RESUMO
BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Animais , Camundongos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Dacarbazina/uso terapêutico , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , O(6)-Metilguanina-DNA Metiltransferase/genética , Estudos Retrospectivos , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Metilação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Metilação de DNA , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: There is lack of consensus regarding optimal adjuvant therapy in elderly glioblastoma (GBM). We have been treating elderly (≥60 years) GBM patients with normofractionated or hypofractionated radiotherapy (RT) plus temozolomide (TMZ) based on Karnofsky performance status (KPS). Herein we report clinical outcomes in this cohort treated at our institute using this approach. METHODS: Medical records of elderly GBM patients (≥60 years) treated between 2013 and 2017 with either normofractionated RT (59.4-60 Gy/30-33 fractions/6-6.5 weeks) or hypofractionated RT (35 Gy/10 fractions/2 weeks) plus TMZ were reviewed retrospectively. Outcomes of interest included progression-free survival (PFS), overall survival (OS), and ≥grade 3 myelotoxicity. Time-to-event outcomes were analyzed with Kaplan-Meier methods, compared using log-rank test, and reported as point estimates with 95% confidence interval (CI). RESULTS: The normofractionated cohort (n = 126) was characterized by a higher proportion of patients younger than age 65 years, KPS ≥70, methylated O6-methylguanine DNA methyltransferase (MGMT), and receiving adjuvant TMZ including extended adjuvant TMZ (>6 cycles) compared with the hypofractionated cohort (n = 20), confirming selection bias. At a median follow-up of 13 months, 1-year Kaplan-Meier estimates of PFS and OS were 43% (95% CI: 36%-52%) and 56% (95% CI: 48%-64%), yielding median PFS and OS of 11.0 months and 13.1 months, respectively. Higher KPS, methylated MGMT, normofractionated RT, and extended adjuvant TMZ emerged as favorable prognostic factors. TMZ was well tolerated with a low risk of ≥grade 3 myelotoxicity. CONCLUSIONS: Our single-institution clinical audit confirms poor survival in elderly GBM with suboptimal performance status but demonstrates acceptably fair outcomes in patients with preserved KPS comparable with the nonelderly cohort.
Assuntos
Glioblastoma , Idoso , Terapia Combinada , Glioblastoma/terapia , Humanos , Índia/epidemiologia , O(6)-Metilguanina-DNA Metiltransferase , Estudos Retrospectivos , Temozolomida/uso terapêuticoRESUMO
OBJECTIVE: To screen ideal lead compounds from a drug library (ZINC15 database) with potential inhibition effect against O6-methylguanine-DNA methyltransferase (MGMT) to contribute to medication design and refinement. METHODS: A series of computer-aided virtual screening techniques were used to identify potential inhibitors of MGMT. Structure-based virtual screening by LibDock was carried out to calculate LibDock scores, followed by absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to demonstrate binding affinity and mechanism between the selected ligands and MGMT protein. Molecular dynamics simulation was performed to evaluate stability of the ligand-MGMT complex under natural circumstances. RESULTS: Two novel natural compounds, ZINC000008220033 and ZINC000001529323, from the ZINC15 database were found to bind with MGMT with a higher binding affinity together with more favorable interaction energy. Also, they were predicted to have less rodent carcinogenicity, Ames mutagenicity, and developmental toxicity potential as well as noninhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis demonstrated that the 2 complexes ZINC000008220033-MGMT and ZINC000001529323-MGMT had more favorable potential energy compared with reference ligand O6-benzylguanine, and they could exist stably in the natural environment. CONCLUSIONS: This study elucidated that ZINC000008220033 and ZINC000001529323 were ideal lead compounds with potential inhibition targeting to MGMT protein. These compounds were selected as safe drug candidates and may contribute a solid basis for MGMT target medication design and improvement.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular/métodos , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , O(6)-Metilguanina-DNA Metiltransferase/química , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
IMPORTANCE: Loss of O6-methylguanine-DNA methyltransferase (MGMT) protein expression has been reported in several malignant tumors and predicts dismal survival outcomes. In gastric cancer, existing studies on this topic are limited and the association between MGMT and fluorouracil-based adjuvant chemotherapy remains obscure. OBJECTIVE: To investigate the postoperative prognostic significance of MGMT in patients with resectable gastric cancer and its responsiveness to fluorouracil-based adjuvant chemotherapy. DESIGN, SETTING, AND PARTICIPANTS: This study recruited 445 consecutive patients with resectable gastric cancer who underwent radical gastrectomy between August 1, 2007, and December 30, 2008, at Zhongshan Hospital at Fudan University in Shanghai, China. Patients were randomly divided into a discovery data set (n = 200) and a validation data set (n = 245), and the range of follow-up time was from 2 to 76 months for the discovery