Anti-cancer action of 4-iodo-3-nitrobenzamide in combination with buthionine sulfoximine: inactivation of poly(ADP-ribose) polymerase and tumor glycolysis and the appearance of a poly(ADP-ribose) polymerase protease

E-ras 20 tumorigenic malignant cells and CV-1 non-tumorigenic cells were treated with a drug combination of 4-iodo-3-nitrobenzamide (INO2BA) and buthionine sulfoximine (BSO). Growth inhibition of E-ras 20 cells by INO2BA was augmented 4-fold when cellular GSH content was diminished by BSO, but the growth rate of CV-1 cells was not affected by the drug combination. Analyses of the intracellular fate of the prodrug INO2BA revealed that in E-ras 20 cells about 50% of the intracellular reduced drug was covalently protein-bound, and this binding was dependent upon BSO, whereas in CV-1 cells BSO did not influence protein binding. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the protein that covalently binds the reduction product of INO2BA, which is 4-iodo-3-nitrosobenzamide. Since only the enzymatically reduced drug INOBA bound covalently to GAPDH, the BSO-dependent covalent protein–drug association indicated an apparent nitro-reductase activity present in E-ras 20 cells, but not in CV-1 cells, explaining the selective toxicity. Covalent binding of INOBA to GAPDH inactivated this enzyme in vitro; INO2BA+BSO also inactivated cellular glycolysis in E-ras 20 cells because it provided the precursor to the inhibitory species: INOBA. Another event that occurred in INO2BA+BSO-treated E-ras 20 cells was the progressive appearance of a poly(ADP-ribose) polymerase protease. This enzyme was partially purified and characterized by the polypeptide degradation product generated from PARP I, which exhibited a 50kDa mass. This pattern of proteolysis of PARP I is consistent with a drug-induced necrotic cell killing pathway.