Correlation Between DNA Methylation Of Secreted Frizzled Related Protein 1 And The Chemoresistant Phenotype Of Human Lung Adenocarcinoma Cells

Background and ObjectiveChemotherapy is effective for the treatment of lung cancer.However,limited response to chemotherapy remains a major impediment in clinic.Both genetic and epigenetic regulation of target genes contributes to the chemoresistant phenotype of cancer cells.Downregulation of secreted frizzled related proteinl(SFRP1),which is induced by the DNA methylation and responsible for Wnt signaling pathway activation,is correlated with the growth,differentiation,apoptosis and invasive phenotype of cancer cells.The aim of this study is to investigate the regulatory effects of SFRP1 on chemosensitivity of human lung adenocarcinoma cells in vitro and in vivo,and to elucidate the underlying mechanisms through aspects such as cell proliferation,apoptosis,cell cycle progress and epithelial-mesenchymal transition(EMT).Materials and Methods1.The 50%inhibitory concentration(IC50)values for docetaxel and taxol of SPC-A1 and SPC-A1/DTX cells were determined by MTT assay.The IC50 values for Taxanes in SPC-A1/DTX cells treated with DNA methyltransferases(DNMT)inhibitor,5-Aza-2'-deoxycytidine(5-Aza-CdR),were also detected by MTT assay.The proliferation of SPC-A1/DTX cells treated with 5-Aza-CdR was assessed by colony formation assay.Apoptotic rate in SPC-A1/DTX cells treated with 5-Aza-CdR was determined by flow cytometry assay.2.The status of DNA methylation in SPC-A1 and SPC-A1/DTX cells was determined by DNA methylation microarray,which was also confirmed by methylation-specific PCR(MSP).The mRNA and protein levels of SFRP1 were determined by real-time PCR and western blot in SPC-A1/DTX cells treated with 5-Aza-CdR.3.Different expression levels of cDNA in SPC-A1 and SPC-A1/DTX cells were determined by cDNA array analysis and confirmed by real-time PCR and western blot.4.The chemosensitivity of SPC-A1/DTX and A549/Taxol cells overexpressing SFRP1(SPC-A1/DTX/SFRP1 and A549/Taxol/SFRP1)or SPC-A1 and A549 cells knockdowning of SFRP1(SPC-A1/shSFRP1 and A549/shSFRP1)was determined by MTT assay.The proliferation ability,cell cycle distribution,and apoptotic rate of these cell lines were detected by colony formation assay and flow cytometry assay.5.SPC-A1/DTX/SFRP1 or A549/Taxol/SFRP1 cells were injected subcutaneously and treated with docetaxel or taxol for 14 days.Tumor tissues were used for hematoxylin and eosin(H&E)staining and immunostaining analysis for proliferating cell nuclear antigen(PCNA).6.The activity of Wnt pathway was detected by real-time PCR and western blot in SPC-A1/DTX/SFRP1 and A549/Taxol/SFRP1 cells.And the effects of SFRP1 on the Wnt pathway were determined by the luciferase assay in SPC-A1/DTX cells.7.The effects of FH535 on the chemosensitivity of SPC-A1/DTX and A549/Taxol cells were determined by MTT assay.The proliferation ability,cell cycle,and apoptotic rate of these cell lines were detected by colony formation assay and flow cytometry assay.8.The expression of SFRP1 was determined by IHC in tumor tissues of 33 patients who had undergone a complete resection for early lung adenocarcinoma(LAD)and received chemotherapy.The probability of survival was plotted by the Kaplan-Meier method.9.The morphologic characters,EMT markers,migration and invasive ability were determined in SPC-A1 and SPC-A1/DTX cells.And the effects of SFRP1 on the EMT phenotype of SPC-A1/DTX cells were also determined.10.The morphologic characters,EMT markers,migration and invasive ability were determined in TGF-?-treated A549 cells.And the effects of SFRP1 on the EMT phenotype induced by TGF-? in A549 cells were also determined.Results1.The IC50 values of SPC-A 1/DTX cells for docetaxel and taxol were significantly increased compared with that of parental SPC-A1 cells,while pre-treatment of 5-azacytidine(5-Aza-CdR)significantly decreased the IC50 value of SPC-A1/DTX cells for docetaxel or taxol.In addition,treatment of 5-Aza-CdR could inhibit the proliferating ability and induce apoptosis enhancement in SPC-A1/DTX cells.2.18 hypermethylated genes(?