group and 2 to 79 months for the validation group. The immunoreactivity for MGMT in cancer cells was reviewed under a light microscope by 2 pathologists who were blinded to the clinicopathological data. The association of MGMT expression with clinicopathological characteristics and measures and prognosis was inspected. Data and specimens were collected from patients from the date of surgery to April 25, 2014. Data analysis took place from May 9, 2016, to July 15, 2016. MAIN OUTCOMES AND MEASURES: Estimates of overall survival on the basis of MGMT expression and hazard ratio (HR) for estimates of overall mortality risk. RESULTS: Of the 445 patients included in the study, 315 (70.8%) were men, and the mean (SD) age of all patients was 60 (12) years. Positive expression of MGMT indicated better overall survival for patients with stage II or III gastric cancer in both the discovery data set (HR, 0.52; 95% CI, 0.32-0.84; P = .003) and the validation data set (HR, 0.63; 95% CI, 0.43-0.93; P = .01). Multivariate analysis identified MGMT expression and TNM stage as 2 independent prognostic factors for overall survival. In stage II disease, the benefit from fluorouracil-based adjuvant chemotherapy was superior among MGMT-positive patients (HR, 0.35; 95% CI, 0.13-0.95; P = .007 for interaction) compared with MGMT-negative patients. CONCLUSIONS AND RELEVANCE: Positive expression of MGMT in gastric cancer was identified as an independent, favorable prognostic factor. Incorporating MGMT expression into the current TNM staging system could lead to better prognostic accuracy. These findings should be confirmed within the framework of randomized clinical trials associated with genomic DNA sequencing studies.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Biomarcadores Tumorais/metabolismo , Fluoruracila/uso terapêutico , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Quimioterapia Adjuvante , Feminino , Gastrectomia/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/cirurgiaRESUMO
Pine needle extracts of Pinus morrisonicola (Hayata) are commonly used as a functional health beverage. However, it remains unclear what the mechanism is underlying the antitumor activity of pine needle extract. The aims of present study were to investigate the anti-glioblastoma effects of pine needle extracts as well as its bioactive compounds. From three different solvent extracts of pine needles, the water extract displayed the strongest cytotoxicity effects on GBM8901 glioblastoma cells. The isolated compounds were identified as pinocembrin, chrysin, and tiliroside. Chrysin was the most active ingredient of pine needle extract for the induction of apoptosis and suppression of migration and invasion. It also markedly inhibited temozolomide (TMZ)-induced autophagy and O(6)-methylguanine-DNA methyltransferase (MGMT) expression. Because both autophagy and MGMT overexpression have been implicated to TMZ-induced drug resistance in glioblastoma, our results showed that pine needle extract and chrysin may serve as a potential anticancer agent against glioblastoma, especially with regard to sensitizing glioblastoma cells resistant to TMZ.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Dacarbazina/análogos & derivados , Glioblastoma/enzimologia , O(6)-Metilguanina-DNA Metiltransferase/genética , Pinus/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dacarbazina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/fisiopatologia , Humanos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Extratos Vegetais/química , TemozolomidaRESUMO
Global hypomethylation, gene-specific methylation, and genome instability are common events in tumorigenesis. To date, few studies have examined the aberrant DNA methylation patterns in coke oven workers, who are highly at risk of lung cancer by occupational exposure to polycyclic aromatic hydrocarbons (PAHs). We recruited 82 PAH-exposed workers and 62 unexposed controls, assessed exposure levels by urinary 1-hydroxypyrene, and measured genetic damages by comet assay, bleomycin sensitivity, and micronucleus assay. The PAHs in coke oven emissions (COE) were estimated based on toxic equivalency factors. We used bisulfite-PCR pyrosequencing to quantitate DNA methylation in long interspersed nuclear element-1 (LINE-1) and O(6)-methylguanine-DNA methyltransferase (MGMT). Further, the methylation alteration was also investigated in COE-treated human bronchial epithelial (16HBE) cells. We found there are higher levels of PAHs in COE. Among PAH-exposed workers, LINE-1 and MGMT methylation levels (with CpG site specificity) were significantly lowered. LINE-1, MGMT, and its hot CpG site-specific methylation were negatively correlated with urinary 1-hydroxypyrene levels (r = -0.329, p < 0.001; r = -0.164, p = 0.049 and r = -0.176, p = 0.034, respectively). In addition, LINE-1 methylation was inversely associated with comet tail moment and micronucleus frequency, and a significant increase of micronucleus in low MGMT methylation group. In vitro study revealed that treatment of COE in 16HBE cells resulted in higher production of BPDE-DNA adducts, LINE-1 hypomethylation, hypomethylation, and suppression of MGMT expression. These findings suggest hypomethylation of LINE-1 and MGMT promoter could be used as markers for PAHs exposure and merit further investigation.
