The Effect Of HDAC Inhibitor SAHA On Erlotinib-resistance In Human Lung Adenocarcinoma Cells

Background: Epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKIs)have been widely used in non small cell lung cancer(NSCLC)treatment,such as erlotinib and gefitinib.However,despite impressive clinical success,particularly in patients harboring specific somatic activating EGFR gene mutations in their cancer cells,most patients,if not all,eventually experience relapse because of acquired drug resistance after an initial response,seriously affecting the curative effect of the treatment of EGFR-TKI.Experimental data have suggested that HDACs are involved in mammary tumorigenesis at multiple levels.HDACs participate in the negative regulation of genes such as ones encoding cell cycle inhibitors,differentiation factors,and pro-apoptotic factors.In addition,the expression of genes associated with angiogenesis along with cell invasion and migration are enhanced by HDACs.Thus,HDACs play important roles in cancer development by regulating the expression of numerous genes involved in both cancer initiation and progression.Histone deacetylase inhibitors(HDACIs),a class of antitumor agents that are able to affect multiple genes and pathways based on the functions of the epigenetic enzymes they regulate.SAHA is a broad-spectrum of HDACI,which has also been the first FDA-approved HDAC inhibitor for treatment of cutaneous T-cell lymphoma.In this study,we established a human lung adenocarcinoma cell line PC-9/ER that is resistant to Erlotinib,and then detected the difference of the expression of PTEN in PC-9/ER and pc-9.The results show that PTEN played an important role in the formation ofdrug resistance.Objective: To detect the effect of SAHA on the proliferation,apoptosis and drug resistance of PC-9 / ER cells.Method:1.To establish an Erlotinib-resistance PC-9ER subline,parental PC-9 cells were treated with 10 nmol/L of Erlotinib for 48 h in medium containing 10% fetal bovine serum.They were then washed and cultured in drug-free medium until surviving cells were 80% confluent.These cells were then re-exposed to increasing concentrations of Erlotinib until the cells were able to grow in 1 ?mol/L of Erlotinib.2.PC-9 and PC-9/ER were treated with Erlotinib for 48h;the inhibition of cell proliferation was determined by the MTT assay.3.PC-9/ER cells were treated with SAHA,Erlotinib alone or in their combination for48 h,and then the inhibitory effects were assessed by MTT assay.4.The influence on PC-9/ER cells apoptosis was determined by Annexin V-FITC / PI when treated with SAHA,Erlotinib alone or in their combination for 48 h.5.The expression of PTEN in PC-9 and PC-9/ER cells were detected by Western blotting analysis.6.PC-9/ER cells were treated with SAHA,Erlotinib alone or in combination for 48 h,and then the expression of PTEN were detected by Western blotting analysis.Results:1.The sensitivity of PC-9/ER cells to Erlotinib was significantly lower than that of PC-9 cells,the resistance factor is more than 100,which indicated that the establishment of Erlotinib-resistance PC-9/ER cells was successful.2.SAHA could inhibit the proliferation of PC-9/ER alone and partially reverse theresistance to Erlotinib of PC-9/ER,when combined with Erlotinib.The reversal fold(RF)is 3.01,and the relative reversal rate(RRR %)is 66.8%.3.Annexin V-FITC/PI apoptosis assay results showed that SAHA combined with Erlotinib could significantly increase the apoptosis rate of PC-9/ER cells when compared with the treatment with individual agent alone(P<0.05).4.The expression level of PTEN in PC-9/ER and PC-9 cells were detected by Western Blot analysis.The results showed that the expression level of PTEN in PC-9/ER was significantly down regulated,compared with that in PC-9 cells.5.After the treatment with SAHA,Erlotinib alone or their combinations for 48 h,the expression level of PTEN in PC-9/ER cells were detected by Western Blot analysis.The expression level of PTEN in the SAHA group and SAHA combined with Erlotinib were higher than that in the Erlotinib group and control group.Conclusion:1.SAHA combined with Erlotinib can significantly inhibit the proliferation of PC-9/ER cells.2.SAHA can induce the apoptosis of PC-9/ER cells.3.Loss of PTEN plays an important role in the development of Erlotinib resistance;4.SAHA can up-regulate the expression of PTEN protein and promote apoptosis,which can improve the drug resistance of Erlotinib.