Effects of different ABGC2 and MDR1 inhibitors on gefitinib accumulation and efflux.

<p>Time course of 10 nM [³H]gefitinib (A) and 1 μM Hoechst 33342 (B) accumulation in H460 cells, incubated in the presence or absence of 10 μM Fumitremorgin C. [³H]gefitinib was expressed as pmol/mg of protein and Hoechst 33342 accumulation was expressed as absorbance per μg of protein. Each point represents the mean (± SD) of four independent determinations (* P<0.05; ***P < 0.001). (C) H460 cells were incubated with 0.1 μM [³H]gefitinib for 4 hours in the absence or in the presence of 10 μM Fumitremorgin C (F) or 1 μM Ko143 (Ko) (ABCG2 inhibitors), or 10 μM PSC833 (PSC) (MDR1 inhibitor). At the end of the treatment, [³H]gefitinib accumulation was measured. After 4 hours of incubation in the presence of 0.1 μM [³H]gefitinib, the cells were washed, incubated in drug-free culture medium with or without the inhibitors and the percentage of efflux was calculated after 1 hour of incubation (D) or at the indicated times (E). (F) SKLU-1, H292, SKMES-1and H460 were incubated with 0.1 μM [³H]gefitinib for 4 hours, in the presence or absence of 10 μM Fumitremorgin C. The percentage of efflux was calculated after a further incubation of 5min in gefitinib-free normal growth medium, in the presence or in the absence of the inhibitor. (G-H) Intracellular level of gefitinib in H460 cell line was evaluated by LC-MS/MS, in the same condition described in panels C-D. Values given are the means (± SD) of three independent determinations; one-way ANOVA followed by Bonferroni post-test.