Nitric oxide and BCNU chemoresistance in C6 glioma cells: role of S-nitrosoglutathione

Inducible nitric oxide synthase (iNOS or NOS2) is expressed in malignant glioma. Previously we noted that C6 glioma cells overexpressing NOS2 displayed chemoresistance against 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and other chloroethylnitrosourea derivatives with carbamoylating action. Herein we report experimental evidence supporting the contention that this NOS2 effect is mediated, at least in part, by S-nitrosoglutathione (GSNO), a potent antioxidant derived from interaction of NO and glutathione. Out of three NO donors tested, only GSNO was effective in protecting glioma cells against BCNU cytotoxicity. Furthermore, the protective effect of GSNO, similar to that of NOS2, was confined to carbamoylating, but not alkylating action. Experimental manipulations that were expected to increase or decrease cellular GSNO stores, as confirmed by immunocytochemical staining using a GSNO-specific antibody and HPLC analysis of GSNO contents in culture medium, led respectively to enhanced or reduced chemoresistance against carbamoylating cytotoxicity. Finally, neocuproine, a selective cuprous ion chelator known to neutralize GSNO actions, abolished NOS2-mediated chemoresistance against carbamoylating agents. Our results reveal a novel action of NOS2/GSNO that may potentially contribute to the development of chemoresistance against BCNU, which remains a mainstay in chemotherapy for glioblastoma multiforme.     

In vitro study of astrocytic tumour metabolism by proton magnetic resonance spectroscopy

In vivo magnetic resonance spectroscopy (MRS) studies of glial brain tumours reported that higher grade of astrocytoma is associated with increased level of choline-containing compounds (Cho) and decreased levels of N-acetylaspartate (NAA) and creatine and phosphocreatine (Cr). In this work, we studied the metabolism of glioma tumours by in vitro proton magnetic resonance spectroscopy (1H-MRS). 1H-MR spectra were recorded in vitro from perchloric acid extracts of astrocytoma (WHO II) and glioblastoma multiforme (WHO IV) samples. We observed differences between astrocytoma and glioblastoma multiforme in the levels of Cho, alanine, lactate, NAA, and glutamate/glutamine. In astrocytoma samples, we found higher MR signal of NAA and lower signal of Cho and alanine. MR spectra of glioblastoma samples reported significantly higher levels of lactate and glutamate/glutamine. In contrast, levels of Cr were the same in both tumour types. We also determined NAA/Cr and Cho/Cr ratios in the tumour samples. The NAA/Cr ratio was higher in astrocytomas than in glioblastomas multiforme. Conversely, the Cho/Cr ratio was higher in glioblastoma multiforme. The results indicate that MRS is a promising method for distinguishing pathologies in human brain and for pre-surgical grading of brain tumours.     

Mammalian class theta GST and differential susceptibility to carcinogens: a review

Glutathione S-transferases (GSTs) are an important part of the cellular detoxification system and, perhaps, evolved to protect cells against reactive oxygen metabolites. Theta is considered the most ancient among the GSTs and theta-like GSTs are found in mammals, fish, insects, plants, unicellular algae, and bacteria. It is thought that an ancestral theta-gene underwent an early duplication before the divergence of fungi and animals and further duplications generated the variety of the other classes of GSTs (alpha, mu, phi, etc.). The comparison of the aminoacidic homologies among mammals suggests that a duplication of an ancient GST theta occurred before the speciation of mammals and resulted in the subunits GSTT1 and GSTT2. The ancestral GST theta has a dehalogenase activity towards several halogenated compounds, such as the dichloromethane. In fact, some aerobic and anaerobic methylotrophic bacteria can use these molecules as the sole carbon and energy source. The mammalian GST theta cannot sustain the growth of bacteria but still retains the dehalogenating activity. Therefore, although mammalian GST theta behaves as a scavenger towards electrophiles, such as epoxides, it acts also as metabolic activator for halogenated compounds, producing a variety of intermediates potentially dangerous for DNA and cells. For example, mice exposed to dichloromethane show a dose-dependent incidence of cancer via the GSTT1-1 pathway. Because GSTT1-1 is polymorphic in humans, with about 20% of Caucasians and 80% of Asians lacking the enzyme, the relationship between the phenotype and the incidence of cancer has been investigated extensively in order to detect GSTT1-1-associated differential susceptibility towards endogenous or exogenous carcinogens. The lack of the enzyme is related to a slightly increased risk of cancer of the bladder, gastro-intestinal tract, and for tobacco-related tumors (lung or oral cavity). More pronounced risks were found in males with the GSTT1-null genotype for brain diseases and skin basal cell carcinomas not related to sunlight exposures. Moreover, there was an increased risk of kidney and liver tumors in humans with the GSTT1-1 positive genotype following exposures to halogenated solvents. Interestingly, the liver and kidney are two organs that express the highest level of GST theta in the human body. Thus, the GSTT1-1 genotype is suspected to confer decreased or increased risk of cancer in relation to the source of exposure; in vitro studies, mostly conducted on metabolites of butadiene, confirm the protective action of GSTT1-1, whereas, thus far, experimental studies prove that the increasing risk is limited.     

