1alpha,25(OH)2D3 INDUCES AUTOPHAGY IN ENDOTELIAL TUMOR CELLS EXPRESSING VGPCR

ALEJANDRA SUARES; VERONICA GONZALEZ PARDO; CINTHYA TAPIA

San Carlos de Bariloche

Congreso; SISTAM 2018; 2018

The Kaposi?s Sarcoma-associated Herpesvirus G Protein-Coupled Receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi?s sarcoma. Persistent expression and activity of vGPCR is required for NF-B pathway activation and tumor maintenance in endothelial cells. We have previously demonstrated that 1α,25(OH)2D3 inhibits vGPCR cell growth, NF-B activity and induces apoptosis in a VDR dependent manner. In this work, we studied whether 1α,25(OH)2D3 induces autophagy as part of its antineoplastic mechanism of action. Firstly, BECN1 expression, a gene involved in autophagy and apoptosis pathways, was evaluated by qRT-PCR (6-48 h) and Western blot (6-24 h) analysis after 1α,25(OH)2D3 treatment (10 nM). Results shown that BECN1 expression was found up-regulated after 12 h of 1α,25(OH)2D3 treatment. Next, to connect apoptotic events with autophagy, we studied whether Bcl-2 protein forms complexes with BECN1 to trigger mitochondrial membrane depolarization. On the contrary, no association between BECN1 and Bcl-2 was observed by co-immunoprecipitation after 16 h of 1α,25(OH)2D3 treatment. Secondly, Akt/mTOR pathway regulation by 1α,25(OH)2D3 was investigated. Proliferation assays indicated that vGPCR cell number decreased in presence of PI3K/Akt pathway (LY 294002) likewise 1α,25(OH)2D3 (10 nM, 48 h). Also, Akt protein phosphorylation was found decreased in time (6-48 h) and dose response studies (0.1-100 nM) by 1α,25(OH)2D3. Decrement in Akt phosphorylation was accompanied by a reduced mTOR phosphorylation and increased LC-3II protein levels after 48 h of 1α,25(OH)2D3 treatment. Moreover, when autophagy flow was inhibited by Cloroquine (CQ, 1 μM), LC-3II protein levels also increased and this event was enhanced by 1α,25(OH)2D3. For far, these results suggest that 1α,25(OH)2D3 triggers autophagy provoking Bcl-2/BECN1 association and inhibiting Akt/mTOR pathway. Supported by grants from CONICET 11220150100057CO and FONCyT PICT-2013-0552 to Verónica González-Pardo