RESUMO
BACKGROUND: The aim of this study is to evaluate local control rates after stereotactic body radiotherapy (SBRT) in recurrent spinal metastasis after external beam radiotherapy (EBRT) and new spinal metastatic lesions. MATERIAL AND METHODS: Retrospective review of medical records and radiological data was performed on 54 retreatment and 131 initial SBRT patients. To compare various fractionation schedules, the biologically effective dose (BED) was applied. SBRT dose was calculated with linear-quadratic model and normalized to a 2-Gy equivalent dose (nBED, α/ß =2 Gy for spinal cord, α/ß =10 Gy for tumor). Doses to a point within the spinal cord that received the maximum dose (Pmax) were checked. Local control failure was defined as progression by imaging study. Overall survival, progression free survival, delivered radiation dose to tumor and spinal cord, and spinal cord Pmax nBED were compared in two groups. RESULTS: The mean delivered radiation doses to tumor margin during SBRT were 51.1 Gy2/10 (retreatment) and 50.7 Gy2/10 (initial treatment). Mean survival was 29.6 months (overall)/20.7 months (retreatment)/ 32.4 months (initial treatment). Mean progression free period was 23.9 months (overall)/18.0 months (retreatment)/ 26.0 months (initial treatment). Radiological control rates of retreatment and initial treatment group were 96%/95% at six months, 81%/89% at 12 months and 79%/90% at 24 months. Among 54 retreatment lesions, 13 lesions showed local control failure during follow-up. With regard to spinal cord radiation dose during SBRT, Spinal cord Pmax nBED was 46.2 Gy2/2 (retreatment) and 48.7 Gy2/2 (initial treatment). In retreatment group, total nBED to spinal cord was a mean of 83.4 Gy2/2. There was no case of radiation myelopathy detected. CONCLUSIONS: Retreatment of spinal metastases using SBRT provided effective local control without neurological complications.
Assuntos
Neoplasias da Mama/cirurgia , Neoplasias da Próstata/cirurgia , Radiocirurgia , Neoplasias da Coluna Vertebral/secundário , Neoplasias da Coluna Vertebral/cirurgia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Feminino , Seguimentos , Humanos , Masculino , Prontuários Médicos , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Lesões por Radiação , Tolerância a Radiação , Eficiência Biológica Relativa , Retratamento , Estudos Retrospectivos , Neoplasias da Coluna Vertebral/mortalidade , Taxa de SobrevidaRESUMO
This study evaluated clinical outcome and safety of radiosurgery using the Cyberknife for treatment of benign spinal tumors. The authors treated 30 benign spinal tumors in 20 patients with the Cyberknife (Accuray, Inc., Sunnyvale, CA, USA) from 2002 to 2008. Among these there were 20 neurogenic tumors, eight hemangioblastomas, and two meningiomas. Four patients with neurofibromatosis (NF) type 2 and four patients with Von Hippel Lindau disease were also included. Radiosurgery was done as primary treatment for 22 lesions, for postoperative residual tumor control for four lesions, and for the remaining four lesions with image-based progression after initial subtotal resection. The distribution of lesions was cervical (18 tumors), thoracic (six), and cauda equina level (six). Follow-up data included imaging studies, clinical findings, and radiotherapy data. Tumor volume ranged from 0.04 to 33.65 cm³ (mean, 4.52 cm³). A 14-33 Gy marginal dose was delivered in 1-5 fractions. The mean follow-up period was 35.6 months (range, 12-84 months). On follow-up, most lesions decreased in size (57%) or remained unchanged (33%). Two lesions initially decreased, then increased later. One lesion increased without response. With regard to clinical aspects, radicular pain and myelopathic pain improved after radiosurgery in most cases (94%). Motor weakness recovered in two out of five patients and recovery of sensory change occurred in four out of ten patients. In two patients, symptoms were aggravated by tumor enlargement and the occurrence of new lesion. Mean spinal cord volumes receiving more than 10 and 8 Gy were 0.40 ± 0.4 and 0.81 ± 0.7 cm³, respectively. Stereotactic radiosurgery (SRS) using the Cyberknife showed the ability to control benign spinal tumors without complication in most cases.
