The role of glutathione (GSH) in the effectiveness of and resistance to 7 platinum compounds [5 Pt(II) and 2 Pt(IV) drugs] was evaluated in a 8.6-fold cisplatin (CDDP)-resistant human small cell lung cancer cell line (GLC4/CDDP), the parent GLC4 line, a 3.7-fold CDDP-resistant human embryonal carcinoma cell line (Tera-CP), and the parent Tera line (NTera2/D 1). Resistance factors for both CDDP-resistant cell lines were determined after continuous incubation (4 days) with CDDP. Continuous incubation with the other studied platinum drugs revealed complete cross-resistance for carboplatin (CBDCA) and zeniplatin but less for enloplatin (ENLO) and iproplatin in both models. Tetraplatin and lobaplatin showed, respectively, partial and complete cross-resistance in GLC4/CDDP but no cross-resistance in Tera-CP. GSH level, but not glutathione S-transferase activity, of the 4 cell lines correlated with platinum drug concentrations inhibiting cell survival by 50% after continuous incubation (r = 0.86, P < 0.05). GSH depletion by aL-buthionine-S,R-sulfoximine (BSO) increased sensitivity, as measured after a 4-h exposure to the drugs, of GLC4/CDDP for CDDP 2.0-fold, for CBDCA 1.7-fold, for zeniplatin 1.7-fold, and almost to the level of the sensitive GLC4 for ENLO, whereas no effect was observed for iobaplatin and the Pt(IV) compounds iproplatin and tetraplatin. BSO-modulating effect was higher in the sensitive GLC4 line for most compounds; therefore reduction of resistance could be achieved only for CDDP and ENLO. In contrast to GLC4, no modulation occurred in Tera. In Tera-CP BSO increased sensitivity for CDDP 1.5-fold, for CBDCA 1.9-fold, and for zeniplatin 1.2-fold; no effect was observed for ENLO, lobaplatin, and the Pt(IV) compounds. Reduction of CDDP resistance by BSO was known to occur with identical cellular platinum levels and higher Pt-DNA binding in GLC4/ CDDP. However, pretreatment with BSO followed by 4 h ENLO incubation increased cellular platinum levels in both GLC4 and GLC4/ CDDP while Pt-DNA binding remained unchanged. In conclusion, GSH reflected sensitivity to platinum-containing drugs. However, since the involvement of GSH differed between the models and the various platinum drugs, the effect of modulation with BSO was unpredictable. I N T R O D U C T I O N The development of acquired resistance to the useful antineoplastic agent C D D P 3 and the severity of toxicity associated Received 6/22/92; accepted 10/2/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was partly presented at the 82nd Annual Meeting of the American Association for Cancer Research, Houston, TX, May 15-18, 1991, and partly supported by Grants GUKC 90-18 and 91-09 from the Dutch Cancer Foundation. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: CDDP, cisplatin, cis-diamminedichloroplatinum(II); GSH, glutathione; GST, glutathione S-transferase; CBDCA, carboplatin, cisdiamminecyclobutane-l,l-dicarboxylatoplatinum(II); ZENI, zeniplatin, CL286,558; ENLO, enloplatin, CL287,110; CHIP, iproplatin, cis-dichlorobisisopropylaminetrans-hydroxyplatinum(IV); TETRA, tetraplatin, tetrachloro(DL-trans)-l,2-diamminecyclohexaneplatinum(IV); LOBA, iobaplatin, D19466, 1,2-diamminomethylcyclobutaneplatinum(II)qactate; DACH, diamminecyclohexane; ICso, concentration inhibiting cell survival by 50%; BSO, DL-buthionine-S,R-suifoximine; FCS, fetal calf serum; PBS, phosphate-buffered saline (0.14 M NaCI, 2.7 mM KCI, 6.4 mM Na2HPO4.2H20, and 1.5 mM KI-I2PO4, pH 7.4); DME, Dulbecco's modified Eagle's medium; RF, resistance factor; DMF, dose modifying factor. with this drug encouraged the development of new platinumcontaining drugs. In vitro studies have demonstrated that cellular resistance to C D D P is multifactorial. Mechanisms involved include: altered membrane transport; inactivation of the drug by cellular thiols such as GSH and metallothioneins; decreased Pt -DNA binding; and/or an increased Pt -DNA repair (1). The role of the GSH system in C D D P resistance has received much attention, because CDDP as well as other platinum-containing compounds are electrophilic agents which are most reactive towards sulfurcontaining nucleophiles. A correlation between the GSH level and sensitivity to, e.g., CDDP has been reported in human tumor cell lines expressing a range of in vitro sensitivities to CDDP (2, 3). Elevated GSH levels have been found in C D D P resistant cell lines (4-13), and BSO, a specific inhibitor of the GSH synthesis, can act as an useful modulator of resistance to nucleophilic agents such as CDDP in vitro (6, 9). The exact mechanism, however, by which GSH influences platinum-induced cytotoxicity and participates in resistance to various platinum-containing drugs is, as yet, not fully elucidated and may differ between various test models. The present study evaluated the role of GSH in the effectiveness of and the resistance to seven platinum-containing drugs in two models of CDDP resistance: a human embryonal carcinoma cell line, Tera, representing a sensitive tumor in the clinic; and a human small cell lung cancer cell line GLC4, representing a sensitive tumor type prone to resistance in the clinic, as well as their sublines with in vitro-acquired C D D P resistance, Tera-CP and GLC4/CDDP. Compared to the parent line, GSH levels were 1.4and 2.5-fold higher in, respectively, Tera-CP and GLC4/ CDDP, and GST activity was 1.5-fold higher in Tera-CP but the same in GLC4/CDDP. Platinum drug cytotoxicity was tested in the absence and presence of BSO pretreatment. Five Pt(II) drugs, namely, CDDP, the less toxic CBDCA, three platinum compounds recently introduced in clinical studies (ZENI, ENLO, and LOBA), as well as two Pt(IV) drugs, CHIP and one of the DACH compounds (TETRA) were studied (Fig. 1). For ENLO, the platinum drug most effectively modulated by BSO in this study, the role of GSH was further investigated by measuring the effect of BSO-induced GSH depletion on cellular platinum levels and Pt -DNA binding. MATERIALS AND M E T H O D S Chemicals. CDDP, CBDCA, and CHIP were kindly provided by Bristol Myers S.A.E. (Weesp, the Netherlands), ZENI and ENLO by Lederle Laboratories (Pearl River, NY), LOBA by ASTA (Frankfurt, Germany), and TETRA by Upjohn (Kalamazoo, MI). RPMI 1610 was obtained from Life Technologies (Paisley, United Kingdom) and FCS from Sanbio (Uden, the Netherlands). DME and Ham's F-12 medium were purchased from Flow Laboratories (Irvine, United Kingdom). GSH, BSO, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were obtained from Sigma (St. Louis, MO), and dimethyl sulfoxide from Merck (Darmstadt, Germany).