ONCOLOGY REPORTS 33: 1465-1474, 2015 Erastin sensitizes glioblastoma cells to temozolomide by restraining xCT and cystathionine-γ-lyase function LIanGyu CHen1, XInXInG LI1, LIBo LIu2,3, Bo yu1, yIXue Xue2,3 and yunHuI LIu1 1Department of neurosurgery, Shengjing Hospital of China Medical university; 2Department of neurobiology, College of Basic Medicine, China Medical university; 3Institute of Pathology and Pathophysiology, China Medical university, Shenyang, Liaoning, P.R. China Received September 19, 2014; accepted December 4, 2014 DoI: 10.3892/or.2015.3712 Abstract. Glioblastoma multiforme (GBM) is one of the decrease in GSH levels as result of co-treatment with erastin, most common encephalic malignant tumors. Due to a high which inhibited cysteine uptake from xCT transporter and recurrence rate and a lack of effective treatments, the average suppressed CTH activity, leading to impaired transformation survival rate remains low. Temozolomide (TMZ), a class of from methionine to cysteine. In conclusion, to GBM therapy alkylating agent, is widely used as a first-line therapeutic drug with a drug combination of TMZ and erastin may be beneficial. during the adjuvant treatment for GBM patients. However, most patients exhibit a palpable resistance to TMZ treat- Introduction ment. Additionally, the underlying mechanism remains to be clarified. In this study, glutathione (GSH) and reactive oxygen Glioblastoma multiforme (GBM) is one of the most common species (ROS) levels were found to be closely associated with gliomas, and is extremely lethal in all central nervous the sensitivity of GBM cells to TMZ. We also found that TMZ system tumors (1). GBM infiltrates into normal brain tissues markedly induced xCT, the subunit of glutamate/cystine trans- where a tumor and the normal brain tissue have no clear - porter system xc expression, which together with the GSH demarcation, due to glioma cells having a high mobility and synthesis was increased while the TMZ-inducible ROS level possessing strong invasive properties (2,3). Thus, it is difficult was decreased in GBM cells. In addition, the cystathionine to completely remove the tumor through surgical resection. γ-lyase (CTH) acivity, a key enzyme in the transsulfuration Additionally, GBM has been proven to resist radio- and pathway was enhanced by TMZ, which insured a cysteine chemotherapy (4). Although patients undergo the most aggres- supply and GSH synthesis in a compensatory manner when sive regimens of debulking surgeries, radiotherapy together xCT was blocked. Thus, the individual inhibition of xCT by with adjuvant chemotherapy results in a median survival of siRNA and a pharmacological inhibitor (sulfasalazine) only ~14 months (5). At present, there are no effective methods partially inhibited GSH synthesis and moderately enhanced to prevent a relapse of the tumor by residual neoplastic cells the GBM cell sensitivity to TMZ. However, the TMZ-induced following surgery and radiotherapy. cytotoxicity was markedly increased along with a marked Temozolomide (TMZ) is a class of alkylating agent approved by the Food and Drug Administration (FDA). TMZ is widely used as a standard-of-care during clinical treatment. However, it only results in a slight increase of overall survival of GBM patients. Furthermore, most patients are resistant to Correspondence to: Professor Yunhui Liu, Department of Neuro- TMZ in the clinic (6). O6-methylguanine produced by DNA surgery, Shengjing Hospital of China Medical university, no. 36 methyl transferase mainly mediates the cytotoxicity of TMZ Sanhao Street, Heping District, Shenyang, Liaoning 110004, P.R. China and triggers cell cycle-dependent DNA damage, ensuring E-mail: [email protected] cell death. Thus, O6-methylguanine-DNA methyl transferase (MGMT) limits the therapeutic effect of TMZ by removing Abbreviations: GBM, glioblastoma multiforme; FDa, Food and Drug 6 administration; TMZ, temozolomide; MGMT, o 6-methylguanine-DNA O -methylguanine (7,8). However, findings of previous GBM methyl transferase; nrf2, nF-e2 related factor 2; aTF4, activating trans- cell line studies showed that the activity of MGMT was not cription factor 4; SaSP, sulfasalazine; eRa, erastin; CTH, cystathionine entirely consistent with the resistance of GBM to TMZ, i.e., γ-lyase; CBS, Cystathionine β-synthase; DFo, deferoxamine mesylate; even in MGMT-silenced GBM cells, the effect of TMZ was PPG, propargylglycine; GSH, glutathione; RoS, reactive oxygen species limited (8). The exact cell death pathway induced by TMZ and the molecular mechanisms affecting the efficacy of TMZ in Key words: glioblastoma, erastin, temozolomide, xCT, cystathionine- GBM cells remain to be determined. γ-lyase - The glutamate/cystine antiporter system xc is an obli- gate sodium-independent amino acid antiporter, comprising 12-pass transmembrane transporter protein xCT (SLC7A11) which is connected to the 4F2 cell surface antigen 4F2hc 1466 chen et al: Erastin enHanCeS TeMoZoLomide Sensitivity - (CD98/SLC3A2) by a disulfide bridge (9,10). System xc (DMSO) was obtained from Wako Pure Chemical (Osaka, transports