was demonstrated as a classâ… selective histone deacetylase inhibitor recently.In this article,the effect of VPA-Na on the cell morphology,cell growth,apoptosis and cell cycle of MCF-7 were observed or detected,the activities and the protein expression of Caspase3,Caspase8,Caspase9 were analysed,and the protein and mRNA expression of Cyclin A,Cyclin D1,Cyclin E,P21^Waf/cip1were also detected to investigate the mechanisms.Method;Effect of VPA-Na on the morphology of MCF-7In our study,MCF-7 breast carcinoma cells were cultured with VPA-Na,then stained and the cell growth condition was observed by invert microscope.Effect of VPA-Na on the cell growth,apoptosis and cell cycle of MCF-7(1)Detection of cell growth inhibition VPA-Na was added into the cell culture system,with the final concentrations of 0.75mmol/L,1.5mmol/L,2.0mmol/L,2.5mmol/L,3.0mmol/L,3.5mmol/L,4.0mmol/L.VPA-Na was substituted by PBS for the control.The cell growth inhibition rate was examined by MTT assay.(2)Detection of cell apoptosis VPA-Na was added into the cell culture system,with the final concentrations of 0.75mmol/L,1.5mmol/L,2.0mmol/L,2.5mmol/L,3.0mmol/L,3.5mmol/L,4.0mmol/L.VPA-Na was substituted by PBS for the control.The apoptosis was detected by flow cytometry with Annexin V/PI and PI assays.(3)Analyse of cell cycle VPA-Na was added into the cell culture system,with the final concentrations of 0.75mmol/L,1.5mmol/L,2.0mmol/L,2.5mmol/L,3.0mmol/L,3.5mmol/L,4.0mmol/L. VPA-Na was substituted by PBS for the control.The apoptosis was detected by flow cytometry with Annexin V/PI and PI assays.Molecular mechanisms of the effect of VPA-Na on MCF-7(1)Detection of activity and protein expression of Caspase The activities and protein expressions of Caspase3,Caspase8,Caspase9 were detected by spectrophotometry and indirect immunofluorescence technique respectively.(2) Analyse of protein and mRNA expression of Cyclin The protein and mRNA expressions of Cyclin A,Cyclin D1,Cyclin E,P21^Waf/cip1were analyzed by indirect immunofluorescence technique and RT-PCR.Result;Effect of VPA-Na on the morphology of MCF-7After treated with different concentration of VPA-Na for 24h,48h,72h,96h,the population decreased and shape,size changed; karyopyknosis and endochylema decrease was found too.Effect of VPA-Na on the cell growth,apoptosis and cell cycle of MCF-7(1)Detection of cell growth inhibition The inhibition rate increased significantly(p＜0.001),and the increasement dependent on VPA-Na dose and action time.(2)Detection of cell apoptosis The apoptosis rates increased.The minimum apoptosis rate of test groups was 6.8% and the maximum apoptosis rate was 28.8% that were higher than the control groups and a dose and time dependent tendency was also found.(3)Analyse of cell cycle The percentages of G1,S,M phrase in cell cycle remained the same in all control groups,while VPA-Na at concentrations between 0.75 and 4.0 mmol/L induced a significant growth arrest in G₁ phrase(p＜0.001)and the effect was different as the dose and time changed.Molecular mechanisms of the effect of VPA-Na on MCF-7Caspase3,Caspase9 were significantly activated in cells treated for 48 hrs with 1.5,3.0 mmol/L VPA-Na compared with control groups and Caspase3,Caspase9 protein were up-regulated too(p＜0.001).However,no statistically significant increase in caspase 8 activity and protein was found.P21^Waf/cip1was up-regulated both at the mRNA and at the protein level,and Cyclin D1 were down-regulated both at the mRNA and the protein level(p＜0.001).Conversely,mRNA expression of Cyclin E,Cyclin A was unchanged upon treatment with VPA-Na; nor did it show any difference in protein level(p＞0.05).Conclusion;Above all,VPA-Na's effect on cell growth include inhibit cell growth,apoptosis induction and cell cycle arrest in G1 phrase.The mechanism underlying its effect on apoptosis induction,the intrinsic pathway(Cytochrome C pathway)was involved,for caspase-9,which finally induces caspase-3,was clearly activated.The mechanism underlying its effect on cell cycle arrest in G1 phrase induction include,we assessed, up-regulate P21^Waf/cip1mRNA and protein expression which can bind CDKs competitively with Cyclins,thus reduce Cyclin-CDK complexes and induce G1 phrase arrest; Down-regulate Cyclin D1 mRNA and protein expression to inhibit CyclinD1-CDK4/CDK6 pathway activity,cause cells can not get through the G1/S check point.VPA-Na can suppress breast carcinoma cell growth,arrest the cell cycle and induce apoptosis at 0.75-4.0mmol/L,so it can be used as an option for the treatment of breast carcinomas.