Int. J. Cancer: 71, 246–250 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer THE CELL-SPECIFIC ANTI-PROLIFERATIVE EFFECT OF REDUCED GLUTATHIONE IS MEDIATED BY g-GLUTAMYL TRANSPEPTIDASE-DEPENDENT EXTRACELLULAR PRO-OXIDANT REACTIONS Paola PEREGO1, Aldo PAOLICCHI2, Roberto TONGIANI2, Alfonso POMPELLA3, Patrizia TONARELLI2, Nives CARENINI1, Simona ROMANELLI1 and Franco ZUNINO1* 1Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy 2Università degli Studi di Pisa, Pisa, Italy 3Istituto di Patologia Generale, Siena, Italy We have shown earlier that extracellular GSH can exert a cell-specific growth-inhibitory effect on human tumor cells. In the present study, 2 human ovarian carcinoma cell lines (A2780 and IGROV-1) were used to investigate the biochemical basis of the GSH growth-inhibitory effect. Whereas cells were resistant, A2780 cells were sensitive to a 1 hr exposure to GSH, as assessed by the growth inhibition assay. Analysis of relevant GSH-dependent enzymes indicated that A2780 cells had low level of GSH S-transferase, glutathione reductase and g-glutamyl transpeptidase (g-GT) activities in comparison with those of IGROV-1 cells, and GSH peroxidase activity was undetectable in A2780 cells. The GSH effect was reversed by catalase and by dithiothreitol, indicating the occurrence of oxidative phenomena resulting in the impairment of critical cellular thiols. Indeed treatment of cells with H2O2 also resulted in growth inhibition, which was more marked in A2780 cells. The g-glutamyl acceptor glycylglycine, a cosubstrate for g-GT, potentiated the growth-inhibitory effect of GSH, which in contrast was decreased by the g-GT inhibitors, serine-borate complex and acivicin, suggesting that the production of reactive forms of oxygen (probably H2O2) was mediated by cysteinyl-glycine after GSH hydrolysis. The results support that the growth-inhibitory effect of low GSH concentration is the result of oxidative damage related to extracellular GSH metabolism. Int. J. Cancer 71:246– 250, 1997. r 1997 Wiley-Liss, Inc. The intracellular tripeptide thiol glutathione (GSH) plays an important role in a series of physiologic functions related to its anti-oxidant properties, including modulation of the redox status of cellular proteins, protection from oxidative stress and detoxication of electrophilic compounds (Meister, 1994). GSH has been implicated in modulating tumor-cell sensitivity to ionizing radiation, alkylating agents and platinum compounds (Meijer et al., 1990). Indeed, tumor cell lines selected in vitro for resistance to cisplatin or alkylating agents may have high levels of GSH or exhibit high capacity for GSH synthesis (Godwin et al., 1992). However, the exact mechanism by which GSH influences the cytotoxicity of DNA-damaging agents has not been fully elucidated. Drug transport in resistant tumor cell lines over-expressing the multidrugresistance-associated protein MRP has been reported to be regulated by intracellular GSH levels (Versantvoort et al., 1995). However, the relevance of alterations of GSH metabolism in the development of clinical resistance remains uncertain, since a marked increase in GSH levels is not a frequent event in human tumors (Pratesi et al., 1995). Exogenous GSH is currently considered a promising chemoprotector against cisplatin-induced nephrotoxicity and neurotoxicity, and it does not decrease the cytotoxic and anti-tumor activity of the platinum compound (Zunino et al., 1989; Hamers et al., 1993). A striking observation of drug/GSH combination studies is a cellspecific anti-proliferative effect of GSH itself at pharmacologically relevant concentrations (Zunino et al., 1993; Tedeschi et al., 1991). A tumor growth-inhibitory effect of exogenous GSH has been described (Novi, 1980; Cook et al., 1984; Karmali, 1984). In particular, Novi (1980) found that GSH may prevent tumor growth or cause tumor regression during aflatoxin-induced hepatocarcino- genesis in rats, although others could not confirm such observations (Neal and Legg, 1983). The cellular and molecular basis of the anti-proliferative effects of GSH remains uncertain. With the goal of investigating the mechanisms helping to determine cellular response to exogenous GSH, we used 2 ovarian carcinoma cell lines provided with differential response to the thiol. On the basis of the data obtained, we propose that the growthinhibitory effect of exogenous GSH is mediated by oxidative phenomena, and that