RESUMEN
BACKGROUND: Breast cancer, lung cancer and melanoma metastasize to the meninges in 5-15% of patients. The identification of specific biomarkers of disease may allow for earlier diagnosis and treatment. Preclinical evidence suggests the possible relevance of SDF-1 and VEGF in the homing and neoangiogenesis of metastases. We chose to measure these molecules in the cerebrospinal fluid (CSF) of melanoma, breast, and lung cancer patients being evaluated for neoplastic meningitis (NM). MATERIALS AND METHODS: We collected CSF from patients with these cancers who were being evaluated for possible NM. CSF was assayed for SDF-1 and VEGF levels using Enzyme-linked Immunosorbent Assay (ELISA) assays. RESULTS: CSF samples from 89 patients met criteria for analysis, including 41 with breast cancer, 35 with lung cancer and 13 with melanoma. Twenty-five percent (22/89) of all samples were positive for malignant cells; 8/41 (20%) from breast cancer, 10/35 (29%) from lung cancer and 4/13 (31%) from melanoma. CSF VEGF levels were available from 83 patients, and were elevated (>20 pg/ml) in 15/22 (68%) of patients with positive CSF cytology and normal (<20 pg/ml) in 59/61 (97%) of patients with negative CSF cytology. The two patients with negative CSF cytology who also had elevated CSF VEGF levels had MRI evidence of NM. CSF SDF-1 levels were available from 81 patients, and were elevated (>950 pg/ml) in 11/18 (61%) of patients with positive CSF cytology and normal (<950 pg/ml) in 57/63 (90%) of patients with negative CSF cytology. CONCLUSIONS: Elevated CSF levels of VEGF are sensitive and highly specific for the diagnosis of NM from breast cancer, lung cancer and melanoma, and may serve as a useful biomarker of NM in high risk patients. CSF SDF-1 levels add little to the diagnostic information provided by CSF VEGF. Evaluation of CSF VEGF levels as a trigger for early treatment in high risk breast cancer, lung cancer and melanoma patients at risk for NM, is warranted.
Asunto(s)
Neoplasias de la Mama/líquido cefalorraquídeo , Quimiocina CXCL12/líquido cefalorraquídeo , Neoplasias Pulmonares/líquido cefalorraquídeo , Melanoma/líquido cefalorraquídeo , Meningitis/líquido cefalorraquídeo , Factor A de Crecimiento Endotelial Vascular/líquido cefalorraquídeo , Adulto , Anciano , Biomarcadores de Tumor/líquido cefalorraquídeo , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/patología , Femenino , Humanos , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/patología , Masculino , Melanoma/complicaciones , Melanoma/patología , Meningitis/etiología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/líquido cefalorraquídeo , Estadificación de Neoplasias , Pronóstico , Sensibilidad y EspecificidadRESUMEN
The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53's ability to induce cell cycle arrest versus apoptosis, particularly because wild-type p53 cells are resistant to Ad-p53-induced apoptosis. Here we use Ad-p53 constructs to test the hypothesis that simultaneous phosphorylation of p53 at threonine 18 (Thr18) and serine 20 (Ser20) is causally associated with p53-mediated apoptosis. Studies using phosphorylation-specific antibodies demonstrated that p53-induced apoptosis correlates with phosphorylation of p53 at Thr18 and Ser20 but not with carboxy-terminal phosphorylation (Ser392). To prove a causal relationship between apoptosis and Thr18 and Ser20 phosphorylation of p53, the effects of an adenoviral p53 construct that was not phosphorylated (Ad-p53) was compared with a Thr18/Ser20 phosphomimetic construct (Ad-p53-18D20D) in wild-type p53 gliomas. Whereas treatment with Ad-p53 resulted only in cell cycle arrest, treatment with Ad-p53-18D20D induced dramatic apoptosis. Microarray and Western blot analyses showed that only Ad-p53-18D20D was capable of inducing expression of apoptosis-inducing proteins. Chromatin immunoprecipitation assays indicated that the protein product of Ad-p53-18D20D, but not Ad-p53, was capable of binding to apoptosis-related genes. We thus conclude that phosphorylation of Thr18 and Ser20 is sufficient for inducing p53-mediated apoptosis in glioma cells. These results have implications for p53 gene therapy and inform other strategies that aim to restore p53 function.
