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Objective: Overexpression of ABCB1 (P-glycoprptein, P-gp) is one of the main reasons of multidrug resistance (MDR) in cancer cells.Trametinib, a small-molecule MEK kinase inhibitor, is currently used for the treatment of melanoma in clinic.In this study, we investigated the effect of trametinib on ABCBl-mediated drug resistance.Methods: The cytotoxicity was measured by MTT assay.The drug-efflux activity of P-gp was assessed by rhodamine 123 or doxorubicin accumulation assay.The cell cycle distribution and cell apoptosis were detected using PI and Annexin V-FITC/PI staining by flow cytometric analysis.The expression levels of proteins were analyzed by western blot.The therapeutic effect in vivo was detected by mouse xenograft model.Results: Trametinib significantly potentiated the drug sensitivity, induction of cell cycle arrest and apoptosis of established ABCB1 substrates and increased the accumulation of rhodamine 123 or doxorubicin in ABCB1-overexpressing cells.Furthermore, trametinib did not clearly alter the sensitivity of non-ABCB1 substrates in all cells.In addition, the results of ATPase assays show that trametinib stimulated the ATPase activity of ABCB 1.However, trametinib at a concentration that produced a reversal of MDR totally inhibited the phosphorylation of ERK, but did not obviously alter the ABCB1 expression levels.Importantly, trametinib remarkably enhanced the effect of vincristine against the ABCBl-overexpressing cancer cell xenografts in nude mice.Conclusions: Trametinib antagonizes ABCB 1-mediaed MDR through direct inhibition of the drug efflux function of ABCB 1, but not by suppression of MEK kinase activity or downregulation of ABCB1 expression.These fimdings may be useful for cancer combinational therapy with trametinib in the clinic.