MHC class I-related chain molecules A and B (MICA and B) expressed on the cell-surface of tumor cells are ligands for an activating receptor, NKG2D, expressed on natural killer (NK) cells and stimulate the NK cell-mediated cytotoxicity. On the other hand, the soluble form of MICA and B produced by proteolytic cleavage of cell-surface MIC interferes with NK cell-mediated cytotoxicity. We investigated effect of sodium valproate (VPA), a histone deacetylase inhibitor, on the production of cell-surface and soluble MICA and B and NK cell-mediated cytotoxicity in four human osteosarcoma cells. VPA at 0.5 and 1.0 mM induced acetylation of histones bound to MICA and B gene promoters, increased cell-surface but not soluble MICA and B, and augmented the susceptibility of osteosarcoma cells to NK cell-mediated cytotoxicity. The present results indicate that VPA sensitizes human osteosarcoma cells to cytotoxicity of NK cells.

Valproic acid, a histone deacetylase inhibitor, increases the expression of cell surface MHC class I-related chain molecules (MICs) A and B (MICA and B) in osteosarcoma cells and decreases their secretion of soluble MICA and MICB, which are produced by the proteolytic cleavage of cell surface MICs. Osteosarcoma cells have been reported to produce high levels of matrix metalloproteinase (MMP)-2 and -9. In this study, we investigated the involvement of MMP-2 and -9 in the inhibitory action of valproic acid (VPA) on the proteolytic cleavage of cell surface MICs using the U-2 OS and SaOS-2 osteosarcoma cell lines. VPA caused a marked decrease in the expression of MMP-9 mRNA in the U-2 OS and SaOS-2 cells and in the expression of MMP-2 mRNA in the U-2 OS cells, but only a slight decrease in the expression of MMP-2 mRNA in the SaOS-2 cells. The transfection of small interfering RNA (siRNA) for MMP-9 decreased the secretion of soluble MICA and MICB by both U-2 OS and SaOS-2 cells, but that of siRNA for MMP-2 did not. The present study therefore demonstrates that the downregulation of MMP-9 mRNA by VPA plays a role in the inhibitory action of VPA on the secretion of soluble MICA and MICB in osteosarcoma cells.

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We investigated the effects of valproic acid (VPA), a histone deacetylase inhibitor, in combination with hydralazine, a DNA methylation inhibitor, on the expression of cell-surface Fas and MHC-class I-related chain molecules A and B (MICA and B), the ligands of NKG2D which is an activating receptor of NK cells, and on production of their soluble forms in HOS, U-2 OS and SaOS-2 human osteosarcoma cell lines. We also examined the susceptibility of these cells to Fas- and NK cell-mediated cell death. VPA did not increase the expression of Fas on the surface of osteosarcoma cells, while hydralazine did, and the combination of VPA with hydralazine increased the expression of cell-surface Fas. In contrast, the combination of VPA with hydralazine did not increase the production of soluble Fas by osteosarcoma cells. Both VPA and hydralazine increased the expression of cell-surface MICA and B in osteosarcoma cells, and their combination induced a greater increase in their expression. VPA inhibited the production of both soluble MICA and MICB by osteosarcoma cells while hydralazine produced no effect. Both VPA and hydralazine enhanced the susceptibility of osteosarcoma cells to Fas- and NK cell-mediated cell death and the combination of VPA with hydralazine further enhanced the effects. The present results suggest that combined administration of VPA and hydrazine is valuable for enhancing the therapeutic effects of immunotherapy for osteosarcomas.

Vascular endothelial growth inhibitor (VEGI; also referred to as TNFSF15 or TL1A) is involved in the modulation of vascular homeostasis. VEGI is known to operate via two receptors: Death receptor‑3 (DR3) and decoy receptor‑3 (DcR3). DR3, which is thus far the only known functional receptor for VEGI, contains a death domain and induces cell apoptosis. DcR3 is secreted as a soluble protein and antagonizes VEGI/DR3 interaction. Overexpression of DcR3 and downregulation of VEGI have been detected in a number of cancers. The aim of the present study was to investigate the effects of sodium valproate (VPA), a histone deacetylase inhibitor, in combination with hydralazine hydrochloride (Hy), a DNA methylation inhibitor, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Combination treatment with Hy and VPA synergistically induced the expression of VEGI and DR3 in both OS and HMVE cells, without inducing DcR3 secretion. In addition, it was observed that the combination of VPA and Hy significantly enhanced the inhibitory effect on vascular tube formation by VEGI/DR3 autocrine and paracrine pathways. Furthermore, the VEGI/VEGF‑A immune complex was pulled down by immunoprecipitation. Taken together, these findings suggest that DNA methyltransferase and histone deacetylase inhibitors not only have the potential to induce the re‑expression of tumor suppressor genes in cancer cells, but also exert anti‑angiogenic effects, via enhancement of the VEGI/DR3 pathway and VEGI/VEGF‑A interference.