Assuntos
Instabilidade Genômica/efeitos dos fármacos , Linfócitos/fisiologia , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Compostos Policíclicos/toxicidade , Regiões Promotoras Genéticas/fisiologia , Adulto , Biomarcadores , Western Blotting , Células Cultivadas , China , Instabilidade Cromossômica/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Metilação de DNA , Humanos , Masculino , Metalurgia , Metilação , Mutagênicos/toxicidade , Exposição Ocupacional , Reação em Cadeia da Polimerase , Pirenos/urina , Aço , Sulfitos/farmacologiaRESUMO
Drug treatment of malignant gliomas is limited by the intrinsic resistance of glioma stem cells (GSCs) to chemotherapy. GSCs isolated from human glioblastoma multiforme (GBM) expressed metabotropic glutamate receptors (mGlu3 receptors). The DNA-alkylating agent, temozolomide, killed GSCs only if mGlu3 receptors were knocked down or pharmacologically inhibited. In contrast, mGlu3 receptor blockade did not affect the action of paclitaxel, etoposide, cis-platinum, and irinotecan. mGlu3 receptor blockade enabled temozolomide toxicity by inhibiting a phosphatidylinositol-3-kinase/nuclear factor-κB pathway that supports the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers resistance against DNA-alkylating agents. In mice implanted with GSCs into the brain, temozolomide combined with mGlu3 receptor blockade substantially reduced tumor growth. Finally, 87 patients with GBM undergoing surgery followed by adjuvant chemotherapy with temozolomide survived for longer time if tumor cells expressed low levels of mGlu3 receptors. In addition, the methylation state of the MGMT gene promoter in tumor extracts influenced survival only in those patients with low expression of mGlu3 receptors in the tumor. These data encourage the use of mGlu3 receptor antagonists as add-on drugs in the treatment of GBM, and suggest that the transcript of mGlu3 receptors should be measured in tumor specimens for a correct prediction of patients' survival in response to temozolomide treatment.
Assuntos
Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Aminoácidos/toxicidade , Animais , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Quimioterapia Adjuvante , Terapia Combinada , Metilação de DNA/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/mortalidade , Humanos , Camundongos , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/citologia , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/genética , Transdução de Sinais , Taxa de Sobrevida , Temozolomida , Transplante Heterólogo , Células Tumorais Cultivadas , Xantenos/toxicidadeRESUMO
DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable. Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O(6)-methylguanine-DNA-methyltransferase (MGMT), which repairs the O(6)-alkylguanine-type of adducts induced in DNA by alkylating genotoxins; and (2) apurinic/apyrimidinic endonuclease 1 (APE 1), which participates in base excision repair (BER) by causing a rate-limiting DNA strand cleavage 5' to the abasic sites. The MGMT assay makes use of the fact that: (a) the enzyme works by irreversibly transferring the alkyl group from the O(6) position of guanine to a cystein residue in its active site and thereby becomes inactivated and (b) that the free base O(6)-benzylguanine (BG) is a very good substrate for MGMT. In the new assay, cell extracts are incubated with BG tagged with biotin and the resulting MGMT-BG-biotin complex is immobilized on anti-MGMT-coated microtiter plates, followed by quantitation using streptavidin-conjugated alkaline phosphatase and a chemiluminescence-producing substrate. A one-step/one-tube phenotypic assay for APE1 activity has been developed based on the use of a fluorescent molecular beacon (partially self-complementary oligonucleotide with a hairpin-loop structure carrying a fluorophore and a quencher at each end). It also contains a single tetrahydrofuran residue (THF) which is recognized and cleaved by APE1, and the subsequently formed single-stranded oligomer becomes a fluorescence signal emitter. Both assays are highly sensitive, require very small amounts of protein extracts, are relatively inexpensive and can be easily automated. They have been extensively validated and are being used in the context of large-scale molecular epidemiology studies.
