Upregulation of NKG2D ligands and enhanced natural killer cell cytotoxicity by hydralazine and valproate

Abstract

Abstract. Natural killer cells play a role in the immune antitumor response by recognizing and eliminating tumor cells through the engagement of NKG2D receptors with their ligands on target cells. This work aimed to investigate whether epigenetic drugs are able to increase MICA and MICB expression as well as NK cell cytotoxicity. Prostate, colon, breast and cervical cancer cell lines were analyzed for the expression of MICA and MICB at the mRNA and protein levels by RT-PCR, Western blot, flow cytometry and ELISA. The activating mark H3K4m2 at the MICA and MICB promoters was investigated by ChIP assays. Cytotoxicity of NK cells against the target epithelial cancer cells was investigated with the CD107 cytotoxicity assay. The results show that hydralazine and valproic acid not only increase the expression of MICA and MICB ligands of target cells, but also reduce their shedding to the supernatant. This upregulation occurs at the transcriptional level as revealed by increase of the H3K4 activating mark at the promoter of MICA and MICB genes. These effects are paralleled by increased cytotoxicity of NK cells, which was attenuated at different degrees by using blocking antibodies against the NKG2D receptor and ligands. In conclusion, our results demonstrate the ability of hydralazine and valproate to increase the NK activity against epithelial cancer cell lines and suggest that these drugs could reduce the levels of soluble MICA and MICB helping in avoiding tumor-induced suppression of NK cytotoxicity against the tumor.

DNA Methylation-Independent Reversion of Gemcitabine Resistance by Hydralazine in Cervical Cancer Cells

“…Of note, the METADRUG™ program also indicated that hydralazine could act positively in the regulation of H3K4 methylation which was not observed in our study, however, recent data from our group indicate that in several cancer cell lines treatment with hydralazine and valproic acid led to over-expression of MIC A and MIC B ligands which was accompanied by an increase in H3K4 methylation at these gene promoters suggesting that hydralazine could have this effect upon this histone mark [54].…”

Downregulation of miR-302c and miR-520c by 1,25(OH)2D3 treatment enhances the susceptibility of tumour cells to natural killer cell-mediated cytotoxicity

“…Both miR-302c and miR-520c have the same ‘seed' sequence and have the same targets (Figure 4A). Of all the hypothetical targets of miR-302c and miR-520c, MICA, MICB, and ULBP2 were of the most interest because these are NKG2D ligands reported to be associated with NK cell-mediated cytotoxicity (Chavez-Blanco et al , 2011; Fernandez-Messina et al , 2012; Hilpert et al , 2012). To further identify whether MICA, MICB, and ULBP2 responded to miR-302c and miR-520c through direct 3′-UTR interactions, we cloned the wt 3′-UTRs of the putative miR-302c and miR-520c targets (MICA-wt, MICB-wt, and ULBP2-wt) and mutated sequences (MICA-mt1+2, MICB-mt1+2, and ULBP2-mut) individually into a reporter plasmid (pSi-CHECK2) downstream of the Renilla luciferase gene (Figure 4A, P <0.01).…”

Role of Innate Immunity against Human Papillomavirus (HPV) Infections and Effect of Adjuvants in Promoting Specific Immune Response

“…For example, the administration of hydralazine and valproate can increase the expression of MICA and MICB ligands in the CaSki cervical cancer cell line and reduce their shedding to the supernatant, allowing NK attack; while cells without hydralazine and valproate are not susceptible to NK attack [57] (Table 1). …”