Blockade of DNA methylation enhances the therapeutic effect of gefitinib in non-small cell lung cancer cells

Abstract

The sensitivity of lung cancer to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has been found to be associated with mutations in the tyrosine kinase domain of EGFR. However, not all mutations are sensitive to gefitinib. While CpG island methylation in the promoter region of the EGFR gene and transcriptional silencing are common in solid tumors, the role of the EGFR gene promoter methylation in affecting resistance to TKIs in non-small cell lung cancer (NSCLC) remains unknown. In this study, we examined the correlation between EGFR gene promoter methylation and the therapeutic effect of gefitinib in NSCLC cells. Three NSCLC cell lines with different EGFR mutation statuses and levels of sensitivity to EGFR-TKIs were used in this study: H1650 (del E746-A750), H1299 (wild-type EGFR) and PC-9 (del E746-A750). Cells were treated with gefitinib or 5-aza-2'-deoxy cytidine (5-aza-CdR), a methylation inhibitor, alone or in combination. Subsequently, the methylation status of the EGFR gene promoter was examined by methylation-specific PCR (MSP). Cell survival and apoptosis assays were performed using the Cell Counting Kit-8 (CCK-8) and flow cytometry. In addition, western blot analysis and quantitative real-time PCR were used to examine the expression levels of EGFR protein and mRNA. Our study showed that the promoter region of the EGFR gene in PC-9 cells was unmethylated, and that the cells were sensitive to gefitinib. By contrast, the promoter region of the EGFR gene in the H1650 and H1299 cells was methylated, and the cells were resistant to gefitinib. Of note, the combination treatment with 5-aza-CdR and gefitinib further enhanced the growth inhibitory effects and led to the induction of apoptosis, while a significant reduction in the expression of EGFR protein and mRNA was observed in the H1650 and H1299 cells. These results suggest that blockade of DNA methylation may enhance the antitumor effects of EGFR-TKIs and gefitinib in NSCLC cells. Thus, EGFR gene promoter methylation may be a potential mechanism for acquired resistance to gefitinib.

Enhanced MAPK signaling drives ETS1-mediated induction of miR-29b leading to downregulation of TET1 and changes in epigenetic modifications in a subset of lung SCC

“…31 We provide further evidence that TET1 is downregulated through MAPK signaling and that this occurs through the novel mechanism of ETS1-mediated upregulation of miR-29b, which targets TET1 for downregulation through an interaction with its 3′UTR (Figures 4 and 5), leading to downstream effects on 5-hmC levels (Figures 6 and 7). Several studies have shown synergy between histone deacetylase inhibition and MAPK inhibition 40, 41 as well as synergy between DNA-hypomethylating agents like 5-azacytidine and MAPK inhibitors, 42, 43 suggesting that DNA demethylation enhances the efficacy of MAPK inhibitors. More recently TET upregulation has been shown to be essential for active demethylation induced by 5-azacytidine.…”

An improved fluorescence polarization assay in 5'-nuclease reaction for gene promoter methylation detection

“…It is of clinical interest that http://dx.doi.org/10.1016/j.jbiotec.2015.07.003 0168-1656/© 2015 Elsevier B.V. All rights reserved. NSCLC cells with EGFR promoter hypermethylation are resistant to EGFR inhibitor (Anglim et al, 2008;Jakopovic et al, 2013;Li et al, 2013;Montero et al, 2006).…”

Study of the methylation patterns of the EGFR gene promoter in non-small cell lung cancer

“…Among the studies conducted on the treatment of NSCLC, studies involving the epidermal growth factor receptor (EGFR) target are widely respected. Although abnormal EGFR expression is ubiquitous in NSCLC patients (Easwaran and Baylin, 2012), there has been little investigation of the cause of the abnormal expression and its significance in clinical diagnosis and treatment (Li et al, 2013).…”