Cucurbitacin D Overcomes Gefitinib Resistance by Blocking EGF Binding to EGFR and Inducing Cell Death in NSCLCs

Abstract

In this study, the mechanism of the anticancer effect through which cucurbitacin D (CuD) can overcome gefitinib resistance in NSCLC was investigated. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay, and cell migration and growth were observed by wound healing and colony formation assays, respectively. Levels of EGFR family members, protein kinase B, extracellular signal-regulated kinase, poly(ADP-ribose) polymerase, and G2/M phase-related proteins were detected by Western blot analysis. Immunofluorescence analysis was used to detect the intracellular expression of p-EGFR. Induction of apoptosis and cell cycle arrest was measured by flow cytometry. Solid-phase binding assays were used to determine binding to the EGFR family. CuD inhibits the phosphorylation of EGFR in gefitinib-resistant NSCLC cells and induces cell death via cell cycle arrest and apoptosis. CuD treatment or EGFR knockdown also suppressed the growth of gefitinib-resistant NSCLC cells. In addition, CuD overcame resistance by blocking EGF binding to EGFR in gefitinib-resistant NSCLC cells. In conclusion, we demonstrate that CuD overcomes gefitinib resistance by reducing the activation of EGFR-mediated survival in NSCLC and by inhibiting the combination of EGF and EGFR.