Effect of glutathione depletion on antitumor drug toxicity (apoptosis and necrosis)in U-937 human promonocytic cells. The role of intracellular oxidation

Abstract

Treatment with the DNA topoisomerase inhibitors etoposide, doxorubicin, and camptothecin, and with the alkylating agents cisplatin and melphalan, caused peroxide accumulation and apoptosis in U-937 human promonocytic cells. Preincubation with the reduced glutathione (GSH) synthesis inhibitorl-buthionine-(S,R)-sulfoximine (BSO) always potentiated peroxide accumulation. However, although GSH depletion potentiated the toxicity of cisplatin and melphalan, occasionally switching the mode of death from apoptosis to necrosis, it did not affect the toxicity of the other antitumor drugs. Hypoxia or preincubation with antioxidant agents attenuated death induction, apoptotic and necrotic, by alkylating drugs. The generation of necrosis by cisplatin could not be mimicked by addition of exogenous H2O2 instead of BSO and was not adequately explained by caspase inactivation nor by a selective fall in ATP content. Treatment with cisplatin and melphalan caused a late decrease in mitochondrial transmembrane potential (ΔΨm), which was much greater during necrosis than during apoptosis. The administration of the antioxidant agents N-acetyl-l-cysteine and butylated hydroxyanisole after pulse treatment with cisplatin or melphalan did not affect apoptosis but attenuated necrosis. Under these conditions, both antioxidants attenuated the necrosis-associated ΔΨm decrease. These results indicate that oxidation-mediated alterations in mitochondrial function regulate the selection between apoptosis and necrosis in alkylating drug-treated human promonocytic cells.

Apoptosis and necrosis are two different forms of cell death with well defined morphological characteristics (1-3). Among other aspects, during apoptosis the cells undergo nuclear and cytoplasmic shrinkage, the chromatin is condensed and partitioned into multiple fragments, and the cells are finally broken into multiple membrane-surrounded bodies (apoptotic bodies). However, the plasma membrane retains the integrity during the process. By contrast, necrosis is characterized by cell swelling, lysis of intracellular organella, and rapid disintegration of the plasma membrane. Apoptosis seems to be clearly advantageous for the organism, because the elimination of the apoptotic cells or the resulting apoptotic bodies by phagocytosis prevents the release of intracellular content and the consequent damage of the surrounding tissue, as it occurs during necrosis. Hence, it seems very important to elucidate the mechanisms that regulate apoptosis and necrosis and the factors that may decide the selection between one or the other mode of death.

One of the most complex aspects in the regulation of cell death is the role of intracellular oxidation. It was initially proposed that oxidation could be a general mediator of apoptosis (4). In fact, (i) exposure to reactive oxygen species (ROS),1 such as hydrogen peroxide (H2O2) or nitric oxide (NO), induces apoptosis in different cell types (5, 6); (ii) many apoptotic inducers, which are not ROS themselves, cause intracellular oxidation,e.g. growth factor deprivation, glucocorticoids, UV irradiation, and some cytotoxic drugs (7-11); and (iii) overexpression of Bcl-2 reduces both ROS generation and apoptosis induction by different stimuli (8, 12). However, the relationship between oxidation and apoptosis is far from being clear. In fact, (i) some forms of apoptosis may take place under very low oxygen tensions, in which ROS generation is expected to be absent or greatly reduced (13, 14), or in the presence of antioxidants (13); (ii) pre-exposure to hyperoxia inhibited H2O2-provoked apoptosis in lung adenocarcinoma cells (15); and (iii) low ROS concentrations may promote proliferation and prevent apoptosis in some cell models (16, and references therein). An additional factor of interest is given by the fact that the intensity of oxidation may be determinant for the mode of death. For instance, treatment with H2O2provoked apoptosis or necrosis, depending on the concentration used (5), and the administration of low concentrations of H2O2 sufficed to inhibit apoptosis and cause necrotic-like death in antitumor drug-treated Burkitt's lymphoma cells (17).

It is known that the toxicity of antitumor drugs may largely depend on the intracellular level of reduced glutathione (GSH). Thus, depletion of GSH by prolonged incubation withl-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase, increased the lethality of the DNA topoisomerase I inhibitor CPT-11 in V79 hamster lung fibroblasts (18), of the DNA topoisomerase II inhibitor etoposide in K562 human erythroleukemia cells (19), and of the anthracycline doxorubicin in different cell types (20-23). The influence of GSH was particularly evident in the case of alkylating agents, where BSO was occasionally able to change the mode of death from apoptosis to necrosis (24, 25). Because GSH is the main antioxidant system in the cell, a possible explanation is that GSH depletion facilitates ROS accumulation in cells treated with antitumor drugs (26), which in turn increases their lethality.

To test the validity of this hypothesis, in the present work we comparatively examined the capacity of BSO to modulate ROS production and cell death in U-937 human promonocytic cells treated with different antitumor drugs. The effects of exogenous H2O2and antioxidant agents, and the possible role of oxidation-related events, such as caspase inactivation, ATP depletion, and mitochondrial dysfunction, were also considered