Glutathione Depletion, Pentose Phosphate Pathway Activation, and Hemolysis in Erythrocytes Protecting Cancer Cells from Vitamin C-induced Oxidative Stress * □ S Received for publication, July 17, 2016, and in revised form, September 12, 2016 Published, JBC Papers in Press, September 22, 2016, DOI 10.1074/jbc.C116.748848 Zhuzhen Z. Zhang ‡ , Eunice E. Lee ‡ , Jessica Sudderth § , Yangbo Yue ‡ , Ayesha Zia ¶ , Donald Glass ‡ , Ralph J. Deberardinis §¶ , and Richard C. Wang ‡1 From the Departments of ‡ Dermatology and ¶ Pediatrics, the University of Texas Southwestern Medical Center, Dallas, Texas 75390, the § Children’s Medical Center Research Institute, the University of Texas Southwestern Medical Center, Dallas, Texas 75390, and the  Eugene McDermott Center for Human Growth and Development, the University of Texas Southwestern Medical Center, Dallas, Texas 75390 Edited by Jeffrey Pessin The discovery that oxidized vitamin C, dehydroascorbate (DHA), can induce oxidative stress and cell death in cancer cells has rekindled interest in the use of high dose vitamin C (VC) as a cancer therapy. However, high dose VC has shown limited effi- cacy in clinical trials, possibly due to the decreased bioavailabil- ity of oral VC. Because human erythrocytes express high levels of Glut1, take up DHA, and reduce it to VC, we tested how eryth- rocytes might impact high dose VC therapies. Cancer cells are protected from VC-mediated cell death when co-cultured with physiologically relevant numbers of erythrocytes. Pharmacolog- ical doses of VC induce oxidative stress, GSH depletion, and increased glucose flux through the oxidative pentose phosphate pathway (PPP) in erythrocytes. Incubation of erythrocytes with VC induced hemolysis, which was exacerbated in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD) patients and rescued by antioxidants. Thus, erythrocytes protect cancer cells from VC-induced oxidative stress and undergo hemolysis in vitro, despite activation of the PPP. These results have implica- tions on the use of high dose VC in ongoing clinical trials and highlight the importance of the PPP in the response to oxidative stress. Consistent with its function as a potent reducing agent essential for numerous biological reactions, VC 2 can be readily oxidized to dehydroascorbate (DHA) in aqueous solution. DHA is transported intracellularly by Glut1 (solute carrier fam- ily 2, facilitated glucose transporter member 1) (1), after which it is recycled to reduced VC at the expense of cellular antioxi- dants, such as GSH (2, 3). This paradoxical role for VC as a source of oxidative stress has been found to be selectively toxic to some cancer cells, particularly to those overexpressing Glut1 (4, 5). Although the benefits of VC supplementation as a cancer therapy have been inconsistent (6), higher doses of intravenous VC have shown more promise, and several clinical trials are ongoing (7). Like many cancer cells, human erythrocytes (RBCs) express very high levels of Glut1. The abundance of the Glut1 trans- porter in erythrocyte membrane allows RBCs to mediate the transport of glucose and DHA at rates that far exceed their capacity to utilize it (3). There is substantial evidence that erythrocytes participate in VC recycling in vivo (8, 9). In addi- tion, erythrocytes have also been found to protect both tissues and cells from H 2 O 2 -mediated damage through their high capacity redox systems (10). We hypothesized that erythrocytes might protect cancer cells from VC-mediated toxicity through similar mechanisms. Indeed, we find that physiologically rele- vant numbers of erythrocytes protected HCT116, A375, and SK-MEL-28 cells from VC-mediated toxicity. Erythrocytes incubated with VC showed higher levels of oxidative stress and shifted glucose metabolism toward the oxidative pentose phos- phate pathway (PPP), which produces NADPH for ROS miti- gation. Finally, we find that VC induced hemolysis of erythro- cytes, which was exacerbated by chemical or genetic inhibition of glucose-6-phosphate dehydrogenase (G6PD) and rescued by incubation with -mercaptoethanol. This study broadens our current understanding of VC metabolism by erythrocytes and has implications for the use and monitoring of VC in the clinical setting. Results Erythrocytes Protect Cancer Cells from VC- and H 2 O 2 -in- duced Cell Death—The selective toxicity of VC to KRAS (proto- oncogene v-Raf murine sarcoma viral oncogene homolog B) and BRAF (proto-oncogene v-Raf murine sarcoma viral onco- gene homolog B) mutant colorectal cancer cells is due, in part, to the high expression levels of Glut1 and preferential uptake of oxidized DHA by the facilitative glucose transporter (5). Con- sistent with their very high expression of Glut1, erythrocytes have also been shown to transport DHA efficiently (11). We compared the kinetics of DHA uptake by erythrocytes and HCT116 at physiological concentrations of glucose and found * This work was supported by National Institutes of Health Grant R01 CA157996 (to R. J. D.), K23 AR069728 (to D. G.), K23 HL132054 (to A. Z.), and K08 CA164047 (to R. C. W.). This work was also supported by Burroughs Wellcome Fund Career Award for Medical Scientists (CAMS) (Grant 1010978) and Disease Oriented Clinical Scholar Awards (to R. C. W.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. □ S This article contains supplemental data. 1 To whom correspondence should be addressed: Dept. of Dermatology, NL08.110FB, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dal- las, TX 75390-9069. Tel.: 214-648-3430; Fax: 214-648-5554; E-mail: richard.wang@utsouthwestern.edu. 2 The abbreviations used are: VC, vitamin C; DCFDA, 2',7'-dichlorofluorescin diacetate; DHA, dehydroascorbate; DHEA, dehydroepiandrosterone; PPP, pentose phosphate pathway; G6PD, glucose-6-phosphate dehydroge- nase; ROS, reactive oxygen species; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; -ME, -mercaptoethanol; MTBSTFA, N- tert-butyldimethylsilyl-N-methyltrifluoroacetamide; AA, ascorbic acid. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 44, pp. 22861–22867, Ocotber 28, 2016 © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. OCOTBER 28, 2016 • VOLUME 291 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 22861 ACCELERATED COMMUNICATION by guest on May 22, 2020 http://www.jbc.org/ Downloaded from