Assuntos
Radiocirurgia , Neoplasias da Medula Espinal/cirurgia , Adolescente , Adulto , Idoso , Feminino , Hemangioblastoma/patologia , Hemangioblastoma/cirurgia , Humanos , Coreia (Geográfico) , Masculino , Pessoa de Meia-Idade , Neurilemoma/patologia , Neurilemoma/cirurgia , Neurofibroma/patologia , Neurofibroma/cirurgia , Recuperação de Função Fisiológica , Neoplasias da Medula Espinal/patologia , Resultado do Tratamento , Adulto JovemRESUMO
A prospective randomized controlled multicenter phase III trial was conducted to evaluate the effects of neoadjuvant chemotherapy with nimustine (ACNU)-cisplatin (CDDP) when used in conjunction with radiotherapy plus adjuvant temozolomide in patients with newly diagnosed glioblastoma. The study population was randomly assigned into one treatment and one control group. Both groups received radiotherapy followed by six cycles of adjuvant oral temozolomide (150-200 mg/m(2)) for 5 days every 28 days after surgery. Prior to radiotherapy, the treatment group also received two cycles, 6 weeks apart, of neoadjuvant chemotherapy with ACNU (40 mg/m(2)/day) and CDDP (40 mg/m(2)/day) infused continuously for 72 h. The primary end-point was median survival time. The study has closed after interim analysis with a total of 82 patients (48.8% of target number) due to unacceptable high frequency of toxicity profiles in spite of the promising actuarial survival outcome. Median survival time was 28.4 months [90% confidence interval (CI), 21.1 months to not available] in the treatment group and 18.9 months (90% CI, 17.1-27.4 months) in the control group (P = 0.2). The 2-year survival rate and progression-free survival time were 50.9% and 6.6 months (90% CI, 3.5-9.5 months) in the treatment group and 27.8% and 5.1 months (90% CI, 3.8-8.8 months) in the control group. Grade 3 or 4 toxicity was documented in 26 (68.4%) patients in the treatment group, including three neutropenic fever and one death from sepsis, while grade 3 or 4 toxicity occurred in 6 patients (15.8%) in the control group. The high frequency of serious hematological toxicity with ACNU-CDDP neoadjuvant chemotherapy followed by radiotherapy and adjuvant temozolomide limits its usage as primary treatment for glioblastoma. Future studies should aim to identify a subpopulation at reduced risk for ACNU-CDDP toxicity so that the potential of this protocol can be realized.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Terapia Combinada , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Adolescente , Adulto , Idoso , Quimioterapia Adjuvante/métodos , Dacarbazina/uso terapêutico , Progressão da Doença , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Temozolomida , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: The aim of the present study was to obtain information about distribution, radiation dosimetry, toxicity, and pharmacokinetics of O-[18F]fluoromethyl-d-tyrosine (d-18F-FMT), an amino acid PET tracer, in patients with brain tumors. PATIENTS AND METHODS: A total of 6 healthy controls (age = 19-25 years, 3 males and 3 females) with brain PET images and radiation dosimetry and 12 patients (median age = 60 years, 6 males and 6 females) with primary (n = 5) or metastatic brain tumor (n = 7) were enrolled. We acquired 60-minute dynamic brain PET images after injecting 370 MBq of d-18F-FMT. Time-activity curves of d-18F-FMT uptake in normal brain versus brain tumors and tumor-to-background ratio were analyzed for each PET data set. RESULTS: Normal cerebral uptake of d-18F-FMT decreased from 0 to 5 minutes after injection, but gradually increased from 10 to 60 minutes. Tumoral uptake of d-18F-FMT reached a peak before 30 minutes. Tumor-to-background ratio peaked at less than 15 minutes for 8 patients and more than 15 minutes for 4 patients. The mean effective dose was calculated to be 13.2 µSv/MBq. CONCLUSIONS: Using d-18F-FMT as a PET radiotracer is safe. It can distinguish brain tumor from surrounding normal brain tissues with a high contrast. Early-time PET images of brain tumors should be acquired because the tumor-to-background ratio tended to reach a peak within 15 minutes after injection.
