Lomustine and nimustine exert efficient antitumor effects against glioblastoma models with acquired temozolomide resistance.
ABSTRACT: Glioblastomas (GBM) often acquire resistance against temozolomide (TMZ) after continuous treatment and recur as TMZ-resistant GBM (TMZ-R-GBM). Lomustine (CCNU) and nimustine (ACNU), which were previously used as standard therapeutic agents against GBM before TMZ, have occasionally been used for the salvage therapy of TMZ-R-GBM; however, their efficacy has not yet been thoroughly examined. Therefore, we investigated the antitumor effects of CCNU and ACNU against TMZ-R-GBM. As a model of TMZ-R-GBM, TMZ resistant clones of human GBM cell lines (U87, U251MG, and U343MG) were established (TMZ-R-cells) by the culture of each GBM cells under continuous TMZ treatment, and the antitumor effects of TMZ, CCNU, or ACNU against these cells were analyzed in vitro and in vivo. As a result, although growth arrest and apoptosis were triggered in all TMZ-R-cells after the administration of each drug, the antitumor effects of TMZ against TMZ-R-cells were significantly reduced compared to those of parental cells, whereas CCNU and ACNU demonstrated efficient antitumor effects on TMZ-R-cells as well as parental cells. It was also demonstrated that TMZ resistance of TMZ-R-cells was regulated at the initiation of DNA damage response. Furthermore, survival in mice was significantly prolonged by systemic treatment with CCNU or ACNU but not TMZ after implantation of TMZ-R-cells. These findings suggest that CCNU or ACNU may serve as a therapeutic agent in salvage treatment against TMZ-R-GBM.
Project description:The outcome of cancer therapy strongly depends on the complex network of cell signaling pathways, including transcription factor activation following drug exposure. Here we assessed whether and how the MAP kinase (MAPK) cascade and its downstream target, the transcription factor AP-1, influence the sensitivity of malignant glioma cells to the anticancer drugs temozolomide (TMZ) and nimustine (ACNU). Both drugs induce apoptosis in glioma cells at late times following treatment. Activation of the MAPK cascade precedes apoptosis, as shown by phosphorylation of Jun kinase (JNK) and c-Jun, a main component of AP-1. Pharmacological inhibition and siRNA mediated knockdown of JNK and c-Jun reduced the level of apoptosis in LN-229 glioma cells treated with TMZ or ACNU. Analyzing the underlying molecular mechanism, we identified the pro-apoptotic gene BIM as a critical target of AP-1, which is upregulated following TMZ and ACNU. Importantly, shRNA mediated downregulation of BIM in the malignant glioma cell lines LN-229 and U87MG led to an attenuated cleavage of caspase-9 and, consequently, reduced the level of apoptosis following TMZ and ACNU treatment. Overall, we identified JNK/c-Jun activation and BIM induction as a late pro-apoptotic response of glioma cells treated with alkylating anticancer drugs.
Project description:Chloroethylnitrosoureas (CENUs), which are bifunctional alkylating agents widely used in the clinical treatment of cancer, exert anticancer activity by inducing crosslink within a guanine-cytosine DNA base pair. However, the formation of dG-dC crosslinks can be prevented by O6-alkylguanine-DNA alkyltransferase (AGT), ultimately leading to drug resistance. Therefore, the level of AGT expression is related to the formation of dG-dC crosslinks and the sensitivity of cells to CENUs. In this work, we determined the CENU-induced dG-dC crosslink in mouse L1210 leukemia cells and in human glioblastoma cells (SF-763, SF-767 and SF-126) containing different levels of AGT using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The results indicate that nimustine (ACNU) induced more dG-dC crosslinks in L1210 leukemia cells than those induced by carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS). This result was consistent with a previously reported cohort study, which demonstrated that ACNU had a better survival gain than BCNU, CCNU and FTMS for patients with high-grade glioma. Moreover, we compared the crosslinking levels and the cytotoxicity in SF-763, SF-767 and SF-126 cells with different AGT expression levels after exposure to ACNU. The levels of dG-dC crosslink in SF-126 cells (low AGT expression) were significantly higher than those in SF-767 (medium AGT expression) and SF-763 (high AGT expression) cells at each time point. Correspondingly, the cytotoxicity of SF-126 was the highest followed by SF-767 and SF-763. The results obtained in this work provided unequivocal evidence for drug resistance to CENUs induced by AGT-mediated repair of DNA ICLs. We postulate that the level of dG-dC crosslink has the potential to be employed as a biomarker for estimating drug resistance and anticancer efficiencies of novel CENU chemotherapies.