Asunto(s)
Apoptosis/fisiología , Terapia Genética/métodos , Serina/metabolismo , Treonina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenoviridae , Western Blotting , Ciclo Celular/fisiología , Línea Celular Tumoral , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Inmunoprecipitación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Reacción en Cadena de la PolimerasaRESUMEN
Multiple genetic aberrations in human gliomas contribute to their highly infiltrative and rapid growth characteristics. Focal adhesion kinase (FAK) regulates tumor migration and invasion. Insulin-like growth factor-I receptor (IGF-IR), whose expression correlates with tumor grade, is involved in proliferation and survival. We hypothesized that inhibiting the phosphorylation of FAK and IGF-IR by NVP-TAE226 (hereafter called TAE226), a novel dual tyrosine kinase inhibitor of FAK and IGF-IR, would suppress the growth and invasion of glioma cells. In culture, TAE226 inhibited extracellular matrix-induced autophosphorylation of FAK (Tyr(397)). TAE226 also inhibited IGF-I-induced phosphorylation of IGF-IR and activity of its downstream target genes such as MAPK and Akt. TAE226 retarded tumor cell growth as assessed by a cell viability assay and attenuated G(2)-M cell cycle progression associated with a decrease in cyclin B1 and phosphorylated cdc2 (Tyr(15)) protein expression. TAE226 treatment inhibited tumor cell invasion by at least 50% compared with the control in an in vitro Matrigel invasion assay. Interestingly, TAE226 treatment of tumor cells containing wild-type p53 mainly exhibited G(2)-M arrest, whereas tumor cells bearing mutant p53 underwent apoptosis. Induction of apoptosis by TAE226 was substantiated by detection of caspase-3/7 activation and poly(ADP-ribose) polymerase cleavage and by an Annexin V apoptosis assay. More importantly, TAE226 treatment significantly increased the survival rate of animals in an intracranial glioma xenograft model. Collectively, these data show that blocking the signaling pathways of FAK and IGF-IR with TAE226 has the potential to be an efficacious treatment for human gliomas.
Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Glioma/enzimología , Glioma/patología , Inhibidores de Proteínas Quinasas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Masculino , Ratones , Ratones Desnudos , Proteínas Mutantes/metabolismo , Invasividad Neoplásica , Fosforilación/efectos de los fármacos , Análisis de Supervivencia , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The abnormal function of tyrosine kinase receptors is a hallmark of malignant gliomas. Tie2 receptor tyrosine kinase is a specific endothelial cell receptor whose function is positively regulated by angiopoietin 1 (Ang1). Recently, Tie2 has also been found in the nonvascular compartment of several tumors, including leukemia as well as breast, gastric, and thyroid cancers. There is, however, little information on the function of the Ang1/Tie2 pathway in the non-stromal cells within human tumors. We found that surgical glioblastoma specimens contained a subpopulation of Tie2+/CD31- and Tie2+/GFAP+ cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. Furthermore, analysis of a tissue array consisting of 116 human glioma samples showed that Tie2 expression in the neoplastic glial cells was significantly associated with progression from a lower to higher grade. Importantly, Ang1 stimulation of Tie2+ glioma cells resulted in increased adherence of the cells to collagen I and IV, suggesting that Tie2 regulates glioma cell adhesion to the extracellular matrix. Conversely, the down-regulation of Tie2 levels by small interference RNA or the addition of soluble Tie2 abrogated the Ang1-mediated effect on cell adhesion. In studying the expression of cell adhesion molecules, we found that Tie2 activation was related to the up-regulation of integrin beta1 levels and the formation of focal adhesions. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop in malignant gliomas and that a Tie2-dependent pathway modulates cell-to-extracellular matrix adhesion, providing new insights into the highly infiltrative phenotype of human gliomas.
