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Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines.

Galetti M, Petronini PG, Fumarola C, Cretella D, La Monica S, Bonelli M, Cavazzoni A, Saccani F, Caffarra C, Andreoli R, Mutti A, Tiseo M, Ardizzoni A, Alfieri RR - PLoS ONE (2015)

Bottom Line: Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.

ABSTRACT

Background: BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.

Aim: The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes.

Methods and results: Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.

Conclusions: Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0141795.g001: 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).

Mentions: We first evaluated ABCG2 expression as total protein level (Fig 1A) and plasma membrane level (Fig 1B) in a panel of NSCLC cell lines showing sensitivity (HCC827, H292) or resistance (H460, H1299, SKMES-1, A549, Calu-1 and SKLU-1) to gefitinib. ABCG2 expression, evaluated by Western blotting and flow cytometry, varied widely among the analyzed cell lines, with H460 and SKMES-1 cells expressing the highest levels.


Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines.

Galetti M, Petronini PG, Fumarola C, Cretella D, La Monica S, Bonelli M, Cavazzoni A, Saccani F, Caffarra C, Andreoli R, Mutti A, Tiseo M, Ardizzoni A, Alfieri RR - PLoS ONE (2015)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4633241&req=5

pone.0141795.g001: 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).
Mentions: We first evaluated ABCG2 expression as total protein level (Fig 1A) and plasma membrane level (Fig 1B) in a panel of NSCLC cell lines showing sensitivity (HCC827, H292) or resistance (H460, H1299, SKMES-1, A549, Calu-1 and SKLU-1) to gefitinib. ABCG2 expression, evaluated by Western blotting and flow cytometry, varied widely among the analyzed cell lines, with H460 and SKMES-1 cells expressing the highest levels.

Bottom Line: Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy.

ABSTRACT

Background: BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.

Aim: The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes.

Methods and results: Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.

Conclusions: Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

No MeSH data available.


Related in: MedlinePlus