Project description:Different chemotherapy drugs are generally introduced in clinical practices combining with therapy for glioma treatment. However, these chemotherapy drugs have rarely been compared with each other and the optimum drug still remains to be proved. In this research, medical databases were consulted, PubMed, Embase and Cochrane Library included. As primary outcomes, hazard ratio (HR) of overall survival (OS) and progression-free survival (PFS) with their corresponding 95% credential intervals (CrI) were reported. A network meta-analysis was conducted; the surface under the cumulative ranking curve (SUCRA) was utilized for treatment rank and a cluster analysis based on SUCRA values was performed. This research includes 14 trials with 3,681 subjects and eight interventions. In terms of network meta-analysis, placebo was proved to be inferior to the combination of temozolomide (TMZ), nimustine (ACNU) and cisplatin (CDDP). Also, bevacizumab (BEV) in conjunction with TMZ were significantly more effective than placebo with an HR of 0.40. The estimated probabilities from SUCRA verified the above outcomes, confirming that the combination of TMZ, ACNU and CDDP exhibited the highest ranking probability of 0.889 with respect to OS, while BEV in combination with TMZ - with a probability of 0.772 - ranked the first place with respect to PFS. According to the results of this network meta-analysis, the combination of (1) TMZ, ACNU and CDDP; (2) BEV in combination with TMZ and (3) cilengitide in combination with TMZ, are considered as the preferable choices of chemotherapy drugs for glioma treatment.
Project description:AbstractBackgroundTreatment options for patients suffering brainstem gliomas are quite limited as surgery is not an option against intrinsic tumors at brainstem and chemotherapy generally failed to demonstrate its efficacy. Intracerebral convection-enhanced delivery (CED) is a novel approach for administering chemotherapy to patients with brain tumors. We present the results of phase I trial of CED of nimustine hydrochloride (ACNU), designed to determine the maximum tolerable concentration of ACNU, for patients with recurrent brainstem gliomas.MethodsSixteen patients, aged 3–81 years old, suffering from recurrent brainstem gliomas, including diffuse intrinsic pontine glioma patients as well as patients with recurrent gliomas that originated from non?brainstem sites, were enrolled in this trial between February 2011 and April 2016. The dose/concentration escalation trial included 3 dose/concentration groups (0.25, 0.5, and 0.75 mg/mL, all at 7 mL) to determine the safety and tolerability of CED of ACNU. Real-time monitoring of drug distribution was performed by mixing gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA) in the infusion solution. CED of ACNU was given in combination with oral or intravenous temozolomide chemotherapy.ResultsCED of ACNU demonstrated antitumor activity, as assessed by radiographic changes and prolonged overall survival. The recommended dosage was 0.75 mg/mL. Drug-associated toxicity was minimal.ConclusionsIntracerebral CED of ACNU under real-time monitoring of drug distribution, in combination with systemic temozolomide, was well tolerated among patients with recurrent brainstem gliomas. The safety and efficacy observed suggest the clinical benefits of this strategy against this devastating disease. Based on this phase I study, further clinical development of ACNU is warranted.