Asunto(s)
Adhesión Celular/fisiología , Matriz Extracelular/fisiología , Glioma/patología , Integrina beta1/fisiología , Neuroglía/metabolismo , Receptor TIE-2/metabolismo , Expresión Génica , Humanos , Neovascularización Patológica , Receptor TIE-2/genética , Células Tumorales CultivadasRESUMEN
Accumulating evidence suggests that glutamate plays a key role in the proliferation and invasion of glioblastoma tumors. Astrocytic tumors have been shown to release glutamate at high levels, which may stimulate tumor cell proliferation and motility via activation of glutamate receptors. Excess glutamate has also been found to facilitate tumor invasion by causing excitotoxic damage to normal brain thereby paving a pathway for tumor migration. Results from tissue microarray analyses showed decreased excitatory amino acid transporter-2 (EAAT-2) expression in high-grade glial tumors compared with low-grade astrocytomas and normal brain. EAAT-2 expression was inversely correlated with tumor grade, implicating its potential role in glial tumor progression, which was reflected by an undetectable level of EAAT-2 protein in glioma cell lines. In this study, we sought to investigate the effect of reconstituted EAAT-2 on glioma cell growth in vitro and in vivo by adenoviral-mediated gene transfer. Infection of glioma cells with Ad-EAAT-2 resulted in a physiologic level of functional EAAT-2, and a subsequent dose-dependent reduction in cell proliferation in all glioma cell lines tested compared with controls. Interestingly, results from analyses of Annexin V staining, detection of poly(ADP-ribose)polymerase cleavage and caspase-3 activation all indicated that Ad-EAAT-2 infection elicited apoptosis in glioma cells. Ex vivo experiments in nude mice showed a total suppression of tumor growth at sites that received Ad-EAAT-2-infected cells. Collectively, our results uncovered a new function of EAAT-2 in controlling glioma proliferation. Further studies will improve our knowledge of the role of glutamate in glioma growth and may provide useful prognostic information and alternative therapeutic targets for the treatment of glioma.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/prevención & control , Transportador 2 de Aminoácidos Excitadores/farmacología , Adenoviridae/genética , Animales , Anexina A5/metabolismo , Astrocitoma/metabolismo , Astrocitoma/patología , Astrocitoma/prevención & control , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Caspasa 3 , Caspasas/metabolismo , Proliferación Celular , Progresión de la Enfermedad , Activación Enzimática , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/prevención & control , Humanos , Técnicas In Vitro , Ratones , Ratones Desnudos , Análisis por Micromatrices , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Tumorales Cultivadas/trasplanteRESUMEN
Therapeutic replacement of the p53 gene using an adenovirus vector (Ad-p53) may be an effective alternative to conventional therapies for the treatment of glioma. We have previously demonstrated that the introduction of Ad-p53 into glioma cells containing mutant p53 induces apoptosis, whereas glioma cells containing wild-type p53 are resistant. However, Ad-p53 will enhance the radiosensitivity of wild-type p53 glioma cells by increasing their tendency for apoptosis. The mechanism underlying these different responses to Ad-p53 has not been elucidated to date. Because phosphorylation of p53 at serines 15, 20, and 392 may play a role in regulating p53-mediated apoptotic activity, we determined the phosphorylation status of exogenous p53 in mutant and wild-type gliomas after Ad-p53 transfer. Monolayer cultures of glioma cell lines expressing mutant p53 (U251 and U373) or wild-type p53 (U87 and D54) were infected with Ad-p53 and analyzed by Western blotting. High levels of exogenous p53 were detected in both cell lines after Ad-p53 transfer. However, only apoptotic mutant p53 cells expressed high levels of phospho-Ser15-p53 and phospho-Ser20-p53. The levels of phospho-Ser15-p53 and phospho-Ser20-p53 were very low in wild-type p53 cells after Ad-p53 infection alone. When wild-type p53 glioma cells were exposed to radiation after Ad-p53 infection, phospho-Ser15-p53 and phospho-Ser20-p53 were detected at high levels, and the cells subsequently underwent apoptosis; no change in serine 392 was detected. The induction of apoptosis and the expression of phospho-Ser15 and phospho-Ser20 in these cells were also enhanced by the combination of Ad-p53 and other DNA-damaging agents such as cisplatin and bichloroethyl nitrosourea. Furthermore, the expression of phospho-Ser15-p53 and phospho-Ser20-p53 correlated with the amount of apoptosis; the apoptotic activity of p53 in glioma cells was partially inhibited by a mutation of p53 at serine 15. These results suggest that phosphorylation of p53 at serine 15 and serine 20 is critical for apoptosis induction in p53 gene therapy for gliomas.
