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The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors

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Abstract

The clinical benefit of selective BRAF inhibitor therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying the acquired resistance to a BRAF inhibitor by comparing the signaling pathways in the parental A375P cells and the resistant subline (A375P/Mdr). We demonstrate that MAPK re-activation does not contribute to the mechanism of resistance to UAI-201 of A375P/Mdr cells. The relative quantitative analysis using the 2−ΔΔCt method revealed that the BRAF inhibitor resistance observed in A375P/Mdr cells is not mediated through the overexpression of MDR proteins. In particular, we found that the expression of N-Ras was upregulated in BRAF inhibitor-resistant A375P/Mdr cells compared with A375P cells. In fact, siRNA-mediated N-Ras knockdown partially conferred UAI-201 sensitivity to A375P/Mdr cells, implying that N-Ras upregulation confers acquired resistance to BRAF inhibition. Notably, the flow cytometric analysis of the N-Ras-knockdown A375P/Mdr cells revealed that UAI-201 causes a significant accumulation of cells in the G 0/G 1 phase with a concomitant decrease in the number of cells in the S and G 2/M phases. However, platelet-derived growth factor receptor β (PDGFRβ) knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI-201, although a remarkable increase in PDGFRβ was observed in the A375P cells after UAI-201 treatment. Taken together, our results suggest that N-Ras is worth targeting to improve the therapeutic outcome of melanomas with acquired resistance to BRAF inhibitors.

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Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2A10058897).

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  1. Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, 12-1 Songdo-dong, Yeonsu-gu, Incheon, 406-772, Republic of Korea

    Jun-Ho Ahn & Michael Lee

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  1. Jun-Ho Ahn
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  2. Michael Lee
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Correspondence to Michael Lee.

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Ahn, JH., Lee, M. The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors. Mol Cell Biochem 392, 239–247 (2014). https://doi.org/10.1007/s11010-014-2034-2

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  • DOI: https://doi.org/10.1007/s11010-014-2034-2

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