O6-methylguanine-DNA-methyltransferase expression and gene polymorphisms in relation to chemotherapeutic response in metastatic melanoma

In a retrospective study, O(6)-methylguanine-DNA-methyltransferase (MGMT) expression was analysed by immunohistochemistry using monoclonal human anti-MGMT antibody in melanoma metastases in patients receiving dacarbazine (DTIC) as single-drug therapy or as part of combination chemotherapy with DTIC-vindesine or DTIC-vindesine-cisplatin. The correlation of MGMT expression levels with clinical response to chemotherapy was investigated in 79 patients with metastatic melanoma. There was an inverse relationship between MGMT expression and clinical response to DTIC-based chemotherapy (P=0.05). Polymorphisms in the coding region of the MGMT gene were also investigated in tumours from 52 melanoma patients by PCR/SSCP and nucleotide sequence analyses. Single-nucleotide polymorphisms (SNPs) in exon 3 (L53L and L84F) and in exon 5 (I143V/K178R) were identified. There were no differences in the frequencies of these polymorphisms between these melanoma patients and patients with familial melanoma or healthy Swedish individuals. Functional analysis of variants MGMT-I143V and -I143V/K178R was performed by in vitro mutagenesis in Escherichia coli. There was no evidence that these variants decreased the MGMT DNA repair activity compared to the wild-type protein. All melanoma patients with the MGMT 53/84 polymorphism except one had tumours with high MGMT expression. There was no significant correlation between any of the MGMT polymorphisms and clinical response to chemotherapy, although an indication of a lower response rate in patients with SNPs in exon 5 was obtained. Thus, MGMT expression appears to be more related to response to chemotherapy than MGMT polymorphisms in patients with metastatic melanoma.     

Phase I trial of temozolomide plus O6-benzylguanine 5-day regimen with recurrent malignant glioma

This phase I clinical trial conducted with patients who had recurrent or progressive malignant glioma (MG) was designed to determine the maximum tolerated dose (MTD) and toxicity of three different 5-day dosing regimens of temozolomide (TMZ) in combination with O⁶-benzylguanine (O⁶-BG). Both TMZ and O⁶-BG were administered on days 1–5 of a 28-day treatment cycle. A bolus infusion of O⁶-BG was administered at 120 mg/m² over 1 h on days 1, 3, and 5, along with a continuous infusion of O⁶-BG at 30 mg/m²/day. TMZ was administered at the end of the first bolus infusion of O⁶-BG and then every 24 h for 5 days during the continuous infusion of O⁶-BG. Patients were accrued to one of three 5-day dosing regimens of TMZ. Twenty-nine patients were enrolled into this study. The dose-limiting toxicities (DLTs) were grade 4 neutropenia, leukopenia, and thrombocytopenia. The MTD for TMZ for the three different 5-day dosing schedules was determined as follows: schedule 1, 200 mg/m² on day 1 and 50 mg/m²/day on days 2–5; schedule 2, 50 mg/m²/day on days 1–5; and schedule 3, 50 mg/m²/day on days 1–5 while receiving pegfilgrastim. Thus, the 5-day TMZ dosing schedule that maximized the total dose of TMZ when combined with O⁶-BG was schedule 1. This study provides the foundation for a phase II trial of O⁶-BG in combination with a 5-day dosing schedule of TMZ in TMZ-resistant MG.     

DNA sequence context affects repair of the tobacco-specific adduct O(6)-[4-Oxo-4-(3-pyridyl)butyl]guanine by human O(6)-alkylguanine-DNA alkyltransferases

The repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) protects cells from the mutagenic and carcinogenic effects of alkylating agents by removing O(6)-alkylguanine adducts from DNA. Recently, we established that AGT protects against the mutagenic effects of pyridyloxobutylation resulting from the metabolic activation of the tobacco-specific nitrosamines (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine by repairing O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine (O(6)-pobG). There have been several epidemiologic studies examining the association between the I143V/K178R AGT genotype and lung cancer risk. Two studies have found positive associations, suggesting that AGT proteins differ in their repair of DNA damage caused by TSNA. However, it is not known how this genotype alters the biochemical activity of AGT. We proposed that AGT proteins may differ in their ability to remove large O(6)-alkylguanine adducts, such as O(6)-pobG, from DNA. Therefore, we examined the repair of O(6)-pobG by wild-type (WT) human, I143V/K178R, and L84F AGT proteins when contained in multiple sequence contexts, including the twelfth codon of H-ras, a mutational hotspot within this oncogene. The AGT-mediated repair of O(6)-pobG was more profoundly influenced by sequence context than that of O(6)-methylguanine. These differences are not the result of secondary structure (hairpin) formation in DNA. In addition, the I143V/K178R variant seems less sensitive to the effects of sequence context than the WT or L84F proteins. These studies indicate that the sequence dependence of O(6)-pobG repair by human AGT (hAGT) varies with subtle changes in protein structure. These data establish a novel functional difference between the I143V/K178R protein and other hAGTs in the repair of a toxicologically relevant substrate, O(6)-pobG.     