Project description:Resistance to chemotherapy remains a major challenge in the treatment of human glioblastoma (GBM). Glycogen synthase kinase-3? (GSK-3?), a positive regulator of NF-?B-mediated survival and chemoresistance of cancer cells, has been identified as a potential therapeutic target in human GBM. Our objective was to determine the antitumor effect of GSK-3 inhibitor 9-ING-41 in combination with chemotherapy in patient-derived xenograft (PDX) models of human GBM. We utilized chemoresistant PDX models of GBM, GBM6 and GBM12, to study the effect of 9-ING-41 used alone and in combination with chemotherapy on tumor progression and survival. GBM6 and GBM12 were transfected by reporter constructs to enable bioluminescence imaging, which was used to stage animals prior to treatment and to follow intracranial GBM tumor growth. Immunohistochemical staining, apoptosis assay, and immunoblotting were used to assess the expression of GSK-3? and the effects of treatment in these models. We found that 9-ING-41 significantly enhanced 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) antitumor activity in staged orthotopic GBM12 (no response to CCNU) and GBM6 (partial response to CCNU) PDX models, as indicated by a decrease in tumor bioluminescence in mouse brain and a significant increase in overall survival. Treatment with the combination of CCNU and 9-ING-41 resulted in histologically confirmed cures in these studies. Our results demonstrate that the GSK-3 inhibitor 9-ING-41, a clinical candidate currently in Investigational New Drug (IND)-enabling development, significantly enhances the efficacy of CCNU therapy for human GBM and warrants consideration for clinical evaluation in this difficult-to-treat patient population.
Project description:Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system. Temozolomide (TMZ)-based adjuvant treatment has improved overall survival, but clinical outcomes remain poor; TMZ resistance is one of the main reasons for this. Here, we report a new phosphatidylinositide 3-kinase inhibitor, XH30; this study aimed to assess the antitumor activity of this compound against TMZ-resistant GBM. XH30 inhibited cell proliferation in TMZ-resistant GBM cells (U251/TMZ and T98G) and induced cell cycle arrest in the G1 phase. In an orthotopic mouse model, XH30 suppressed TMZ-resistant tumor growth. XH30 was also shown to enhance TMZ cytotoxicity both in vitro and in vivo. Mechanistically, the synergistic effect of XH30 may be attributed to its repression of the key transcription factor GLI1 via the noncanonical hedgehog signaling pathway. XH30 reversed sonic hedgehog-triggered GLI1 activation and decreased GLI1 activation by insulin-like growth factor 1 via the noncanonical hedgehog signaling pathway. These results indicate that XH30 may represent a novel therapeutic option for TMZ-resistant GBM.
Project description:BACKGROUND:Glioblastomas (GBM) are therapy-resistant tumors with a profoundly immunosuppressive tumor microenvironment. Chemotherapy has shown limited efficacy against GBM. Systemic delivery of chemotherapeutic drugs is hampered by the difficulty of achieving intratumoral levels as systemic toxicity is a dose-limiting factor. Although some of its effects might be mediated by immune reactivity, systemic chemotherapy can also inhibit induced or spontaneous antitumor immune reactivity. Convection-enhanced delivery of temozolomide (CED-TMZ) can tentatively increase intratumoral drug concentration while reducing systemic side effects. The objective of this study was to evaluate the therapeutic effect of intratumorally delivered temozolomide in combination with immunotherapy and whether such therapy can generate a cellular antitumor immune response. METHODS:Single bolus intratumoral injection and 3-day mini-osmotic pumps (Alzet®) were used to deliver intratumoral TMZ in C57BL6 mice bearing orthotopic gliomas. Immunotherapy consisted of subcutaneous injections of irradiated GL261 or KR158 glioma cells. Tumor size and intratumoral immune cell populations were analyzed by immunohistochemistry. RESULTS:Combined CED-TMZ and immunotherapy had a synergistic antitumor effect in the GL261 model, compared to CED-TMZ or immunotherapy as monotherapies. In the KR158 model, immunization cured a small proportion of the mice whereas addition of CED-TMZ did not have a synergistic effect. However, CED-TMZ as monotherapy prolonged the median survival. Moreover, TMZ bolus injection in the GL261 model induced neurotoxicity and lower cure rate than its equivalent dose delivered by CED. In addition, we found that T-cells were the predominant cells responsible for the TMZ antitumor effect in the GL261 model. Finally, CED-TMZ combined with immunotherapy significantly reduced tumor volume and increased the intratumoral influx of T-cells in both models. CONCLUSIONS:We show that immunotherapy synergized with CED-TMZ in the GL261 model and cured animals in the KR158 model. Single bolus administration of TMZ was effective with a narrower therapeutic window than CED-TMZ. Combined CED-TMZ and immunotherapy led to an increase in the intratumoral influx of T-cells. These results form part of the basis for the translation of the therapy to patients with GBM but the dosing and timing of delivery will have to be explored in depth both experimentally and clinically.