Assuntos
Enzimas Reparadoras do DNA/análise , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/análise , O(6)-Metilguanina-DNA Metiltransferase/análise , Extratos Celulares , Dano ao DNA , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Epidemiologia Molecular/tendências , Mutagênicos/toxicidade , Estudos de Validação como AssuntoRESUMO
Genotyping of putative determinants of temozolomide (TMZ)-induced life-threatening bone marrow suppression was performed in two patients with glioma treated with adjuvant TMZ and radiation therapy. DNA was extracted from the patients' mononuclear cells and genotyping of O-methylguanine-DNA-methyltransferase (MGMT), multidrug resistance (MDR1; also known as ABCB1), NQO1, and GSTP1 genes and analysis for the epigenetic silencing of specific MGMT gene promoters were carried out to evaluate the possible genetic determinants of increased risk of severe TMZ-induced myelosuppression. Although both patients were heterozygous for all ABCB1 single nucleotide polymorphisms and for rs12917 and rs1803965 in the MGMT gene, patient 1 was heterozygous for rs1695 in GSTP1 and rs2308327 in the MGMT gene. This patient also exhibited GG genotype for the MGMT single nucleotide polymorphisms, rs2308321, which is noteworthy for its 0.7% frequency globally. Epigenetic silencing of MGMT gene was not detected in either patient. Two single nucleotide polymorphisms identified in patient 1 (missense I143V and K178R polymorphisms; rs2308321 and rs2308327, respectively) have recently been shown to correlate with an increased risk of severe TMZ-induced myelosuppression. The polymorphisms identified in patient 2 have not been associated with an increased risk of severe TMZ-induced myelosuppression. Genotyping analyses of larger patient populations administered TMZ are required to validate the genetic determinants of severe TMZ-induced myelosuppression.
Assuntos
Antineoplásicos Alquilantes/efeitos adversos , Dacarbazina/análogos & derivados , Glioma/tratamento farmacológico , Transtornos Mieloproliferativos/induzido quimicamente , Transtornos Mieloproliferativos/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Antineoplásicos Alquilantes/farmacologia , Dacarbazina/efeitos adversos , Dacarbazina/uso terapêutico , Feminino , Glioma/enzimologia , Glioma/genética , Glutationa S-Transferase pi/genética , Humanos , Masculino , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias Neuroepiteliomatosas/complicações , Neoplasias Neuroepiteliomatosas/terapia , O(6)-Metilguanina-DNA Metiltransferase/genética , Pacientes , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , TemozolomidaRESUMO
The purpose of this study was to assess the potential of boron neutron capture therapy (BNCT), with a 6-h infusion of the boron carrier l-boronophenylalanine as a fructose preparation (BPA-f), as first-line radiotherapy for newly diagnosed glioblastoma multiforme (GBM). Patient survival data from a Phase II study using BNCT were compared with retrospective data from the two arms of a Phase III study using conventional radiotherapy (RT) in the reference arm and using RT plus concomitant and adjuvant medication with temozolomide (TMZ) in the experimental arm, and were also compared with small subgroups of these patients for whom the methylation status of the MGMT (O(6)-methylguanine-DNA methyltransferase) DNA repair gene was known. Differences in the baseline characteristics, salvage therapy after recurrence and levels of severe adverse events were also considered. The results indicate that BNCT offers a treatment that is at least as effective as conventional RT alone. For patients with an unmethylated MGMT DNA repair gene, a possible clinical advantage of BNCT over RT/TMZ was suggested. BNCT is a single-day treatment, which is of convenience to patients, with mild side effects, which would offer an initial 6 weeks of good-quality life during the time when patients would otherwise be undergoing daily treatments with RT and TMZ. It is suggested that the use of BNCT with a 6-h infusion of BPA-f should be explored in a stratified randomised Phase II trial in which patients with the unmethylated MGMT DNA repair gene are offered BNCT in the experimental arm and RT plus TMZ in the reference arm.
Assuntos
Compostos de Boro/uso terapêutico , Terapia por Captura de Nêutron de Boro , Frutose/análogos & derivados , Glioblastoma/radioterapia , Adulto , Idoso , Antineoplásicos Alquilantes/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Intervalo Livre de Doença , Frutose/uso terapêutico , Glioblastoma/tratamento farmacológico , Humanos , Pessoa de Meia-Idade , O(6)-Metilguanina-DNA Metiltransferase/genética , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Temozolomida , Resultado do Tratamento , Adulto JovemRESUMO
Epigenetic alteration of DNA methylation plays an important role in the regulation of gene expression associated with chemosensitivity of human hepatocellular (HCC) carcinoma cells. With the aim of improving the chemotherapeutic efficacy for HCC, the effect of the naturally occurring compound n-butylidenephthalide (BP), which is isolated from a chloroform extract of Angelica sinensis, was investigated. In both HepG2 and J5 HCC cell lines, a synergistic antiproliferative effect was observed when a low dosage of BP was combined with the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is an alkylating agent, and it prompts us to examine one of DNA repair genes, O(6)-methylguanine methyltransferase (MGMT). It was evident from methylation-specific polymerase chain reaction (PCR) analysis that BP/BCNU combined treatment caused a time- and concentration-dependent enhancement of MGMT promoter methylation. Overexpression of MGMT could abolish BP-induced growth inhibition in the J5 tumor cell line as measured by colony formation assay. When BP was combined with BCNU and administered, it showed significant antitumor effects in both HepG2 and J5 xenograft tumors as compared with the use of only one of these drugs. The BCNU-induced apoptosis and inhibited MGMT protein expression in HCC cells, both in vitro and in vivo, resulting from the combination treatment of BP and BCNU suggest a potential clinical use of this compound for improving the prognosis for HCCs.
Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/fisiopatologia , Carmustina/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Metilação de DNA , Neoplasias Hepáticas/fisiopatologia , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Anidridos Ftálicos/administração & dosagem , Angelica sinensis/química , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Extratos Vegetais/administração & dosagemRESUMO
Micro contact printing (microCP) has been established as a simple technique for high-resolution protein patterning for micro- and nanoarrays. However, as biochemical assays based on immobilized protein arrays progress from immunoassays to more delicate functional assays, the demand for methods of miniaturized, gentle, and oriented immobilization, which are applicable to many different target proteins, becomes larger. In this study, we present a novel microCP templated assembly approach, based on a recombinant SNAP-FLAG-HIS 10 (SFH) immobilization vehicle, which exploits the recently developed SNAP-tag protein. The SNAP-tag is derived from the human DNA repair protein hAGT, which covalently transfers the alkyl group of benzyl guanine (BG) substrates onto itself. We have designed a model SFH cassette carrying three tags (SNAP-tag, FLAG-tag, and HIS-tag), each of which can be used for fluorescence labeling or surface immobilization. When patterns of streptavidin modified with BG-biotin (streptavidin-BG) are stamped onto a surface, the SFH can subsequently assemble on the ligand pattern from solution, functioning as a general immobilization vehicle for high-resolution patterning of any protein expressed in the SFH cassette, in a gentle and oriented manner. Alternatively, the SFH can be site-selectively biotinylated using BG-biotin and, subsequently, assemble on stamped streptavidin. We exploit several ways to biotinylate the SFH protein via the SNAP-tag, promoting its templated assembly on micropatterns of streptavidin in four complementary formats. Quantitative analysis of the obtained patterns, revealed by immunostaining, indicates that all four approaches resulted in proper SFH immobilization and antibody recognition, demonstrating the versatility of the SFH cassette and the potential for high resolution patterning applications. Also, our data confirm that streptavidin can be stamped directly on surfaces, without loss of activity. While three strategies resulted in similar patterning efficiencies, one particular approach--namely templated assembly of SFH directly on streptavidin-BG patterns--resulted in an order of magnitude increase in patterning efficiency.