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.     

Study of the association between glutathione S-transferase (GSTM1, GSTT1, GSTP1) polymorphisms with type II diabetes mellitus in southern of Iran

Diabetes Mellitus is characterized by chronic hyperglycemia and associated with an increased production of reactive oxygen species (ROS). Oxidative stress is the result of accumulation of free radicals in tissues which specially affects beta cells in pancreas. Glutathione S-transferases (GSTs) are a family of antioxidant enzymes that include several classes of GSTs. These enzymes have important roles in decreasing of ROS species and act as a kind of antioxidant defense. To investigate the association between GSTs polymorphism with type 2 diabetes mellitus (T2DM), we investigated the frequency of GSTM1, T1 and P1 genotypes in patients with T2DM and controls. The genotypes of GSTT1, M1 and P1 were determined in 171 clinically documented T2DM patients and 169 normal cases (as controls) by multiplex polymerase chain reaction and PCR–RFLP. In diabetic patients, the frequency of GSTM1-null genotype was significantly (OR?=?1.74; 95?% CI?=?1.13–2.69, P?=?0.016) higher than that in control. However, the frequency of GSTT1 (OR?=?1.29; 95?% CI?=?0.07–2.14, P?=?0.367) and GSTP1 (OR?=?0.83; 95?% CI?=?0.53–1.30, P?=?0.389) genotypes were not significantly different comparing both groups. Also, the frequency of both GSTT1-null and GSTM1-null genotypes in patients (19.88?%) was significantly higher compared to controls with the same genotypes (11.83?%, P?=?0.022). Our results indicated that GSTM1 and GSTT1 genotypes might be involved in the pathogenesis of T2DM in south Iranian population.     

First step toward the “fingerprinting” of brain tumors based on synchrotron radiation X-ray fluorescence and multiple discriminant analysis

Synchrotron-radiation-based X-ray fluorescence was applied to the elemental microimaging of neoplastic tissues in cases of various types of brain tumors. The following cases were studied: glioblastoma multiforme, gemistocytic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ganglioglioma, fibrillary astrocytoma, and atypical transitional meningioma. Apart from neoplastic tissue, the analysis included areas of tissue apparently without malignant infiltration. The masses per unit area of P, S, Cl, K, Ca, Fe, Cu, Zn, Br, and Rb were used to construct a diagnostic classifier for brain tumors using multiple discriminant analysis. It was found that S, Cl, Cu, Fe, K, Br, and Zn are the most significant elements in the general discrimination of tumor type. The highest similarity in elemental composition was between atypical transitional meningioma and fibrillary astrocytoma. The smallest differentiation was between glioblastoma multiforme and oligodendroglioma. The mean percentage of correct classifications, estimated according to the a posteriori probabilities procedure, was 99.9%, whereas the mean prediction ability of 87.6% was achieved for ten new cases excluded previously from the model construction. The results showed that multiple discriminant analysis based on elemental composition of tissue may be a potentially valuable method assisting differentiation and/or classification of brain tumors.     

Neuronatin in a subset of glioblastoma multiforme tumor progenitor cells is associated with increased cell proliferation and shorter patient survival

Glioblastoma multiforme is the most common and malignant primary brain tumor. Recent evidence indicates that a subset of glioblastoma tumor cells have a stem cell like phenotype that underlies chemotherapy resistance and tumor recurrence. We utilized a new "multidimensional" capillary isoelectric focusing nano-reversed-phase liquid chromatography platform with tandem mass spectrometry to compare the proteomes of isolated glioblastoma tumor stem cell and differentiated tumor cell populations. This proteomic analysis yielded new candidate proteins that were differentially expressed. Specifically, two isoforms of the membrane proteolipid neuronatin (NNAT) were expressed exclusively within the tumor stem cells. We surveyed the expression of NNAT across 10 WHO grade II and III gliomas and 23 glioblastoma (grade IV) human tumor samples and found NNAT was expressed in a subset of primary glioblastoma tumors. Through additional in vitro studies utilizing the U87 glioma cell line, we found that expression of NNAT is associated with significant increases in cellular proliferation. Paralleling the in vitro results, when NNAT levels were evaluated in tumor specimens from a consecutive cohort of 59 glioblastoma patients, the presence of increased levels of NNAT were found to be a an independent risk factor (P?=?0.006) for decreased patient survival through Kaplan-Meier and multivariate analysis. These findings indicate that NNAT may have utility as a prognostic biomarker, as well as a cell-surface target for chemotherapeutic agents.     

