Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O⁶-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.     

Effectiveness of interferon-beta and temozolomide combination therapy against temozolomide-refractory recurrent anaplastic astrocytoma

Background

Malignant gliomas recur even after extensive surgery and chemo-radiotherapy. Although a relatively novel chemotherapeutic agent, temozolomide (TMZ), has demonstrated promising activity against recurrent glioma, the effects last only a few months and drug resistance develops thereafter in most cases. Induction of O⁶-methylguanine-DNA methyltransferase (MGMT) in tumors is considered to be responsible for resistance to TMZ. Interferon-beta has been reported to suppress MGMT in an experimental glioma model. Here we report a patient with TMZ-refractory anaplastic astrocytoma (AA) who was treated successfully with a combination of interferon-beta and TMZ.

Case presentation

A patient with recurrent AA after radiation-chemotherapy and stereotactic radiotherapy was treated with TMZ. After 6 cycles, the tumor became refractory to TMZ, and the patient was treated with interferon-beta at 3 × 10⁶ international units/body, followed by 5 consecutive days of 200 mg/m² TMZ in cycles of 28 days. After the second cycle the tumor decreased in size by 50% (PR). The tumor showed further shrinkage after 8 months and the patient's KPS improved from 70% to 100%. The immunohistochemical study of the initial tumor specimen confirmed positive MGMT protein expression.

Conclusion

It is considered that interferon-beta pre-administration increased the TMZ sensitivity of the glioma, which had been refractory to TMZ monotherapy.

     

Resveratrol restores sensitivity of glioma cells to temozolamide through inhibiting the activation of Wnt signaling pathway

Malignant gliomas are aggressive primary neoplasms that originate in the glial cells of the brain or the spine with notable resistance to standard treatment options. We carried out the study with the aim to shed light on the sensitization of resveratrol to temozolomide (TMZ) against glioma through the Wnt signaling pathway. Initially, glioma cell lines with strong resistance to TMZ were selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the glioma cells were subjected to resveratrol, TMZ, Wnt signaling pathway inhibitors, and activators. Cell survival rate and inhibitory concentration at half maximum value were detected by MTT, apoptosis by flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, in vitro proliferation by hanging drop method and β-catenin translocation into nuclei by TOP/FOP-FLASH assay. The expressions of the Wnt signaling pathway-related and apoptosis-related factors were determined by western blot analysis. Nude mice with glioma xenograft were established to detect tumorigenic ability. Glioma cell lines T98G and U138 which were highly resistant to TMZ were selected for subsequent experiments. Resveratrol increased the efficacy of TMZ by restraining cell proliferation, tumor growth, and promoting cell apoptosis in glioma cells. Resveratrol inhibited Wnt2 and β-catenin expressions yet elevated GSK-3β expression. Moreover, the Wnt signaling pathway participates in the sensitivity enhancing of resveratrol to TMZ via regulating O ⁶-methylguanine-DNA methyltransferase (MGMT) expression. Resveratrol sensitized TMZ-induced glioma cell apoptosis by repressing the activation of the Wnt signaling pathway and downregulating MGMT expression, which may confer new thoughts to the chemotherapy of glioma.     

Inhibition of Casein Kinase II by CX-4945, But Not Yes-associated protein (YAP) by Verteporfin,Enhances the Antitumor Efficacy of Temozolomide in Glioblastoma

Overcoming temozolomide (TMZ) resistance in glioma cancer cells remains a major challenge to the effective treatment of the disease. Increasing TMZ efficacy for patients with glioblastoma (GBM) is urgently needed because TMZ treatment is the standard chemotherapy protocol for adult patients with glioblastoma. O⁶-methylguanine-DNA-methyltransferase (MGMT) overexpression is associated with TMZ resistance, and low MGMT is a positive response marker for TMZ therapy. Here, we used 3 glioma cell lines (SF767, U373, and LN229), which had different levels of TMZ sensitivity. We found TMZ sensitivity is positively correlated with MGMT expression and multidrug-resistance protein ABC subfamily G member 2 (ABCG2) in these cells. CK2-STAT3 signaling and Hippo-YAP signaling are reported to regulate MGMT expression and ABCG2 expression, respectively. We combined CK2 inhibitor CX-4945 and YAP inhibitor verteporfin with TMZ treatment. We found that CX-4945 but not verteporfin can sensitize TMZ-resistant cells SF767 to TMZ and that CX-4945 and TMZ combinational treatment was effective for glioma treatment in mouse models compared with TMZ alone.ImplicationsA combination of CK2 inhibitor with TMZ may improve the therapeutic efficiency of TMZ toward GBM with acquired resistance.     