Assuntos
Imunoensaio/métodos , Biotina/química , Biotinilação , Clonagem Molecular , Reparo do DNA , Escherichia coli/metabolismo , Humanos , Ligantes , Microscopia Confocal , Microscopia de Fluorescência , Biologia Molecular , O(6)-Metilguanina-DNA Metiltransferase/química , Proteínas Recombinantes/química , Estreptavidina/química , Propriedades de SuperfícieRESUMO
VNP40101M is a novel alkylating agent that yields two reactive compounds (a chloroethylating species and methylisocyanate) and has demonstrated activity against a wide spectrum of tumor xenografts. VNP40101M was tested against an in vivo panel of five pediatric brain tumor xenografts at a dose of 18 mg/kg/day administered for 5 days. O-6-methylguanine-DNA methyltransferase (MGMT) levels of xenografts were assessed by Western blot analysis. Only one xenograft (GBM2), which lacked detectable MGMT expression, demonstrated an objective response to VNP40101M. VNP4010M antitumor activity was observed only in the absence of MGMT expression, with resistance to VNP4010M seen even with low MGMT expression.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Hidrazinas/farmacologia , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Antineoplásicos Alquilantes , Avaliação Pré-Clínica de Medicamentos , Feminino , Glioblastoma , Glioma , Hidrazinas/administração & dosagem , Camundongos , Camundongos Endogâmicos , Sulfonamidas/administração & dosagem , Transplante Heterólogo , Resultado do TratamentoRESUMO
O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme whose expression is controlled by its promoter methylation. A cell that expresses a low amount of MGMT is known to be more sensitive to the antiproliferative effects of alkylating agents. We have previously shown that the colorectal cancer patients treated with 5-fluorouracil (5-FU) as adjuvant chemotherapy had a better prognosis when the tumor revealed hypermethylation in its MGMT promoter. Therefore, we sought to investigate the relationship between the expression levels of MGMT and the anti-tumor effect of 5-FU in vitro by using two colon adenocarcinoma and four oral cancer cell lines with a variety of MGMT expression. We also investigated the effects of MGMT depletion by O6-benzylguanine (O6-BG), a potent inhibitor of MGMT. The 5-FU treatment uniformly depleted protein and mRNA expression of MGMT in all cell lines examined. Cell lines expressing low levels of MGMT were sensitive to 5-FU. On the other hand, cells expressing high levels of MGMT were less sensitive to 5-FU. The 5-FU treatment exhibited a better antiproliferative effect on the cells expressing high levels of MGMT by the pretreatment of O6-BG. Depletion of MGMT by O6-BG enhanced the anti-tumor effect of 5-FU. Assessment of the levels of MGMT expression in cancer cells and the control of its expression could contribute to the effective chemotherapy by 5-FU especially in patients who previously were considered as low-responsive individuals whose tumors have high levels of MGMT.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/enzimologia , Inibidores Enzimáticos/farmacologia , Fluoruracila/farmacologia , Guanina/análogos & derivados , Neoplasias Bucais/enzimologia , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Guanina/farmacologia , Humanos , O(6)-Metilguanina-DNA Metiltransferase/análise , O(6)-Metilguanina-DNA Metiltransferase/metabolismoRESUMO
O6-alkylguanines are potent mutagenic, pro-carcinogenic and cytotoxic lesions induced by exogenous and endogenous alkylating agents. A facilitated elimination of these lesions by increasing the activity of O6-methylguanine-DNA methyltransferase (MGMT) is likely to be a beneficial chemoprevention strategy, which, however, has not been examined. Because, a marginal enhancement of this protein may be adequate for genomic protection, we studied alterations in MGMT activity and expression in human peripheral blood lymphocytes and cancer cell lines induced by water-soluble and alcohol-soluble constituents of several plants with established antioxidant and medicinal properties. Both the ethanolic and aqueous extracts from neem (Azadirachta indica), holy basil (Ocimum sanctum), winter cherry (Withania somnifera), and oregano (Origanum majorana) increased the levels of MGMT protein and its demethylation activity in a time-dependent manner with a maximum of 3-fold increase after 72-h treatment. The extracts from gooseberry (Emblica officinalis), common basil (Ocimum basilicum), and spearmint (Mentha viridis) were relatively less efficient in raising MGMT levels. Increased levels of MGMT mRNA accounted at least, in part, for the increased activity of the DNA repair protein. The herbal treatments also increased glutathione S-transferase-pi (GSTP1) expression, albeit to a lesser extent than MGMT. These data provide the first evidence for the upregulation of human MGMT by plant constituents and raise the possibility of rational dietary approaches for attenuating alkylation-induced carcinogenesis. Further, they reveal the putative antioxidant responsiveness of the MGMT gene in human cells.
Assuntos
Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Magnoliopsida/química , Neoplasias/prevenção & controle , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Extratos Vegetais/uso terapêutico , Linhagem Celular Tumoral , Quimioprevenção , Etanol/química , Expressão Gênica/efeitos dos fármacos , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Neoplasias/enzimologia , Neoplasias/genética , O(6)-Metilguanina-DNA Metiltransferase/análise , O(6)-Metilguanina-DNA Metiltransferase/genética , Extratos Vegetais/química , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Água/químicaRESUMO
To find the possible association of gene methylation of p16(INK4a) and O(6)-Methylguanine-DNA Methyltransferase (O(6)-MGMT) with occupational exposure to radon, 91 male miners from a uranium mine in China were divided into 4 groups according to the cumulative doses of radon exposure from 2 to 425 WLM (working-level months), and aberrant promoter methylation of p16(INK4a) and O(6)-MGMT genes in sputum samples was determined by a specific PCR assay. The results revealed that the methylated rates of 16(INK4a) gene (z=2.844, P=0.005) and O(6)-MGMT gene (z=3.034, P=0.002), and the total methylated rate of these two genes (z=3.859, P=0.0001) increased significantly with the cumulative doses of radon among the miners. This methylation could be applied as a potential marker for the detection of early DNA damage induced by occupational radon exposure.
