RESUMO
Methylation-specific polymerase chain reaction (MSP) for the promoter methylation status of O(6)-methylguanine-DNA-methyltranferase (MGMT) gene theoretically provides a positive or negative result. However, the faint MSP product is difficult to interpret. The aim of this study was to evaluate the significance of faint MSP product in glioblastoma (GBM). Critical concentrations of methylated control DNA, i.e., 100, 1, 0.5 and 0 % were run parallel with 116 newly diagnosed GBMs in order to standardize the interpretation and to distinguish positive (+), equivocal (±), and negative (-; unmethylated) results. Cases with the faint MSP product and its intensity between those of 1 and 0.5 % DNA controls were considered equivocal (±). MGMT methylation quantifications were also determined by quantitative real-time MSP (qMSP) and pyrosequencing (PSQ), and protein expression was detected by immunohistochemistry. There were significant correlations between MSP and all the aforementioned studies. The concordance rates between the MSP+ and qMSP+ cases, as well as the MSP- and qMSP- cases were 100 %, and the MSP± cases comprised 76.5 % of qMSP+ cases and 23.5 % of qMSP- cases. PSQ study showed that heterogeneous methylation was more frequently encountered in the MSP± cases. Multivariate analyses disclosed that although the overall survival of the MSP± cases was indistinct from that of the MSP+ cases, its progression free survival was significantly worse and was indistinct from that of the MSP- cases. In conclusion, GBMs with faint MGMT MSP products should be distinguished from MSP+ cases as their behaviors were different.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/genética , Glioblastoma/mortalidade , Reação em Cadeia da Polimerase/métodos , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Seguimentos , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Taxa de Sobrevida , Proteínas Supressoras de Tumor/metabolismo , Adulto JovemRESUMO
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Patients diagnosed with GBM have a poor prognosis, and it has been reported that tumor malignancy and GBM recurrence are promoted by STAT3 signaling. As resveratrol (RV), a polyphenol in grapes, is reported to be a potent and non-toxic cancer-preventive compound, the aim of this study was to investigate the therapeutic effect and molecular mechanisms of RV on GBM-derived radioresistant tumor initiating cells (TIC). Firstly, our results showed that primary GBM-CD133(+) TIC presented high tumorigenic and radiochemoresistant properties as well as increased protein levels of phosphorylated STAT3. We consistently observed that treatment with shRNA-STAT3 (sh-STAT3) or AG490, a STAT3 inhibitor, significantly inhibited the cancer stem-like cell properties and radioresistance of GBM-CD133(+) in vitro and in vivo. Furthermore, treatment of GBM-CD133(+) with 100 µM RV induced apoptosis and enhanced radiosensitivity by suppressing STAT3 signaling. Microarray results suggested that RV or AG490 inhibited the stemness gene signatures of GBM-CD133(+) and facilitated the differentiation of GBM-CD133(+) into GBM-CD133(-) or astrocytoma cells. Finally, xenotransplant experiments indicated that RV or sh-STAT3 therapy could significantly improve the survival rate and synergistically enhance the radiosensitivity of radiation-treated GBM-TIC. In summary, RV can reduce in vivo tumorigenicity and enhance the sensitivity of GBM-TIC to radiotherapies through the STAT3 pathway.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Fator de Transcrição STAT3/antagonistas & inibidores , Estilbenos/farmacologia , Idoso , Animais , Antineoplásicos Fitogênicos/farmacologia , Astrocitoma/tratamento farmacológico , Astrocitoma/patologia , Astrocitoma/radioterapia , Neoplasias Encefálicas/patologia , Quimiorradioterapia/métodos , Feminino , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Resveratrol , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The O-methylguanine-DNA-methyltranferase (MGMT) status is a powerful predictor of response to temozolomide for newly diagnosed glioblastoma (GBM) patients, and it is commonly assessed by immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP), quantitative real-time MSP (qMSP), and/or pyrosequencing (PSQ). This study was to compare their predictive power of prognosis in 121 newly diagnosed GBM patients using multivariate Cox regression with bootstrapping. MGMT status tested by IHC, MSP, qMSP, or PSQ all showed significant correlation with the progression-free survival and overall survival of GBM patients. The predictive power of IHC for progression-free survival and overall survival was lower than those of the methylation assays, but their differences were not significant. Performing additional methylation assay, especially PSQ, could better predict the prognosis of patients with IHC- tumors. MGMT status tested by IHC, MSP, qMSP, or PSQ all showed prognostic significance. An additional MGMT methylation assay, of which PSQ appeared to be the best, could improve the predictive power for GBM patients with MGMT IHC- tumors.
Assuntos
Neoplasias Encefálicas , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Glioblastoma , Proteínas de Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Intervalo Livre de Doença , Feminino , Glioblastoma/enzimologia , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Taxa de SobrevidaRESUMO
Positron emission tomography (PET) allows examination of a variety of physiological parameter, including blood flow, glucose, amino acid and oxygen metabolism. However, correlation of PET scan findings and the degree of malignancy of intracerebral tumors continues to be controversial. Nine patients with primarily diagnosed intraparenchymal brain tumors were included in this study. We performed 11C-methionine-PET (met-PET) prior to surgical treatment and the differential absorption ratio (DAR) was calculated. All patients underwent open or stereotactic surgery and specimens for pathological diagnosis were obtained. The biological activity of each tumor was determined by calculation of the proliferation index from MIB-1 immunohistochemistry. The DAR of met-PET for individual tumors correlated with the histological diagnosis and degree of malignancy and this was further confirmed by good correlation with the MIB-1 proliferation index. We conclude that met-PET may be a reliable and effective preoperative evaluation to determine the type and malignancy of intraparenchymal brain lesions.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Metionina/farmacocinética , Tomografia por Emissão de Pósitrons , Idoso , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Antígeno Ki-67/metabolismo , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Tomografia Computadorizada de Emissão/métodos , TrítioRESUMO
We evaluated the predictive value of O6-methylguanine-DNA methyltransferase (MGMT) protein expression and MGMT promoter methylation status in glioblastomas (GBM) treated with temozolomide (TMZ) in a Taiwan medical center. Protein expression by immunohistochemical analysis (IHC) and MGMT promoter methylation detected by methylation-specific polymerase chain reaction (MSP) were performed in a series of 107 newly diagnosed GBMs. We used endothelial cells as an internal reference for IHC staining because the staining intensities of the MGMT-expressing cells in different specimens varied considerably; a positive result was defined as the staining intensity of the majority of tumor cells similar to that of the adjacent endothelial cells. Immunostainings for microglial/endothelial markers were included as part of the MGMT IHC evaluation, and in cases that were difficult to interpret, double-labeling helped to clarify the nature of reactive cells. The MGMT protein expression was reversely associated with MGMT promoter methylation status in 83.7% of cases (MSP/IHC and MSP/IHC; Pearson r=-0.644, P<0.001). Twenty-two of 24 (91.7%) IHC tumors did not respond to TMZ treatment. Combining MSP and IHC results, all the 15 MSP/IHC GBMs were TMZ resistant. The MGMT status detected by either IHC or MSP was significantly correlated with the TMZ treatment response (both P<0.001) and survival of GBM patients (both P<0.05).
Assuntos
Neoplasias Encefálicas/diagnóstico , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Endotélio Vascular/patologia , Glioblastoma/diagnóstico , Histiócitos/patologia , Proteínas Supressoras de Tumor/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Alquilantes/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Histiócitos/efeitos dos fármacos , Histiócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Coloração e Rotulagem/métodos , Taxa de Sobrevida , Taiwan/epidemiologia , Temozolomida , Resultado do Tratamento , Proteínas Supressoras de Tumor/genética , Adulto JovemRESUMO
BACKGROUND: 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin-9-ylamino)phenyl]urea (BO-1051) is an N-mustard DNA alkylating agent reported to exhibit antitumor activity. Here we further investigate the effects of this compound on radiation responses of human gliomas, which are notorious for the high resistance to radiotherapy. METHODS: The clonogenic assay was used to determine the IC50 and radiosensitivity of human glioma cell lines (U87MG, U251MG and GBM-3) following BO-1051. DNA histogram and propidium iodide-Annexin V staining were used to determine the cell cycle distribution and the apoptosis, respectively. DNA damage and repair state were determined by γ-H2AX foci, and mitotic catastrophe was measure using nuclear fragmentation. Xenograft tumors were measured with a caliper, and the survival rate was determined using Kaplan-Meier method. RESULTS: BO-1051 inhibited growth of human gliomas in a dose- and time-dependent manner. Using the dosage at IC50, BO-1051 significantly enhanced radiosensitivity to different extents [The sensitizer enhancement ratio was between 1.24 and 1.50 at 10% of survival fraction]. The radiosensitive G2/M population was raised by BO-1051, whereas apoptosis and mitotic catastrophe were not affected. γ-H2AX foci was greatly increased and sustained by combined BO-1051 and γ-rays, suggested that DNA damage or repair capacity was impaired during treatment. In vivo studies further demonstrated that BO-1051 enhanced the radiotherapeutic effects on GBM-3-beared xenograft tumors, by which the sensitizer enhancement ratio was 1.97. The survival rate of treated mice was also increased accordingly. CONCLUSIONS: These results indicate that BO-1051 can effectively enhance glioma cell radiosensitivity in vitro and in vivo. It suggests that BO-1051 is a potent radiosensitizer for treating human glioma cells.
Assuntos
Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Compostos de Mostarda Nitrogenada/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/efeitos da radiação , Relação Dose-Resposta a Droga , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Modelos Biológicos , Radiossensibilizantes/farmacologia , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A chemoenzymatic approach has been developed to synthesize poly(ethylene glycol)-based amphiphilic copolymers under mild reaction conditions that self-assemble in aqueous media to form polymeric nanomicelles in the range of 20-50 nm. The supramolecular organization of polymeric nanomicelles was studied by 1H NMR longitudinal relaxation time (T1) and light scattering techniques (static and dynamic). Interestingly, the enzyme novozyme-435 plays an important role in controlling the polymerization and distribution of polymer chains, which is critical for the formation of nanomicelles with unimodal distributions. The methodology developed is highly flexible as it allows the introduction of various functionalities in the polymeric nanomicelles. These self-organized nanomicelles are highly efficient drug delivery vehicles for hydrophobic and partially hydrophilic drugs, both transdermally and orally, as they have the ability to encapsulate guest molecules during self-organization. In vivo studies by encapsulating anti-inflammatory agents (aspirin and naproxen) in these polymeric nanomicelles and by applying topically resulted in significant reduction in inflammation. The % reduction in inflammation using polymeric nanomicelles containing aspirin and naproxen was 62 and 64%, respectively.