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

替莫唑胺治疗胶质母细胞瘤的耐药性产生机制研究进展

胶质母细胞瘤作为胶质瘤中恶性程度最高的原发性脑部肿瘤,具有治愈率低、复发率高、呈浸润性生长等特点,在不使用化疗药物的情况下,患者中位生存期仅为12.1个月。胶质母细胞瘤患者的标准治疗方法以手术切除为主,放化疗为辅,其中替莫唑胺（temozolomide,TMZ）作为一种新型的口服烷化剂,是目前用于胶质瘤化学治疗的一线药物。但经过替莫唑胺治疗后,患者中位生存期仅提高了2个月,主要原因为胶质母细胞瘤可对TMZ产生耐药性。胶质母细胞瘤对TMZ产生的耐药机制主要为DNA修复机制,其包括了O⁶?甲基鸟嘌呤DNA甲基转移酶（O⁶?methyl guanine DNA methyltransferase,MGMT）对药物作用位点进行的直接修复、错配修复（mismatch repair,MMR）及碱基切除修复（base excision repair,BER）,这些修复机制可修复TMZ引起的DNA损伤,从而降低肿瘤细胞对TMZ敏感性。通过对近年来胶质母细胞瘤的TMZ耐药机制的研究进展进行介绍,旨在为发展新的治疗手段提供理论基础。     

RANTES-mediated control of excitatory amino acid release in mouse spinal cord

O⁶‐methylguanine‐DNA methyltransferase (MGMT) is a DNA‐repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth‐depressing transfection of a T98G glioma cell line with liver‐type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA‐transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non‐transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100–1000 μM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 μM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.     

Rational incorporation of selenium into temozolomide elicits superior antitumor activity associated with both apoptotic and autophagic cell death

Background

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.

Principal Findings

This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.

Conclusions

Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.     

Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells

We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O₆-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.     

Chemotherapeutic resistance in anaplastic astrocytoma cell lines treated with a temozolomide–lomeguatrib combination

The treatment of anaplastic astrocytoma (AA) is controversial. New chemotherapeutic approaches are needed for AA treatment. Temozolomide (TMZ) is one of the chemotherapeutic drugs for the treatment of AA. The cytotoxic effects of TMZ can be removed by the MGMT (O(6)-methylguanine-DNA methyltransferase) enzyme. Then, chemotherapeutic resistance to TMZ occurs. MGMT inhibition by MGMT inactivators (such as lomeguatrib) is an important anticancer therapeutic approach to circumvent TMZ resistance. We aim to investigate the effect of TMZ–lomeguatrib combination on MGMT expression and TMZ sensitivity of SW1783 and GOS-3 AA cell lines. The sensitivity of SW1783 and GOS-3 cell lines to TMZ and to the combination of TMZ and lomeguatrib was determined by a cytotoxicity assay. MGMT methylation was detected by MS-PCR. MGMT and p53 expression were investigated by real-time PCR after drug treatment, and the proportion of apoptotic cells was analyzed by flow cytometry. When the combination of TMZ–lomeguatrib (50 μM) was used in AA cell lines, IC₅₀ values were reduced compared to only using TMZ. MGMT expression was decreased, p53 expression was increased, and the proportion of apoptotic cells was induced in both cell lines. The lomeguatrib–TMZ combination did not have any effect on the cell cycle and caused apoptosis by increasing p53 expression and decreasing MGMT expression. Our study is a pilot study investigating a new therapeutic approach for AA treatment, but further research is needed.     

