Abstract
Abstract Tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, are therapeutic target drugs used in clinic lung cancer therapy. Patients with NSCLC harboring EGFR-activating mutations have better clinical responses to EGFR-TKI. However, the development of drug resistance is a major problem in clinical applications of TKI. Imbalance of nitric oxide (NO) has been reported to be closely correlated with cancer progression, yet its linkage to TKI resistance remains unknown. This study was thus designed to investigate the role of NO in the development of acquired resistance during TKI therapy by using an EGFR-TKI sensitive human non-small cell lung cancer (NSCLC) line, PC9, and its derivative PC9/gefB4 resistant cell line. MTT assay confirmed that PC9/gefB4 cells exhibited significant resistance to erlotinib as compared to the sensitive PC9 cells. Immunoblotting showed that the effect of erlotinib on the inhibition of ERK phosphorylation in PC9 cells was stronger than that in PC9/gefB4 cells. By using a specific fluorescence dye to capture NO in flow cytometry analysis, we found that erlotinib could markedly induce NO levels in PC9, but not in PC9/gefB4. Co-treatment with PTIO, a NO scavenger, decreased the erlotinib-induced cytotoxicity in PC9 cells. By contrast, exposing PC9/gefB4 cells to DETA-NONOate, increased the cellular NO, and decreased the phospho-ERK levels and cell viability. As epithelial–mesenchymal transition (EMT) may play an important role in developing EGFR-TKI resistance, the relationship among NO levels, the expression of E-Cadherin (an epithelial marker) and Slug (a mesenchymal marker) in erlotinib/NO modulators-treated cells, were investigated. Immunoblotting showed that erlotinib could induce the expression of Slug and decrease the expression of E-Cadherin in PC9 cells, while PTIO co-treatment decreased the expression of erlotinib-induced Slug in PC9 cells. By contrast, DETA-NONOate enhanced Slug expression and decreased E-Cadherin expression in both PC9 and PC9/gefB4. Furthermore, erlotinib significantly increased the inducible NO synthase (iNOS) levels in PC9, but not in PC9/gefB4. Co-administering L-NIL, a specific iNOS inhibitor, could prevent the erlotinib-induced cytotoxicity, decrease Slug, and enhance E-Cadherin expression levels. Moreover, DETA-NONOate could promote PC9 cell migration as determined by the transwell assay. Together, results obtained in this study suggest that erlotinib-induced NO may contribute to cell death; yet it also stimulates EMT process, thereby enhancing the metastasis of cancer cells. The results also suggest that the increase of NO release in combined with depletion of Slug may be a new therapeutic strategy for EGFR-TKI resistant lung cancer. However, why erlotinib can not induce iNOS and NO release in PC9/gefB4 cells remains to be investigated in the future.