Fig 1.
Characterization of ABCG2 expression and activity in NSCLC cell lines.
(A) Cells were lysed and 50μg of proteins loaded to assess ABCG2 protein expression by western blot analysis. Data are from a representative experiment. Each experiment, repeated three times, yielded similar results. (B) ABCG2 protein levels on cell surface were quantified by flow-cytometry and expressed as molecular equivalent of fluorochrome (MEF) as described in the Methods Section. (C) Cells were loaded with 1 μM Hoechst 33342 in the presence or in the absence of 10 μM Fumitremorgin C. After 4 hours of incubation, Hoechst 33342 was removed and its fluorescence was determined by luminometer. Relative ABCG2 activity was defined as the ratio of Hoechst 33342 accumulation per μg of protein between Fumetrimorgin C treated cells and untreated cells and was expressed as fold increase. The mean values of three independent measurements (± SD) are shown (***P < 0.001).
Fig 2.
Effect of gefitinib on ABCG2 activity and protein expression.
H460 cells were loaded with 1 μM Hoechst 33342 in the presence of the indicated concentrations of gefitinib. (A) After 4 hours, Hoechst 33342 was removed and fluorescent dye accumulation was expressed as absorbance per μg of protein. (B) The percentage of efflux was calculated after a further incubation of 1 hour in phenol red-free medium in the presence of the indicated concentration of gefitinib. Values are the means (± SD) of three independent determinations; one-way ANOVA followed by Bonferroni post-test (*** P < 0.001) (C) H460 cells were treated with 5 μM gefitinib for 4 hours; then cell surface expression of ABCG2 was evaluated by flow cytometry and the quantification is reported as molecular equivalent of fluorochrome (MEF). The mean values of three independent measurements (± SD) are shown.
Fig 3.
Effects of different ABGC2 and MDR1 inhibitors on gefitinib accumulation and efflux.
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.
Fig 4.
Effect of ABCG2 silencing on [3H]gefitinib accumulation, efflux, and uptake.
H460 cells were transfected with ABCG2 siRNA (1:1:1 mixture of #HSS114013, #HSS114014 and #HSS114015) or control siRNA (scr) for 48 hours and then analyzed for ABCG2 expression by Western blotting (A) or ABCG2 activity (B). Cells were incubated for 4 hours with 1 μM Hoechst 33342 in the presence or in the absence of Fumetrimorgin C and the relative ABCG2 activity was calculated as the ratio of Hoechst 33342 accumulation per μg of protein between Fumitremorgin C treated cells and untreated cells, and expressed as fold increase. Data are expressed as mean (± SD) of three different experiments (***P < 0.001). In H460 transfected cells, radiolabeled gefitinib accumulation was measured after 4 hours of treatment with 0.1 μM [3H]gefitinib (C), then the medium was replaced with fresh medium and the percentage of efflux was calculated after 5, 10, 20 and 30 min in the presence or absence of 1 μM Ko143 (***P < 0.001) (D). Initial velocity (5 min) of [3H] gefitinib uptake was measured after 4 hours of 0.1 μM gefitinib treatment (E). Each bar represents the mean (± SD) of four independent determinations. (** P < 0.01).
Fig 5.
Effect of ABCG2 overexpression on gefitinib accumulation, efflux, and uptake.
Characterization of HEK293/R2 overexpressing cells: (A) Cells were lysed and 50μg of proteins for H292, H460 and HEK293 or 10μg for HEK293/R2 cells loaded to assess ABCG2 protein expression by Western blot analysis. Different amounts of proteins were loaded to avoid the signal saturation in the sample from ABCG2 overexpressing cells. Data are from a representative experiment. Each experiment, repeated twice, yielded similar results. (B) ABCG2 protein levels on cell surface were quantified by flow-cytometry and expressed as molecular equivalent of fluorochrome (MEF) as described in the Methods Section. (C) Cells were loaded with 1 μM Hoechst 33342 in the presence or in the absence of 10 μM Fumitremorgin C. After 4 hours of incubation, Hoechst 33342 was removed and its fluorescence was determined by luminometer. Relative ABCG2 activity was defined as the ratio of Hoechst 33342 accumulation per μg of protein between Fumetrimorgin C treated cells and untreated cells and was expressed as fold increase. The mean values of three independent measurements (± SD) are shown. The cellular accumulation (D), efflux (E) and uptake (F) of 0.1 μM [3H]gefitinib was determined in overexpressing ABCG2 transporter HEK293/R2 cells and in the parental cells (HEK293), after 4 hours of gefitinib treatment. The percentage of efflux was calculated after 5 min of incubation with fresh medium, while initial velocity (5 min) of [3H] gefitinib uptake was measured after 4 hours of 0.1 μM gefitinib treatment (***P < 0.001).
Fig 6.
Effect of ABCG2 overexpression on the expression of SLC transporter genes and MPP, glucose and glutamine transport.
Down-regulation of SLC transporter expression (SLC22A1, SLC22A3, SLC2A1, SLC38A2, SLC3A2, SLC7A5, and SLC7A6) in HEK293/R2 cells versus HEK293 was evaluated by PCR drug transporter array (A). Fold decrease expression was determined by the ΔΔCT method normalized to five housekeeping genes. Values given are the means (± SD) of three independent experiments (*P<0.05; **P<0.01). Initial velocity (5 min) of [3H]MPP (B), [3H]2-deoxy-D-glucose (C) and [3H]Glutamine (D) uptake was determined and values given are the means (± SD) of three independent determinations (*P<0.05; **P<0.01).