Poster presentation, P165

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

Involvement of autophagy in the apoptosis induced by Terfenadine, an H1 histamine receptor antagonist, in human melanoma cells

BACKGROUND AND OBJECTIVES: Previously we demonstrated that terfenadine acts as a potent apoptosis inducer in melanoma cells. Deeping on the apoptotic mechanisms activated by terfenadine we found that terfenadine induced autophagy. The aim of this study was to know the role of the autophagy in the cell death induced by terfenadine. METHODS: Cells treated with terfenadine for 8 hours were observed by Electron microscopy. LC3B-II protein expression, wich is localized on the autophagic vacuoles, was analyzed by Western Blot. Intracellular ROS levels were measured by flow cytometry after labeling cells with carboxi-H2DCFDA. Finally, to investigate how autophagy inhibition could affect terfenadine-induced cell death, we down-regulated Atg-7 by siRNA transfection assays. Seventy two hours after transfection, cell lysates were obtained to verify the Atg7 protein knockdown by Western Blotting. The viability experiments in the presence of terfenadine were performed by XTT assay after 48 hours of transfection. RESULTS: Electron microscopy examination revealed a massive vacuolization of the cytoplasm and autophagic vacuoles of multiple membranes. Western Blotting analysis revealed that terfenadine could rapidly elicit accumulation of LC3B-II. ROS production assays demonstrated that autophagy triggered by terfenadine in a ROS-dependent or ROS-independent manner according to the culture conditions. Moreover, Atg 7 down-regulated using siRNA assays partially prevented cell death induced by terfenadine at initial times of treatment, in both A375 and HT144 cells, but not when terfenadine had finally killed most of the cells. CONCLUSION: These results indicate that autophagy acts by promoting cell death in terfenadine-treated melanoma cells. This study has been supported by grants from the University of the Basque Country (GIC 07/25-IT-423-07 and the Healthy Department of the Basque Country). Erika Alonso-Tejerina was supported by a research fellowship from the Gangoiti Foundation.

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