Anti-Tumoral Effect of the Non-Nucleoside DNMT Inhibitor RG108 in Human Prostate Cancer Cells

Abstract

Background: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. Methods:The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-/J2, by quantitative methylation-specific PCR and RT-PCR, respectively.Results: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-/J2 promoter hypermethylation levels, although mRNA reexpression was only attained GSTP1 and APC.Conclusions: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.

Anti-neoplastic properties of hydralazine in prostate cancer

“…The drug displayed an EC50 of 63 μM in LNCaP and 30 μM in DU145 (Supplementary Figure 1). To investigate the effects of hydralazine on the malignant phenotype of PCa, four human PCa cell lines (LNCaP, 22Rv1, DU145, and PC-3) were exposed to two different concentrations of this drug (20 and 40 μM) or to the vehicle (PBS) during 14 days, as previously described for RG108 [11]. Cell viability was evaluated at days 0, 1, 2, 3, 7, 10, and 14.…”

“…Specific GSTP1, APC and RARβ2 primers and TaqMan probes were designed using the Methyl Primer Express Software v1.0 (Applied Biosystems). β-actin ( ACTB ) was used as an internal reference gene to normalize for DNA input and all qMSP reactions were performed as previously described [11]. Methylation levels for each sample were derived from calibration curves constructed using serial dilutions of bisulfite modified CpGenomeTM Universal Methylated DNA (Millipore, Billerica, MA).…”

“…Recently, we reported RG108 as an effective tumor growth suppressor in PCa cell lines. However, this drug is substantially more effective in androgen-responsive than in castration-resistant cell lines [11]. Among non-nucleoside analogues, hydralazine, a potent arterial vasodilator approved by FDA for treatment of severe hypertension, heart failure and hypertension in pregnancy, has been described as a weak non-nucleoside DNA methylation inhibitor [12-14].…”

Effects of DNA methyltransferase inhibitor RG108 on methylation in buffalo adult fibroblasts and subsequent embryonic development following somatic cell nuclear transfer

“…To improve nuclear transfer efficiency, several epigenetic remodeling drugs, such as the DNA methylation inhibitors AzC and RG108 have been used in efforts to ameliorate the development of cloned embryos (Ding et al, 2008). The latter, a new type of non-cytidine DNA methylation inhibitor, can markedly reduce DNA methylation in tumor cells and parthenogenetic embryos in vitro and in vivo (Graça et al, 2014;Zhang et al, 2015a). It has been demonstrated that treatment of lung adenocarcinoma cells with RG108 decreases methylation of the RASSF1A gene, inducing apoptosis (Rui and Wei, 2009).…”

DNA Methylation and Its Implications and Accessibility for Neuropsychiatric Therapeutics

“…As discussed above, the existing literature regarding how DNA methylation is altered in the CNS is much more broad and well developed than literature describing drugs designed to target these mechanisms. Indeed, literature describing the use of drugs that target DNA methylation in any human pathology is essentially absent outside of developing efforts using DNMT inhibitors to slow cancer progression (101–104). …”