Assuntos
Genes p16/efeitos da radiação , Metilação/efeitos da radiação , Mineração , O(6)-Metilguanina-DNA Metiltransferase/genética , Exposição Ocupacional , Urânio , Adulto , China , Humanos , Masculino , Pessoa de Meia-Idade , O(6)-Metilguanina-DNA Metiltransferase/efeitos da radiação , Radônio/análiseRESUMO
PURPOSE: An inverse correlation has been established between tumor levels of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) and a positive outcome after alkylator chemotherapy. Quantitative imaging of AGT could provide important information for patient-specific cancer treatment. Several radiolabeled analogues of O6-benzylguanine (BG), a potent AGT inactivator, have been developed and shown to be capable of labeling pure AGT protein. Herein, two of these analogues--O6-3-[*I]iodobenzylguanine ([*I]IBG) and O6-3-[*I]iodobenzyl-2'-deoxyguanosine ([*I]IBdG)--were further evaluated in two murine xenograft models. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, also was investigated as an alternative agent. METHODS: Several biodistribution studies of radioiodinated IBG and IBdG were performed in TE-671 human rhabdomyosarcoma and DAOY human medulloblastoma murine xenograft models. Mice were treated with BG or its nucleoside analogue dBG to deplete the tumor AGT content. The effect of unlabeled IBG and that of 7,8-benzoflavone (BF), an inhibitor of the cytochrome P-450 isozyme CYP1A2, on the tumor uptake of the tracers was determined. The uptake of (AcO)2-[131I]IBdG along with that of [125I]IBdG in DAOY cells in vitro was determined in the presence and absence of a nucleoside transporter inhibitor, dipyridamole. RESULTS: Pretreatment of mice either with BG or dBG failed to reduce tumor levels of [*I]IBG or [*I]IBdG even though such treatments completely depleted tumor AGT content. Treatment of mice with BF increased tumor uptake of [125I]IBG by 56%; however, differentiation of tumors with and without AGT still was not possible. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, had a higher uptake in vitro in DAOY tumor cells. However, its uptake, like that of [125I]IBdG, was blocked by dipyridamole. CONCLUSIONS: Taken together, these results suggest that labeled agents that are more specific for cellular AGT and that are more metabolically stable are needed.
Assuntos
Guanina/análogos & derivados , Radioisótopos do Iodo/farmacocinética , Meduloblastoma/diagnóstico por imagem , Rabdomiossarcoma/diagnóstico por imagem , Animais , Benzoflavonas/farmacologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Guanina/farmacocinética , Humanos , Masculino , Meduloblastoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Cintilografia , Rabdomiossarcoma/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
PURPOSE: We have previously shown the reactivation of some methylation-silenced genes in cancer cells by (-)-epigallocatechin-3-gallate, the major polyphenol from green tea. To determine whether other polyphenolic compounds have similar activities, we studied the effects of soy isoflavones on DNA methylation. EXPERIMENTAL DESIGN: Enzyme assay was used to determine the inhibitory effect of genistein on DNA methyltransferase activity in nuclear extracts and purified recombinant enzyme. Methylation-specific PCR and quantitative real-time PCR were employed to examine the DNA methylation and gene expression status of retinoic acid receptor beta (RARbeta), p16INK4a, and O6-methylguanine methyltransferase (MGMT) in KYSE 510 esophageal squamous cell carcinoma cells treated with genistein alone or in combination with trichostatin, sulforaphane, or 2'-deoxy-5-aza-cytidine (5-aza-dCyd). RESULTS: Genistein (2-20 micromol/L) reversed DNA hypermethylation and reactivated RARbeta, p16INK4a, and MGMT in KYSE 510 cells. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARbeta by genistein were also observed in KYSE 150 cells and prostate cancer LNCaP and PC3 cells. Genistein (20-50 micromol/L) dose-dependently inhibited DNA methyltransferase activity, showing substrate- and methyl donor-dependent inhibition. Biochanin A and daidzein were less effective in inhibiting DNA methyltransferase activity, in reactivating RARbeta, and in inhibiting cancer cell growth. In combination with trichostatin, sulforaphane, or 5-aza-dCyd, genistein enhanced reactivation of these genes and inhibition of cell growth. CONCLUSIONS: These results indicate that genistein and related soy isoflavones reactivate methylation-silenced genes, partially through a direct inhibition of DNA methyltransferase, which may contribute to the chemopreventive activity of dietary isoflavones.