the differential sensitivity of the studied cell lines may be accounted for by their different characteristics in GSH-centered anti-oxidant enzymatic activities. MATERIAL AND METHODS Cell lines The 2 ovarian carcinoma cell lines used in the study, IGROV-1 and A2780 (Bénard et al., 1985; Behrens et al., 1987) originated from untreated patients. They were cultured in RPMI-1640 medium without antibiotics, supplemented with 10% FCS. Reagents GSH was kindly supplied by Boehringer Mannheim (Milan, Italy). Immediately before use, GSH was freshly dissolved in water (30 mg/ml) and further diluted in saline. Oxidized glutathione (GSSG), catalase, dithiothreitol (DTT) and glycylglycine were purchased from Sigma (St. Louis, MO). Acivicin (NC-163501) was provided by the NCI (Bethesda, MD). Growth-inhibition assay The anti-proliferative effects of GSH on human carcinoma cells were assessed as described (Perego et al., 1996). Briefly, cells in the logarithmic phase of growth were harvested and seeded in duplicate into 6-well plates (960 mm2, Costar, Pleasanton, CA). Then, 24 hr after seeding, a freshly prepared solution of GSH and/or other compounds was added to the medium and cells were incubated for 1 hr. The medium was replaced with fresh medium and cells were harvested 72 hr after the exposure, and counted with a Coulter counter (PBI Electronics, Luton, UK). IC50 is defined as the inhibitory concentration causing 50% decrease of cell growth compared with untreated control. Activity of GSH-dependent enzymes All enzymatic activities were assayed as described (Pratesi et al., 1995). Frozen cell pellets were thawed and homogenized on ice in 0.05 M Tris-HCl buffer, pH 7.4, by 20 strokes of a tight-fitting Contract grant sponsors: Associazione Italiana per la Ricerche sul Cancro, Ministero della Sanitá, Consiglio Nazionale delle Ricerche (Finalized Project ACRO). *Correspondence to: Division of Experimental Oncology B, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. Fax: 39-2-2390764. Received 5 August 1996; revised 13 November 1996 ANTI-PROLIFERATIVE EFFECT OF REDUCED GLUTATHIONE 247 Dounce homogenizer. Aliquots of crude homogenates were used for the determination of g-GT activity with g-glutamyl-pnitroaniline and glycilglycine as substrates. The remaining cell homogenate was centrifuged at 105,000 g for 60 min, and the supernatants were used for the determination of GSH S-transferase, GSH peroxidase, glutatione reductase and g-glutamylcysteine synthetase. The results are expressed as nmol/min/mg protein, with the exception of g-glutamylcysteine synthetase, which was expressed as µg of inorganic phosphate (Pi) produced/min/mg protein. RESULTS AND DISCUSSION Growth-inhibitory effects of GSH were studied in 2 human ovarian carcinoma cell lines after 1-hr exposure to GSH. The dose-response curves (Fig. 1a) indicated that the A2780 line was sensitive to GSH treatment in the concentration range 10 to 300 µg/ml. In contrast, IGROV-1 cells were insensitive to GSH. The anti-proliferative effect was selectively shown with reduced GSH; the oxidized form of the thiol (GSSG) was ineffective in a wide range of concentrations (Fig. 1b). A peculiar feature of the A2780 cell response to GSH exposure was a biphasic profile, since strong inhibition (up to 80%) was observed at 100 to 300 µg/ml GSH, whereas higher concentrations were ineffective. The differential effect in the 2 cell lines was not related to intracellular accumulation of GSH. In fact, following 1-hr exposure to a GSH concentration (100 µg/ml) causing growth inhibition in A2780 cells, the intracellular non-protein thiol level was appreciably increased only in IGROV-1 cells (Tedeschi et al., 1991). This increase is allegedly explained by the markedly higher (approximately 5-fold) expression of g-GT in IGROV-1 than in A2780 cells (Table I). In fact, the role of g-GT in favoring the cellular uptake of GSH precursor amino acids is well established (Hanigan and Ricketts, 1993). To investigate the biochemical basis of the varying effect of GSH on the 2 ovarian carcinoma lines, an analysis of the GSHdependent enzyme activities in IGROV-1 and A2780 cells was undertaken. As shown in Table I, IGROV-1 cells had GSH S-transferase activity 3-fold higher than A2780 cells. Higher activity was also found in IGROV-1 cells for the other analyzed enzymes (GSH peroxidase, glutathione reductase and g-GT), with the exception of g-glutamylcysteine synthetase, whose