title: Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkappaB.
abstract: In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFkappaB DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB protein levels and p65-NFkappaB nuclear translocation during hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFkappaB to DNA, suggesting that most sulfhydryls in p65-NFkappaB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFkappaB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFkappaB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.
authors.affiliation: Department of Anatomy, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Suparna
authors.initials: S
authors.lastname: Qanungo
authors.affiliation: Department of Pharmacology, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: David W
authors.initials: DW
authors.lastname: Starke
authors.affiliation: Department of Pharmacology, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Harish V
authors.initials: HV
authors.lastname: Pai
authors.affiliation: Department of Pharmacology, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: John J
authors.initials: JJ
authors.lastname: Mieyal
authors.affiliation: Department of Anatomy, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106. Electronic address: axn25@case.edu.
authors.forename: Anna-Liisa
authors.initials: AL
authors.lastname: Nieminen
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D002384:Catalysis; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia; D010088:Oxidoreductases / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; C516005:GLRX protein, human; D054477:Glutaredoxins; C498143:RELA protein, human; D051996:Transcription Factor RelA; D004247:DNA; D010088:Oxidoreductases; D005978:Glutathione; D010100:Oxygen