Asunto(s)
Apoptosis/fisiología , Genes p53/genética , Terapia Genética/métodos , Glioma/patología , Proteína p53 Supresora de Tumor/metabolismo , Adenoviridae/genética , Antineoplásicos/farmacología , Apoptosis/genética , Carmustina/farmacología , Cisplatino/farmacología , Daño del ADN , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Glioma/genética , Glioma/metabolismo , Glioma/terapia , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The phosphatidylinositol 3-kinase pathway is an important regulator of a wide spectrum of tumor-related biological processes, including cell proliferation, survival, and motility, as well as neovascularization. Protein kinase B/Akt is activated in a complex manner through the phosphorylation of protein kinase B/Akt on Thr308 and Ser473. Although protein-dependent kinase-1 has been shown to phosphorylate Akt at Thr308, it is not clear whether there is a distinct kinase that exclusively phosphorylates Akt at Ser473. A possible candidate is integrin-linked kinase (ILK), which has been shown to phosphorylate Akt at Ser473 in vitro. ILK is a multidomain focal adhesion protein that is believed to be involved in signal transmission from integrin and growth factor receptors. Further, ILK is implicated in the regulation of anchorage-dependent cell growth/survival, cell cycle progression, invasion and migration, and tumor angiogenesis. In this study, we tested the hypothesis that ILK inhibition would inhibit these processes in gliomas in which it is constitutively expressed. We found that a newly developed small-molecule compound (QLT0267) effectively inhibited signaling through the ILK/Akt cascade in glioma cells by blocking the phosphorylation of Akt and downstream targets, including mammalian target of rapamycin and glycogen synthase kinase-3beta. Treatment of glioma cells with 12.5 micromol/L QLT0267 inhibited cell growth by 50% at 48 hours. An anchorage-dependent cell growth assay confirmed the cell growth-inhibitory effect of QLT0267. Further, the decrease in cell growth was associated with a dramatic accumulation of cells in the G2-M phase of the cell cycle. Although the cell growth-inhibitory effects of the ILK inhibitor were achieved only at a high concentration, the QLT0267 was able to reduce cellular invasion and angiogenesis at much lower concentrations as shown by in vitro invasion assays and vascular endothelial growth factor secretion. Thus, blocking the ILK/Akt pathway is a potential strategy for molecular targeted therapy for gliomas.
Asunto(s)
Glioblastoma/enzimología , Glioblastoma/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Western Blotting , Ciclo Celular , División Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Fase G2 , Glioma/terapia , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Serina/química , Treonina/química , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vitronectina/química , Vitronectina/metabolismoRESUMEN
MBP-1, a cellular factor, appears to be involved in multiple functions, including transcriptional modulation, apoptosis and cell growth regulation. In this study, we have investigated the signaling pathway involved in MBP-1 mediated apoptotic cell death. Human carcinoma cells infected with a replication deficient adenovirus expressing MBP-1 (AdMBP-1) induced apoptosis, when compared with cells infected by replication-defective adenovirus (dl312) as a negative control. Transduction of MBP-1 in carcinoma cells releases cytochrome c from mitochondria into the cytosol leading to activation of procaspase-9, procaspase-3 and PARP cleavage. We previously observed that MBP-1 mediated apoptosis can be protected by Bcl-2, although MBP-1 does not share a homology with the BH domain of the Bcl-2 family member of proteins. To further understand the mechanism of MBP-1 mediated apoptosis, we examined whether MBP-1 modulates the Bcl-2 gene family. Our results demonstrated that human breast carcinoma cells infected with AdMBP-1 selectively reduced Bcl-xL mRNA and protein expression when compared with dl312 infected negative control cells. An in vitro transient reporter assay also suggested repression of the Bcl-x promoter activity by MBP-1. Additional studies indicated that MBP-1 modulates Ets family protein function, thereby downregulating Bcl-xL expression. Taken together, our results suggest that MBP-1 selectively represses Bcl-xL expression in MCF-7 cells and induces mitochondrial involvement in the apoptotic process.