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

We evaluated the pharmacodynamic effects of the O⁶-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O⁶-methylguanine (O⁶-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O⁶-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O⁶-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O⁶-meG in PBMC DNA during treatment.     

A phase II trial of O 6-benzylguanine and carmustine in patients with advanced soft tissue sarcoma

Purpose: Tumor resistance to alkylating agents such as carmustine (BCNU) has been found to be associated with intracellular expression of O ⁶ -methylguanine-DNA methyltransferase (MGMT). Administration of O ⁶-benzylguanine (O ⁶-BG), a substrate that inactivates MGMT, may help overcome chemotherapy resistance. We performed a phase II study to explore the activity of O ⁶-BG in combination with BCNU in patients with advanced soft tissue sarcoma. Experimental design: Informed consent was obtained from patients with metastatic soft tissue sarcoma naïve to systemic chemotherapy (adjuvant chemotherapy allowed). Patients received O ⁶ -BG 120 mg/m² I.V. followed by BCNU 40 mg/m² I.V. Treatment was repeated every 6 weeks until disease progression or development of unacceptable toxicity. Results: No objective responses were observed in 12 enrolled patients. Four patients exhibited stable disease lasting 11–25+ weeks. The median overall survival was 16.9 months (95% CI, 2.9–NR). The most common grade 3–4 toxicities were neutropenia, thrombocytopenia, and anemia. Depletion of MGMT activity was demonstrated in peripheral blood mononuclear cells. Immunohistochemical estimation of MGMT expression from archival tissue ranged from 20 to 99% positive staining cells. Conclusions: Observed toxicities were consistent with previous studies of O ⁶-BG plus BCNU. The degree of MGMT expression was variable in this small sample of heterogeneous sarcomas. Further development of this regimen and dose for the treatment of soft tissue sarcoma is not warranted due to the lack of objective responses.     

MGMT gene variants,temozolomide myelotoxicity and glioma risk. A concise literature survey including an illustrative case

Temozolomide may cause thrombocytopenia or neutropenia in 3–4% of glioblastoma patients, respectively. However, pancytopenia is rarely reported. MGMT (O6-methylguanine-DNA-methyltransferase) enzyme repairs temozolomide-induced DNA mutations and associates both with antitumour efficacy and myelosuppression. Many studies on the effects of MGMT gene-methylation on temozolomide’s effects exist, but much fewer publications concerning MGMT variants were documented. A full sequencing of the MGMT gene was performed in a female glioblastoma patient, who developed pancytopenia following temozolomide treatment. Results indicated the presence of all the rs2308321 (I143 V), rs2308327 (K178R) and rs12917 (L84F) MGMT-variants, which were previously associated with temozolomide myelotoxicity. rs12917 (L84F) variant was reported as associating with lesser risk of gallbladder tumours, yet with higher risk of non-Hodgkin lymphomas related with exposure to chlorinated solvents or hair dyes. DNA repair proteins may exert diverging effects on DNA injuries caused by different chemicals and therefore exerting complex effects on myelotoxicity, antitumour activity and carcinogenesis.     

Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy

DNA adducts at the O6-position of guanine are a result of the carcinogenic, mutagenic and cytotoxic actions of methylating and chloroethylating agents. The presence of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) renders cells resistant to the biological effects induced by agents that attack at this position. O6-Benzylguanine (O6-BG) is a low molecular weight substrate of AGT and therefore, results in sensitizing cells and tumors to alkylating agent-induced cytotoxicity and antitumor activity. Presently, chemotherapy regimens of O6-BG in combination with BCNU, temozolomide and Gliadel are in clinical development. Other ongoing clinical trials include expression of mutant AGT proteins that confer resistance to O6-BG in bone marrow stem cells, in an effort to reduce the potential enhanced toxicity and mutagenicity of alkylating agents in the bone marrow. O6-BG has also been found to enhance the cytotoxicity of agents that do not form adducts at the O6-position of DNA, including platinating agents. O6-BG's mechanism of action with these agents is not fully understood; however, it is independent of AGT activity or AGT inactivation. A better understanding of the effects of this agent will contribute to its clinical usefulness and the design of better analogs to further improve cancer chemotherapy.     