extracellular cystine into cells in exchange for Japan). CellROX Orange reagent was purchased from Life intracellular glutamate at a ratio of 1:1, and maintaining Technologies (Tokyo, Japan). Anti-xCT antibody (ab37185) intracellular cysteine pools is important (10). Cysteine is a was obtained from abcam (Cambridge, Ma, uSa). anti-nrf2 crucial material in glutathione (GSH) synthesis, which is antibody (sc-722), anti-ATF4 antibody (sc-200) and anti- indispensable for maintaining intracellular redox balance and LaminB antibody (sc-56144) were purchased from Santa Cruz drug metabolism (11,12). xCT expression is mediated by the Biotechnology, Inc. (Dallas, TX, uSa). oxidative stress-response transcription factor NF-E2 related to factor 2 (Nrf2) and activation of transcription factor 4 Cell culture. The human a172, u87-MG and T98G malig- (ATF4) (13). xCT is expressed in many types of malignan- nant glioblastoma multiforme cells were purchased from the cies and is associated with tumor growth and metastasis. It american Tissue Culture Collection (Rockville, MD, uSa). is also associated with resistance to chemotherapy and poor GBM-n6 and GBM-n15, isolated from a grade Iv human survival (13-17). Accordingly, xCT has been considered as glioblastoma, were a generous gift from Dr Dongcheng Wang a potential therapeutic target (13,14,16,17). Sulfasalazine at Shandong university. (SaSP) is a sulfa drug used for inflammatory bowel diseases The cells were maintained in DMEM containing 10% fetal and rheumatoid arthritis treatment and is a widely recognized bovine serum (Gibco, Grand Island, NY, uSa) with 100 U/ml xCT-specific inhibitor (18). although it has been suggested penicillin and 100 µg/ml streptomycin (Gibco). The cells were that the inhibitory effect of SASP on xCT can suppress GBM cultured at 37˚C with 5% Co2 and saturated humidity. cell growth, the combination of SASP combined with TMZ for GBM treatment in the clinic has yielded controversial Cloning of CTH cDNA and stable transfection. The over- results (19,20). Erastin (ERA) is a voltage-dependent anion expression vector of CTH was constructed by using the primer channels (vDaC)-binding small molecule that is selectively 5'-CGT CCC AGC ATG CAG AAG AA-3' and 5'-CAG TTA lethal to some cancer cells (21). Compared to SASP, ERA exerts TTC AGA AGG TCT GGC CC-3'. The constructs containing a stronger inhibitory effect on xCT (22). In addition, eRa is CTH cDNA were then subcloned into the pIReS2-eGFP able to inhibit the activity of certain GSH-related enzymes, expression vector (Clontech) as previously reported (30). For such as glutathione peroxidase 4 (GPx4), resulting in more stable transfection, GBM-N15 cells were transfected with lethal oxidative damage to cells (23). ERA can cause a unique linearized constructs by using FuGENE transfection reagent form of cell death on iron-dependent tumor cells as compared (Roche Applied Science) according to the manufacturer's to conventional apoptosis, necrosis and autophagy (22). instructions. After 48 h of transfection, the cells were seeded in In many types of cells cysteine is derived from an imported 35-mm dish at a density of 5x104 cells/dish. The following day, xCT process, however, there is also a trans sulfuration pathway the culture media was refreshed containing 500 µg/ml G418 in which methionine is transferred to cysteine via catalysis (Life Technologies) for antibiotic selection. after five weeks by cystathionine β-synthase (CSE) and cystathionine-γ-lyase of culturing the selected antibiotics, survival-transfected cells (CTH) (24,25). In brain carcinoma cells such as glioma cells were collected. Stable-transfected cells were used within and astrocytes, most cysteine originates from the reduction 15 passages as previously reported (30). process of cystine imported by xCT under normal conditions. However, when xCT is blocked or GSH is decreased, the trans- Determination of intracellular reactive oxygen species sulfuration pathway is activated, which insures the cysteine (ROS). u87, T98G and GBM-n15 cells were plated in 12-well supply for GSH synthesis in a compensatory manner (26,27). plates at a density of 5.0x105 cells/well and cultured over- Furthermore, CTH activity may limit cysteine synthesis via night. The cells were treated with DMSO or 400 µM TMZ the methionine transsulfuration pathway (28,29). However, for 3 h. In the experiment in which TMZ and eRa or SaSP whether transsulfuration and CTH are involved in TMZ resis- were used as co-treatment methods, 5.0x105 cells/well of tance remains to be elucidated. GBM-N15 cells were plated in 12-well plates. After 24 h, In this study, we demonstrated that GBM cells were sensi- the culture medium was refreshed with 400 µM TMZ and tive to TMZ with a downregulation of the GSH level. TMZ 5 µM ERA or 0.3 mM SASP. After 3-h incubation, the culture enhanced xCT expression in an Nrf2- and ATF4-dependent medium was replaced with fresh culture medium containing manner. Moreover, it activated the transsulfuration pathway in 5 µM CellRox Orange Reagent, and the cells were incubated GBM cells by enhancing CTH activity.