RESUMO
Vestibular schwannomas (VSs) arise from Schwann cells (SCs) and result from the loss of function of merlin, the protein product of the NF2 tumor suppressor gene. In contrast to non-neoplastic SCs, VS cells survive long-term in the absence of axons. We find that p75(NTR) is overexpressed in VSs compared with normal nerves, both at the transcript and protein level, similar to the response of non-neoplastic SCs following axotomy. Despite elevated p75(NTR) expression, VS cells are resistant to apoptosis due to treatment with proNGF, a high affinity ligand for p75(NTR) . Furthermore, treatment with proNGF protects VS cells from apoptosis due to c-Jun N-terminal kinase (JNK) inhibition indicating that p75(NTR) promotes VS cell survival. Treatment of VS cells with proNGF activated NF-κB while inhibition of JNK with SP600125 or siRNA-mediated knockdown reduced NF-κB activity. Significantly, proNGF also activated NF-κB in cultures treated with JNK inhibitors. Thus, JNK activity appears to be required for basal levels of NF-κB activity but not for proNGF-induced NF-κB activity. To confirm that the increase in NF-κB activity contributes to the prosurvival effect of proNGF, we infected VS cultures with Ad.IκB.SerS32/36A virus, which inhibits NF-κB activation. Compared with control virus, Ad.IκB.SerS32/36A significantly increased apoptosis including in VS cells treated with proNGF. Thus, in contrast to non-neoplastic SCs, p75(NTR) signaling provides a prosurvival response in VS cells by activating NF-κB independent of JNK. Such differences may contribute to the ability of VS cells to survive long-term in the absence of axons.
Assuntos
NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroma Acústico/fisiopatologia , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , NF-kappa B/agonistas , NF-kappa B/antagonistas & inibidores , Fator de Crescimento Neural/metabolismo , Precursores de Proteínas/metabolismo , Ratos , Receptores de Fatores de Crescimento , Células de Schwann/fisiologia , Nervo Isquiático/fisiologiaRESUMO
Monitoring the long-term radiotherapy-associated molecular changes in low-grade gliomas (LGGs) facilitates the understanding of LGG response to radiotherapy. In this study, we used immunohistochemistry to analyze the expression of Ki-67, tumor protein P53 (TP53), P21, and P27 in 8 paired WHO grade II astrocytoma samples. The interval between radiotherapy (RT) and the second surgery was more than 3 months in all cases. The average Ki-67 labeling index (LI) was 5.3% in pre-RT samples and 11.54% in post-RT samples. Ki-67 LI was higher in the primary tumors that underwent malignant transformation observed at the second surgery after radiation. Post-RT Ki-67 LI decreased in 2 cases with an interval of less than 12 months between RT and the second surgery. TP53 expression was found in 3 out of 4 pre-RT samples with malignant transformation and in 1 out of 4 pre-RT samples without malignant transformation. Post-RT TP53 increased in 2 cases in which increased expression of P21 or P27 was also observed. Our study suggests that radiotherapy can inhibit WHO grade II astrocytoma proliferation as reflected by Ki-67 LI, but the effect attenuates with time. In addition, there is a tendency of malignant transformation for WHO grade II astrocytomas with a high Ki-67 level or TP53 expression in initial samples.
Assuntos
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Antígeno Ki-67/metabolismo , Adulto , Astrocitoma/patologia , Astrocitoma/radioterapia , Astrocitoma/cirurgia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirurgia , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Proteína Supressora de Tumor p53/metabolismoRESUMO
We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.
RESUMO
OBJECT: Astrocytoma may progress rapidly or remain stable for many years. To clarify whether molecular characteristics could be prognostic factors, several cell cycling-associated molecular alterations in the diffuse astrocytoma have been investigated. METHODS: Thirty-three patients in whom WHO Grade II astrocytoma had been initially diagnosed were assigned to 1 of 3 groups. Group 1 consisted of 10 patients with malignant progression; the tumor had recurred within 5 years and histological analysis had confirmed that the tumor progressed to Grade III or IV. Group 2 consisted of 10 patients in whom there was no malignant progression; the tumor recurred within 5 years, but histological analysis confirmed that the tumor remained at Grade II. Group 3 consisted of 13 patients who did not experience recurrence within 5 years. Expression of Ki 67, TP53, p27, and p21 was examined using immunohistochemical analysis for the tumor samples obtained during the first and second (in recurrent cases) surgeries. Exons 5, 7, and 8 of TP53 were scanned by DNA sequencing. RESULTS: The Ki 67 labeling index expression was significantly higher in Group 1 (even though it was similar between initial and recurrent tumors) than that of Group 3 (p < 0.05). However, there was no difference between Group 2 (both initial and recurrent tumors) and Group 3. The TP53 protein accumulation was also higher in Group 1 than in Group 2 or 3 (p < 0.05); a difference in TP53 expression was not found between Groups 2 and 3. The p27 and p21 was expressed in all cases, but no predictive values were found. The p53 mutation was found only in 6 cases in Group 1. CONCLUSIONS: Overexpression of TP53, TP53 mutation, and Ki 67 labeling index could be molecular markers in astrocytomas predicting malignant progression.
Assuntos
Astrocitoma/química , Astrocitoma/patologia , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/química , Neoplasias Encefálicas/patologia , Inibidor de Quinase Dependente de Ciclina p21/análise , Antígeno Ki-67/análise , Antígeno Nuclear de Célula em Proliferação/análise , Proteína Supressora de Tumor p53/análise , Adulto , Sequência de Bases , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Recidiva Local de Neoplasia , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/genéticaRESUMO
Serous cystadenofibromas are uncommon benign ovarian lesions, consisting of both fibrous and epithelial components, that are usually cystic but may contain solid or papillary architecture that can be confused with a malignancy on imaging. Papillary architecture seen on frozen section may also falsely steer the pathologist in the direction of a diagnosis of a borderline serous tumor. Overcalling the lesion may lead to more aggressive surgery than necessary, so extensive tissue sampling and consideration of this entity is important in possibly avoiding this mistake.
Assuntos
Cistoadenofibroma/diagnóstico , Neoplasias Ovarianas/diagnóstico , Ovário/patologia , Lesões Pré-Cancerosas/diagnóstico , Cistoadenofibroma/patologia , Cistoadenofibroma/cirurgia , Diagnóstico Diferencial , Feminino , Secções Congeladas , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/cirurgia , Ovário/cirurgia , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/cirurgiaRESUMO
Vasculogenic mimicry (VM) has been observed in melanoma and in some nonmelanoma tumor types. It is unknown whether a similar VM phenomenon exists in astrocytoma. The present study was to examine 45 astrocytomas (including World Health Organization grade II 15 cases, grade III 15 cases, and grade IV 15 cases) by CD34 endothelial marker periodic acid-Schiff (PAS) dual staining to see if VM existing in these tumors. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and stained positively for PAS, laminin, and endothelial marker. PAS-positive pattern of VM was found in two grade IV astrocytomas. Channels stained positively for PAS, laminin, and negatively for CD34 of the VM entrapped in the tumor tissue. Erythrocytes could be observed in some of these channels. In these networks of PAS-positive pattern, spots of weak reaction for CD34 were observed, suggesting the incorporation of VM channel and normal vessel. Furthermore, in astrocytoma, especially glioblastoma, focus of anaplastic tumor cells appeared with CD34 expression, whereas some tumor cells lost glial fibrillary acid protein expression. It is assumed that genetically deregulated tumor cells in astrocytoma could lose the astrocyte-specific protein and express inappropriate markers not expected in cells of astrocyte lineage. The present results suggest that VM phenomenon exists in some malignant astrocytoma.