New (alternative) temozolomide regimens for the treatment of glioma

One barrier to successful treatment of malignant glioma is resistance to alkylating agents such as temozolomide. The cytotoxic activity of temozolomide and other alkylating agents is believed to manifest largely by the formation of O(6)-methylguanine DNA adducts. Consequently, the primary mechanism of resistance to temozolomide is a function of the activity of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT). Fortuitously, MGMT is inactivated after each reaction (i.e., suicide enzyme). Therefore, if the rate of DNA alkylation were to outpace the rate of MGMT protein synthesis, the enzyme could, in theory, be depleted. Several studies have shown that prolonged exposure to temozolomide can deplete MGMT activity in blood cells, a process that could potentially increase the antitumor activity of the drug. To date, however, there are limited data demonstrating the depletion of MGMT activity in tumor tissue exposed to temozolomide. A variety of dosing schedules that increase the duration of exposure and the cumulative dose of temozolomide are currently being investigated for the treatment of glioma, with the goal of improving antitumor activity and overcoming resistance. These alternative dosing regimens have been shown to deplete MGMT activity in peripheral blood mononuclear cells, but the regimen that provides the best balance between enhanced antitumor activity and acceptable hematologic toxicity has yet to be determined.     

O6-benzylguanine and BCNU in multiple myeloma: a phase II trial

Purpose Carmustine (BCNU) is known to have modest activity in multiple myeloma; however, resistance to BCNU manifests by the activity of O⁶-methylguanine methyltransferase (MGMT). The objective of this study was to determine the safety and efficacy of depletion of MGMT activity in plasma cells using O⁶-benzylguanine (O⁶-BG) with BCNU in patients with multiple myeloma. Methods Patients with previously treated or untreated multiple myeloma were eligible. Cycles of O⁶-BG at a dose of 120 mg/m² and BCNU at a dose of 40 mg/m² were repeated every 6 weeks. Results Seventeen patients were enrolled on the study, with a median follow-up of 24.5 (range 5–69) months. One complete response (7%) and 3 partial responses (20%) were observed. Nine patients (60%) had stable disease. Bone marrow studies demonstrated 94% depletion of MGMT activity in CD38+ marrow cells. The most frequent grade 3 and 4 adverse events were neutropenia (71%), lymphocytopenia (53%), and thrombocytopenia (53%). Conclusions Chemotherapy utilizing the MGMT inhibitor O⁶-benzylguanine and BCNU results in inhibition of MGMT activity in malignant plasma cells and produces meaningful responses in a modest proportion of patients with multiple myeloma. Hematologic toxicity with this regimen is significant and dose-limiting.     

Association of genetic variants of O6-methylguanine-DNA methyltransferase with risk of lung cancer in non-Hispanic Whites.

O6-methylguanine, a methylated damage lesion in DNA, correlates with spontaneous G:C --> A:T transition mutations and leads to activation of oncogene K-ras or dysfunction of the tumor suppressor gene p53. O6-methylguanine-DNA methyltransferase (MGMT) is critical for repairing damage to the O6-position of guanine. Therefore, we tested our hypothesis that genetic variants of MGMT are associated with increased lung cancer risk in a Caucasian population of 1,121 lung cancer patients and 1,163 matched cancer-free controls. We genotyped four potentially functional single nucleotide polymorphisms (SNPs) of MGMT: exon 3 codon 84C --> T (L84F), exon 5 codon 143A --> G (I143V), and two promoter SNPs 135G --> T and 485C --> A. The allele frequency distributions of the SNPs of codon 84C --> T and the promoter 135G --> T in the cases were borderline different from that in the controls. After defining the minor allele (T for codon 84C --> T and G for codon 143A --> G) as the variant allele, we categorized the MGMT genotypes as either 0 variants (84CC-143AA) or 1-4 variants. Compared with 0 variants, those with 1-4 variants showed a statistically significantly increased risk of lung cancer (P = 0.040). Further stratification analysis showed that this increased risk was more pronounced in women, current smokers, and non-small cell lung cancer. We did not find any association between the MGMT promoter SNPs and lung cancer risk. Our findings suggest that non-synonymous SNPs in MGMT are associated with modestly increased risk of lung cancer in Caucasians and need to be further investigated.     