Assuntos
Anticarcinógenos/farmacologia , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA , Neoplasias Esofágicas/tratamento farmacológico , Genisteína/farmacologia , Glycine max/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Receptores do Ácido Retinoico/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Decitabina , Relação Dose-Resposta a Droga , Neoplasias Esofágicas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoflavonas/química , Isoflavonas/farmacologia , Isotiocianatos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfitos/farmacologia , Sulfóxidos , Chá , Tiocianatos/farmacologiaRESUMO
O(6)-methylguanine DNA methyl-transferase (MGMT) is the only known critical gene involved in cellular defense against alkylating agents in the DNA direct reversal repair (DRR) pathway. Three single nucleotide polymorphism (SNP) coding for non-conservative amino acid substitutions have been identified [C250T (Leu84Phe), A427G (Ile143Val) and A533G (Lys178Arg)]. To examine the importance of the DRR pathway in risk for breast cancer and the potential interaction with cigarette smoking and dietary antioxidants, we genotyped for these variants using biospecimens from the Long Island Breast Cancer Study Project. Genotyping was performed by a high throughput assay with fluorescence polarization and included 1067 cases and 1110 controls. Overall, there was no main effect between any variant genotype, haplotype or diplotype and breast cancer risk. Heavy smoking (>31 pack-year) significantly increased breast cancer risk for women with the codon 84 variant T-allele [odds ratio, OR = 3.0, 95% confidence interval (95% CI) = 1.4-6.2]. An inverse association between fruits and vegetables consumption and breast cancer risk was observed among women with the wild-type genotype for codon 84 (OR = 0.8, 95% CI = 0.6-0.9 for > or =35 servings of fruits and vegetables per week and CC genotype versus those with <35 servings per week and CC genotype). The association between fruits and vegetables consumption and reduced breast cancer risk was apparent among women with at least one variant allele for codon 143 (OR = 0.6, 95% CI = 0.5-0.9 for > or =35 servings of fruits and vegetables per week and AG or GG genotype versus those with <35 servings per week and AA genotype). Similar patterns were observed for dietary alpha-carotene and supplemental beta-carotene, but not for supplemental vitamins C and E. These data suggest that polymorphisms in MGMT may modulate the inverse association previously observed between fruits and vegetables consumption, dietary antioxidants and breast cancer risk, and support the importance of fruits and vegetables on breast cancer risk reduction.
Assuntos
Antioxidantes/metabolismo , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Dieta , O(6)-Metilguanina-DNA Metiltransferase/genética , Polimorfismo de Nucleotídeo Único , Fumar , Adulto , Idoso , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Suplementos Nutricionais , Feminino , Fluorescência , Frutas , Genótipo , Haplótipos/genética , Humanos , Menopausa , Pessoa de Meia-Idade , New York/epidemiologia , Fatores de Risco , VerdurasRESUMO
O(6)-Benzylguanine derivatives with suitable radionuclides attached to the benzyl ring are potentially useful in the noninvasive imaging of the DNA repair protein, alkylguanine-DNA alkyltransferase (AGT). Previously, O(6)-3-[(131)I]iodobenzylguanine ([(131)I]IBG) was prepared using a two-step approach; we now report its synthesis in a single step by the radioiododestannylation of O(6)-3-(trimethylstannyl)benzylguanine in 85-95% radiochemical yield. The in vitro specific uptake of [(131)I]IBG in DAOY human medulloblastoma cells, in TE-671 human rhabdomyosarcoma cells and a CHO cell line transfected to express AGT was linear (r(2) = 0.9-1.0) as a function of cell density. After intravenous injection of [(131)I]IBG in athymic mice bearing TE-671 xenografts, tumor uptake was 1.38 +/- 0.34% ID/g at 0.5 h and declined at 2 and 4 h. Preadministration of O(6)-(3-iodobenzyl)guanine (IBG) at 0.5 h increased uptake not only in tumor but also in several normal tissues. Notable exceptions were thyroid (p < 0.05), lung (p <0.05) and stomach. After intratumoral injection of [(131)I]IBG in the same xenograft model, the uptake in tumors that were depleted of AGT by BG treatment (165.8 +/- 27.5% ID/g) was about 60% of that in control mice (272.4 +/- 48.2% ID/g; p < 0.05).