Effects of vitamin D binding protein-derived macrophage activating factor (GcMAF) on human neuroblastoma cells and predicted molecular interaction with the vitamin D receptor

Background: Vitamin D binding protein-derived Macrophage Activating Factor (GcMAF) proved effective in a variety of experimental and human carcinomas. In this study we evaluated its effects on human neuroblastoma cells and we studied the predicted molecular interactions with the vitamin D receptor (VDR). Materials and Methods: Human neuroblastoma cells (cell line SH-SY5Y, ATCC) were treated with increasing concentration (0.4–40 ng/ml) of purified GcMAF (Immuno Biotech) for 24–48–72 h. Cell viability and proliferation were assessed by MTT assay (Sigma-Aldrich). cAMP levels were measured by competitive EIA assay (Abnova) after 15 min challenge with GcMAF. Cells were observed by light microscopy (Optika, Nikon) at 24–48–72 h. Results: GcMAF decreased human neuroblastoma cell viability and induced morphological alterations that were interpreted as induction of apoptosis. These data are consistent with the hypothesis that GcMAF exerts a direct effect on cancer cells in addition to its immunostimulating properties. GcMAF stimulated the rapid formation of cAMP that could be responsible for the induction of apoptosis. We also determined the predicted molecular interactions between GcMAF and VDR that could be responsible for the observed non-genomic effects. GcMAF and VDR share 20% identity of amino acid (aa) sequence in the tract coded for by exons 1, 2, 3, 4, 5, i.e. aa 1–197. A higher degree of identity (40%) was observed in the tract coded for by exons 6, 7, 8, i.e. aa 217–330. The first 23 aa of the NH-terminus of GcMAF and the last 23 aa of VDR are highly hydrophobic and could represent a site of interaction for the two proteins. Conclusions: The proposed mode of interaction between GcMAF and VDR could explain the observed association between VDR gene polymorphisms and the responsiveness of human monocytes to GcMAF. This interaction is likely to occur at the level of the plasma membrane thanks to the hydrophobic interactions between the two proteins and it could be facilitated by the presence of vitamin D binding to both proteins, with GcMAF on the outer side of the membrane and VDR on the inner side. In addition, since it had been previously observed that GcMAF induced the formation of cAMP also in normal human monocytes, it could be concluded that the cAMP signalling pathway is involved in the early event of GcMAF-stimulated cell responses independently of the cell type, possibly through interaction with VDR as part of the non-genomic responses to vitamin D-axis components.