Antitumor effect of sarcnu in a 06-methylguanine-DNA methyltransferase positive human glioma xenograft model

Objective: To assess whether novel analogue of nitrosoureas, 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), has antitumor effect to 0⁶-methylguanine-DNA methyltransferase (MGMT) positive tumorsin vivo. Methods: MGMT positive human glioma cell line SF-767 xenografts in nude mice were treated with SarCNU. The antitumor efficacy of SarCNU was compared with the results of 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment with or without 0⁶-benzylguanine (0⁶-BG) preadministration. Results: Since the SF-767 is MGMT strongly positive, BCNU treatment alone did not result in a satisfactory anticancer effect. As expected, 0⁶-BG by depleting MGMT activity, significantly enhanced BCNU antitumor efficacy (P<0.001). More interestingly, SarCNU treatment alone had a better antitumor effect than O⁶-BG plus BCNU treatment (F=51.7,P=0.00036). Conclusion: Since SarCNU enters cells via extraneuronal monoamine transporter (EMT), the enhanced antitumor activity of SarCNU in this MGMT positive human tumor xenograft model may be due to the presence of EMT in SF-767. SarCNU may be used as an alternative treatment for MGMT positive tumors, specifically for tumors expressing EMT. This work was supported by the National Natural Science Foundation of China (No. 39670735) and a private donation from Helen and Nicki Lang, Quebec, Canada.     

Direct reversal of DNA damage by mutant methyltransferase protein protects mice against dose-intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells

Direct reversal of O6 adducts caused by chemotherapy agents is accomplished in mammalian cells by the protein O6-methylguanine DNA methyltransferase (MGMT). Some tumors overexpress MGMT and are resistant to alkylator therapy. One future approach to treatment of these tumors may rely on concurrent pharmacological depletion of tumor MGMT with O6-benzylguanine (6-BG) and protection of sensitive tissues, such as hematopoietic stem and progenitor cells, using genetic modification with 6-BG-resistant MGMT mutants. We have used retroviral-mediated gene transfer to transduce murine hematopoietic bone marrow cells with MGMT point mutants showing resistance to 6-BG depletion in vitro. These mutants include proline to alanine and proline to lysine substitutions at the 140 position (P140A and P140K, respectively), which show 40- and 1000-fold resistance to 6-BG compared with wild-type (WT) MGMT. Lethally irradiated mice were reconstituted with murine stem cells transduced with murine stem cell virus retrovirus expressing each mutant, WT MGMT, or mock-infected cells and then treated with a combination of 30 mg/kg 6-BG and 10 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with 40 mg/kg BCNU alone. Compared with mice treated with BCNU alone, significant myeloid toxicity and death occurred in mice reconstituted with mock-infected or WT MGMT (<0.1 probability of survival) or the P140A mutant (0.13 probability of survival) MGMT cDNAs. In contrast, after an initial period of mild cytopenia, mice reconstituted with the P140K mutant (0.83 probability of survival) recovered nearly normal blood counts, even during continued treatment. Comparison of peripheral blood neutrophils after completion of 5 weekly treatments in these animals showed a direct correlation between the treatment and in vivo selection for progeny of transduced cells (pretreatment, approximately 8-12% transduced cells; no treatment, approximately 6% transduced cells; BCNU only, 51% transduced cells; 6-BG/BCNU, 93% transduced cells). To determine whether this selection occurred at the stem cell level, bone marrow from each treatment group was infused into secondary recipients. Whereas animals that received bone marrow from untreated animals reconstituted with 2% transduced cells, animals receiving marrow from 6-BG/BCNU-treated animals reconstituted with 94% transduced cells, demonstrating nearly complete selection for stem cells in the primary animals. Mice reconstituted with marrow from animals treated with BCNU only demonstrated 23% transduced cells, consistent with partial selection of stem cells in the primary mice. The levels of transduced cells also correlated with survival during a second round of intensive combination chemotherapy (probability of survival: 6-BG/BCNU, 1.0; BCNU alone, >0.70; no treatment, <0.1). These data demonstrate that mutant MGMT expressed in the bone marrow can protect mice from time- and dose-intensive chemotherapy and that the combination of 6-BG and BCNU leads to uniform selection of transduced stem cells in vivo in mice.     

