Abstract
We have previously shown that cultured malignant mesothelioma cells contain elevated manganese superoxide dismutase (MnSOD) mRNA levels and activities compared with non-malignant mesothelial cells. As many cytotoxic drugs generate both superoxide and hydrogen peroxide, we assessed the relative significance of catalase and the glutathione redox cycle, as well as glutathione S-transferase (GST), in protecting these cells against hydrogen peroxide and epirubicin toxicity. Mesothelioma cell lines containing high (M38K cells) and low (M14K cells) MnSOD, and non-malignant MeT-5A mesothelial cells were selected for the study. M38K cells were the most resistant of these three cell types to hydrogen peroxide (0.1-0.5 mM, 4 h) and epirubicin (0.1-0.5 microg ml(-1), 48 h) as judged by lactate dehydrogenase (LDH) release and by high-energy nucleotide (ATP, ADP, AMP) depletion. Total glutathione was higher in M38K cells (63.8 +/- 20.3 nnmol mg(-1) protein) than in M14K (25.2 +/- 8.2 nmol mg[-1]) or MeT-5A cells (23.5 +/- 4.5 nmol mg[-1]). Furthermore, GST specific activity was higher in M38K cells (111.3 +/- 15.8 U mg[-1]) than in M14K cells (77.4 +/- 6.6 U mg[-1]) or in MeT-5A cells (68.8 +/- 7.6 U mg[-1]). Western blotting indicated the presence of GST-pi in all these cells, the reactivity again being highest in M38K cells. Depletion of glutathione by buthionine sulphoximine and inhibition of catalase by aminotriazole enhanced hydrogen peroxide toxicity in all cell types, while only the depletion of glutathione increased epirubicin toxicity. We conclude that simultaneous induction of multiple antioxidant enzymes can occur in human mesothelioma cells. In addition to the high MnSOD activity, hydrogen peroxide scavenging antioxidant enzymes, glutathione and GST can partly explain the high hydrogen peroxide and epirubicin resistance of these cells in vitro.
Highlights
We have previously shown that human mesothelioma cells contain high manganese superoxide dismutase (MnSOD) levels and that these mesothelioma cells are more resistant than non-malignant mesothelial cells against the exogenous oxidant menadione (Kinnula et al, 1996)
We investigated the relative role of the most important hydrogen peroxide-scavenging AOEs catalase and glutathione peroxidase, glutathione and the detoxification enzyme glutathione S-transferase (GST) on oxidant and drug resistance in human mesothelioma cells that had been established from the tumours of untreated mesothelioma patients
M38K cells were most resistant to hydrogen peroxide when assessed by lactate dehydrogenase (LDH) release (Figure 1) or depletion of high-energy nucleotides
Summary
Cell culturePreviously characterized (Pelin-Enlund et al, 1990) continuous mesothelioma cell line cells (M38K, M14K) were cultured on uncoated plastic plates in RPMI 1640 medium (Gibco Europe, Paisley, UK) supplemented with heat-inactivated 10% fetal calf serum and the antibiotics streptomycin and penicillin. Cells were pretreated with 20 mm aminotriazole (ATZ) for 60 min to inactivate catalase or with 0.2 mm or 1 mM buthionine sulphoximine (BSO) for 16-18 h to inhibit y-glutamylcysteine synthetase and to cause glutathione depletion. Both these inhibitors are widely used, specific and their effects have been confirmed in earlier investigations (Margoliash et al, 1960; Buckley et al, 1991; Kinnula et al, 1992ab). The ATZ treatment was repeated after 24 h for 60 min, and the last 16-h incubation with epirubicin was conducted in the presence of BSO
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