Asunto(s)
Apoptosis , Grupo Citocromo c/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Células 3T3 , Animales , Caspasa 3 , Caspasa 9 , Caspasas/metabolismo , Proteínas de Unión al ADN/genética , Activación Enzimática , Precursores Enzimáticos/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción , Células Tumorales Cultivadas , Proteína bcl-XRESUMEN
Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 x 10(8) PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Adenovirus Humanos/genética , Glioma/patología , Glioma/virología , Animales , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Eliminación de Gen , Humanos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Trasplante HeterólogoRESUMEN
p16 is an important regulator of the cell cycle at the G(1) phase. Frequent aberration of p16 in nasopharyngeal carcinoma (NPC) suggests a role for this tumor suppressor gene in disease development. p16 gene transfer has been demonstrated to be effective in various human cancer models, including breast, lung, and prostate, causing cell cycle arrest, apoptosis, and tumor growth delay. We investigated the potential of adenoviral-mediated p16 therapy, in combination with ionizing radiation (RT), in two distinct NPC models. Two deltaE1 adenoviral vectors were employed: one carrying the human p16 gene (adv.p16), and the other a beta-galactosidase reporter gene (adv.beta-gal), both driven by the cytomegalovirus (CMV) promoter. Two NPC cell lines with differential endogenous p16 expression, CNE-1 (low) and CNE-2Z (high), were evaluated for protein expression, cytotoxicity, cell cycle analysis, apoptosis, and senescence. The CNE-1 cells were exquisitely sensitive to adv.p16, with 0.1% survival level after gene therapy [25 plaque-forming unit (pfu)/cell], which further decreased to 0.01% with the addition of RT (2 Gy). This reduction in survival was effected through necrosis, G1 arrest, and senescence. In contrast, CNE-2Z cells were resistant to adv.p16 gene transfer, with 75% surviving at an equivalent viral dose. This differential sensitivity was recapitulated in vivo in that adv.p16-treated CNE-1 cells formed no tumors in severe-combined-immunodeficiency (SCID) mice, followed for over 100 days. In contrast, tumor formation was detected 40 days after implantation of adv.p16-treated CNE-2Z cells. In conclusion, adv.p16 gene transfer appears to be highly effective against NPC that lack functional p16, which is the situation in the majority of NPC patients.
Asunto(s)
Carcinoma/terapia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Terapia Genética/métodos , Neoplasias Nasofaríngeas/terapia , Adenoviridae/genética , Animales , Apoptosis , Western Blotting , Carcinoma/genética , Ciclo Celular , Línea Celular Tumoral , Citomegalovirus/genética , Fragmentación del ADN , Citometría de Flujo , Genes Reporteros , Humanos , Cinética , Ratones , Ratones SCID , Neoplasias Nasofaríngeas/genética , Necrosis , Trasplante de Neoplasias , Proteína de Retinoblastoma/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Malignant gliomas are devastating diseases that localize within the central nervous system and are notoriously invasive. Despite recent advances in established treatment modalities such as postoperative radiotherapy and chemotherapy for gliomas, no definitive improvement in survival has been observed. However, progress in the understanding of the biology of these tumors allows for the development of translational research projects and new therapeutic approaches such as gene therapy. One of the most recent strategies is based on the use of targeted oncolytic adenoviruses. The progress of the oncolytic adenoviral system relies on the knowledge of the molecular biology of both the adenovirus and cancer. This review outlines the main strategies currently used to improve adenoviral infection, to restrict adenoviral replication to tumor cells, and to optimize the anticancer effect of oncolytic adenoviruses. Specifically, we discuss the concepts of conditionally replicative adenoviruses, tropism modifications used to efficiently redirect infectivity to cancer cells, and the transcription/transduction systems that limit the adenovirus to the target host cell. Mastery of the mechanisms of adenoviral infection and replication will lead to a full realization of the potential for adenoviruses as critical anticancer tools, and may result in the improvement of the prognosis of patients with brain tumors.