Polymorphisms in MGMT and DNA repair genes and the risk of esophageal adenocarcinoma

Rates of adenocarcinoma of the esophagus (EAC) and esophago-gastric junction (EGJAC) have increased rapidly in recent decades. The primary risk factors, gastro-esophageal acid reflux and smoking, are potentially genotoxic through the generation of N-nitroso compounds. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is the major cellular defense against alkylating DNA damage. We compared patients with EAC (n = 263) or EGJAC (n = 303) with matched population controls (n = 1,337) for the frequency of 5 MGMT single nucleotide polymorphisms (SNPs) (rs12269324, rs12268840, L84F, I143V, K178R), as well as SNPs in DNA repair genes ERCC1 (N118N), XRCC1 (Q399R) and XPD (K751Q). Relative risks were estimated using multivariable logistic regression. Potential biological interaction was assessed through the synergy index S. Each MGMT SNP conferred increased risks of EAC but not EGJAC; strongest associations were found for the 2 variant MGMT alleles rs12268840 and I143V (p = 0.005 and p < 0.001, respectively). Homozygous carriers of MGMT rs12268840 with frequent acid reflux had significantly higher risks of EAC (OR 15.5, 95% CI 5.8-42) than expected under an additive model, consistent with biological interaction (S = 3.3, 95% CI 1.1-10). Modest, nonsignificant interactions with smoking were also observed. Homozygous variant ERCC1 genotype was associated with reduced risks of EAC (OR 0.6, 95% CI 0.4-1.1), while the homozygous variant XRCC1 genotype conferred higher risks of EGJAC (OR 1.6, 95% CI 1.1-2.4). No associations with EAC or EGJAC were observed with XPD (rs13181). In summary, MGMT SNPs are associated with increased risks of EAC. Exposure to acid reflux, and possibly smoking, confer markedly higher risks among homozygous variant genotype carriers.     

GM-CSF在替莫唑胺抗高级别胶质瘤中的作用

目的 探讨粒-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GMCSF）在替莫唑胺（temozolomide, TMZ）抗高级别胶质瘤中的作用及机制。方法 选取6例高级别胶质瘤患者来源的肿瘤组织培养肿瘤细胞,待细胞状态稳定后采用MTT法进行细胞增殖毒性实验,流式细胞仪检测细胞周期变化和细胞凋亡情况,甲基化特异性PCR和免疫组织化学染色法分别检测六氧甲基鸟嘌呤DNA甲基转移酶（O6-methylguanine-DNA methyltransferase, MGMT）基因启动子甲基化状态和MGMT蛋白表达水平。 结果 MTT实验显示,GM-CSF处理组的细胞存活率与对照组相比均有不同程度的增加。MGMT基因启动子甲基化的3例细胞,GM-CSF+TMZ组的细胞存活率比TMZ组显著降低（P<0.05）,另3例MGMT启动子非甲基化细胞,GM-CSF+TMZ组与TMZ组相比,其存活率差异均无统计学意义（P>0.05）。6例细胞GM-CSF组的G1期细胞比例均比对照组降低,而S期细胞比例GMCSF处理组较对照组显著增加（P<0.05）。流式细胞仪凋亡检测显示,MGMT启动子甲基化的3例细胞,GM-CSF+TMZ组凋亡率与单药TMZ组凋亡率相比均显著增加（P<0.05）,而MGMT非甲基化细胞GM-CSF+TMZ组与单药TMZ组凋亡率相比无统计学差异。结论 GM-CSF可通过诱导高级别胶质瘤细胞快速进入细胞周期,显著提高TMZ对MGMT基因启动子甲基化的高级别胶质瘤细胞的杀伤作用,而对MGMT基因启动子非甲基化高级别胶质瘤细胞的作用不明显。     

O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients

O⁶-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (χ² test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76–17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45–2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.     