Targeted nitric oxide delivery preferentially induces glioma cell chemosensitivity via altered p53 and O6‐Methylguanine‐DNA Methyltransferase activity

Glioblastoma multiforme is the most common malignant central nervous system tumor, and also among the most difficult to treat due to a lack of response to chemotherapeutics. New methods of countering the mechanisms that confer chemoresistance to malignant gliomas could lead to significant advances in the quest to identify novel drug combinations or targeted drug delivery systems for cancer therapy. In this study, we investigate the use of a targeted nitric oxide (NO) donor as a pretreatment to sensitize glioma cells to chemotherapy. The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells, and we have developed CTX–NO, a glioma‐specific, NO‐donating CTX derivative. Pretreatment of cells with CTX–NO followed by 48‐h exposure to either carmustine (BCNU) or temozolomide (TMZ), both common chemotherapeutics used in glioma treatment, resulted in increased efficacy of both therapeutics. After CTX–NO exposure, both T98G and U‐87MG human malignant glioma cells show increased sensitivity to BCNU and TMZ. Further investigation revealed that the consequences of this combination therapy was a reduction in active levels of the cytoprotective enzyme MGMT and altered p53 activity, both of which are essential in DNA repair and tumor cell resistance to chemotherapy. The combination of CTX–NO and chemotherapeutics also led to decreased cell invasion. These studies indicate that this targeted NO donor could be an invaluable tool in the development of novel approaches to treat cancer. Biotechnol. Bioeng. 2013; 110: 1211–1220. © 2012 Wiley Periodicals, Inc.     

Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT,MMR, P-Glycoprotein and CD133 Expression

Background

The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated.

Methods

Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed.

Results

Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC₅₀ showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC₅₀ did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC₅₀, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines.

Conclusions

These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.     

NETRIN-4 Protects Glioblastoma Cells FROM Temozolomide Induced Senescence

Glioblastoma multiforme is the most common primary tumor of the central nervous system. The drug temozolomide (TMZ) prolongs lifespan in many glioblastoma patients. The sensitivity of glioblastoma cells to TMZ is interfered by many factors, such as the expression of O-6-methylguanine-DNA methyltransferase (MGMT) and activation of AKT signaling. We have recently identified the interaction between netrin-4 (NTN4) and integrin beta-4 (ITGB4), which promotes glioblastoma cell proliferation via activating AKT-mTOR signaling pathway. In the current work we have explored the effect of NTN4/ITGB4 interaction on TMZ induced glioblastoma cell senescence. We report here that the suppression of either ITGB4 or NTN4 in glioblastoma cell lines significantly enhances cellular senescence. The sensitivity of GBM cells to TMZ was primarily determined by the expression of MGMT. To omit the effect of MGMT, we concentrated on the cell lines devoid of expression of MGMT. NTN4 partially inhibited TMZ induced cell senescence and rescued AKT from dephosphorylation in U251MG cells, a cell line bearing decent levels of ITGB4. However, addition of exogenous NTN4 displayed no significant effect on TMZ induced senescence rescue or AKT activation in U87MG cells, which expressed ITGB4 at low levels. Furthermore, overexpression of ITGB4 combined with exogenous NTN4 significantly attenuated U87MG cell senescence induced by TMZ. These data suggest that NTN4 protects glioblastoma cells from TMZ induced senescence, probably via rescuing TMZ triggered ITGB4 dependent AKT dephosphorylation. This suggests that interfering the interaction between NTN4 and ITGB4 or concomitant use of the inhibitors of the AKT pathway may improve the therapeutic efficiency of TMZ.     

