| Apoptosis is also known as programmed cell death,which plays an essential role in eliminating excessive,damaged or harmful cells.Previous work demonstrated that the anticancer drugs could induce cytotoxicity by inducing cell apoptosis.In recent years,several reports documented modulated expression of DNA methyltransferases(DNMTs)and inhibition of DNMT1 could induce the cell apoptosis in a variety of tumors.We proposed that the expression of DNMTs could be regulated,which might affect the growth of tumor cells and clinical outcomes in the process of chemotherapy drug treatment for lung cancer.Purpose:This paper investigates the role of Cis-platin(CDDP)in cell apoptosis of lung cancer cell lines,investigates the expression of DNMT1 in CDDP-induced cell apoptosis in lung cancer cells,dissects the regulatory mechanism of the DNMT1 expression in CDDP-induced cell apoptosis in lung cancer cells,elucidates the regulation of CDKN1A by DNA methylation which affected by DNMT1 and MAPKs pathway and investigates the potential role of MAPKs and DNMT1 in lung cancer occurrence,development and treatment.Methods:1)Lung cancer cell lins SKMES-1 and H520 were treated with CDDP at different concentrations and CCK8 assay was introduced to detect the cell viability of the cells,Transwell assay was employed to measure the cell migration potential of the cells,TUNEL assay was applied to test the cell apoptosis of the cells,western blot assay was used to determine the biomarker of cell apoptosis cleaved caspase 3 in the cells.2)Lung cancer cell lins SKMES-1 and H520 were treated with CDDP at different concentrations and western blot assay was used to measure the expression of DNMT1,DNMT3 A and DNMT3B in the cells.3)We constructed the overexpression clone of DNMT1 and transfected them into the SKMES-1 and H520 cells,qPCR and western blots assay were used to measure the expression of DNMT1.Transfected the overexpression cloned for DNMT1 into the SKMES-1 and H520 cells treated with CDDP nd CCK8 assay was introduced to detect the cell viability of the cells;Transwell assay was employed to measure the cell migration potential of the cells;Western blots applied to examine the cell apoptosis of the cells.4)Lung cancer cell lins SKMES-1 and H520 were treated with CDDP at different concentrations and western blot assay was used to measure the expression of the protein levels and phosphorylation statuses of three well-characterizedMAPK subfamilies:JNK,ERK,and p38 in the cells.After induction of apoptosis,inhibitors of phosphorylated proteins of JNK,ERK and p3 8 were added,and western blot was applied to detect the protein levels of DNA methyltransferase I and the phosphorylation statuses of three well-characterized MAPK subfamilies:JNK,ERK,and p38 in SKMES-1 and H520 cells.5)After induction of apoptosis,inhibitors of phosphorylated proteins of JNK,ERK and p38 were added,and CCK8 assay was introduced to detect the cell viability of the cells,Transwell assay was employed to measure the cell migration potential of the cells,TUNEL assay was applied to test the cell apoptosis of the cells.6)After induction of apoptosis,qPCR and western blots assay was employed to measure the expression of CDKN1 A;After induction of the DNMT1 inhibitor,qPCR and western blots assay was applied to measure the expression of CDKN1A;After overexpression of DNMT1,qPCR and western blots assay was used to measure the expression of CDKN1A;After induction of apoptosis in the cells overexpressed with DNMT1,qPCR and western blots assay was used to measure the expression of CDKN1A;After induction of apoptosis in the cells overexpressed with DNMT1,sodium bisulfite DNA sequencing for DNA methylation assay was introduced to measure the methylation state of CpGs in promoter region of CDKN1A;7)After induction of apoptosis,inhibitors of phosphorylated proteins of JNK,ERK and p3 8 were added,and then,qPCR and western blots assay was introduced to measure the expression of DNMT1 and CDKN1A.8)Immunohistochemistry assay was employed to measure the compared the protein levels of CDKN1A,DNA methyltransferase I and the phosphorylation statuses of three well-characterized MAPK subfamilies:JNK,ERK,and p38 level in clinical lung cancer samples and paired adjacent normal tissues.Results:1)Chemotherapeutic drug CDDP inhibited the cell viability and cell migration potential of SKMES-1 and H520 cells;CDDP induced cell apoptosis in SKMES-1 and H520 cells,and promoted the expression of the apoptotic marker gene cleaved Caspase-3 in SKMES-1 and H520 cells.2)Chemotherapeutic drug CDDP inhibited the expression of DNA methyltransferase I without affecting the expression of DNA methyltransferase 3 A and DNA methyltransferase 3B in SKMES-1 and H520 cells.3)The overexpression clone of DNA methyltransferase I overexpressed DNA methyltransferase I in lung cancer cell lines SKMES-1 and H520;Overexpression of DNA methyltransferase I inhibits the chemotherapeutic drug CDDP-induced repression of cell viability and cell migration potential in SKMES-1 and H520 cells;Overexpression of DNA methyltransferase I inhibits the chemotherapeutic drug CDDP-induced inducetion of cell apoptosis in SKMES-1 and H520 cells.4)Chemotherapeutic drug CDDP promoted the avtivation of MAPKs pathway in SKMES-1 and H520 cells.Phosphorylated JNK inhibitor SD100625,phosphorylated ERK inhibitor PD98059,and phosphorylated p38 inhibitor SB203580,effectively inhibited the expression of their phosphorylated targets respectively,and restore the expression of DNMT1 in SKMES-1 and H520 cells treated with chemotherapeutic drug CDDP.5)The inhibitor of MAPKs pathway,SD100625,PD98059,SB203580,effectively enhanced the cell viability and cell migration potential of SKMES-1 and H520 cells treated with chemotherapeutic drug CDDP.At the same time,these inhibitors can effectively inhibite the cell apoptosis in SKMES-1 and H520 cells.6)Chemotherapeutic drug CDDP promoted the expression of CDKN1A;after induction of the DNMT1 inhibitor,AZA promoted the expression of CDKN1A;overexpression of DNMT1 promoted the expression of CDKN1 A;overexpression of DNMT1 inhibited the chemotherapeutic drug CDDP-induced promotion of CDKN1A expression;chemotherapeutic drug CDDP idecreased the DNA methylation of CpGs in the promoter region of CDKN1A and the overexpression of DNMT1 restored that in lung cancer cells.7)inhibitors of phosphorylated proteins of JNK,ERK and p38 restored the role of chemotherapeutic drug CDDP in lung cancer cells.8)Increased CDKN1A expression was showed in cancer tissues in 11 of 15 patient samples,compared with those in paired normal tissues;decreased DNMT1 expression was displayed in cancer tissues in 11 of 15 patient samples and conversely,increased p-JNK,p-ERK,p-p38 was found in 11 of 15 patient samples,compared with those in paired normal tissues.Conclusion:This study demonstrates that the chemotherapeutic drug cisplatin induces apoptosis in lung cancer cells;the chemotherapeutic drug CDDP inhibits the expression of DNA methyltransferase I in lung cancer cells;the overexpression of DNA methyltransferase I inhibits the action of the chemotherapeutic drug CDDP in lung cancer cells;MAPKs participated in the regulation of DNMT1 expression in apoptosis;chemotherapeutic drug CDDP promoted the CDKN1A expression via inhibiting the DNMT1 expression which induced the reduction of the DNA methylation of CpGs in the promoter region of CDKN1A and meanwhile,overexpression of DNMT1 restored that;the inhibitor of MAPKs pathway restored the chemotherapeutic drug CDDP-induced promotion of CDKN1A expression;the expression of CDKN1A,DNMT1 and MAPKs pathway is abnormal in clinical lung cancer patients. |
PDF Full Text Request