姜黄素抑制MGMT基因表达增强恶性胶质瘤对替莫唑胺化疗的敏感性

目的 探讨姜黄素对高水平O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)的调控作用及其对恶性胶质瘤化疗敏感性的影响.方法 通过实时荧光定量PCR(qRT-PCR)测定姜黄素、替莫唑胺单药和二者联合对MGMT表达阳性恶性胶质瘤C6及U87细胞株、复发或耐药恶性胶质瘤原代细胞MGMT表达水平的影响.CCK-8检测细胞增殖变化,流式细胞检测细胞凋亡的变化.结果 与姜黄素(C6:0.64 ±0.03;U87:0.63±0.06;原代细胞:0.51±0.07)、替莫唑胺(C6:0.53 ±0.06;U87:0.51±0.04;原代细胞:0.79±0.03)单药比较,姜黄素、替莫唑胺二者联合(C6:0.14±0.01;U87:0.12±0.03;原代细胞:0.29±0.02)能明显降低C6及U87细胞株、复发或耐药恶性胶质瘤原代细胞株中MGMT的表达,组间差异具有统计学意义(C6:F=23.675,P=0.006;U87:F=29.021,P=0.001;原代细胞株:F=25.534,P=0.001).与姜黄素、替莫唑胺单药比较,二者联合均能抑制细胞的增殖,半数抑制浓度(IC50)值降低,差异具有统计学意义(C6:F=6.731,P=0.012;U87:F=17.321,P=0.008;原代细胞株:F=18.857,P=0.007).姜黄素和替莫唑胺二者联合作用后细胞的凋亡率较单药作用后明显增加,组间差异具有统计学意义(C6:F=25.871,P=0.001;U87:F=6.847,P=0.009;原代细胞株:F =36.641,P=0.000).结论 姜黄素与替莫唑胺能协同降低MGMT的表达,增强恶性胶质瘤对替莫唑胺的化疗敏感性,为替莫唑胺耐药的恶性胶质瘤的治疗提供新的思路和方法.     

Expression of O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea resistance of human brain tumors.

Northern blot analysis with O6-methylguanine-DNA methyltransferase (MGMT) cDNA as a probe was used to analyze the MGMT activity regulating drug resistance of human cells to chloroethylnitrosoureas (CENUs). By this method, the expression levels of MGMT mRNA in six human glioma cell lines and 12 human brain tumor tissues from surgical specimens were determined. These MGMT mRNA levels were compared with the SD10 values of the tumor cells, estimated by cell survival assay, which indicated their resistance to the anticancer drug, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Human brain tumors that were highly resistant to ACNU, such as glioblastoma Gbl1 and metastatic brain tumor Col1 with SD10 values (microM) of above 100, expressed markedly increased amounts of 0.95 kb MGMT mRNA. In contrast, tumor cells such as U-87MG, U-251MG, U-343MG, U-373MG and SF-126 with SD10 values of under 14 indicating low resistance to ACNU scarcely synthesized any MGMT mRNA. These results indicated that the level of expression of MGMT mRNA in human brain tumors determined by Northern blot analysis truly reflects their cellular resistance to ACNU. Thus the Northern method with MGMT cDNA probe reported here is a practical and reliable method for estimation of cellular resistance to CENUs such as ACNU and for screening the chemotherapeutic response to CENUs of human brain tumors.     

Activity of irinotecan and temozolomide in the presence of O6-methylguanine-DNA methyltransferase inhibition in neuroblastoma pre-clinical models

Background:

The combination of temozolomide (TMZ) and irinotecan is a regimen used in neuroblastoma patients with recurrent disease. O⁶-methylguanine-DNA methyltransferase (MGMT) may have a function in resistance to TMZ. Using neuroblastoma pre-clinical models, we determined whether the inhibition of MGMT by O⁶-benzylguanine (O6-BG) could enhance the anti-tumour activity of TMZ and irinotecan.

Methods:

The cytotoxicity of TMZ and irinotecan, either alone or in combination, was measured in five neuroblastoma cell lines in the presence or absence of O6-BG with a fluorescence-based cell viability assay (DIMSCAN). Anti-tumour activity was measured in three neuroblastoma xenograft models.

Results:

MGMT mRNA and protein were expressed in 9 out of 10 examined cell lines. Pretreatment of cells with 25 μ O6-BG decreased MGMT protein expression and enhanced The TMZ cytotoxicity by up to 0.3–1.4 logs in four out of five tested cell lines. TMZ (25 mg kg⁻¹ per day for 5 days every 3 weeks for four cycles) did not significantly improve mice survival, whereas the same schedule of irinotecan (7.5 mg kg⁻¹ per day) significantly improved survival (P<0.0001) in all three xenograft models. Combining O6-BG and/or TMZ with irinotecan further enhanced survival.

Conclusion:

Our in vitro and in vivo findings suggest that irinotecan drives the activity of irinotecan and TMZ in recurrent neuroblastoma. Inhibitors of MGMT warrant further investigation for enhancing the activity of regimens that include TMZ.