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

P. Bauer, Jerome Mendeleyeva, Eva Kirsten, John A. Comstock, Alaeddin Hakam, Kalman G. Buki, Ernest Kun

Research output: Contribution to journalArticle

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Abstract

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 50 kDa mass. This pattern of proteolysis of PARP I is consistent with a drug-induced necrotic cell killing pathway.

Original languageEnglish
Pages (from-to)455-462
Number of pages8
JournalBiochemical Pharmacology
Volume63
Issue number3
DOIs
Publication statusPublished - Feb 1 2002

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Buthionine Sulfoximine
Poly(ADP-ribose) Polymerases
Glycolysis
Tumors
Peptide Hydrolases
Glyceraldehyde-3-Phosphate Dehydrogenases
Neoplasms
Drug Combinations
Pharmaceutical Preparations
Proteolysis
Protein Binding
Proteins
Prodrugs
Enzymes
iniparib
Toxicity
Oxidoreductases
Cells
Association reactions
Growth

Keywords

  • 4-Iodo-3-nitrobenzamide reduction
  • Inhibition of glycolysis
  • Poly(ADP-ribose) polymerase protease induction
  • Selective tumor necrosis

ASJC Scopus subject areas

  • Pharmacology

Cite this

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. / Bauer, P.; Mendeleyeva, Jerome; Kirsten, Eva; Comstock, John A.; Hakam, Alaeddin; Buki, Kalman G.; Kun, Ernest.

In: Biochemical Pharmacology, Vol. 63, No. 3, 01.02.2002, p. 455-462.

Research output: Contribution to journalArticle

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abstract = "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 50 kDa mass. This pattern of proteolysis of PARP I is consistent with a drug-induced necrotic cell killing pathway.",
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AB - 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 50 kDa mass. This pattern of proteolysis of PARP I is consistent with a drug-induced necrotic cell killing pathway.

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