Assuntos
Neoplasias Encefálicas , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Adulto , Encéfalo/diagnóstico por imagem , Neoplasias Encefálicas/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Tirosina , Adulto JovemRESUMO
The Hsp90-associated protein p23 modulates Hsp90 activity during the final stages of the chaperone pathway to facilitate maturation of client proteins. Previous reports indicate that p23 cleavage induced by caspases during cell death triggers destabilization of client proteins. However, the specific role of truncated p23 (Delta p23) in this process and the underlying mechanisms remain to be determined. One such client protein, hTERT, is a telomerase catalytic subunit regulated by several chaperone proteins, including Hsp90 and p23. In the present study, we examined the effects of p23 cleavage on hTERT stability and telomerase activity. Our data showed that overexpression of Delta p23 resulted in a decrease in hTERT levels, and a down-regulation in telomerase activity. Serine phosphorylation of Hsp90 was significantly reduced in cells expressing high levels of Delta p23 compared with those expressing full-length p23. Mutation analyses revealed that two serine residues (Ser-231 and Ser-263) in Hsp90 are important for activation of telomerase, and down-regulation of telomerase activity by Delta p23 was associated with inhibition of cell growth and sensitization of cells to cisplatin. Our data aid in determining the mechanism underlying the regulation of telomerase activity by the chaperone complex during caspase-dependent cell death.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteínas de Choque Térmico HSP90/metabolismo , Telomerase/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Fosforilação , Estabilidade Proteica , Telomerase/genéticaRESUMO
Epidermal growth factor receptor (EGFR) is activated by ionizing radiation (IR), but the molecular mechanism for this effect is unknown. We have found that intracellular generation of nitric oxide (NO) by NO synthase (NOS) is required for the rapid activation of EGFR phosphorylation by IR. Treatment of A549 lung cancer cells with IR increased NOS activity within minutes, accompanied by an increase of NO. 2-Phenyl-4,4,5,5,-tetramethylimidazolline-1-oxyl-3-oxide, an NO scavenger, and NG-monomethyl-l-arginine, an NOS inhibitor, abolished the increase in intracellular NO and activation of EGFR by IR. In addition, an NO donor alone induced EGFR phosphorylation. Transient transfection with small interfering RNA for endothelial NOS reduced IR-induced NO production and suppressed IR-induced EGFR activation. Overexpression of endothelial NOS increased IR-induced NO generation and EGFR activation. These results indicate a novel molecular mechanism for EGFR activation by IR-induced NO production via NOS.
Assuntos
Receptores ErbB/metabolismo , Óxido Nítrico/biossíntese , Radiação Ionizante , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de SinaisRESUMO
Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. Treatment of cancer cells with HDAC blockers, such as suberoylanilide hydroxamic acid (SAHA), leads to the activation of apoptosis-promoting genes. To enhance proapoptotic efficiency, SAHA has been used in conjunction with radiation, kinase inhibitors, and cytotoxic drugs. In the present study, we show that at the suboptimal dose of 250 muM, sulindac [2-[6-fluoro-2-methyl-3-[(4-methylsulfinylphenyl)methylidene]inden-1-yl]-acetic acid] significantly enhances SAHA-induced growth suppression and apoptosis of A549 human non-small cell lung cancer cells, primarily via enhanced collapse of the mitochondrial membrane potential, release of cytochrome c, and caspase activation. Furthermore, sulindac/SAHA cotreatment induced marked down-regulation of survivin at both the mRNA and protein levels and stimulated the production of reactive oxygen species (ROS), which were blocked by the antioxidant N-acetyl-l-cysteine. Overexpression of survivin was associated with reduced sulindac/SAHA-induced apoptosis of A549 cells, whereas suppression of survivin levels with antisense oligonucleotides or small interfering RNA further sensitized cells to sulindac/SAHA-induced cell death. Our results collectively demonstrate that sulindac/SAHA-induced apoptosis is mediated by ROS-dependent down-regulation of survivin in lung cancer cells.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Sulindaco/farmacologia , Anexina A5/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Caspases/análise , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Inibidores de Histona Desacetilases , Humanos , Indicadores e Reagentes/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Propídio/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , VorinostatRESUMO
SUMMARY: In the present study, we show that a combination of sulindac and arsenic trioxide (ATO) induces more extensive apoptosis than either drug alone in H1299 human non-small cell lung carcinoma (NSCLC) cells. Treatment with sulindac/ATO triggered three major apoptotic signaling events, namely, collapse of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases. Furthermore, the sulindac/ATO combination induced reactive oxygen species (ROS) generation, and the antioxidant, N-acetyl-L-cysteine, blocked this apoptotic signaling. The c-Jun NH(2)-terminal kinase (JNK) was activated downstream of ROS production in H1299 cells. Blockage of JNK by pretreatment with SP600125, a pharmacological inhibitor, or transfection with dominant-negative (DN) JNK1 vectors abrogated sulindac/ATO-induced apoptosis, as evident from the disruption of caspase activation. Interestingly, a slower migrating Bcl-xL band was observed on immunoblots after treatment of cells with sulindac/ATO. The band was absent upon the treatment of cell lysates with lambda protein phosphatase. Moreover, confocal microscopy findings disclose that active JNK translocates to mitochondria. Treatment with SP600125 and transfection with DN-JNK blocked Bcl-xL phosphorylation, suggesting that JNK plays an important role in sulindac/ATO-induced Bcl-xL phosphorylation. In conclusion, in H1299 human NSCLC cells, sulindac and ATO synergistically induce a high degree of apoptosis, which is mediated by the ROS-dependent JNK activation pathway via Bcl-xL phosphorylation.