Effects of glutathione S-transferase T1 and M1 deletions on advanced carotid atherosclerosis, oxidative, lipid and inflammatory parameters

Glutathione S-transferases (GSTs) carry out a wide range of functions in cells, such as detoxification of endogenous compounds, removal of reactive oxygen species, and even catalysis of reactions in metabolic pathways beyond detoxification. Based on previous research, GSTM1 and GSTT1 might modify the risk of atherosclerosis. The aim of our study was to analyze the possible association of GSTM1 and GSTT1 gene polymorphisms with the occurrence of carotid plaque (CP); and biochemical parameters of oxidative stress, lipid profile and inflammation, in 346 consecutive patients with advanced atherosclerosis that underwent endarterectomy. A multiplex polymerase chain reaction (PCR) method was used to detect the deletions in GSTM1 and GSTT1 genes in the genomic DNA in 346 patients and 330 controls. The adjusted OR for CP presence (adjusted for age, gender, smoking, hypertension, BMI, HDLC, TG) was 0.24, 95 %CI 0.08–0.7, p < 0.01 for GSTT1 null and 1.13, 95 %CI 0.62–2.07, p = 0.7 for GSTM1 null genotype. We found significantly lower plasma lipoprotein (a) (Lp(a)) levels in GSTT1 null compared to wild-type genotype carriers in patient group (20.68 ± 26.02 mg/dl vs. 40.66 ± 42.89 mg/dl, mean ± SD, p = 0.04). The serum interleukin-6 (IL-6) values were significantly influenced by both GST polymorphisms in patients with CP. Our results, showing the significant reduction of GSTT1 deletions in patients with CP, suggest involvement of GSTs in carotid atherosclerosis. This study shows additional view of the possible role of GSTs in advanced chronic inflammatory disease of vascular system, but the confirmation in a larger studies in different populations are needed.     

Genotoxic effects of doxorubicin in cultured human lymphocytes with different glutathione S-transferase genotypes

Doxorubicin (Dox) is a widely used drug in oncology with a broad spectrum of interactions with various cellular components; therefore, it is likely to act through different mechanisms. In clinical practice there is inter-individual variability in cytotoxic drug response and in the occurrence of adverse reactions. Glutathione S-transferases (GSTM1, GSTT1 and GSTP1) are thought to be involved in the detoxification of endogenous and exogenous genotoxicants. The aim of this work is the assessment of a possible influence of polymorphisms in GSTs on the levels of genetic damage induced in vitro by Dox in cultured human lymphocytes. For this purpose, whole blood cultures from individuals with different genotypes for GSTM1, GSTT1 and GSTP1 were exposed to Dox and the cytokinesis-blocked micronucleus (CBMN) assay was used as the endpoint for chromosomal damage in the lymphocytes. Genotyping of GSTM1 and GSTT1 was carried out by multiplex PCR and the GSTP1-Ile105Val polymorphism was determined by PCR/RFLP. The total number of micronuclei present in 1000 binucleated cells and the frequency of micronucleated binucleated lymphocytes in the different individuals were analyzed considering the GSTM1, GSTT1 and GSTP1 genotypes. The results obtained suggest that GSTM1 and GSTT1 deletion polymorphisms do not modify significantly the genotoxic potential of Dox. However, the GSTP1 Ile105Val polymorphism was associated with an increase of micronucleated binucleated cells induced by Dox. Lymphocytes from homozygous individuals for the variant form (Val/Val) presented a significant increase in micronucleated binucleated cells (approximately 1.5-fold; p<0.05) when compared with individuals with at least one wild-type allele. These results suggest a possible role for GSTP1 on the modulation of the genotoxicity induced by Dox, which may be considered in cancer therapy.     

