NY-ESO-1 is a highly immunogenic cancer-testis antigens and it is a potential candidate for immunotherapy. Cellular expression of NY-ESO-1 gene is heterogeneous depending on the demethylation status of its promoter. Exposure to 5-aza-2'-deoxycytidine (5-aza-CdR) has been reported to enhance NY-ESO-1 protein expression. Our aim is to study the effect of 5-aza-CdR on NY-ESO-1 expression in lung cancer cell lines.
Three human lung cancer cell lines were treated with 5µM of 5-aza-CdR. NY-ESO-1 protein expression was analyzed using cellular ELISA. NCI-H1975 and NCI-H522 were tested for their dose response to increasing doses of 5-aza-CdR (2.5 to 10μM). Expression of NY-ESO-1 mRNA was assessed using qRT-PCR. Cellular ELISA, western blot (WB) and flow cytometry (FACS) techniques were used to assess NY-ESO-1 protein expression. Epigenetic modifications of CpG islands in NY-ESO-1 gene were analyzed using bisulfate sequencing. The proteomic profiling of the cells was carried out using label-free mass spectrometry analysis.
NCI-H1975 and NCI-H522 cells had significantly enhanced expression (11 and 1.6 folds) of NY-ESO-1 protein after exposure to 5µM of 5-aza-CdR. Cellular ELISA showed a dose-dependent increase of NY-ESO-1 protein in both cell lines with the highest expression for NCI-H1975 (16 folds vs. control). qRT-PCR data showed a dose-dependent increase in NY-ESO-1 mRNA expression in both cells, with peak expression in NCI-H1975 cells (128 folds) at 10μM of 5-aza-CdR. FACS and WB analysis revealed increased protein expression with increasing concentration of 5-aza-CdR. Moreover, the CpG islands in the NY-ESO-1 gene were significantly hypomethylated in treated cells especially at 5μM. Proteomic profiling resulted in identification of various proteins with differential expression and functions.
We have shown that treatment with 5-aza-CdR enhances the expression of epigenetically silenced NY-ESO-1 antigen in lung cancer cells and this should trigger T cells immune responses against the NY-ESO-1 antigen resulting in tumor suppression. Further in vivo studies are required to apply such treatment to be as an innovative immunotherapeutic approach to treat lung cancer patients.
Medical Research Center, Hamad Medical Corporation, Doha, Qatar.
Medical Research Center, Hamad Medical Corporation, Doha, Qatar.
All authors have declared no conflicts of interest.