Blockage of drug resistance in vitro by disulfiram, a drug used to treat alcoholism.
BACKGROUND
P-glycoprotein (P-gp) pumps a wide range of cytotoxic drugs out of cells. Inhibiting maturation of P-gp would be a novel method for circumventing P-gp-mediated multidrug resistance, which complicates cancer chemotherapy and treatment of patients infected with human immunodeficiency virus. We examined the effect of disulfiram (Antabuse(TM)) on the maturation and activity of P-gp.


METHODS
Embryonic kidney cells were transfected with a complementary DNA for the P-pg gene, and the effects of disulfiram on the sensitivity of the transfected cells to cytotoxic agents were determined. Enzyme assays were used to determine the effects of disulfiram on the verapamil-stimulated adenosine triphosphatase (ATPase) activity of P-gp. Disulfiram modifies cysteine residues, and mutant forms of P-gp that lack individual cysteines were used to determine whether particular cysteine residues mediate disulfiram's effects on P-gp activity. Maturation of recombinant P-gp was followed on immunoblots.


RESULTS
Disulfiram increased the sensitivity of P-gp-transfected cells to vinblastine and colchicine and inhibited P-gp's verapamil-stimulated ATPase activity. Half-maximal inhibition of ATPase activity occurred at 13.5 microM disulfiram. Disulfiram (at 100 microM) inhibited a P-gp mutant by 43% (95% confidence interval [CI] = 37%-48%) when cysteine was present at position 431 only and by 72% (95% CI = 66%-77%) when cysteine was present at position 1074 only. Treatment of P-gp-transfected cells with 50 nM disulfiram blocked maturation of recombinant P-gp.


CONCLUSIONS
Disulfiram can potentially reduce P-gp-mediated drug resistance by inhibiting P-gp activity (possibly via cysteine modification) and/or by blocking its maturation. These results suggest that disulfiram has the potential to increase the efficacy of drug therapies for cancer and acquired immunodeficiency syndrome.
Loo
T W
TW
Departments of Medicine, University of Toronto, ON, Canada.
Clarke
D M
DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000187:drug effects; D000427:Alcohol Deterrents / Q000494:pharmacology; D000444:Aldehyde Dehydrogenase / Q000187:drug effects; D015153:Blotting, Western; D003545:Cysteine / Q000378:metabolism; D018076:DNA, Complementary; D004221:Disulfiram / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000166:cytology; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000427:Alcohol Deterrents; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D000444:Aldehyde Dehydrogenase; D000251:Adenosine Triphosphatases; D003545:Cysteine; D004221:Disulfiram