Figure 3

Production and biological activity of gefitinib metabolites. Cells were incubated with 0.1 μM gefitinib for 0.5, 6 or 24 h and then the M1 content was determined both in the intracellular (A) and extracellular (B) compartment, through LC-MS/MS analysis. M1 levels calculated for each cell line were expressed as pmol/mg of protein (intracellular) or μM (extracellular). Values given are the means (± SD) of three independent determinations C) H322 cells were exposed for 72 h to different concentrations of gefitinib or its metabolites (ranging from 0.05 to 10 μM) and then cell growth was assessed using crystal violet staining. Data are expressed as percent inhibition of cell proliferation versus control cells. The mean values of three independent measurements (± SD) are shown. D) H322 cells were incubated for 0.5 h with the indicated concentrations of gefitinib and metabolites, before stimulation with 0.1 μg/ml EGF for 5 min. Western blot analysis was performed by using monoclonal antibodies directed to p-EGFR(p-Tyr1068) and to EGFR. The immunoreactive spots at each point were quantified by densitometric analysis, ratios of phosphotyrosine/total EGFR were calculated and values expressed as percentage of inhibition versus control. The experiment, repeated three times, yielded similar results. E) H1299 cells were exposed for 72 h to different concentrations of gefitinib or its metabolites (ranging from 1 to 30 μM) and then cell growth was assessed using crystal violet staining. Data are expressed as percent inhibition of cell proliferation versus control cells. The mean values of three independent measurements (± SD) are shown.