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Functional characterization of gefitinib uptake in non-small cell lung cancer cell lines
Maricla Galetti1, Roberta R Alfieri, Andrea Cavazzoni
1Department of Experimental Medicine, University of Parma, Italy.
Abstract:
Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase, has been developed and approved for treatment of advanced non-small cell lung cancer (NSCLC). In this study, we investigated the uptake of gefitinib in gefitinib-sensitive and -resistant NSCLC cell lines. The transport system was temperature-dependent, indicative of an active process and sodium- and potential-independent. Moreover, high cell densities and low extracellular pH significantly reduced the uptake of gefitinib. Inhibitors of the human organic cation transporter 1 (hOCT1) significantly decreased gefitinib uptake; however, gefitinib was not a substrate for hOCT1 or hOCT2 in overexpressing HEK293 cells. Interestingly, gefitinib significantly reduced uptake of the hOCT prototypical substrate MPP suggesting that gefitinib may exert an inhibitory effect on the intracellular accumulation of drugs transported by hOCT1 and hOCT2. After 15min of treatment at 1microM (the maximum plasma concentration of gefitinib obtained at the clinically relevant dose) gefitinib accumulated within the cell in resistant-cell lines at concentrations similar or even higher than in gefitinib-sensitive cells tending to rule out an alteration in drug uptake as a mechanism of resistance to gefitinib treatment. Moreover, our results suggest that the extrusion of lactate by crowded cells may contribute in decreasing the pH, which in turn can influence the uptake of gefinitib and as a result the inhibition of EGFR autophosphorylation.
Insights
Gefitinib uptake in non-small cell lung cancer (NSCLC) is an active process influenced by cell density and pH. Drug resistance is unlikely due to altered gefitinib uptake, suggesting other mechanisms are at play.
Area of Science:
- Pharmacology
- Cancer Biology
- Cellular Transport
Background:
- Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor approved for advanced non-small cell lung cancer (NSCLC).
- Understanding gefitinib's cellular uptake is crucial for optimizing its efficacy and managing treatment resistance.
Purpose of the Study:
- To investigate the cellular uptake mechanisms of gefitinib in NSCLC cell lines.
- To determine if altered drug uptake contributes to gefitinib resistance in NSCLC.
Main Methods:
- Investigated gefitinib uptake in sensitive and resistant NSCLC cell lines under varying temperature, pH, and cell density conditions.
- Utilized human organic cation transporter 1 (hOCT1) and hOCT2 overexpressing HEK293 cells to assess gefitinib's interaction with these transporters.
- Examined the effect of gefitinib on the uptake of a prototypical organic cation substrate (MPP).
Main Results:
- Gefitinib uptake is an active, temperature-dependent, and sodium/potential-independent process.
- High cell density and low extracellular pH significantly reduced gefitinib uptake.
- Gefitinib uptake was inhibited by hOCT1 inhibitors but gefitinib was not a substrate for hOCT1 or hOCT2.
- Gefitinib inhibited the uptake of MPP, suggesting interference with hOCT1/hOCT2 substrates.
- Gefitinib intracellular concentrations were similar or higher in resistant cell lines compared to sensitive ones, ruling out uptake alteration as a resistance mechanism.
Conclusions:
- Gefitinib uptake is modulated by cellular microenvironment factors like pH and cell density.
- Altered cellular uptake is unlikely to be the primary mechanism of gefitinib resistance in NSCLC.
- Extracellular lactate extrusion influencing pH may indirectly affect gefitinib uptake and EGFR inhibition.
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