Project description:Background: Although programmed death-1 (PD-1) blockade is effective in treating several types of cancer, the efficacy of this agent in glioblastoma (GBM) is largely unknown. Methods: We evaluated therapeutic effects of anti-PD-1, temozolomide (TMZ), and their combination in an orthotopic murine GBM model. The phenotype, number, and composition of lymphocytes were evaluated using flow cytometry. Transcriptional profiles of tumor tissues were analyzed using microarrays. Generation of antitumor immunological memory was investigated upon rechallenge. Results: Combined treatment with anti-PD-1 and TMZ yielded synergistic antitumor efficacy in the presence of donor-originated PD-1+CD8+ T cells in vitro, necessitating in vivo validation. Whereas TMZ did not rescue GBM-implanted mice, anti-PD-1 completely eradicated GBM in 44.4% of mice, and combination of anti-PD-1 and TMZ in all mice. Anti-PD-1 significantly increased the number of tumor-infiltrating lymhpocytes (TILs), and reduced frequencies of exhausted T cells and regulatory T cells. However, combining TMZ with anti-PD-1 significantly decreased the number of TILs, which was also observed with TMZ treatment alone. A transcriptome analysis of tumor tissues revealed that anti-PD-1 monotherapy perturbed immune-related genes, distinctly with combined therapy. Upon rechallenge, tumor growth was not observed in mice cured by anti-PD-1 monotherapy, whereas tumors regrew in the combination group. Furthermore, an analysis of peripheral blood revealed that antitumor memory T cells were generated in mice cured by anti-PD-1 monotherapy, not in the combination group. Conclusion: PD-1 blockade induces long-term therapeutic response, and combination with TMZ further enhances antitumor efficacy. However, immunological memory is provoked by anti-PD-1 monotherapy, not by combined therapy.
Project description:Glioblastoma multiforme (GBM), the most common type of primary brain tumor, is universally fatal, with a median survival duration ranging from 12-15 months despite maximum treatment efforts. Temozolomide (TMZ) is the current standard of care for GBM patients; however patients usually develop resistance to TMZ and limits its benefit. The identification of novel synergistic targets in GBM will lead to the development of new targeted drugs, which could be combined with broad-spectrum cytotoxic agents. In this study, we used a high-throughput synthetic lethality screen with a pooled short hairpin DNA repair library, in combination with TMZ, to identify targets that will enhance TMZ-induced antitumor effects. Using an unbiased bioinformatical analysis, we identified BRCA1 as a potential promising candidate gene that induced synthetic lethality with TMZ in glioma sphere-forming cells (GSCs). BRCA1 knockdown resulted in antitumor activity with TMZ in P53 wild-type GSCs but not in P53 mutant GSCs. TMZ treatment induced a DNA damage repair response; the activation of BRCA1 DNA repair pathway targets and knockdown of BRCA1, together with TMZ, led to increased DNA damage and cell death in P53 wild-type GSCs. Our study identified BRCA1 as a potential target that sensitizes TMZ-induced cell death in P53 wild-type GBM, suggesting that the combined inhibition of BRCA1 and TMZ treatment will be a successful targeted therapy for GBM patients.
Project description:In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53(wt)) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53(wt) GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53(null) GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner.