Disulfiram is a DNA demethylating agent and inhibits prostate cancer cell growth

Abstract

Background: The clinical success of the nucleoside analogs 5-aza-cytidine (5-azaC) and 5-aza-2'deoxycytidine (5-aza-dC) as DNA methyltransferase (DNMT) inhibitors has spurred interest in the development of non-nucleoside inhibitors with improved pharmacologic and safety profiles. Because DNMT catalysis features attack of cytosine bases by an enzyme thiol group, we tested whether disulfiram (DSF), a thiol-reactive compound with known clinical safety, demonstrated DNMT inhibitory activity.

Methods: Inhibition of DNMT1 activity by DSF was assessed using methyltransferase activity assays with recombinant DNMT1. Next, prostate cancer cell lines were exposed to DSF and assessed for: i) reduction of global 5-methyl cytosine ((5me)C) content using liquid chromatography/tandem mass spectrometry (LC-MS/MS); ii) gene-specific promoter demethylation by methylation-specific PCR (MSP); and iii) gene-reactivation by real-time RT-PCR. DSF was also tested for growth inhibition using prostate cancer cell lines propagated in vitro in cell culture and in vivo as xenografts in nude mice.

Results: Disulfiram showed a dose-dependent inhibition of DNMT1 activity on a hemimethylated DNA substrate. In prostate cancer cells in culture, DSF exposure led to reduction of global genomic (5me)C content, increase in unmethylated APC and RARB gene promoters, and associated re-expression of these genes, but did not significantly alter prostate-specific antigen (PSA) expression. DSF significantly inhibited growth and clonogenic survival of prostate cancer cell lines in culture and showed a trend for reduced growth of prostate cancer xenografts.

Conclusions: Disulfiram is a non-nucleoside DNMT1 inhibitor that can reduce global (5me)C content, reactivate epigenetically silenced genes, and significantly inhibit growth in prostate cancer cell lines.

Fig. 1

Disulfiram inhibits DNMT1 in vitro and results in reduction of ^5meC content in prostate cancer cells. A: DNMT1enzyme activity assays were performed by incubating recombinant His6-DNMT1 with hemimethylated oligonucleotide substrates and S-adenosyl-L-[methyl-³H] methionine as a methyl group donor in the presence of solvent control alone or DSF at increasing concentrations. Relative enzymatic activity is expressed as percentages of solvent control. B: DNMT1is expressed at high levels in prostate cancer cell lines but not in normal PrECs. Normal PrEC and prostate cancer cell lines (CWR22Rv1, PC3, C4-2B, DU145) were lysed and lysates were subjected to SDS^PAGE separation. Proteins were blotted onto PVDF membranes and were incubated with anti-DNMT1 and anti-Actin-specific antibodies. Desitometric analyses show DNMT1 band intensities normalized to Actin. C: LC-MS/MS analysis was used to determine ^5meC as a fraction of total cytosine content in CWR22Rv1 and PC3 cells treated with DSF for indicated time points. Normal white blood cell (WBC) genomic DNA in which all CpG dinucleotides were methylated to completion with the M.SssI methyltransferase in vitro (WBC-SssI) was used as positive control. *P <0.05.

Fig. 2

DSF treatment leads to de-methylation of methylated promoter regions in prostate cancer cells and results in re-expression of RARB and APC. A: C4-2B and CWR22Rv1 cells were cultured for 2 or 4 weeks in the presence of 0.5 μMDSF (C4-2B for RARB and CWR22Rv1 cells for APC). Total DNA was extracted and promoter methylation status was evaluated with methylation-specific PCR (MSP) using primers specific to the methylated (M) and unmethylated (U) sequence after bisulfite conversion. An increase in the signal with unmethylated-specific primers indicated de-methylation of the locus. B: RNA was extracted from CWR22Rv1 cells treated with 0.5 μMDSF or solvent control for 2 or 4 weeks. APC and RARB transcript levels were determined by quantitative real-time PCR and are shown as relative expression levels normalized to TBP expression.

Fig. 3

Disulfiram inhibits PCa cell growth in vitro and in vivo. A, B: Cells were exposed to DSF at indicated concentrations for 48 hr and cell viability was assessed by MTT and IC50 for DU145, CWR22R1, PC3, and C4-2B were calculated. C: DSF impairs clonogenic survival in prostate cancer cells. CWR22Rv1, DU145, and C4-2B cells were split to clonogenic density and cells were exposed to 100 nMDSF or solvent control. After 14 days, colony formation was assessed. *No colony formation was observed in any of the cell lines subjected to 100 nMDSF. D: Xenograft tumors of C4-2B cells were established in nude mice. When tumors became easily palpable (~0.1mm³) mice were injected i.p. with solvent control or indicated doses of DSF. Changes in tumor volume over time ±SD are shown for each treatment group (n = 8).

Fig. 4

DSF does not affect per-cell PSA secretion. A: PSA producing C4-2B cells were exposed to indicated concentrations of DSF. The conditioned media as well as the total cell lysates were collected after 48 hr and PSA concentrations were determined by ELISA. B: C4-2B cells were exposed to 0.5 μM disulfiram. After 2 weeks PSA concentrations in conditioned medium as well as the cell number of surviving cells were determined. Graph shows PSA levels normalized to cell number in solvent control and DSF-treated cells. C: Serum samples from C4-2B xenograft mice (see Fig. 3D) were collected and PSA concentration was determined by ELISA. Graph shows relative PSA levels normalized to tumor weights.