Abstract
We have previously demonstrated that resveratrol (Resv)-induced cellular apoptosis occurs after formation of reactive oxygen species (ROS) but the role of GSH has not been well defined. Our experimental data enumerated that Resv treatment (50 μm) induced apoptosis in human leukemic monocyte lymphoma cells, which was preceded by cellular GSH efflux. High concentration of extracellular thiol (GSH, N-acetyl cysteine) and two specific inhibitors of carrier-mediated GSH extrusion, methionine or cystathionine, prevented the process of oxidative burst and cell death. This proved that GSH efflux could be a major molecular switch to modulate Resv-induced ROS generation. Spectrofluorometric data depicted that after 6 h of Resv treatment, ROS generation was evident. Pretreatment of cells with intracellular ROS scavenger (polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase) efficiently reduced ROS generation but failed to prevent intracellular GSH depletion. Thus, it suggested that intracellular GSH depletion was independent of ROS production but dependent on GSH extrusion. Furthermore, to bridge the link between GSH efflux and ROS generation, we carried out confocal microscopy of the localization of Bax protein. Microscopic analysis and small interfering RNA treatment emphasized that cellular GSH efflux triggered Bax translocation to mitochondria, which resulted in the loss of mitochondrial membrane potential, ROS generation, and caspase 3 activation and thus triggered apoptosis.
Footnotes
This work was supported by Life Science Research Board, Defense Research and Development Organisation, Government of India (to A.D.); and Department of Science and Technology, Government of India (to P.G.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.171983.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- GSSG
- oxidized glutathione
- Resv
- resveratrol
- ROS
- reactive oxygen species
- COX
- cyclooxygenase
- FBS
- fetal bovine serum
- PCR
- polymerase chain reaction
- PI
- propidium iodide
- TUNEL
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- ΔΨm
- mitochondrial membrane potential
- JC-1
- 5,5′6,6′-tetracholoro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide
- PBS
- phosphate-buffered saline
- CM-H2DCFDA
- 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester
- FACS
- fluorescence-activated cell sorting
- PEG
- polyethylene glycol
- SOD
- superoxide dismutase
- NAC
- N-acetyl cysteine
- BSO
- buthionine sulfoximine
- Cat
- catalase
- siRNA
- small interfering RNA
- DMTU
- dimethylthiourea
- l-NAME
- Nω-nitro-l-arginine methyl ester.
- Received June 24, 2010.
- Accepted September 22, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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