Potentiation of Treosulfan Toxicity by the Glutathione-Depleting Agent Buthionine Sulfoximine in Human Malignant Glioma Cells THE ROLE OF BCL-2 Ulrike Reber,* Ulrich Wu ¨llner,† Martin Trepel,* Ju ¨rgen Baumgart,‡ Jan Seyfried,† Thomas Klockgether,† Johannes Dichgans*† and Michael Weller*§ LABORATORIES OF *MOLECULAR NEURO-ONCOLOGY AND †NEUROPHARMACOLOGY,DEPARTMENT OF NEUROLOGY, UNIVERSITY OF T ¨ UBINGEN,SCHOOL OF MEDICINE,T ¨ UBINGEN,GERMANY; AND ‡MEDAC CO., HAMBURG,GERMANY ABSTRACT. Median survival of human malignant glioma patients is less than one year even with cytoreductive surgery and postoperative radiotherapy. Adjuvant chemotherapy has been rather ineffective. Here, we studied the potentiation by L-buthionine-[S,R]-sulfoximine (BSO), a glutathione-depleting agent, of anticancer drug actions on two human malignant glioma cell lines, LN-229 and T98G. LN-229 has wild-type p53 status, T98G is mutant for p53. Glutathione levels were depleted by BSO with similar kinetics in both cell lines. Only LN-229 cells were growth-inhibited by BSO. BSO had minor effects on the toxicity of doxorubicin, ACNU (1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea, nimustine) and vincris- tine. BSO failed to alter teniposide or cytarabine toxicity. BSO induced prominent sensitization to the alkylating agent, treosulfan, in both cell lines, as assessed by viability assays, in situ DNA end labeling and quantitative DNA fragmentation. Treosulfan is thought to mediate toxicity via formation of reactive epoxides. In the absence of BSO, treosulfan had little acute cytotoxic and moderate antiproliferative effects. Synergistic glioma cell cytotoxicity induced by treosulfan and BSO was not associated with reactive oxygen species formation. Ectopic expression of bcl-2 did not alter basal glutathione levels but attenuated glutathione depletion induced by BSO. Bcl-2 provided only moderate protection from synergistic induction of glioma cell death by treosulfan and BSO. Glutathione depletion may play a role in BSO-mediated chemosensitization, but other mechanisms are probably involved as well. BSO may be a useful agent for glioma cell sensitization to specific chemotherapeutic drugs such as treosulfan. BIOCHEM PHARMACOL 55;3:349 –359, 1998. © 1998 Elsevier Science Inc. KEY WORDS. treosulfan; apoptosis; chemotherapy; malignant glioma; glutathione; BSO Malignant gliomas are the most common intrinsic human brain tumors. Established therapeutic approaches include cytoreductive surgery and postoperative radiotherapy. This therapy results in median survival of less than one year. A minority of glioma patients benefit from adjuvant chemo- therapy. The major drugs used for the adjuvant chemother- apy of malignant gliomas are the nitrosoureas, BCNU¶ (1,3-bis(2-chloroethyl)-1-nitrosourea, carmustine), ACNU (1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloro- ethyl)-3-nitrosourea, nimustine), and, as part of the PCV (procarbacine, CCNU, vincristine) protocol, CCNU (1- (2-chloroethyl)-3-cyclohexyl-1-nitrosourea, lomustine). Part of the failure of chemotherapy may be caused by the blood-brain barrier which shields at least the tumor margins from cancer chemotherapy and by the common practice of concurrent steroid treatment which stabilizes the blood- brain barrier and provides protection from chemotherapy at the subcellular level [1]. The molecular mechanisms underlying intrinsic drug resistance of human malignant glioma cells may involve loss of wild-type p53 activity and enhanced expression of antiapoptotic proteins of the bcl-2 family [2– 4] and of cytoprotective, drug-detoxifying enzymes including the glu- tathione system [5–7]. Thus, elevated glutathione levels or enhanced expression of glutathione transferase (RX: gluta- thione R-transferase; E.C. 2.5.1.18) have been attributed a role in glioma cell resistance to BCNU [5, 8] and the topoisomerase I inhibitor, camptothecin-11 [9]. Sensitiza- tion to BCNU has been observed in some glioma cells after preexposure to the glutathione-depleting agent BSO [8, 10], an inhibitor of glutathione synthase (-L-glutamyl-L- cysteine:glycine ligase, E.C. 6.3.2.3), the rate-limiting en- zyme of glutathione synthesis. A similar BSO-based ap- proach has been used to sensitize human glioma xenografts § Corresponding author: Dr. Michael Weller, Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tu ¨bingen Medical School, Hoppe-Seyler-Strasse 3, 72076 Tu ¨bingen, Germany. TEL. (49) 7071 2982141; FAX (49) 7071 296507; E-mail: michael.weller@uni-tuebingen.de ¶Abbreviations: ACNU, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]- 3-(2-chloroethyl)-3-nitrosourea); BCNU, 1,3-bis(2-chloroethyl)-1-nitro- sourea; BSO, L-buthionine-[S,R]-sulfoximine; CCNU, 1-(2-chloroethyl)- 3-cyclohexyl-1-nitrosourea; DCF-Hr 2 , 2',7'-dichlorodihydrofluorescein diacetate. Received 16 April 1997; accepted 5 August 1997. Biochemical Pharmacology, Vol. 55, pp. 349 –359, 1998. ISSN 0006-2952/98/$19.00 + 0.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0006-2952(97)00480-2