Background and Purpose: The Kaposi sarcoma (KS)‐associated herpesvirus GPCR (vGPCR) is a key molecule in the pathogenesis of KS, where it increases NF‐κB gene expression and activates the NF‐κB pathway. We investigated whether the less calcemic vitamin D analogue TX 527 inhibited the proliferation of endothelial cells transformed by vGPCR by modulation of the NF‐κB pathway.Experimental Approach: Endothelial cells transformed by vGPCR (SVEC‐vGPCR) were treated with TX 527. Proliferation was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt (MTS) and cell cycle by flow cytometry. mRNA and protein levels were measured by real‐time quantitative reverse transcriptase‐PCR (qRT‐PCR) and immunoblot analysis respectively.Key Results: TX 527, similar to bortezomib (0.5 nM), a proteasome inhibitor that inhibits the activation of NF‐κB, reduced proliferation and induced G0/G1 cell cycle arrest in SVEC‐vGPCR. TX 527 like 1α,25(OH)₂D₃, biological active form of vitamin D, decreased the activity of NF‐κB comparable with the effect of bortezomib. Time‐response studies showed that TX 527 significantly decreased NF‐κB and increased IκBα mRNA and protein levels. The increase of IκBα was accompanied by a reduction in p65/NF‐κB translocation to the nucleus. These responses were abolished when vitamin D receptor (VDR) expression was suppressed by stable transfection of shRNA against VDR. In parallel with NF‐κB inhibition, there was a down‐regulation of inflammatory genes such as IL‐6, CCL2/MCP and CCL20/MIP3α.Conclusions and Implications: These results suggest that the anti‐proliferative effects of the vitamin D analogue TX 527 in SVEC‐vGPCR occur by modulation of the NF‐κB pathway and are VDR dependent.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. We have previously shown that 1α,25(OH)2D3 or its less-calcemic analog TX 527 inhibits the proliferation of endothelial cells expressing vGPCR, NF-κB activity and induces apoptosis in a VDR dependent manner. In this work, we further explored whether 1α,25(OH)2D3 or TX 527 regulates PI3K/Akt/mTOR axis and induces autophagy as part of its antineoplastic mechanism of action. Proliferation assays indicated that vGPCR cell number decreased in presence of LY294002 (PI3K/Akt inhibitor) likewise 1α,25(OH)2D3 or TX 527 (10 nM, 48 h). Also, Akt phosphorylation was found decreased in dose (0.1-100 nM) and time response studies (12-72 h) after both compounds treatments. In addition, decreased phosphorylated Akt was significantly observed in the nucleus. Moreover, regulation of Akt phosphorylation was NF-κB and VDR dependent. TNFAIP3/A20, an ubiquitin-editing enzyme, a direct NF-κB target gene and a negative regulator of Beclin-1, was down-regulated whereas Beclin-1 was up-regulated after 10 nM of 1α,25(OH)2D3 or TX 527 treatment. Decrement in Akt phosphorylation was accompanied by a reduced mTOR phosphorylation and an increase in the autophagy marker LC3-II. Since increment in autophagosomes not always indicates increment in autophagy activity, we used Chloroquine (CQ, 1 μM), an inhibitor of autophagy flow, to confirm autophagy after both VDR agonists treatment. In conclusion, VDR agonists, 1α,25(OH)2D3 or TX 527, inhibited PI3K/Akt/mTOR axis and induced autophagy in endothelial cells expressing vGPCR by a VDR-dependent mechanism.