PII S0360-3016(01)01810-7 BIOLOGY CONTRIBUTION REDOX CYCLING BY MOTEXAFIN GADOLINIUM ENHANCES CELLULAR RESPONSE TO IONIZING RADIATION BY FORMING REACTIVE OXYGEN SPECIES DARREN MAGDA,PH.D.,* CHERYL LEPP, M.S.,* NIKOLAY GERASIMCHUK,PH.D.,* INTAE LEE,PH.D.,* JONATHAN L. SESSLER,PH.D., † ALICE LIN, M.S.,* JOHN E. BIAGLOW,PH.D., ‡ AND RICHARD A. MILLER, M.D.* *Pharmacyclics, Inc., Sunnyvale, CA; † University of Texas at Austin, Austin, TX; ‡ University of Pennsylvania, Philadelphia, PA Purpose: To examine the mechanism of radiation enhancement by motexafin gadolinium (Gd-Tex) in vitro. Methods and Materials: Oxidation of ascorbate and NADPH by Gd-Tex was evaluated in a neutral buffer. Growth inhibition of human uterine cancer cell line MES-SA was measured using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) dye. Clonogenic assays were used to measure radiation response in MES-SA, A549 human lung carcinoma, E89, a CHO cell line variant deficient in glucose-6-phosphate dehydro- genase activity, and murine lymphoma cell lines LYAR and LYAS. Results: Gd-Tex catalyzed the oxidation of NADPH and ascorbate under aerobic conditions, forming hydrogen peroxide. Decreased viability was observed in MES-SA cells incubated with Gd-Tex in media containing NADPH or ascorbate. Gd-Tex and ascorbate increased fluorescence in dichlorofluorescin acetate–treated cultures. Synergistic effects on the aerobic radiation response in MES-SA and A549 were seen using Gd-Tex in combi- nation with L-buthionine-(S,R)-sulfoximine (BSO). Incubation with Gd-Tex in the presence of ascorbate in- creased the aerobic radiation response of E89 and the apoptosis-sensitive B-cell line (LYAS). Conclusions: Gd-Tex sensitizes cells to ionizing radiation by increasing oxidative stress as a consequence of futile redox cycling. Optimization of the concentration of ascorbate (or other reducing species) may be required when evaluating Gd-Tex activity in vitro. © 2001 Elsevier Science Inc. Texaphyrin, Radiation, Sensitizer, Ascorbate, Superoxide. INTRODUCTION Compounds that display affinity for electrons can potentiate the biologic effect of ionizing radiation (1). Both indirect and direct mechanisms of action have been proposed: Sen- sitization may occur indirectly by altering levels of radio- protective metabolites (e.g., glutathione), or the sensitizer may interact directly with cellular macromolecules to cause or enhance damage. Examples of radiation sensitizers believed to act by the former mechanism include “dia- mide” (diazenedicarboxylic acid bis[N, N'-dimethylam- ide]), tert-butyl hydroperoxide, L-buthionine-(S,R)-sulfoxi- mine (BSO), and other thiol depleters (2– 6). On the other hand, tirapazamine, nitroimidazoles, and oxygen react di- rectly with DNA or other biologic targets (7–9). In general, “oxygen mimetic” sensitizers of this latter group are most effective under hypoxic conditions, where activity is not masked by the competitive activity of oxygen. Solvated electrons produced by the radiolysis of water may serve to reduce the electron-affinic sensitizer (1). How- ever, even in the absence of ionizing radiation, electron transfer to a radiation sensitizer may occur in the presence of cellular metabolites that have a more negative standard reduction potential, e.g., NADPH, reduced glutathione, fla- vins, or ascorbate (10, 11). This process consumes the reducing metabolite, which must then be replenished. More- over, in the presence of oxygen, further electron transfer can form reduced oxygen species (e.g., superoxide and hydro- gen peroxide) and regenerate the sensitizer. Overall, such “redox cycling” can lead to a condition of oxidative stress. Texaphyrins are porphyrin-like macrocycles that form highly stable complexes with large metal cations (12). Mo- texafin gadolinium (Xcytrin, Gd-Tex, Fig. 1) has been re- ported to enhance the efficacy of radiation in animal tumor models and is currently in Phase III clinical development as an adjuvant to radiation therapy (13–17). Gd-Tex is electron affinic, with a first reduction potential near -50 mV (NHE) (18). To better understand the mechanism of its action as a radiation enhancer, the properties of the complex were examined under in vitro conditions. Our findings lead us to suggest that Gd-Tex sensitizes cells through a novel mech- anism of action, wherein oxidative stress caused by redox cycling leads to an enhanced radiation response. Reprint requests to: Darren Magda, Ph.D., Pharmacyclics, Inc., 995 E. Arques Avenue, Sunnyvale, CA 94085. Received Dec 5, 2000, and in revised form Jul 11, 2001. Accepted for publication Jul 16, 2001. Int. J. Radiation Oncology Biol. Phys., Vol. 51, No. 4, pp. 1025–1036, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/01/$–see front matter 1025