Assuntos
Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Pulmonares/patologia , Óxidos/farmacologia , Sulindaco/farmacologia , Proteína bcl-X/metabolismo , Acetilcisteína/farmacologia , Trióxido de Arsênio , Arsenicais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral/metabolismo , Sinergismo Farmacológico , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Neoplasias Pulmonares/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxidos/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sulindaco/metabolismo , Proteína bcl-X/fisiologiaRESUMO
Redd1, a recently discovered stress-response gene, is regulated by hypoxia via hypoxia-inducible factor 1 (HIF-1) and by DNA damage via p53/p63; however, the signaling pathway by which its expression is induced by hypoxia has not been elucidated. In the present study, we demonstrated that the expression of Redd1 in response to hypoxia (1% O(2)), hypoxia-mimetic agent, cobalt chloride (CoCl(2)) and high cell density (HCD) requires coactivation of HIF-1alpha and Sp1. CoCl(2) and HCD induced the activation of HIF-1alpha and Sp1 in HeLa cells, and siRNAs targeting HIF-1alpha and Sp1 abrogated Redd1 expression. Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 and by a dominant-negative PI3K mutant reduced the expression of Redd1 and activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Also, suppression of Akt activation blocked the expression of Redd1 and the activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Furthermore, we found that the induction of Redd1 expression by CoCl(2) can be mediated by activation of Sp1 in HIF-1alpha-deficient cells but that a higher level of Redd1 expression is achieved when these cells are transfected with HIF-1alpha. These results demonstrate that hypoxic condition-and HCD-induced expression of Redd1 is mediated by coactivation of Sp1 and HIF-1alpha downstream of the PI3K/Akt signaling pathway.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Contagem de Células , Hipóxia Celular , Sequência Consenso , Ativação Enzimática , Regulação da Expressão Gênica , Células HeLa , Humanos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
The net balance of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) system has been known to be a key factor in tumor cell invasion. In the present study, we investigated the molecular mechanisms of anti-invasive and antimigrative activity of transforming growth factor (TGF)-beta1 on HT1080 human fibrosarcoma cells. In in vitro Matrigel invasion and Transwell migration assays, TGF-beta1 dose-dependently inhibited the invasion and migration of HT1080 cells, respectively. Gelatin zymography, Western blot, and real-time PCR analysis showed that TGF-beta1 enhanced the expression and secretion of MMP-2, TIMP-1, and, to a lesser degree, MMP-9 but not membrane type 1-MMP and TIMP-2. The addition of recombinant TIMP-1 protein reduced the Matrigel invasion and Transwell migration of HT1080 cells, similar to TGF-beta1. Because augmentation of TIMP-1 might be the major factor for the anti-invasive and antimigrative activity of TGF-beta1, we investigated possible molecular mechanisms responsible for the expression of TIMP-1 induced by TGF-beta1. Treatment of HT1080 cells with TGF-beta1 rapidly phosphorylated three mitogen-activated protein kinases [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase] and Akt. Among these kinases, the inhibition of only ERK1/2 pathway by PD98059, a specific inhibitor of MAPK/ERK kinase(MEK)-1, and transfection of dominant-negative MEK 1 effectively blocked the TIMP-1 induction by TGF-beta1. Mithramycin, a specific inhibitor of Sp1 transcription factor, but not curcumin, an inhibitor of activator protein-1, and transfection of Sp1 small interfering RNA significantly inhibited the TGF-beta1-induced expression of TIMP-1. In addition, electrophoretic mobility shift assay showed that TGF-beta1 up-regulated Sp1 DNA-binding activity, and PD98059 and mithramycin effectively inhibited these events. Finally, pretreatment of HT1080 cells with PD98059 and mithramycin, but not curcumin, restored the invasive activity of these cells. Taken together, these data suggest that TGF-beta1 modulates the net balance of the MMPs/TIMPs the systems in HT1080 cells for anti-invasion and antimigration by augmenting TIMP-1 through ERK1/2 pathway and Sp1 transcription factor.