Asunto(s)
Adenoviridae , Neoplasias del Sistema Nervioso Central/terapia , Glioma/terapia , Animales , HumanosRESUMEN
Prostate cancer presents with a broad spectrum of biologic behavior, ranging from being an indolent, incidental finding to an aggressively invasive and metastatic disease. An improved understanding of the events involved in prostate cancer progression is critically important to its diagnosis and staging, as well as to the development of new therapies. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of an angiogenic, gene-driven switch. In prostate cancer, one of the most powerful stimulators of angiogenesis is the vascular endothelial growth factor (VEGF). VEGF transcription can be induced by hypoxia through activation of the PI3 kinase pathway and hypoxia-inducible factor alpha. MMAC/PTEN (henceforth referred to as PTEN) is a recently identified tumor suppressor gene residing on chromosome 10q23, which is frequently inactivated in a wide range of human tumors, including advanced prostate cancer. The goal of this study was to determine whether PTEN inhibits angiogenesis by modulating VEGF activity. Our results showed that reintroduction of the PTEN gene into human prostate PC-3 and LNCaP cells decreased VEGF secretion, which was accompanied by various biologic activities, including inhibited endothelial cell growth and migration. PTEN expression also down-regulated VEGF mRNA levels, as detected by RT-PCR analysis. Concomitant with lessened VEGF expression was the reduction of VEGF promoter activity in PTEN-expressing cells. Our findings suggest that PTEN modulates angiogenesis by regulating VEGF expression.
Asunto(s)
Adenocarcinoma/irrigación sanguínea , Factores de Crecimiento Endotelial/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Linfocinas/fisiología , Neovascularización Patológica/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Neoplasias de la Próstata/irrigación sanguínea , Proteínas Supresoras de Tumor/fisiología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Movimiento Celular , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/biosíntesis , Linfocinas/genética , Masculino , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Regiones Promotoras Genéticas , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Recombinantes de Fusión/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Adenoviral p53 gene transfer (Ad-p53) induces apoptosis in glioma cells expressing mutant p53, but fails in cells with wild-type p53. Endogenously, gliomas express varied levels of Fas/CD95, yet constitutively high levels of Fas/CD95 ligand. Because the mechanism behind the differential apoptotic response to Ad-p53 infection remains elusive, we examined how the Fas/CD95 pathway is involved in U87MG (wt-p53), D54 (wt-p53), U251MG (mutant-p53), and U373MG (mutant-p53) glioma cell lines. Ad-p53 infection did not alter the levels of Fas/CD95 ligand in either wild-type or mutant p53-expressing cell lines. In contrast, Ad-p53 infection led to an approximately 3-fold increase in Fas/CD95 mRNA expression in mutant p53-bearing cell lines but not in their wild-type (wt) counterparts, as assessed in an RNase protection assay. Fas/CD95 mRNA induction appeared to be regulated at the transcriptional level because Ad-p53 infection resulted in up to a 4-fold increase in Fas/CD95 promoter reporter activity. Subsequently, flow cytometric analysis revealed a 2- to 4-fold increase in surface Fas/CD95 expression following Ad-p53 infection in mutant-p53-containing cell lines. Use of the protein transport inhibitor Brefeldin A significantly inhibited Ad-p53-induced surface Fas/CD95 expression, but only partially inhibited apoptosis in mutant-p53 cell lines. These results suggest that p53 regulates Fas/CD95 expression at the transcriptional level and through protein trafficking in mutant-p53 cell lines. Fluorogenic activity assays demonstrated that induction of caspase-8 activity following Ad-p53 infection correlated with increases in Fas/CD95 expression. Incubating cells with a caspase-8-specific inhibitor Ac-IETD-CHO prior to Ad-p53 infection inhibited caspase-8 activity and apoptosis. Together, our results suggest that regulation of the Fas/CD95 pathway is partly responsible for Ad-p53-induced apoptosis in glioma cells, which depends on the p53 status of the involved cells. Additionally, the inability of Ad-p53 to activate the Fas/CD95 pathway in wt-p53 glioma cells coincides with their apoptotic-resistant phenotype. Further elucidation of the nature of this resistance could ultimately augment the efficacy of Ad-p53 gene therapy.
