The Study On The Drug Resistance Induction Of Breast Cancer Cell Line By The Overexpression Of HDAC1

Breast cancer is one of the most popular female cancers with high incidence rate,mortality rate and uncertain prognosis.Drug resistance acquired during the chemotherapy is a possible reason which leads to the failure of treatment.There are more than 50%of the patients who suffering breast cancer could not aviod the recurring even after an effective initially chemotherapy.Histone deacetylation regulation is a kind of epigene modification for cells,it involves in the occurrence and development of many malignant tumors.HDAC 1 is a kind of histone deacetylase that has the closest relationship to tumors,related to many biological regulations such as cell cycle,differentiation,proliferation and mitosis.However,there is no information describing the relationship between HDAC 1 and drug resistance induction of breast cancer cells.In this study,first of all,lentivirus vectors were used to express HDAC1 protein in vitro,and then a HDAC1 stable espression MCF-7 cell strain were screened by puromycin.Finally,the anti-apoptosis effect of HDAC 1 was detected.The purpose of this study is that verifing overexpressed HDAC1 could enhence the drug resistance of breast cancer and the HDAC1 inhibitors might used in combination with traditional antitumor agents to promote the treatment efficacy in clinics.Objective:To discover the anti-apoptosis effect of HDAC1 protein,the present study aimed to verify the relationship between HDAC 1 and drug resistance of breast cancer cells.Methods:1.The construction and identification of a HDAC1 recombinant lentiviral vector to overexpress HDAC1 protein in vitro.A lentivirus vector with histone deacetylase 1 gene was constructed by subcloning technique,identified the vector by digesting with EcoR I and Xba I restriction enzymes,PCR and sequencing,then packaging lentivirus,transfecting MCF-7 cell and chosing the MCF-7 cell line that expresed HDAC 1 stably.HDAC 1 expression was assessed by Western blot.Overexpress the HDAC1 by extracting mRNA from human breast cancer cell line MCF-7,identificating the gene through RT-PCR,TA clone,restriction enzymes and DNA sequence analysis;connecting HDAC1 to pCDH-CMV-MCS-EF1-Puro,packaging the viral vectors into packaging cells 293T,transfecting MCF-7 cell line and screening the MCF-7-HDAC1 cell line by puromycin.2.The influence on the drug resistance induction of breast cancer cells line by the overexpression of HDAC 1.To discover the relationship between overexpressed HDAC1 and drug resistance,MCF-7-HDAC 1 and MCF-7-control cells were treated with 3 antitumor drugs:VM-26(teniposide),DDP(c is-d ic hlo ro d ia minep lat inum)and THP(pirarubicin).The cells were divided into 4 groups,the final VM-26 drug concentration of each group is 0.39,0.78,1.56 and 3.12 ?g/mL,and the drug treating time are 48 h.DDP was also set into four final drug concentration at 0.3125,0.625,1.25 and 2.5?g/mL,and the drug treating time are 48 h.THP were set into three final drug concentration at 0.39,0.78 and 1.56 ?g/mL,and the drug treating time are 72 h.MTT assay is arranged to detect the cell viability.Apoptotic rates of the target cells were observed through Hoechst 33258/PI double staining and flow cytometry.Bcl-2/Bax and caspase 3 expression were assessed by western blot.Results:1.The lentiviral expression plasmid carrying HDAC1 gene was identified by double restriction enzyme digestion and DNA sequence.Western blot showed a significantly increase of HDAC 1 expression in the MCF-7-HDAC1 cell line.2.After 48 h or 72 h treated with VM-26,DDP and THP,cell viabilities of both MCF-7-HDAC 1 and MCF-7-control cell lines reduced significantly in dose-dependent manner(P<0.05,P<0.01,P<0.001).However,the cell viabilities of MCF-7-HDAC 1 cells are higher than that of MCF-7-control significantly in each concentration of VM-26,DDP and THP.The IC50(half maximal inhibitory concentration)of MCF-7-HDAC1 vs MCF-7-control are(18.861 ?g/mL vs 14.906?g/mL,18.310 ?g/mL vs 11.589 ?g/mL,8.225 ?g/mL vs 5.144 ?g/mL)respectively.3.Fragmented,condensed nuclei or death cells could be observed in a dose-dependent manner of VM-26,DDP and THP through Hoechst33258/PI double-staining.Compared with MCF-7-control cells,typical apoptotic features of the MCF-7-HDAC 1 cells such as cell shrinkage and detachment,nucleus condensation and cell death were slight much more.Meanwhile,the results of flow cytometry further affirmed these phenomenons(P<0.05,P<0.01,P<0.001).Western blot showed a significant decrease in the Bcl-2 expression and a significant increase in the Bax and caspase 3 expression with the increased concentrations of VM-26,DDP and THP(P<0.05,P<0.01,P<0.001).The Bcl-2/Bax level was significantly down-regulated(P<0.05,P<0.01,P<0.001)and caspase 3 level was obviously up-regulated(P<0.01)in MCF-7-HDAC 1 cells.Conclusion:1.A HDAC1 recombinant lentiviral vector was constructed successfully and a MCF-7-HDAC1 cell strain that over expresing HDAC 1 stably was established.2.The overexpression of HDAC 1 increase cell viability of MCF-7 and reduce appototis induced by VM-26,DDP and THP.3.Under each concentration of VM-26,DDP and THP,the Bcl-2/Bax level decreased and caspase 3 expression increased in MCF-7-HDAC 1 cells,which indicated the overexpressing HDAC 1 induce drug resistance induction of breast cancer cells was mediated by down-regulation of caspase 3 and up-regulation of Bcl-2/Bax expression.