Immunohistochemical localization of glutathione S-transferase-T1 in murine kidney, liver, and lung

 Glutathione S-transferase-mediated metabolism of exogenous compounds usually leads to detoxification, but there are some exceptions. For example, glutathione S-transferase-T1 (GSTT1) can also generate genotoxic metabolites. Studies on the biology of GSTT1 are limited by the lack of specific antibodies recognizing GSTT1 in animal tissues. We localized GSTT1 immunohistochemically in mouse kidney, liver, and lung using a novel antibody targeted against the C-terminus of rat GSTT1 (rGSTT1). The antibody was characterized using immunoblot and shown to specifically recognize rGSTT1 and mouse GSTT1, but not human GSTT1. In kidney, GSTT1 staining was detected only in collecting duct epithelium. In liver, pericentral hepatocytes showed cytoplasmic and nuclear staining. Nuclear staining was also observed in several other hepatocytes without relation to liver zonation. Nuclei and supranuclear cytoplasm of bile duct epithelium and endothelium of interlobular arterioles also reacted strongly. In lung, staining was observed in bronchiolar epithelium and in surrounding muscle cells. Type II pneumocytes and endothelial cells of intrapulmonary capillaries also showed strong positive staining. This report describes the first immunohistochemical localization of GSTT1 in mammalian tissues. The reported location of GSTT1 is consistent with its known metabolic activity toward compounds such as dichloromethane and their metabolism into genotoxic products. Accepted: 11 May 1998     

Minor Modifications of the C-terminal Helix Reschedule the Favored Chemical Reactions Catalyzed by Theta Class Glutathione Transferase T1-1

Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme.     

Prognostic implication of histological oligodendroglial tumor component: clinicopathological analysis of 111 cases of malignant gliomas

The favorable prognosis of high-grade oligodendroglial tumor such as glioblastoma (GBM) with oligodendroglioma component (GBMO) has been suggested; however, the studies which examine the prognostic significance of oligodendroglial tumor were limited. In this study, we performed a histopathology-based reevaluation of 111 cases of high grade gliomas according to the latest World Health Organization (WHO), and compared the clinical outcomes between oligodendroglial tumors and pure astrocytic tumors. The survival analysis revealed that the patients with high grade oligodendroglial tumor including GBMO significantly indicated better prognosis compared to the patients with high grade pure astrocytic tumors (GBM and AA, anaplastic astrocytoma) as expected, and the obtained survival curves were almost identical to those from the patients with conventional Grade III or Grade IV tumors, respectively. Moreover, if the cases of oligodendroglial tumor were histopathologically excluded, the patients with AA exhibited extremely poor prognosis which was similar to that of GBM, suggesting that the histological identification of oligodendroglial tumor component, even partially, prescribe the prognosis of high grade glioma patients. This is the prominent report of retrospective clinicopathological analysis for high-grade gliomas throughout Grade III and IV, especially referring to the prognostic value of histological oligodendroglial tumor component; in addition, our results might offer an alternative aspect for the grading of high-grade astrocytic/oligodendroglial tumors.     

Glutathione S-transferase gene polymorphisms in Turkish patients with diabetes mellitus

Glutathione S-transferases (GSTs) are enzymes involved in the metabolism of many disease-causing electrophilic substrates and protect the cells against oxidative stress. In the present study, we investigated the GSTM1, GSTT1 and GSTP1 gene polymorphisms in diabetic patients and healthy individuals and searched whether polymorphisms in GST genes are associated with diabetes mellitus (DM) in the Turkish population. The study population consisted of 98 unrelated healthy individuals and 98 patients with DM. Genotyping of GSTM1, GSTT1 and GSTP1 genes was performed using real time polymerase chain reaction with a Light Cycler instrument. Patients had a higher frequency of the GSTM1 null genotype than the control group (Odds ratios, OR = 3.7; 95% confidence intervals, CI = 2.05-6.70). However, there was no significant difference in the frequencies of the GSTT1 and GSTP1 gene polymorphisms between the patients and control group. The combined analysis of these three GST genotypes showed a further DM risk increase (OR = 5.7, 95% CI = 1.51-31.07). This is the first study to determine the association of diabetes with GST gene polymorphism in the Turkish population. These results show that GSTM1 null genotype may play a significant role in the aetiopathogeneses of DM and the GSTM1 gene may be a useful marker in the prediction of DM susceptibility of the Turkish population.     