levels were similar in the 2 cell lines. Most interestingly, GSH peroxidase activity was undetectable in the GSH-sensitive A2780 cells, suggesting that the growth-inhibitory effect of GSH in these cells might be related to impairment of the cellular detoxication of peroxides. The fact that intracellular GSH content was not increased in sensitive A2780 cells suggests that the anti-proliferative effect of GSH is mediated by a cell-specific mechanism operating extracellularly, probably at the membrane level. Several lines of evidence indicate that thiols, including GSH, can mediate the production of reactive forms of oxygen, such as H2O2 (Rowley and Halliwell, 1982; Misra, 1974; Tien et al., 1982). We therefore examined the sensitivity of the 2 ovarian carcinoma cell lines to hydrogen peroxide, and indeed, as shown in Figure 2, A2780 cells appeared to be markedly more sensitive to H2O2 (IC50 5 0.026 mM) than IGROV-1 cells (IC50 5 0.14 mM). We thus investigated whether the addition of the H2O2-metabolizing enzyme catalase might protect against the cytotoxic effects of GSH in A2780 cells. When A2780 cells were exposed to GSH in the presence of non-toxic concentrations of catalase, no appreciable decrease of cell growth was detected (Fig. 3a), indicating that the damage induced by GSH was dependent on extracellular production of H2O2. It is in fact known that the scavenging of H2O2 by catalase is restricted to the extracellular space (Gelvan et al., 1995). A major effect of reactive oxygen species, including H2O2, is the oxidation of thiol groups into proteins (Suzuki et al., 1990). The possible involvement of oxidation of cellular thiols in the growthinhibitory effect of GSH was therefore investigated by means of the FIGURE 1 – Effect of reduced (GSH) (a) and oxidized glutathione (GSSG) (b) on human ovarian carcinoma cell lines after 1-hr exposure. Closed circles, A2780 cells; open circles, IGROV-1 cells. Each point represents the average of 3 to 6 independent experiments. Vertical bars, SD. When not indicated, SD was smaller than symbol size. TABLE I – ACTIVITY OF GSH-DEPENDENT ENZYMES IN A2780 AND IGROV-1 HUMAN OVARIAN CARCINOMA CELLS1 Activity S-transferase2 GSH GSH peroxidase2 Glutathione reductase2 g-glutamylcysteine synthase3 g-glutamyl transpeptidase3 Cell lines A2780 IGROV-1 75.30 nd 11.10 1.31 8.86 229.60 8.41 62.30 1.26 43.98 1A representative experiment is reported; SD average 10%.–2Results are expressed as nmol/min/mg.–3Activity is expressed as µg of Pi/min/mg protein.–nd, not detectable. disulfide-reducing agent DTT. Indeed, as shown in Figure 3b, the addition of sub-toxic concentrations of DTT (0.1 mM) was able to suppress the GSH-induced growth inhibition in A2780 cells, indicating that the effect is mediated by the oxidation of critical thiols involved in cellular proliferation. In several other experimental models, the ability of H2O2 to modulate cell proliferation through the oxidation of critical thiols, e.g., of transcription factors, has been in fact documented (Guehmann et al., 1992; Hainaut and Milner, 1993). The biphasic dose-response curve obtained after exposure of A2780 cells to GSH (Fig. 1a) was consistently observed with a 248 PEREGO ET AL. FIGURE 2 – Growth-inhibitory effect of hydrogen peroxide on A2780 and IGROV-1 cells after 1-hr exposure. Closed circles, A2780 cells; open circles, IGROV-1 cells. Data points are the average of 2 independent experiments. Vertical bars, SD. FIGURE 3 – (a) Suppression of GSH-induced growth inhibition in A2780 cells by catalase. (s) GSH alone; (d) GSH 1 500 U/ml catalase; (S) GSH 1 100 U/ml catalase. The effect was measured by growth-inhibition assay after 1-hr simultaneous exposure. Vertical bars, SD. (b) Suppression of GSH-induced growth inhibition in A2780 cells by dithiothreitol. (s) GSH alone; (n) GSH 1 0.1 mM DTT. SD, vertical bars. When not indicated, SD was smaller than symbol size. series of GSH-sensitive cell lines (Zunino et al., 1993). It is therefore likely that, while at lower GSH concentrations a prooxidant, anti-proliferative effect (probably mediated by reactive oxygen species) is operative, at higher GSH concentrations (in the mM range) the intrinsic anti-oxidant potential of GSH can be FIGURE 4 – Potentiation of GSH-induced growth inhibition in A2780 cells by the g-GT co-substrate, glycylglycine (simultaneous combination). (s) GSH alone; (d) GSH 1 glycylglycine (2 mM). FIGURE 5 – Suppression of