Assuntos
Astrocitoma/irrigação sanguínea , Neoplasias Encefálicas/irrigação sanguínea , Antígenos CD34/metabolismo , Astrocitoma/metabolismo , Astrocitoma/patologia , Biomarcadores/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Corantes , Endotélio Vascular/metabolismo , Humanos , Microcirculação , Estadiamento de Neoplasias , Ácido Periódico , Bases de SchiffRESUMO
BACKGROUND: Radiosurgery is increasingly used to treat vestibular schwannomas (VSs). Increasing the sensitivity of VS cells to irradiation (IR) could allow for lower and/or more effective doses of IR, improving safety and efficacy. Persistent c-Jun N-terminal kinase (JNK) activity in VS cells reduces cell death by suppressing the accumulation of reactive oxygen species (ROS), raising the possibility that JNK activity protects against IR-induced VS cell death, which is mediated by ROS. OBJECTIVE: To determine the extent to which JNK signaling contributes to VS cell radiosensitivity. METHODS: Primary human VS cultures, derived from acutely resected tumors, received single doses (5-40 Gy) of gamma irradiation. Histone 2AX phosphorylation, a marker of IR-induced DNA damage, was assayed by Western blot and immunostaining. ROS levels were quantified by measuring 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. Cell apoptosis was determined by terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling. RESULTS: The JNK inhibitors SP6000125 and I-JIP reduced histone 2AX phosphorylation after IR. They also increased H2DCFDA fluorescence in nonirradiated cultures and significantly increased IR-induced (5-10 Gy) H2DCFDA fluorescence 72 hours, but not 2 hours, after IR. Finally, I-JIP (50 µmol/L) significantly increased VS cell apoptosis in cultures treated with 20 to 40 Gy. I-JIP (20 µmol/L), SP600125 (20 µmol/L), and JNK1/2 short interfering RNA knockdown each increased VS cell apoptosis in cultures treated with 30 to 40 Gy, but not lower doses, of IR. CONCLUSION: Inhibition of JNK signaling decreases histone 2AX phosphorylation and increases ROS and apoptosis in VS cells after gamma irradiation. These results raise the possibility of using JNK inhibitors to increase the effectiveness of radiosurgery for treatment of VSs.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Neuroma Acústico/patologia , Apoptose/efeitos da radiação , Histonas/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
Vestibular schwannomas (VSs) result from inactivating mutations in the merlin tumor suppressor gene. The merlin protein suppresses a variety of progrowth kinase-signaling cascades, including extracellular regulated kinase/mitogen-activated protein kinase (ERK/MAPK), c-Jun N-terminal kinase (JNK), and phosphatidyl-inositol 3-kinase (PI3-K)/Akt. Recent studies indicate that ERKs and Akt are active in human VSs, and here we show that JNKs are also persistently active in human VS cells. With use of cultures of human VSs, we investigated the contribution of each of these signals to the proliferative and survival response of VS cells. Inhibition of ERK or Akt signaling reduced VS cell proliferation but did not increase apoptosis, whereas inhibition of JNK with SP600125, I-JIP, or siRNA knock-down reduced VS cell proliferation and survival by inducing apoptosis. By contrast, JNK activity promotes apoptosis in normal Schwann cells. Inhibition of JNK increased the fluorescence intensity of VS cells loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), a fluorescent probe for reactive oxygen species (ROS). Furthermore, ebselen, a ROS scavenger, rescued VS cells with suppressed JNK from apoptosis, suggesting that JNK activity protects VS cells from apoptosis by limiting accumulation of ROS. VS cultures treated with JNK inhibitors demonstrated significantly higher levels of MitoSOX Red fluorescence, implying that persistent JNK activity specifically suppresses superoxide production in the mitochondria. Overexpression of superoxide dismutase 2 (MnSOD; mitochondrial SOD) prevented apoptosis in VS cells with suppressed JNK signaling. Taken together, these results indicate that persistent JNK activity enhances VS cell survival, at least in part, by suppressing accumulation of mitochondrial superoxides.
Assuntos
Apoptose , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Técnicas Imunoenzimáticas , Mitocôndrias , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
HYPOTHESIS: Elevated levels of hsa-microRNA-21 (miR-21) in vestibular schwannomas (VSs) may contribute to tumor growth by downregulating the tumor suppressor phosphatase and tensin homolog (PTEN) and consequent hyperactivation of protein kinase B (AKT), a key signaling protein in the cellular pathways that lead to tumor growth. BACKGROUND: Vestibular schwannomas are benign tumors that arise from the vestibular nerve. Left untreated, VSs can result in hearing loss, tinnitus, vestibular dysfunction, trigeminal nerve dysfunction, and can even become life threatening. Despite efforts to characterize the VS transcriptome, the molecular pathways that lead to tumorigenesis are not completely understood. MicroRNAs are small RNA molecules that regulate gene expression posttranscriptionally by blocking the production of specific target proteins. METHODS: We examined miR-21 expression in VSs. To determine the functional significance of miR-21 expression in VS cells, we transfected primary human VS cultures with anti-miR-21 or control, scrambled oligonucleotides. RESULTS: We found consistent overexpression of miR-21 when compared with normal vestibular nerve tissue. Furthermore, elevated levels of miR-21 correlated with decreased levels of PTEN, a known molecular target of miR-21. Anti-miR-21 decreased VS cell proliferation in response to platelet-derived growth factor stimulation and increased apoptosis, suggesting that increased miR-21 levels contributes to VS growth. CONCLUSION: Because PTEN regulates signaling through the growth-promoting phosphoinositide 3-kinase/AKT pathway, our findings suggest that miR-21 may be a suitable molecular target for therapies aimed specifically at reducing VS growth.
