In this work we present evidence that the antigen-specific recognition of cervical cancer cells by cytotoxic T lymphocytes, is enhanced by the treatment of the cancer cells with the histone deacetylase inhibitor valproic acid alone or in combination with the DNA methylation inhibitor hydralazine. This effect can be attributed to the improved antigen presentation on the cell surface as a result of at least partially from increased transcription of HLA class-I molecules in treated cells. Although up-regulation of these class-I molecules has already been observed to occur after cells are treated with a demethylating agent  or with a histone deacetylase inhibitor  our results demonstrate that in some cell lines and patients the up-regulation is higher with the combination as compared to the individual effect of these drugs. A synegistic effect upon cancer testis antigens NY-ESO-1 and MAGE-A3 expression has been observed with 5-aza-2'-deoxycytidine and depsipeptide  but not upon HLA class-I molecules. Here we demonstrate that hydralazine and valproic acid synergize in this regard. This observation is supported by our previous study in which SW480 cells showed up-regulation of major histocompatibility complex, class-I-related only with the combined treatment but no with hydralazine or valproic acid alone . Interestingly, in CasKi and MS751 cells H/V slightly increase the up-regulation when added to IFN-γ, as compared to IFN-γ alone, a potent and well-known inducer of HLA-class-I expression .
Previous studies have reported that the de novo expression of HLA class-I antigens induced by 5-aza-2'-deoxycytidine seems to be a sporadic phenomenon, since it was observed only in one melanoma cell line  and in a human esophageal cell carcinoma cell line , but not in a panel of HLA class-I-negative or HLA-A2-negative melanoma cells . Consistent with an up-regulatory and not with a the de novo re-expression effect we also observed that these three cervical cell lines showed basal mRNA expression of HLA-A, -B and -C loci as well as constitutive expression of antigen processing components such as LMP-2, LMP-7, LMP-10 catalytic subunits of the proteasome and the transporters TAP-1 and TAP-2 (data not shown). It was of interest the observation that the effect of hydralazine was consistent regarding the lack of effect in the expression of HLA class-I molecules as in the cervical cancer cell lines tested the HLA-A, -B and -C promoters were unmethylated. Interestingly, despite 5-aza-2'-deoxycytidine has shown the ability to demethylate HLA-B locus in a an esophageal carcinoma cell line, both hydralazine and the nucleoside analog which is the prototype demethylating agent failed to demethylate the promoter in the SW480 cell line despite 5-aza-2'-deoxycytidine increased gene expression. This clearly indicates that at least in this model, chromatin remodelling by histone acetylation predominates over methylation regarding the regulation of gene expression.
Besides the well demonstrated antitumor effects of epigenetic therapies achieved by restoring the expression of key genes responsible of the malignant phenotype , the restoration of the defective expression of distinct components of the "tumor recognition complex" through epigenetic targeting of cancer cells results in their efficient recognition and lysis by antigen-specific CTL. In fact, de novo expression of selected cancer tumor antigens induced by 5-aza-2'-deoxycytidine allowed specific CTL recognition of melanoma, lung cancer, esophageal cancer, mesothelioma, renal cell carcinoma and sarcoma cells [34, 37–39]. Furthermore, the up-regulated expression of HLA class-I antigens and allospecificities observed in melanoma cell lines after exposure to 5-aza-2'-deoxycytidine resulted in their increased recognition by a gp 100-specific HLA-A2-restricted CTL clone .
Accordingly, the treatment of Caski and MS751 cell lines with H, VA, IFN-γ or H/VA/IFN-γ enhanced their specific recognition by the patients CTL's raised against specific related peptides (TLGIVCPIC and YMLDLQPETT) of the E7 HPV-16 protein and (KLPDLCTEL) of E6 HPV-18 but no against the control peptide. Interestingly, the cytotoxicity was higher with VA or H/VA and the combination of H/VA/IFN IFN-gamma suggesting that in our system chromatin remodeling by histone HA acetylation could be the key determinant for the enhanced specific recognition of cancer cells by CTLs. In fact, whereas histone acetyltransferases promote CIITA function in transactivation of MHC genes, histone deacetylases  interfere with this CIITA function following IFN-gamma induction. Of note, the observed cytotoxicity was higher with VA than with IFN-gamma. It is known that histone deacetylation impairs the transactivation of MHC genes by IFN-gamma, accordingly, in CaSki and MS751 cells, it seems that H/VA slightly increase the expression.
The role of HPV genome DNA hypermetylation is currently being studied. Existing information suggests that methylation status of viral oncogenes in lesions is perhaps solely the result of their transcriptional activity level and not a causal event for neoplastic progression . Here we also found no changes of HPV-16 E7 on CaSki cells and HPV-18 E6 on MS751. This result is in line with observations that HLA-A*0201-restricted CTL clones against HPV-16 E629–38 that recognize HPV-16 E6 antigens transfected into B lymphoblastoid cells are unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV-16 E6 in these cells was increased by transfection. In addition, the defect in presentation of HPV16 E6 [25–27, 40–43] correlates with low level expression of HLA class-I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines, suggesting that presentation of the HPV16 E6 epitope in cervical carcinoma cell lines is limited by mechanisms other than the level of HPV16 E629–38 epitope availability .
To the best of our knowledge this is the first study showing an up-regulated HLA class-I expression and antigen-specific CTL response in cervical cancer cells following the use of hydralazine and valproic acid. It will be of interest to investigate whether epitopes derived from proteins whose genes have been reactivated by hydralazine and valproic acid, different from those derived from HPV oncogenic proteins can be specific targets for CTL immune recognition. In fact, ongoing laboratory data from our group demonstrate that these drugs have the ability to increase the expression of tumor associated antigens such as MAGE and GAGE families in cervical cancer cell lines (data not shown). In addition, this combination of epigenetic agents may also help to avoid immune evasion strategy of tumors by up-regulating the expression of the major histocompatibility complex, class-I-related, a powerful NKG2D ligand for NK cell-mediated antitumor immunity  as we have observed it in a colon carcinoma cell line treated with hydralazine and valproate .