_beta>0.8)including SFRP1 were identified by DNA methylation microarray assays,which was also confirmed by MSP.Treatment with different concentrations of 5-Aza-CdR could significantly increase the expression of SFRP1 in SPC-A1/DTX cells at both mRNA and protein levels(p<0.01).3.The results of cDNA microarray analysis indicated that a total of 13 genes(more than 15.0-fold change)were differentially expressed and SFRP1 was significantly down-regulated in SPC-A1/DTX cell line(about 24.15-fold change),which were also confirmed by real-time PCR and western blot.4.Compared with SPC-A1/DTX/control and A549/Taxol/control cells,the IC50 values of SPC-A1/DTX/SFRP1 and A549/Taxol/SFRP1 cell line for docetaxel and taxol were significantly decreased(p<0.01).Overexpression of SFRP1 could decrease the proliferating ability,increase the early apoptotic rate and induce the G1 arrest of SPC-A1/DTX and A549/Taxol cells.The IC50 values of SPC-Al/shSFRP1 or A549/shSFRP1 cell line for docetaxel and taxol were increased,while inhibition of SFRP1 could increase the proliferation ability and decrease the G1-phase cells in SPC-A1 and A549 cells.5.Overexpression of SFRP1 increased the chemosensitivity of SPC-A 1/DTX and A549/Taxol cell lines as demonstrated by the tumor size analysis and PCNA staining,which indicated that SFRP1 could inhibit the proliferating rate in vivo.6.Wnt signaling pathway was activated in SPC-A 1/DTX and A549/Taxol cells and SFRP1 could inhibit the Wnt pathway,which was also confirmed by luciferase assay.7.FH535 increased the chemosensitivity of SPC-A 1/DTX and A549/Taxol cell lines to docetaxel and taxol.In addition,FH535 decreased the proliferating ability,induced G1 arrest and increased the early apoptotic rate in SPC-A 1/DTX and A549/Taxol cells.8.LAD patients with high SFRP1 expression had a prolonged disease-free survival than those with low SFRP1 expression,indicating that the expression level of SFRP1 might be positively correlated with the response of LAD patients to taxanes-based adjuvant chemotherapy.9.The phenotypic changes were observed in SPC-A1/DTX cells including loss of cell polarity,increased intercellular separation,and increased formation of pseudopodia.Decreased levels of the epithelial adhesion molecules such as E-cadherin and CK-19,and increased levels of mesenchymal markers such as Vimentin and N-cadherin were detected in SPC-A1/DTX cells compared with SPC-A1 cells.SPC-A1/DTX cells exerted increased migratory and invasive capacity,which was consistent with the EMT phenotype.SFRP1 could also suppress EMT and the invasive ability of SPC-Al/DTX cells.10.TGF-?1 induced the EMT phenotype in A549 cell line as evidenced by the phenotypic changes,including a loss of cell to cell contact and elongated,fibroblastoid morphology,decreased expression of epithelial markers such as E-cadherin and CK-19 and increased expression of mesenchymal markers such as Vimentin and N-cadherin,and increased migratory and invasive capacity.In addition,restoration of SFRP1 inhibited TGF-?1 induced EMT phenotype and inactivated Wnt pathway in A549 cell line.Furthermore,FH535,a small-molecule inhibitor of Wnt pathway,could also suppress TGF-?1 induced EMT in A549 cell line.Conclusions1.Hypermethylation of the CpG islands contributes to downregulation of SFRP1 in SPC-A1/DTX cell line.2.SFRP1 restoration increases the sensitivity of Taxanes-resistant LAD cell lines to Taxanes by inducing apoptosis,G1 phase arrest and reversion of EMT.3.For the first time our results demonstrates the correlation of SFRP1 and chemosensitivity in human LAD,suggesting that SFRP1 might be a novel therapeutic target for the treatment of taxanes-resistant LAD patients.4.SFRP1 plays a critical role in the EMT phenotype,which indicates that SFRP1 might be a potential biomarker for the metastsis of LAD.