Tumor cell anti-oxidant defenses. Inhibition of the glutathione redox cycle enhances macrophage-mediated cytolysis

Abstract

The basis of resistance to oxidative injury was studied in six murine tumor cell lines that differed 54-fold in their resistance to enzymatically generated H(2)0(2). The tumors varied 56.7-fold in their specific activity of catalase, 5.3-fold in glutathione peroxidase (GPO), 3.3-fold in glutathione reductase (GR), and 2.7-fold in glutathione. There was no correlation among the levels of the three enzymes, and tumor cell resistance to lysis by H(2)0(2). However, the logarithm of the flux of H(2)0(2) necessary to cause 50 percent lysis of the tumor cells correlated with their content of glutathione (r = 0.91). The protective role of glutathione was analyzed by blocking GR and GPO, the catalysts of the glutathione redox cycle. This was facilitated by the demonstration that the anti-neoplastic agent 1,3-bis-(2- chloroethyl)-l-nitrosourea (BCNU) was a potent inhibitor of GR in intact tumor cells. BCNU inactivated tumor cell GR with a 50 percent inhibitory dose of 11 μM and a t(l/2) of inhibition of 30 s. Complete inhibition of GR was attained with no effect on GPO or catalase. Tumor cells whose GR was inactivated by BCNU could be lysed by fluxes of H(2)0(2) to which they were otherwise completely resistant. They could be killed by phorbol myristate acetate (PMA)-stimulated, bacilli Calmette-Guerin-activated macrophages in numbers which were otherwise insufficient, and by nonactivated macrophages, which otherwise were ineffective. BCNU-treated target cells were also much more sensitive to antibody-dependent, macrophage-mediated cytolysis. However, such tumor cells were no more sensitive than controls to lysis by alloreactive T cells or by antibody plus complement. Next, we deprived tumor cells of selenium by passage in selenium-deficient mice. GPO was inhibited 85 percent in such cells, with no effect on GR or catalase. Tumor cells with reduced GPO activity were markedly sensitized to lysis by small fluxes of H(2)0(2) or by PMA-stimulated macrophages or granulocytes. In contrast, inhibition of catalase with aminotriazole had no effect on the sensitivity of three tumors to peroxide-mediated lysis, and had modest effects with two others. Thus, the oxidation-reduction cycle of glutathione serves as one of the major defense mechanisms of tumor cells against three related forms of oxidant injury: lysis by fluxes of H(2)0(2), by PMA-triggered macrophages, and by macrophages in the presence of anti-tumor antibody.

Resistance of human tumor cells in vitro to oxidative cytolysis.

“…To determine which extracellularly generated oxygen reduction products rapidly lyse human cells, and therefore which enzymes might be involved in defense against oxidative cytolysis, we compared the susceptibility of fibroblasts, SK-BR-1-III, SK-BR-2-III, erythrocytes, and CAMA-I to H202,°-, and OH. Lysis first was attempted with the H202 generating system of glucose oxidase and glucose, which lysed most murine tumors tested (1). However, fluxes of H202 (nanomoles per minute per 0.2 ml) up to 3 for fibroblasts, 14 for SK-BR-l-III, 15 for SK-BR-2-Ill, 21 for erythrocytes, and 105 for CAMA-I failed to produce substantial lysis (not shown).…”

“…Weiss et al (48) showed that huma umbilical vein endothelial cells were sensitive to lysis by H2C released from stimulated neutrophils. The concentration c H202 generated by the neutrophils was 67 ,M, 1 (26). Even in our study, where human fibroblasts were insensitive to fluxes of H202 attainable with glucose oxidase or xanthine oxidase, the cells were rendered up to 20-fold more sensitive to H202 by inhibition of the GSH redox cycle with BSO or BCNU.…”

“…Hydrogen peroxide, a secretory product of activated macrophages and granulocytes, lyses murine tumor cells under some experimental conditions (1). A major defense of murine tumors against lysis by H202 is the glutathione (GSH)' redox cycle (2-4).…”

Enhancement of Antigen-Specific Immunoglobulin G Production in Mice by Co-Administration of L-Cystine and L-Theanine

“…Glutathione (GSH) is a tripeptide of cysteine, glutamic acid and glycine (L-γ-glutamyl-L-cysteinyl-glycine) and is a ubiquitous major antioxidant in vivo [14,21,26]. The antioxidative activity of GSH is exhibited by supplementing and removing reactive oxygen species generated in the living body, and many recent findings have indicated its importance in the immune system [8,13,26].…”

Phase 2 study of imexon, a prooxidant molecule, in relapsed and refractory B-cell non-Hodgkin lymphoma

“…Previous murine investigations have demonstrated that glutathione depletion increases tumor cell susceptibility to macrophage-mediated oxidative injury. 36 As CD68 is a known prognostic marker in lymphoma and can be evaluated using immunohistochemistry, future investigations of imexon should investigate CD68 expression further.…”