Xanthine oxidase-generated hydrogen peroxide is a consequence, not a mediator of cell death.
Oxidative stress has been associated with a wide range of diseases including atherosclerosis, cancer and Alzheimer's disease. When present in excessive concentrations, reactive oxygen species (ROS) can cause deleterious effects. This has led to the notion that the anticancer effects of various chemotherapeutics may be mediated, at least in part, by an increase in ROS. To investigate the role of xanthine oxidase (XO), a source of hydrogen peroxide, in cell death, MCF7, HeLa and 293T cells were treated with various cell-death-inducing drugs in the presence and absence of allopurinol, a specific inhibitor of XO. In the absence of allopurinol, each drug led to a time- and concentration-dependent increase in percent DNA fragmentation and ROS levels, regardless of the mechanism of cell death incurred, i.e. caspase dependent and caspase independent. By contrast, pretreatment with allopurinol led to dramatically lower ROS levels in all cases, suggesting that XO is a major contributor to oxidative stress. However, allopurinol did not exhibit a protective effect against cell death. Similarly, the administration of siRNA against XO also did not exhibit a protective effect against cell death. The level of oxidative stress was recorded using the ROS sensor CM-H(2) DCFDA and a ratiometric bioluminescent assay that takes advantage of the increased sensitivity of Firefly luciferase to hydrogen peroxide, compared with a stable variant of Renilla luciferase (RLuc), RLuc8. Overall, these findings suggest that XO-generated hydrogen peroxide, and perhaps hydrogen peroxide in general, is a consequence, but not a mediator of cell death.
Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104, USA.
Julie
J
Czupryna
Andrew
A
Tsourkas
D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D008819:Mice, Nude; D009154:Mutation / Q000235:genetics; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D010957:Plasmids; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism
D000970:Antineoplastic Agents; D016166:Free Radical Scavengers; D016877:Oxidants; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014970:Xanthines; D000493:Allopurinol; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D049409:Luciferases, Firefly; D014969:Xanthine Oxidase