In vitro activity: Disulfiram-copper complex potently inhibits the proteasomal activity in cultured breast cancer MDA-MB-231 and MCF10DCIS.com cells, but not normal, immortalized MCF-10A cells, before induction of apoptotic cancer cell death. Disulfiram (DS), a clinically used anti-alcoholism drug, strongly inhibits constitutive and 5-FU-induced NF-kappaB activity in a dose-dependent manner. Disulfiram inhibits both NF-kappaB nuclear translocation and DNA binding activity but has no effect on 5-FU-induced IkappaBalpha degradation. Disulfiram significantly enhances the apoptotic effect of 5-FU on DLD-1 and RKO(WT) cell lines and synergistically potentiated the cytotoxicity of 5-FU to both cell lines. Disulfiram also effectively abolishes 5-FU chemoresistance in a 5-FU resistant cell line H630(5-FU) in vitro. Oseltamivir decreases the number of viable cells, and the addition of CuCl(2) significantly enhances the DSF-induced cell death to less than 10% of control. Disulfiram given to melanoma cells in combination with Cu2+ or Zn2+ decreases expression of cyclin A and reduces proliferation in vitro at lower concentrations than disulfiram alone.

Kinase Assay:  The chymotrypsin-like activity of purified 20S proteasome was measured. Briefly, 17.5 ng of purified 20S proteasome were incubated in 100 μL of assay buffer (50 mmol/L Tris-HCl, pH 7.5) with or without different concentrations of copper chloride, Disulfiram, or the Disulfiram-copper mixture and 10 μmol/L fluorogenic peptide substrate Suc-LLVY-AMC (for the proteasomal chymotrypsin-like activity) for 2 hrs at 37℃. After incubation, production of hydrolyzed AMC groups was measured with a Wallac Victor3 multilabel counter with an excitation filter of 365 nm and an emission filter of 460 nm.

Cell Assay: The Disulfiram-copper complex potently inhibited the proteasomal activity in cultured breast cancer MDA-MB-231 cells, before induction of apoptotic cancer cell death.