Absence of association between GSTM1 and GSTT1 polymorphisms and melanoma susceptibility: a meta-analysis

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that are involved in the metabolism of many xenobiotics, including a wide range of environmental carcinogens. The null genotypes GSTM1 and GSTT1 have been implicated in the development of carcinogenesis. We conducted a meta-analysis to examine the association of GSTM1 and GSTT1 homozygous deletion polymorphisms with melanoma risk. In total, 8 relevant studies were identified in searches of the PubMed and Embase databases: 8 investigated GSTM1 (1349 cases and 1560 controls) and 5 GSTT1 (977 cases and 1060 controls). Fixed- and random-effects models were used to assess the summary odds ratios (ORs). No significant association of the GSTM1 and GSTT1 polymorphisms or the GSTM1-GSTT1 interaction on the risk of melanoma was observed (for GSTM1: OR=1.09; 95% confidence interval, 0.94-1.27; and for GSTT1: OR=0.95; 95% confidence interval, 0.76-1.19). Similarly, no significant association was found in a subgroup analysis of hair color. These results indicate that the GSTM1 and GSTT1 polymorphisms may not be a risk factor for developing melanoma.     

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.     

Establishment of a New Human Glioblastoma Multiforme Cell Line (WJ1) and Its Partial Characterization

(1) A new human glioblastoma multiforme (GBM) cell line, WJ1, was established from the tissue derived from a 29-year-old patient diagnosed with a grade IV GBM. (2) The WJ1 cell line has been subcultured for more than 80 passages in standard culture media without feeder layer or collagen coatings. (3) GBM cells grow in vitro with distinct morphological appearance. Ultrastructural examination revealed large irregular nuclei and pseudo-inclusion bodies in nuclei. The cytoplasm contained numerous immature organelles and a few glia filaments. Growth kinetic studies demonstrated an approximate population doubling time of 60 h and a colony forming efficiency of 4.04%. The karyotype of the cells was hyperdiploid, with a large subpopulation of polyploid cells. Drug sensitivities of DDP, VP-16, tanshinone IIA of this cell line were assayed. They showed a dose- and time-dependent growth inhibition effect on the cells. (4) Orthotopic transplantation of GBM cells into athymic nude mice induced the formation of solid tumor masses about 6 weeks. The cells obtained from mouse tumor masses when cultivated in vitro had the same morphology and ultrastructure as those of the initial cultures. (5) This cell line may provide a useful model in vitro and in vivo in the cellular and molecular studies as well as in testing novel therapies for human glioblastoma multiforme.     

Combined Use of Anticancer Drugs and an Inhibitor of Multiple Drug Resistance-Associated Protein-1 Increases Sensitivity and Decreases Survival of Glioblastoma Multiforme Cells In Vitro

Glioblastoma multiforme (GBM) is a brain tumour characterised by a remarkably high chemoresistance and infiltrating capability. To date, chemotherapy with temozolomide has contributed only poorly to improved survival rates in patients. One of the most important mechanisms of chemoresistance comes about through the activity of certain proteins from the ATP-binding cassette superfamily that extrudes antitumour drugs, or their metabolites, from cells. We identify an increased expression of the multiple drug resistance-associated protein 1 (Mrp1) in glioblastoma multiforme biopsies and in T98G and G44 cell lines. The activity of this transporter was also confirmed by measuring the extrusion of the fluorescent substrate CFDA. The sensitivity of GBM cells was low upon exposure to temozolomide, vincristine and etoposide, with decreases in cell viability of below 20% seen at therapeutic concentrations of these drugs. However, combined exposure to vincristine or etoposide with an inhibitor of Mrp1 efficiently decreased cell viability by up to 80%. We conclude that chemosensitization of cells with inhibitors of Mrp1 activity might be an efficient tool for the treatment of human GBM.     

Pharmacologic blockade of chloride channel synergistically enhances apoptosis of chemotherapeutic drug-resistant cancer stem cells

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is the most commonly used chemotherapeutic agent in the treatment of human glioblastoma multiforme (GBM). However, BCNU chemotherapy fails due to subpopulations of intrinsic resistant-cells within the tumor mass. In our previous study, we dissociated BCNU-resistant cancer stem cells (CSCs) and observed the over-expression of multiple ion channel genes related to drug efflux. In the present study, we identified chloride intracellular channel 1 (CLIC1) in dissociated-BCNU-resistant CSCs using 2-DE and MALDI-TOF/MS analysis. To develop the specific target therapy of BCNU-resistant CSCs, we evaluated the drug-sensitivity of these CSCs using the chloride channel blocker, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). When combined with BCNU, DIDS synergistically increased the apoptotic events of BCNU-resistant CSCs in vitro and augmented BCNU sensitivity ex vivo. These findings suggest that CLIC1 is involved in the resistance of BCNU-resistant CSCs and BCNU/DIDS combined-therapy can provide valuable insight for promoting apoptosis or sensitizing glioblastomas to BCNU chemotherapy.