ORIGINAL ARTICLE Alain-The´ ophile Sane´ Æ Andre´ M. Cantin Benoit Paquette Æ J. Richard Wagner Ascorbate modulation of H 2 O 2 and camptothecin-induced cell death in Jurkat cells Received: 27 December 2003 / Accepted: 24 March 2004 / Published online: 10 June 2004 Ó Springer-Verlag 2004 Abstract The effect of ascorbate on cell death was examined in Jurkat cells (human T-cell leukemia) by incubation with dehydroascorbate (DHA), which is rapidly taken up by cells and efficiently reduced to ascorbate. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of annexin V-labeled cells. In parallel, necrosis was estimated by the release of lactate dehydrogenase. Minor effects on cell death were observed when Jurkat cells were incubated with either DHA alone (100–1,000 lM) or a single dose of 10 lM H 2 O 2 . However, pre-incubation with DHA followed by exposure to H 2 O 2 clearly stimulated both apoptosis and necrosis. In complete contrast, pre-incubation of cells with DHA significantly inhibited apoptosis, but did not affect necrosis, induced by the topoisomerase I inhibitor camptothecin. Our results indicate that intracellular ascorbate can modulate cell death in a manner which depends upon the nature of the apoptotic stimulus, which in turn has critical implications regarding the mechanism and potential application of ascorbate in cancer therapy. Keywords Dehydroascorbate Æ Reactive oxygen species Æ Topoisomerase inhibitors Abbreviations CPT: 20-S-camptothecin lactone Æ DHA: Dehydroascorbate Æ LDH: Lactate dehydrogenase Æ Ac-DEVD-AMC: Ac-Asp-Glu-Val- Asp-amino-4-methylcoumarin Æ PS: Phosphatidyl- serine Æ GSH: Glutathione Æ DTT: Dithiothreitol Introduction The utility of ascorbate (vitamin C) as an effective che- motherapeutic agent is highly controversial. In 1974, Cameron and Campbell reported catastrophic tumor hemorrhage and necrosis in a minority of terminally ill cancer patients treated with ascorbate [7]. Later on, results from double-blind clinical trials revealed no anticancer effects of ascorbate [16]. However, a critical difference between these latter two studies was the method of administration of ascorbate, i.e., intrave- nously vs orally, respectively. Oral administration leads to rapid saturation of ascorbate in plasma, whereas the intravenous method can yield much higher levels in cells and tissues [25]. A number of fundamental studies have underlined the potential benefit of high concentrations of ascorbate in cancer therapy. High concentrations of ascorbate have been reported to be toxic in several cancer cell lines [3, 18, 29, 30, 36] as well as in hollow fiber and ascitic liver tumor (TLT)-bearing mice [8, 39]. The combination of ascorbate and menadione appears to increase the life span of tumor- bearing mice [40]. In addition, cultured tumor cells [13, 37] and xenograph tumors of hematopoietic and epithelial cell lines in mice rapidly incorporate and accumulate ascorbate [1, 13, 37]. This accumulation is explained by initial oxidation of ascorbate near the tumor site followed by rapid incorporation of the oxidized form, dehydro- ascorbate (DHA) [1]. Interestingly, tumor cells accumu- late DHA more than normal cells because the former generate more reactive oxygen species (ROS) and overexpress glucose transporters involved in DHA transport [38, 41, 42]. Much recent interest in ascorbate A.-T. Sane´ Æ J. R. Wagner (&) Centre de recherche sur le vieillissement, Institut Universitaire de Ge´riatrie de Sherbrooke, Pavillion d’Youville, 1036, rue Belve´de`re Sud, Sherbrooke, QC, Canada, J1H 4C4 E-mail: richard.wagner@usherbrooke.ca Tel.: +1-819-8211170 Fax: +1-819-8297141 A. M. Cantin Unite´ de recherche pulmonaire, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC, Canada, J1H 5N4 B. Paquette Æ J. R. Wagner De´partement de me´decine nucle´aire et radiobiologie, Faculte´ de Me´decine, Universite´ de Sherbrooke, Sherbrooke, QC, Canada, J1H 5N4 Cancer Chemother Pharmacol (2004) 54: 315–321 DOI 10.1007/s00280-004-0828-8