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Fator de Transcrição Sp1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Movimento Celular/efeitos dos fármacos , Ativação Enzimática , Flavonoides/farmacologia , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Fosforilação , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1RESUMO
Survivin, a member of the inhibitor of apoptosis protein (IAP) family, may be a good target for cancer therapy because it is expressed in a variety of human tumors but not in differentiated adult tissues. In the present study, we show that a combination of sulindac and arsenic trioxide (ATO) induces more extensive apoptosis than either drug alone in A549 human non-small cell lung carcinoma (NSCLC) cells. Treatment with sulindac/ATO reduced the expression of survivin and promoted major apoptotic signaling events, namely, collapse of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases. Combined sulindac/ATO treatment did not significantly affect the levels of other members of the IAP family (XIAP, cIAP1 and cIAP2), indicating that the effects were specific to survivin. In addition, sulindac/ATO treatment induced the production of reactive oxygen species and the antioxidant N-acetyl-l-cysteine blocked the down-regulation of survivin and induction of apoptotic signaling by the combination of sulindac and ATO. Combined sulindac/ATO treatment also activated p53 expression, and inhibition of p53 expression by small interfering RNA (siRNA) prevented sulindac/ATO-induced down-regulation of survivin, suggesting that survivin expression is negatively regulated by p53. Overexpression of survivin reduced sulindac/ATO-induced apoptosis in A549 cells and reduction of survivin levels by siRNA sensitized the cells to sulindac/ATO-induced cell death. These results demonstrate that, in A549 human NSCLC cells, sulindac/ATO-induced apoptosis is mediated by the reactive oxygen species-dependent down-regulation of survivin.
Assuntos
Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/biossíntese , Óxidos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sulindaco/farmacologia , Trióxido de Arsênio , Arsenicais/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Pulmonares/patologia , Óxidos/administração & dosagem , Sulindaco/administração & dosagem , SurvivinaRESUMO
Cisplatin is a DNA-damaging chemotherapeutic drug that may have a role in the adjuvant chemotherapy of several solid tumors, such as malignant glioblastoma, and the status of p53 tumor suppressor protein is a critical determinant of cisplatin chemosensitivity. In the present study, we showed the relationship of p53 status and chemosensitivity of cisplatin between two human malignant glioblastoma cell lines, A172 and T98G, harboring wild-type and mutant-type p53, respectively. Cisplatin was found to be more cytotoxic to A172 than T98G cells in a time- and concentration-dependent manner. Cisplatin-induced cytotoxicity manifested as apoptosis, characterized by genomic DNA fragmentation, nuclear condensation and an increase in sub-G1 population. Cisplatin induced the accumulation of p53 and p21 proteins in A172 cells, but not in T98G cells. The introduction of the adenovirus-mediated wild-type p53 gene into T98G cells resulted in the decrease of viability as well as the increase in sub-G1 population with p53 accumulation, activation of caspase-3 protease and release of cytochrome c from the mitochondria. These data strongly suggest that the expression of p53 is essential for the cytotoxic effect of cisplatin in human malignant glioblastoma cells, A172 and T98G, and the introduction of apoptotic signal molecules, such as p53, will be beneficial to achieve chemosensitivity in malignant glioma.
