Figure 7
Effects of CYP1A1 inhibition on intracellular level of gefitinib, EGFR autophosphorylation and inhibition of cell growth. A) Calu-3 cells were incubated with 0.1 μM [3H]gefitinib for 0.5, 24, 48 or 72 h in the absence or in the presence of 10 μM α-NAP. Values given of gefitinib content, expressed as pmol/mg of protein, are the means (± SD) of four independent determinations. α-NAP was renewed after 36 h of gefitinib treatment. (***P < 0.001). B) Calu-3 cells were treated with 0.1 μM gefitinib for 24 h in the absence or in the presence of 10 μM α-NAP, then the conditioned media (CM) were collected and extracts were prepared from H322 cells exposed for 2 h to CM. The effect of CM on EGF (0.1 μg/ml for 5 min)-induced EGFR autophosphorylation was examined by Western blotting using monoclonal antibodies directed against pEGFR(Tyr1068) and actin. The experiment, repeated twice, yielded similar results. Line 1: CM from control cells; line 2: CM from gefitinib treated cells; line 3: CM from α-NAP treated cells; line 4: CM from α-NAP and gefitinib treated cells. C) H322 and Calu-3 cells were incubated for 48 h with 0.1 μM gefitinib in the absence or in the presence of 10 μM α-NAP. Before protein extraction cells were stimulated with 0.1 μg/ml EGF for 5 min. Western blot analysis was performed by using monoclonal antibodies directed against pEGFR(Tyr1068), EGFR, p-p44/42 MAPK, p44/42 MAPK, pAKT (Ser473), AKT. The experiment, repeated three times, yielded similar results. α-NAP was renewed after 24 h of gefitinib treatment. D) Calu-3, (E) H322, (F) H292 were exposed for 72 h to different concentrations of gefitinib in the absence or in the presence of 10 μM α-NAP. Cell growth was assessed using crystal violet staining as described in Materials and Methods. Data are expressed as percent inhibition of cell proliferation versus control cells. The mean values of three independent measurements (± SD) are shown. α-NAP was renewed after 36 h of gefitinib treatment (*P < 0.05; **P < 0.01; ***P < 0.001).