Assuntos
Neoplasias dos Nervos Cranianos/patologia , MicroRNAs/biossíntese , Neuroma Acústico/patologia , Nervo Vestibulococlear/patologia , Apoptose/genética , Western Blotting , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Neoplasias dos Nervos Cranianos/genética , Eletroforese em Gel de Poliacrilamida , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , MicroRNAs/genética , Neurofibromatose 2/genética , Neurofibromatose 2/patologia , Neuroma Acústico/genética , PTEN Fosfo-Hidrolase/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
In 1999, Maniotis described a novel process by which tumors develop a highly patterned microcirculation that was independent of angiogenesis: in aggressive primary and metastatic melanomas, tumor cells generate non-endothelial cell-lined microcirculatory channels composed of extracellular matrix and lined externally by tumor cells. They named the process "vasculogenic mimicry" (VM). Folberg used PAS staining to show VM network, and identified 7 morphologic patterns of PAS-positive channels uveal melanomas which were confirmed as tubular type and patterned matrix type. Maniotis suggested PAS-positive patterns of VM in uveal melanoma are indeed a form of tumor microcirculation which is different from angiogenesis, and it is not a stromal host response at the interface between the tumor and the surrounding host stroma. VM has also been observed in carcinomas of the breast, prostate, ovary and lung, glioblastoma, synoviosarcoma, rhabdomyosarcoma, and phaeochromocytoma, and in the process of placenta formation from cytotrophoblasts. The molecular "signature" of aggressive melanoma cells is illustrative of an undifferentiated cell with a gene expression profile that is similar to that of embryonic-like cells. VE-cadherin, EphA2, laminin5 gamma2, matrix metalloproteinases (MMPs), vascular endothelial growth factor-C (VEGF-C), LYVE1, TF and NOTCH are important components of molecular switch of vasculogenic mimicry. The heterogeneity of tumor vasculature and the molecular regulation mechanisms present an opportunity for tumor therapy.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Melanoma/patologia , Neoplasias/patologia , Neovascularização Patológica/patologia , Receptor EphA2/metabolismo , Animais , Antígenos CD/fisiologia , Caderinas/fisiologia , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Humanos , Melanoma/irrigação sanguínea , Melanoma/metabolismo , Microvasos/metabolismo , Microvasos/patologia , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Reação do Ácido Periódico de Schiff , Receptor EphA2/fisiologia , CalininaRESUMO
BACKGROUND & OBJECTIVE: Malignant glioma cells are resistant to most chemotherapeutic agents. Nitrosourea and temozolomide (TMZ) are main agents for treating malignant glioma. Resistance of malignant glioma to these agents is frequently associated with high levels of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). This study was to evaluate the efficacy of individualized chemotherapy, according to chemotherapy sensitivity and resistance assays (CSRAs) and MGMT expression pattern, on malignant glioma, and observe the adverse events. METHODS: The pathologically confirmed malignant glioma patients, treated by operation at Cancer Center of Sun Yat-sen University from Dec. 2001 to Feb. 2006, were enrolled. The fresh tumor tissues obtained during operation were immediately sent for CSRAs using MTT assay. The expression of MGMT protein was detected by immunohistochemistry. After radiotherapy,the patients received chemotherapy according to the results of CSRAs and MGMT expression. The patients were evaluated for response to chemotherapy according to WHO criteria, and for toxicity according to National Cancer Institute (NCI) criteria. RESULTS: Forty-two patients were evaluated for response to chemotherapy. Seven patients received 2 chemotherapy regimens consecutively, therefore, overall 49 cases were evaluable. Of the 49 cases, 6 (12%) achieved complete remission (CR), 10 (20%) achieved partial remission (PR), 20 (41%) had stable disease (SD), and 13 (27%) had progressive disease (PD). The objective response rate (CR and PR) was 33%, and the disease control rate (CR, PR, and SD) was 73%. Hematologic toxicities were the main adverse events observed in this study, included grade IV anemia (1%), grade III-IV leukopenia (28%), and grade III-IV thrombocytopenia (8%). Non-hematologic toxicities mainly included nausea/vomit, fatigue, and alopecia. CONCLUSION: Individualized chemotherapy based on in vitro CSRAs and MGMT expression for patients with malignant glioma could improve overall response rate.