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 (INO(2)BA) and buthionine sulfoximine (BSO). Growth inhibition of E-ras 20 cells by INO(2)BA 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 INO(2)BA 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 INO(2)BA, 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; INO(2)BA+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 INO(2)BA+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.
Bauer
Pal I
PI
Laboratory for Environmental Toxicology and Chemistry, Octamer Inc., Octamer Research Foundation, Romberg Tiburon Centers, San Francisco State University, Tiburon, CA 94920, USA.
Mendeleyeva
Jerome
J
Kirsten
Eva
E
Comstock
John A
JA
Hakam
Alaeddin
A
Buki
Kalman G
KG
Kun
Ernest
E
D000970:Antineoplastic Agents / Q000494:pharmacology*; D000971:Antineoplastic Combined Chemotherapy Protocols; D001549:Benzamides / Q000494:pharmacology*; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D002455:Cell Division / Q000187:drug effects; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D009603:Nitroso Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001549:Benzamides; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D009603:Nitroso Compounds; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C090712:iniparib; D019328:Buthionine Sulfoximine; C076039:3-nitrosobenzamide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011065:Poly(ADP-ribose) Polymerases; D010450:Endopeptidases