Inhibitory Effects And Mechanism Of SAHA Combined With Erlotinib On EGFR-TKI Resistant Lung Cancer Cells

Background:Because of smoking,air pollution,bad habits,aging and other factors,the incidence of lung cancer is increasing year by year,which poses great threat to human health.In the genotyping of tumor tissue,we found that the epidermal growth factor receptor(EGFR)gene mutation in lung cancer patients have a high proportion,so lung cancer patients with EGFR gene mutations were used EGFR Targeted drugs such as Tarceva(erlotinib),Iressa(Gefitinib)for the first time,will have achieved good clinical effects in the early days,but after a period of treatment,drug resistance could not be avoided,resulting in the failure of the treatment.In addition to genetic changes that have an important relationship with the development of tumors,changes in epigenetics also play an important role.Recent studies have confirmed that abnormal epigenetic changes such as DNA methylation and histone modification have a crucial role in the development of cancer.In addition to the regulation of histone acetylation which constitutes the nucleosome structure,the histone deacetylase inhibitor(HDACi)can also regulate the non-histone acetylation modification that has important functions in the cell,and thus affects The biological function of tumor cells and achieves the anti-tumor effect of inhibiting proliferation and promoting apoptosis.In this study,we investigated the effects of SAHA,erlotinib and SAHA combined with erlotinib on H1975 cells,respectively,to observe their effects on cell proliferation,cloning and invasion.To investigate whether HDAC inhibitor vorinostat(SAHA)combined with erlotinib has a synergistic inhibitory effect on lung cancer cells and its molecular mechanism Objective: To explore the inhibitory effect and mechanism of SAHA combined Erlotinib on egfr-tki resistant lung cancer cell line H1975 cells.To find the theoretical basis at the laboratory level,lay the foundation for subsequent animal experimental research and clinical experiments,and ultimately hope to find suitable treatment means for lung cancer patients with EGFR-TKI acquired resistance.Method: 1.CCK-8 assay was used to detect the growth inhibitory effect of SAHA monotherapy,erlotinib monotherapy and SAHA combined with erlotinib on H1975 cell line.2.Plate colony assay was used to detect the formation of cloned rate of H1975 cell line with SAHA monotherapy,erlotinib monotherapy and SAHA combined with erlotinib.3.Transwell chamber was used to detect the invasion rate of SAHA monotherapy,erlotinib monotherapy and SAHA combined with erlotinib.4.Western Blot method was used to detect the expression of PI3 K,AKT,p-akt,mTOR and p-mtor protein in the H1975 cells treated by SAHA monotherapy,erotinib monotherapy,and SAHA combined with erlotinib.Results: 1.The results of CCK-8 assay showed that the IC50 of SAHA after 48 h of H1975 cells was 3.16μmol/L;the IC50 of erlotinib after 48 h of H1975 cells was 19.31μmol/L;Growth inhibition curves showed that both of SAHA and erlotini had a concentration dependent on the growth inhibition of H1975 cells.SAHA combined with erlotinib significantly inhibited the growth of H1975 cells.The CI value was less than 1 at each combination concentration,indicating that SAHA combined with erlotinib had a synergistic effect.2.The clone formation rate of SAHA group was 41.90%,and the formation rate of erlotini formation was 48.11%.However,the combination group of the two drugs was 17.89%,and the combination group was more significant than the single drug group to inhibit the clone formation of tumor cells.The difference was statistically significant(F=29.350,P<0.001).3.Compared with single-drug group cells,the number of perforated cells of Matrigel gel was significantly decreased in Transwell small ventricular invasion experiment,and the difference was statistically significant(F=29.350,P<0.05).4,Western blot SAHA combined with erlotinib on the expression level of H1975 cells in PI3 K,AKT,p-AKT,mTOR and p-mTOR protein compared with erlotinib or SAHA monotherapy group were significantly decreased,the difference was statistically significant(P<0.01)Conclusion: SAHA combined with erlotinib had synergistic inhibitory effects on the growth,cloning,and invasion of H1975 cells.It may be related to the inhibition effects of the PI3K/AKT/mTOR signaling pathway.