Assuntos
Neoplasias Encefálicas/patologia , Cisplatino/farmacologia , Glioblastoma/patologia , Proteína Supressora de Tumor p53/biossíntese , Apoptose , Caspase 3 , Caspase 9 , Caspases/metabolismo , Citocromos c/metabolismo , Dano ao DNA , Ativação Enzimática , Genes p53 , Humanos , Transdução Genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Glioblastoma is a severe type of primary brain tumor and its invasion is strongly correlated with the secretion of matrix metalloproteinases (MMPs). To investigate a role of PTEN, a tumor suppressor gene, in the regulation of hyaluronic acid (HA)-induced invasion of glioma cells, we examined the secretion of MMP-9 in various glioma cells with or without a functional PTEN gene. The secretion of MMP-9 in glioma cells lacking functional PTEN (U87MG, U251MG, and U373MG) was induced by HA, although not in wildtype (wt)-PTEN-harboring cells (LN229, LN18, and LN428). In addition, stable expression of wt-PTEN into U87MG cells significantly decreased the secretion of HA-induced MMP-9 and basal levels of MMP-2, inhibiting the activation of focal adhesion kinase and extracellular signal-regulated kinase 1/2, whereas the secretion levels of the tissue inhibitor of metalloproteinase-1 and -2 were increased, finally resulting in the inhibition of invasion by HA in vitro. Ectopic expressions of adenoviral (Ad)-wt-PTEN and -lipid phosphatase-deficient (G129E)-PTEN, but not both protein and -lipid phosphatase-deficient (C124S)-PTEN, reduced MMP-9 secretion and invasion by HA. These results were also confirmed by expressions of Ad-wt-PTEN and Ad-G129E-PTEN in other glioblastoma cells lacking functional PTEN, U251MG, and U373MG. These findings strongly suggest the possibility that PTEN may block HA-induced MMP-9 secretion and invasion through its protein phosphatase activity.
Assuntos
Glioblastoma/enzimologia , Ácido Hialurônico/antagonistas & inibidores , Metaloproteinase 9 da Matriz/biossíntese , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Ácido Hialurônico/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Invasividade Neoplásica , PTEN Fosfo-Hidrolase , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/metabolismo , Proteínas ras/fisiologiaRESUMO
Although mechanisms of arsenic trioxide (As(2)O(3))-induced cell death have been studied extensively in hematologic cancers, those in solid cancers have yet to be clearly defined. In this study, we showed that the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for As(2)O(3)-induced cell death in human cervical cancer cells. We also showed that reactive oxygen species (ROS)-mediated poly(ADP-ribose) polymerase-1 (PARP-1) activation is necessary for AIF release from mitochondria. The treatment of human cervical cancer cells with As(2)O(3) induces dissipation of mitochondrial membrane potential (Deltapsi(m)), translocation of AIF from mitochondria to the nucleus, and subsequent cell death. Small interfering RNA targeting of AIF effectively protects cervical cancer cells against As(2)O(3)-induced cell death. As(2)O(3) also induces an increase of intracellular ROS level and a marked activation of PARP-1. N-acetyl-l-cystein, a thiol-containing antioxidant, completely blocks As(2)O(3)-induced PARP-1 activation, Deltapsi(m) loss, nuclear translocation of AIF from mitochondria, and the consequent cell death. Furthermore, pretreatment of 1,5-dihydroxyisoquinoline or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, PARP-1 inhibitors, effectively attenuates the loss of Deltapsi(m), AIF release, and cell death. These data support a notion that ROS-mediated PARP-1 activation signals AIF release from mitochondria, resulting in activation of a caspase-independent pathway of cell death in solid tumor cells by As(2)O(3) treatment.
