Disulfiram is a DNA demethylating agent and inhibits prostate cancer cell growth.
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.
Lin
Jianqing
J
Chemical Therapeutics Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
Haffner
Michael C
MC
Zhang
Yonggang
Y
Lee
Byron H
BH
Brennen
W Nathaniel
WN
Britton
Justin
J
Kachhap
Sushant K
SK
Shim
Joong Sup
JS
Liu
Jun O
JO
Nelson
William G
WG
Yegnasubramanian
Srinivasan
S
Carducci
Michael A
MA
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation; D004221:Disulfiram / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008297:Male; D051379:Mice; D017430:Prostate-Specific Antigen / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology
D004791:Enzyme Inhibitors; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; D017430:Prostate-Specific Antigen; D004221:Disulfiram
Mol Cancer Ther. 2004 Sep;3(9):1049-60
15367699
Mol Biosyst. 2005 Jul;1(2):127-34
16880974
Cancer Lett. 2009 Jun 8;278(1):82-7
19168279
J Natl Cancer Inst. 2006 Oct 18;98(20):1443-4
17047192
Cancer. 1981 Apr 1;47(7):1739-42
6164472
Front Biosci. 2007 May 01;12:4254-66
17485372
N Engl J Med. 2003 Nov 20;349(21):2042-54
14627790
Clin Cancer Res. 2009 Oct 1;15(19):6070-8
19789329
Cancer Res. 2001 Feb 15;61(4):1477-85
11245454
Trends Pharmacol Sci. 2004 Nov;25(11):551-4
15491775
Cancer Res. 2006 Dec 15;66(24):11659-67
17178860
Nat Rev Genet. 2009 Nov;10(11):805-11
19789556
Cancer Treat Rep. 1977 Jan-Feb;61(1):51-4
67893
Hum Mol Genet. 2001 Apr;10(7):687-92
11257100
Nature. 2004 May 27;429(6990):457-63
15164071
Clin Genitourin Cancer. 2007 Dec;5(7):457-9
18272030
J Biochem Mol Biol. 2007 Nov 30;40(6):1069-76
18047805
Curr Pharm Biotechnol. 2006 Oct;7(5):315-21
17076647
Clin Cancer Res. 2005 May 15;11(10):3604-8
15897554
Annu Rev Pharmacol Toxicol. 1981;21:575-96
7016017
J Natl Cancer Inst. 2005 Jan 19;97(2):103-15
15657340
J Mol Med (Berl). 2007 Oct;85(10):1137-48
17571247
Chembiochem. 2002 Apr 2;3(4):274-93
11933228
Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9821-6
8790415
J Natl Cancer Inst. 2000 Jun 7;92(11):898-902
10841824
Int J Cancer. 2006 Mar 15;118(6):1577-80
16206267
Biochem Pharmacol. 1997 Feb 21;53(4):511-8
9105402
Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827-31
1542678
Endocrinology. 2009 Sep;150(9):3991-4002
19520778
N Engl J Med. 2008 Mar 13;358(11):1148-59
18337604
Cancer. 2006 Apr 15;106(8):1794-803
16532500
Cancer Res. 2006 Jan 1;66(1):385-92
16397253
Science. 2002 Sep 13;297(5588):1807-8; discussion 1807-8
12229925
J Natl Cancer Inst. 2005 Oct 19;97(20):1498-506
16234563
Cancer Res. 2006 Nov 1;66(21):10425-33
17079463
Carcinogenesis. 2010 Jan;31(1):27-36
19752007
Cell. 1983 May;33(1):9-10
6205762
Acta Psychiatr Scand Suppl. 1992;369:15-26
1471547
J Biol Chem. 2005 May 6;280(18):18302-10
15755728
J Biol Chem. 2005 Dec 9;280(49):40749-56
16230360
Biochem Pharmacol. 2007 Jan 1;73(1):25-33
17026967
Cancer J. 2007 Jan-Feb;13(1):17-22
17464242
Proc Natl Acad Sci U S A. 1984 Nov;81(22):6993-7
6209710
Oncogene. 2002 Aug 12;21(35):5483-95
12154409
Mol Pharmacol. 2003 Nov;64(5):1076-84
14573756
J Biol Chem. 2008 Aug 8;283(32):22089-96
18524780
Cancer Res. 2004 Mar 15;64(6):1975-86
15026333
Int J Cancer. 2003 Apr 20;104(4):504-11
12584750
J Clin Oncol. 2008 Mar 1;26(7):1148-59
18309951
Genes Dev. 1999 Jan 1;13(1):26-34
9887097
Anticancer Drugs. 1997 Apr;8(4):358-68
9180389
Cancer Res. 2008 Nov 1;68(21):8954-67
18974140
Drug Saf. 1999 May;20(5):427-35
10348093
Hum Mol Genet. 2000 Oct;9(16):2395-402
11005794
Biochem Biophys Res Commun. 2001 Nov 30;289(2):586-90
11716515
Front Biosci. 2005 Sep 01;10:2932-9
15970547
J Mol Biol. 1996 Sep 6;261(5):634-45
8800212
Cancer Res. 2000 Sep 15;60(18):5165-70
11016644
Nucleic Acids Res. 2000 May 15;28(10):2108-13
10773079
Clin Cancer Res. 2001 Oct;7(10):3047-55
11595694
Biochemistry. 1991 Nov 19;30(46):11018-25
1932026
Prostate. 2007 Jul 1;67(10):1099-110
17477369
Cancer Res. 2001 Dec 15;61(24):8611-6
11751372
Endocr Relat Cancer. 2009 Jun;16(2):401-13
19153211
Mol Carcinog. 2002 Mar;33(3):163-71
11870882