Preparation of temozolomide-loaded polymer particles and study of their antitumor activity in models of glioma and melanoma

Temozolomide (TMZ) is a cytostatic drug used in treatment of patients with malignant gliomas and melanomas. The development of innovative formulations for delivery of TMZ into target cells in order to reduce its systemic toxicity and gain prolonged effect is a topical problem of modern biotechnology and pharmacology. The article presents experimental data on development of the TMZ polymer-based formulation. The TMZ formulation presents nanoparticles with average size of 300 nm exhibiting high in vitro cytotoxic activity against melanoma and glioma cells (cell lines C6, U377MG, B16, and Mel-10). Melanoma (B16 cell line) bearing mice (C57Bl/61) treated by TMZ polymer nanoparticles revealed inhibition of tumor growth and increase of the animal lifespan. Also, loading of TMZ into the polymer particles was shown to decrease acute toxicity of the drug compared to the intact drug.     

Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo

Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3–7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors.Malignant gliomas are the most common and aggressive type of primary brain tumor in adults. Current therapy includes surgery for tumor resection, followed by radiotherapy and/or concomitant adjuvant chemotherapy with temozolomide (TMZ) or chloroethylating nitrosoureas (CNUs). However, these protocols have limited success, and patients diagnosed with glioma have a dismal prognosis, with a median survival of 15 months and a 5-year survival rate of ~2%.¹ Several molecular mechanisms for cell resistance to these agents have been described. Because both are alkylating agents, the repair enzyme O₆-methylguanine-DNA methyltransferase (MGMT) is certainly a first barrier that is associated with increased tumor resistance.^{2, 3} The p53 status has also been proposed to act in an opposite manner in glioma cell resistance to TMZ or CNUs. Although p53 mutation is shown to be more resistant to TMZ treatment, owing to the induction of cell death,⁴ the p53 protein protects glioma cells after CNU treatment, most likely by improving other DNA repair systems.⁵Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors, such as ovarian, lung and testicular cancer,⁶ and it is used for adjuvant therapy in gliomas.⁷ Cisplatin is a molecule formed by one platinum ion that is surrounded by four ligands at the cis position: two chloride atoms and two amine molecules. The mechanism of action of cisplatin is mainly based on DNA damage. Once inside the cell, cisplatin becomes activated by the substitution of one or two chloride atoms by water, a process known as aquation. Owing to this process, the drug becomes positively charged and interacts with the DNA molecule, inducing the formation of DNA adducts. Activated cisplatin preferentially binds to purine bases in the nucleophilic N7 sites, where the majority of adducts occur between two guanines on the same strand, whereas ~3–5% of cisplatin adducts react with purines at the opposite strands, forming interstrand crosslinks (ICLs). The DNA lesions, in turn, trigger a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis.⁸Although relatively efficient, resistance to cisplatin, either intrinsic or acquired, during cycles of therapy is common, and overcoming tumor resistance remains the major challenge for cisplatin anticancer therapy. Cellular cisplatin resistance is a multifactorial phenomenon that may include decreased drug uptake, enhanced DNA repair capacity and higher glutathione (GSH) concentration.⁹GSH is a highly abundant, low-molecular-weight peptide in the cell, and it is well known for its critical importance in maintaining the cellular oxidative balance as a free radical scavenger. Additionally, GSH has a protective role against xenobiotic agents once its highly reactive thiol group binds and inactivates those agents. In fact, the GSH content and glutathione S-transferase (GST) have long been associated with cisplatin resistance in numerous cell lines and tumor tissues.^{9, 10, 11}Considering these possible pathways, it is not clear, however, which one determinates cisplatin resistance in glioma cells. Aiming to better understand the molecular mechanisms of resistance to this drug, four human glioma cell lines with different p53 status were investigated. We showed that cellular resistance was found to be independent of p53 as well as of the DNA repair capacity of the cells. On the other hand, the GSH levels within the cell were shown to act as a decisive resistance barrier to cisplatin, reducing the induction of DNA damage in the treated cells. Also, both in an in vitro and in vivo model depletion of GSH by an inhibitor (buthionine sulfoximine, BSO) sensitized the glioma cell lines to cisplatin. Interestingly, BSO also potentiated TMZ cytotoxicity. Thus, combination with BSO, cisplatin and TMZ turned out to be an extremely powerful approach to improve cytotoxicity in glioma, thus providing an exciting alternative for glioma treatment.