Assuntos
Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Caspases/fisiologia , Cloretos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/patologia , Acetilcisteína/farmacologia , Apoptose/fisiologia , Fator de Indução de Apoptose , Arsenicais/antagonistas & inibidores , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cloretos/antagonistas & inibidores , Ativação Enzimática , Feminino , Flavoproteínas/genética , Flavoproteínas/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismoRESUMO
PURPOSE: In the field of cancer research, there has been a paucity of interest in necrosis, whereas studies focusing on apoptosis abound. In neuro-oncology, this is particularly surprising because of the importance of necrosis as a hallmark of glioblastoma (GBM), the most malignant and most common primary brain tumor, and the fact that the degree of necrosis has been shown to be inversely related to patient survival. It is therefore of considerable interest and importance to identify genes and gene products related to necrosis formation. EXPERIMENTAL DESIGN: We used a nylon cDNA microarray to analyze mRNA expression of 588 universal cellular genes in 15 surgically resected human GBM samples with varying degrees of necrosis. Gene expression was correlated with the degree of necrosis using rank correlation coefficients. The expression of identified genes was compared with their expression in tissue samples from 5 anaplastic astrocytomas (AAs). Immunostaining was used to determine whether genes showing the most positive correlation with necrosis were increasingly expressed in tumor tissues, as grade of necrosis increased. RESULTS: The hybridization results indicated that 26 genes showed significant correlation with the amount of necrosis. All 26 genes had functions associated with either Ras, Akt, tumor necrosis factor alpha, nuclear factor kappaB, apoptosis, procoagulation, or hypoxia. Nine genes were positively correlated with necrosis grade, and 17 genes were negatively correlated with necrosis grade. There were significant differences in the median expression levels of 3 of the 26 genes between grade III necrosis GBM and anaplastic astrocytoma (AA) samples; all but 1 of the genes had elevated expression when comparing necrosis grade III with AA samples. Two factors, the ephrin type A receptor 1 and the prostaglandin E(2) receptor EP4 subtype, not previously considered in this context, were highlighted because of their particularly high (positive) correlation coefficients; immunostaining showed the products of these two genes to be localized in perinecrotic and necrotic regions and to be overexpressed in grade III GBMs, but not AAs. These two molecules also showed significant correlation with survival of GBM patients (P = 0.0034) in a combined model. CONCLUSIONS: The application of cDNA expression microarray analysis has identified specific genes and patterns of gene expression that may help elucidate the molecular basis of necrogenesis in GBM. Additional studies will be required to further investigate and confirm these findings.
Assuntos
Neoplasias Encefálicas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Glioblastoma/genética , Humanos , Necrose , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismoRESUMO
There are several currently employed classification systems for diffuse gliomas that sort tumors based on histological features. Contemporary molecular techniques, however, offer the promise of improved tumor classification and resultant patient stratification for treatment and prognosis. In particular, gene expression profiling has shown exceptional promise for providing an alternative and more objective molecular approach to glioma classification. In this study, we used cDNA array technology to profile the gene expression of 30 primary human glioma tissue samples comprising 4 different glioma subtypes as defined by current World Health Organization (WHO 2000) criteria: glioblastoma (GM, WHO grade IV), anaplastic astrocytoma (AA, WHO grade III), anaplastic oligodendroglioma (AO, WHO grade III), and oligodendroglioma (OL, WHO grade II). Gene expression data alone were used to group the tumors using multidimensional scaling, which is an unsupervised statistical method. Results show that impressive separation of the 4 glioma subtypes can be achieved solely on the basis of molecular data. In addition, a subcluster of 3 glioblastomas was identified as distinct from other GMs and from the oligodendroglial tumors. These 3 patients have shown extended survival compared to other GMs in the study. Survival analysis of the full data set revealed a good correlation with the molecular classification. Results of this proof-of-principle study demonstrate that molecular profiling alone can recapitulate conventional histologic classification and grading with high fidelity. In addition, results show that the molecular approach to tumor classification can generate clinically meaningful patient stratification, and, more importantly, is an efficient class-discovery tool for human gliomas, permitting the identification of previously unrecognized, clinically relevant tumor subsets.
Assuntos
Algoritmos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Análise Mutacional de DNA/métodos , Processamento Eletrônico de Dados/métodos , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , Glioma/patologia , Análise de Sequência com Séries de Oligonucleotídeos/tendências , Análise Mutacional de DNA/instrumentação , Processamento Eletrônico de Dados/instrumentação , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Taxa de SobrevidaRESUMO
Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies.
