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: Suparna Qanungo, David W Starke, Harish V Pai, John J Mieyal, Anna-Liisa Nieminen
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 282 Issue 25 Pg. 18427-18436 (Jun 22 2007) ISSN: 0021-9258 [Print] United States
• PMID: 17468103 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Antineoplastic Agents
• GLRX protein, human
• Glutaredoxins
• RELA protein, human
• Transcription Factor RelA
• DNA
• Oxidoreductases
• Glutathione
• Oxygen

Topics

• Antineoplastic Agents (pharmacology)
• Apoptosis
• Catalysis
• Cell Line, Tumor
• Cell Nucleus (metabolism)
• Cytoplasm (metabolism)
• DNA (chemistry)
• Glutaredoxins
• Glutathione (metabolism)
• Humans
• Hypoxia
• Oxidoreductases (metabolism)
• Oxygen (metabolism)
• Transcription Factor RelA (metabolism)
• Transcriptional Activation
• Transfection