The MEK1/2 inhibitor AS703026 circumvents resistance to the BRAF inhibitor PLX4032 in human malignant melanoma cells

摘要

BACKGROUND:: Although inhibitors of the proto-oncogene BRAF have shown excellent antitumor activity against malignant melanoma, their efficacy is limited by the development of acquired drug resistance, a process in which reactivation of MAP kinase (MEK) is known to play an important role. In this study, we evaluated the efficacy of AS703026, a new MEK inhibitor, in BRAF inhibitor-resistant melanoma cell lines. METHODS:: Two melanoma cells lines, RPMI-7951 and SK-MEL5, harboring an activating mutation of BRAF (V600E) were treated with the BRAF inhibitor PLX4032 to select a BRAF inhibitor-resistant cell line for further study. Cell viability assay was determined with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium] assay and trypan blue exclusion method; apoptosis assay was performed by annexin-V staining. Knockdown of BRAF was investigated by small interfering RNA. RESULTS:: RPMI-7951 cells exhibited an increased sensitivity to combined treatment with PLX4032 and AS703026 compared to either drug alone. Consistent with this, the combination of PLX4032 and AS703026 significantly induced apoptosis, whereas each drug used alone did not, as demonstrated by a flow cytometric analysis of annexin-V/propidium iodide-stained cells and Western blot analysis of cleaved caspase-3. Notably, immunoblot analyses also showed a depletion of phosphorylated-ERK with combined drug treatment. In addition, AS703026 synergized with small interfering RNA-mediated downregulation of BRAF to produce results similar to those of combined treatment with PLX4032 and AS703026. CONCLUSIONS:: Our results suggest that combined treatment with AS703026 and a BRAF inhibitor overcomes the resistance to BRAF inhibitors in malignant melanoma cells harboring a mutant form of BRAF.