International Journal of Radiation Oncology*Biology*Physics
Biology contributionRedox cycling by motexafin gadolinium enhances cellular response to ionizing radiation by forming reactive oxygen species
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: Sensitization may occur indirectly by altering levels of radioprotective 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′-dimethylamide]), tert-butyl hydroperoxide, L-buthionine-(S,R)-sulfoximine (BSO), and other thiol depleters 2, 3, 4, 5, 6. On the other hand, tirapazamine, nitroimidazoles, and oxygen react directly with DNA or other biologic targets 7, 8, 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). However, 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, flavins, or ascorbate 10, 11. This process consumes the reducing metabolite, which must then be replenished. Moreover, in the presence of oxygen, further electron transfer can form reduced oxygen species (e.g., superoxide and hydrogen 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). Motexafin gadolinium (Xcytrin, Gd-Tex, Fig. 1) has been reported 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, 14, 15, 16, 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 mechanism of action, wherein oxidative stress caused by redox cycling leads to an enhanced radiation response.
Section snippets
Cell lines and materials
Reagents were purchased from Sigma Chemicals, unless otherwise indicated. The diacetate form of motexafin gadolinium was prepared as previously described (18).
Human uterine cancer cells MES-SA were provided by B. Sikic (Stanford School of Medicine) (19). The derivation and characterization of Chinese hamster ovary cell lines E89 and K1 have been described previously (20). Murine B-lymphocytes LYAR and LYAS were provided by D. Voehringer (Stanford School of Medicine) (21). Human lung cancer line
Oxidation of NADPH by Gd-Tex
Co-incubation of NADPH with Gd-Tex converted the cofactor to its oxidized form, at catalytic concentrations of complex. The initial rate of oxidation correlated with cofactor concentration; i.e., the reaction displayed saturation kinetics (Fig. 2). Values of kcat and KM are compiled in Table 1.
The above studies were conducted under aerobic conditions. Carrying out analogous studies under an inert atmosphere led to rapid complex degradation, as evidenced by bleaching of the characteristic
Discussion
Co-incubation of NADPH (or NADH) with catalytic amounts of Gd-Tex rapidly converted this cofactor to its oxidized form in buffer (Fig. 2). This led to the production of hydrogen peroxide under aerobic conditions (Fig. 3). Similarly, ascorbic acid can serve as a source of reducing equivalents, to form superoxide and, ultimately, hydrogen peroxide (Fig. 3). Such “futile” redox cycling has frequently been encountered as a property of electron-affinic molecules (25). The specific rate at which this
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