Assuntos
Arsenicais/farmacologia , Carcinoma/terapia , Terapia Combinada/métodos , Óxidos/farmacologia , Radiossensibilizantes/farmacologia , Neoplasias do Colo do Útero/terapia , Animais , Trióxido de Arsênio , Arsenicais/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Óxidos/uso terapêutico , Radiossensibilizantes/uso terapêutico , Resultado do Tratamento , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We demonstrate here that selective activation of endogenous members of the caspase family and cleavage of substrates responsible for the maintenance of nuclear functional and structural integrity are major effectors of antigen receptor (AgR)- and ionomycin-triggered apoptosis in Ramos-Burkitt lymphoma (Ramos-BL) B cells. Ramos-BL B cells express significant proenzyme levels of caspase-2, -3, -7 and -8, low levels of caspase-6 and are caspase-1-negative. However, while anti-IgM and ionomycin trigger for significant activation of caspase-3, -7 and -8 at 12-16 h and at 4 h post-stimulation respectively, both anti-IgM and ionomycin fail to activate caspase-2 indicating that AgR- and ionomycin-triggered Ramos-BL B cell apoptosis is mediated by the selective activation of, at least, caspase-3, -7 and -8. Anti-IgM triggers for cleavage of the resident nuclear proteins poly(ADP-ribose) polymerase (PARP) at 8 h, lamins B1 and B2 from 12 to 16 h; likewise, ionomycin triggers for degradation of PARP at 2 h, lamins B1 and B2 at 4 h. Signal transduction through CD40 rescues Ramos-BL B cells from AgR- and ionomycin-triggered apoptosis at a very early stage of the apoptotic process by inhibiting both the early cleavage of PARP as well as the activation of caspase-3, -7 and -8 and cleavage of lamin B1; CD40-mediated rescue occurs upstream of CD40-induced expression of Bcl-2 and increased expression of Bcl-xL. In such cellular populations subject to regulation through apoptosis, dysregulation of the apoptotic mechanisms can have devastating consequences by contributing to the pathogenesis of malignancy as well as to lymphoproliferative and autoantibody disorders. An understanding of the role played by caspases in the execution of apoptosis may provide insight into the pathogenesis of these disease states and thereby provide targets for novel therapeutic strategy.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/citologia , Linfoma de Burkitt/metabolismo , Antígenos CD40/metabolismo , Caspases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Apoptose/fisiologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfoma de Burkitt/patologia , Antígenos CD40/imunologia , Caspase 1 , Ciclo Celular/efeitos dos fármacos , Criança , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Células HL-60 , Humanos , Imunoglobulina M/farmacologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Células Jurkat , Lamina Tipo B/metabolismo , Masculino , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-XRESUMO
Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia (APL) through induction of apoptosis. To investigate the potential therapeutic usage of As2O3 in cervical cancer and its possible mechanisms, human cervical cancer cell line HeLa was employed. The cells underwent apoptosis in response to As2O3, accompanied by a decrease of mitochondrial membrane potential and caspase-3 activation. Overexpression of Bcl-2, however, prevented the dissipation of mitochondrial membrane potential, subsequently protecting the cells from As2O3-induced apoptosis. As2O3 increased cellular content of reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), and the antioxidant N-acetyl-L-cysteine completely suppressed As2O3-induced apoptosis. Furthermore, incubation of the cells with catalase resulted in significant suppression of As2O3-induced apoptosis. The above results indicate that the induction of HeLa cell apoptosis by As2O3 involved an early decrease in cellular mitochondrial membrane potential and increase in ROS content, predominantly H2O2, followed by caspase-3 activation and DNA fragmentation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Caspases/metabolismo , Células HeLa/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Óxidos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Trióxido de Arsênio , Catalase/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Células HeLa/metabolismo , Células HeLa/patologia , Humanos , Peróxido de Hidrogênio/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/fisiologiaRESUMO
In the present study, we investigated the effect of tetraarsenic oxide (As4O6, 2,4,6,8,9,10-Hexaoxa-1,3,5,7-tetraarsatricyclo[3.3.1.13,7]decane) upon induction of apoptosis in arsenic trioxide (diarsenic oxide, As2O3) resistant U937 leukemic cells. As4O6 induced apoptosis in U937 leukemic cells at much lower concentrations than As2O3 via an early increase of cellular reactive oxygen species (ROS), and a decrease in cellular mitochondrial membrane potential, followed by cytochrome c release and caspase-3 activation. As4O6 generated ROS and induced caspase-3 activation more potently than As2O3 in U937 cells. Incubation of the cells with N-acetyl-L-cysteine and catalase resulted in significant suppression of As4O6-induced apoptotic cell death. These results show that the generation of ROS leads to the consequences associated with apoptosis induced by As4O6. In conclusion, As4O6 might be a new arsenic compound which may induce apoptosis in U937 leukemic cells by activating unique apoptotic signaling mediated by ROS more potently than As2O3, and deserves further evaluation.