Antineoplastic effect of 1α,25(OH)2D3 in spheroids from endothelial cells transformed by Kaposi’s sarcoma-associated herpesvirus G protein coupled receptor

Abstract

The Kaposi´s sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi´s sarcoma. In endothelial cells, tumor maintenance and NF-κB activation depends on vGPCR constitutive expression and activity. We have previously demonstrated that 1α,25(OH)2D3 induces apoptosis in a VDR dependent manner, inhibits vGPCR cell growth and NF-κB activity. In this study, we developed a method to obtain multicellular spheroids (MCS) from endothelial cells expressing vGPCR in order to test whether MCS have a similar response to 2D-cultures after 1α,25(OH)2D3 treatment. Firstly, we found that vGPCR MCS started to form at 2nd day-growth, reaching a diameter up to 300 μm at 7th day-growth, whereas cells without vGPCR expression (SVEC) developed spheroids earlier and remained smaller throughout the period monitored. Secondly, vGPCR MCS size and architecture were analyzed during 1α,25(OH)2D3 (0.1-100 nM, 48 h) treatment. We found that once treated with 10 nM of 1α,25(OH)2D3 the initials MCS began a slight disaggregation with no changes in size; whereas at the higher dose (100 nM) the architecture of MCS was found completely broken. Furthermore, VDR mRNA expression increased significantly and this change was accompanied by a reduction of HIF-1α, an increase of VEGF, p21 and Bim mRNA expression. Finally, results from Western blot analysis showed that 1α,25(OH)2D3 decreased Akt and ERK1/2 protein phosphorylation. In conclusion, these data have revealed that 1α,25(OH)2D3 inhibits vGPCR MCS proliferation and induces apoptosis similar to vGPCR cells growing in 2D-cultures.