Fig. 8

Acidic transformation of gefitinib increases the sensitivity of NSCLC cells. a Design strategy for the alkaline-neutralizing gefitinib analog (Gefi-2OH). The pKa values of gefitinib and Gefi-2OH were estimated by a freely available package, Marvin by ChemAxon, based on the Hammett-Taft approach. b A549 and H460 cells were treated with DMSO, gefitinib, or Gefi-2OH for 12 h and then incubated with LysoTracker Blue for 2 h. Fluorescence microscopy was used to detect the number of acidic vesicles. Scale bar: 10 μm. The quantification of acidic vesicle compartments per cell is expressed as the mean ± SD (n = 10 cells, three independent experiments). Asterisks indicate significant differences (Student’s t test, p < 0.05). ND, not detected. c H460 cells were treated with gefitinib or Gefi-2OH for 24 h, and the expression of SQSTM1, CTSB, and LC3 was measured by western blotting. d A549 and H460 cells were treated with different concentrations of gefitinib or Gefi-2OH for 48 h, and then, the cell viability was measured by MTS assay. e A549 and H460 cells were treated with gefitinib or Gefi-2OH for 24 h. The annexin V-PI staining assay was performed after the indicated treatment, and the relative percentage of apoptotic cells is summarized