Fig. 4

Inhibition of cell viability and reduction of colony growth of NSCLC cells resistant to anticancer drugs by eliciting apoptosis by EV206 treatment. (a) The efficacy of EV206 in reducing the viability of H1299/CsR (cisplatin-resistant), H1299/PmR (pemetrexed-resistant), H460/PcR (paclitaxel-resistant), H226B/PcR (paclitaxel-resistant), and PC9/ER (erlotinib-resistant) cells was examined using a crystal violet assay (mean ± SD, n = 5 or 6/group). Cells were treated with various concentrations of EV206 for 48 h. (b) The effect of EV206 on colony formation by drug-resistant NSCLC cells under anchorage-dependent (AD) conditions was examined using an AD colony formation assay (mean ± SD, n = 3/group). (c) The effect of EV206 on colony formation by drug-resistant NSCLC cells in an anchorage-independent (AID) setting was assessed using a soft agar AID colony formation assay (mean ± SD, n = 3–5/group). (d, e) The effect of EV206 on apoptosis in drug-resistant NSCLC cells was assessed by cell cycle analysis using flow cytometry (d) and western blotting (e). (e, bottom) Western blotting results were quantified using densitometric analysis (mean ± SD, n = 3/group). The cells were treated with various concentrations of EV206 for 48 h. (f) The pro-apoptotic effect of EV206 (0.5 µM) in comparison with that of evodiamine (Evo, 0.5 µM) was determined by western blotting. Cells were treated with these compounds for 48 h. *p < 0.05; **p < 0.01; ***p < 0.001, determined by one-way ANOVA with Dunnett’s post-hoc test by comparison with the vehicle-treated control group (EV206 0 µM).