As shown in Figure 7B selleck screening library in H322 cells EGFR autophosphorylation was unaffected when cells were treated with gefitinib conditioned medium collected from Calu 3 in the absence of a NAP, in contrast when the inhibitor was present in the gefitinib conditioned medium, EGFR autophosphorylation was completely inhibited. These results strongly suggest that in sensitive cells the metabolites released into the medium were ineffective in EGFR inhibition. The high and constant drug level inside the cells obtained in the presence of a NAP maintained a signifi cant inhibition of EGFR p44/42 MAPK and AKT phos phorylation even after a prolonged period of treatment when compared with cells incu Inhibitors,Modulators,Libraries bated with gefitinib alone.

Sensitive cell lines were then treated with gefitinib in the presence of 10 uM a NAP for 72 h in order to evaluate the effects of CYP1A1 inhibition on efficacy of gefitinib in inhibiting cell proliferation. In the presence of the inhibitor the Inhibitors,Modulators,Libraries IC50 for gefitinib, evaluated by crystal violet staining and confirmed Inhibitors,Modulators,Libraries by cell counting and MTT assay, was reduced 15, 3 and 6 times in Calu 3, H322 and H292 cells respectively. Overall, these results show that inhibition of CYP1A1 is associated with reduced gefitinib metabolism, increased intracellular gefitinib content and increased drug efficacy in cultured NSCLC cells. Discussion The cytochrome P450 system consists of a large number of enzyme subfamilies involved in the oxidative metabo lism of xenobiotics including drugs. They are expressed mainly in the liver, but extra hepatic expression of a number of these enzymes does occur.

Although the primary site of gefitinib metabolism is the liver, tumor cell metabolism Inhibitors,Modulators,Libraries can significantly affect treatment effec tiveness. However, to our knowledge, no studies have been performed addressing gefitinib metabolism in lung tumor cells. The present Inhibitors,Modulators,Libraries study shows that the drop in gefitinib con tent observed in EGFR wild type gefitinib sensitive cell lines after 24 h of treatment was mainly due to gefitinib metabolism by CYP1A1 activity and not related to a time dependent modification of influx or efflux processes. Our results indicate that there is a significant difference between gefitinib sensitive and resistant cell lines with regard to drug metabolism. Surprisingly, only sensitive cells were able to metabolize gefitinib and as a conse quence, after 24 h of treatment, gefitinib disappeared both inside and outside the cells. till The majority of radiolabeled gefitinib metabolites were present in the extracellular compartment as not well defined metabolites since we could barely detect the M1 metabolite and M2 or M3 were undetectable.