GSH-induced growth inhibition in A2780 cells by the g-GT inhibitors (simultaneous combination). (a) GSH alone (s); GSH 1 serine-borate complex, 1 mM (S) or 10 mM (d). (b) GSH alone (s); GSH 1 acivicin, 30 µg/ml (S) or 100 µg/ml (d). prevalent, thus counteracting its own growth-inhibitory effect. Thus, a plausible explanation of the unusual dose-response curve is the involvement of thiol products of GSH catabolism in promoting oxidative damage at low GSH concentrations. This interpretation is also consistent with a lack of cellular effect of GSSG. Indeed, the GSH hydrolysis by isolated g-GT has been shown to favor the production of reactive forms of oxygen through Fe31 reduction mediated by cysteinyl-glycine produced by enzymatic reaction ANTI-PROLIFERATIVE EFFECT OF REDUCED GLUTATHIONE (Stark et al., 1988, 1993). In our cellular system sensitive to GSH, the putative involvement of plasma-membrane g-GT activity in mediating the GSH anti-proliferative effect was therefore investigated by using modulators of the enzyme activity in combination with GSH. As shown in Figure 4, when glycylglycine, a g-glutamyl acceptor known to accelerate g-GT activity (Huseby and Strömme, 1974), was combined with GSH, the growth-inhibitory effect of GSH on A2780 cells was remarkably increased, in conditions where glycylglycine did not affect cell growth per se. Conversely, when g-GT activity was competitively inhibited with the serineborate complex (Tate and Meister, 1978), A2780 cells were protected from the GSH inhibitory effect (Fig. 5a). Partial protection was also observed when the non-competitive inhibitor acivicin was employed at sub-toxic levels (Fig. 5b). Taken together, these results support the interpretation that the effect of GSH on A2780 cells is mediated by its g-GT-dependent metabolism. It is thus conceivable that competitive inhibition exerted on g-GT by GSH itself at high concentrations (Hanigan et al., 1994b) may contribute to the biphasic effect of GSH. In conclusion, the present study indicates that (i) GSH, despite being a major and well-established factor of cellular anti-oxidant defense systems, may exert a cell-specific growth-inhibitory effect at an extracellular level; (ii) the effect is mediated by the formation of reactive oxygen species, such as H2O2; and (iii) GSH metabolism by plasma-membrane g-GT activity potentiates the effect. The molecular basis for the observed cell specificity of the GSH growth-inhibitory effect probably lies in the differential arrays of anti-oxidant enzymes displayed by the studied cell lines. In particular, the marked depression of GSH peroxidase, glutathione reductase and GSH S-transferase activity detected in A2780 cells 249 may help to explain the selective susceptibility of these cells to the g-GT-mediated pro-oxidant effects of GSH. An alternative explanation may be that oxidative stress, however generated, may have varying effects on the proliferative capacity of different cell lines, depending on the cell-specific expression of oxidant-sensitive membrane receptors and/or other members of the signal transduction chain (Van der Vliet and Bast, 1992). The present observations in the A2780 ovarian carcinoma cell line are consistent with the interpretation given for the mutagenicity and genotoxicity of GSH in different biological systems (Ross et al., 1986; Thust and Bach, 1985). Again, such effects have been shown to involve the production of reactive forms of oxygen and/or other radicals (Stark et al., 1994) and to be potentiated by GSH metabolism mediated by g-GT (Stark et al., 1988). Finally, since GSH is often used in combination with cisplatin in high-dose therapy of advanced ovarian cancer (Di Re et al., 1993), a tumor frequently expressing high g-GT levels (Hanigan et al., 1994a), the cell specificity of the g-GT-mediated GSH growthinhibitory effect may have important clinical implications. Identification of the cellular targets of oxidative damage caused by extracellular GSH may be of considerable biologic significance in elucidating the regulatory mechanisms of cell proliferation, and may have implications for pharmacologic intervention. ACKNOWLEDGEMENTS This work was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro, Ministero della Sanità, and the Consiglio Nazionale delle Ricerche (Finalized Project ACRO). We thank Ms. L. Zanesi and Ms. B. Johnston for editorial assistance. 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