Asunto(s)
Adenoviridae/genética , Neoplasias Encefálicas/metabolismo , Genes p53 , Glioma/metabolismo , Receptor fas/biosíntesis , Apoptosis , Brefeldino A/farmacología , Caspasa 8 , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Proteína Ligando Fas , Citometría de Flujo , Genes Reporteros , Humanos , Cinética , Glicoproteínas de Membrana/metabolismo , Mutación , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , TransfecciónRESUMEN
Regulated RNA translation is critical to provide proteins needed to maintain persistent modification of synaptic strength, which underlies the molecular basis of long-term memory (LTM). Cytoplasmic polyadenylation element-binding proteins (CPEBs) are sequence-specific RNA-binding proteins and regulate translation in various tissues. All four CPEBs in vertebrates are expressed in the brain, including the hippocampal neurons, suggesting their potential roles in translation-dependent plasticity and memory. Although CPEB1 and CPEB3 have been shown to control specific kinds of hippocampus-related LTM, the role of CPEB2 and CPEB4 in learning and memory remains elusive. Thus, we generated CPEB4 knockout (KO) mice and analyzed them using several behavioral tests. No difference was found in the anxiety level, motor coordination, hippocampus-dependent learning and memory between the KO mice and their wild-type (WT) littermates. Electrophysiological recordings of multiple forms of synaptic plasticity in the Schaffer collateral pathway-CA1 neurons also showed normal responses in the KO hippocampal slices. Morphological analyses revealed that the CPEB4-lacking pyramidal neurons possessed slightly elongated dendritic spines. Unlike its related family members, CPEB1 and CPEB3, CPEB4 seems to be dispensable for hippocampus-dependent plasticity, learning and memory.
Asunto(s)
Conducta Animal/fisiología , Región CA1 Hipocampal , Memoria/fisiología , Plasticidad Neuronal/genética , Proteínas de Unión al ARN/genética , Animales , Aprendizaje/fisiología , Ratones , Ratones Noqueados , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Several small molecules that inhibit the PI3 kinase (PI3K)-Akt signaling pathway are in clinical development. Although many of these molecules have been effective in preclinical models, it remains unclear whether this strategy alone will be sufficient to interrupt the molecular events initiated and maintained by signaling along the pathways because of the activation of other pathways that compensate for the inhibition of the targeted kinase. In this study, we performed a synthetic lethality screen to identify genes or pathways whose inactivation, in combination with the PI3K inhibitors PX-866 and NVPBEZ-235, might result in a lethal phenotype in glioblastoma multiforme (GBM) cells. We screened GBM cells (U87, U251, and T98G) with a large-scale, short hairpin RNA library (GeneNet), which contains 43 800 small interfering RNA sequences targeting 8500 well-characterized human genes. To decrease off-target effects, we selected overlapping genes among the 3 cell lines that synergized with PX-866 to induce cell death. To facilitate the identification of potential targets, we used a GSE4290 dataset and The Cancer Genome Atlas GBM dataset, identifying 15 target genes overexpressed in GBM tissues. We further analyzed the selected genes using Ingenuity Pathway Analysis software and showed that the 15 genes were closely related to cancer-promoting pathways, and a highly interconnected network of aberrations along the MYC, P38MAPK, and ERK signaling pathways were identified. Our findings suggest that inhibition of these pathways might increase tumor sensitivity to PX-866 and therefore represent a potential clinical therapeutic strategy.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Genes Letales , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Gonanos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , ARN Interferente Pequeño/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Combinación de Medicamentos , Diseño de Fármacos , Sinergismo Farmacológico , Humanos , Transducción de SeñalRESUMEN
Aberrant genetic alternations in human gliomas, such as amplification of epidermal growth factor receptor, mutation and/or deletion of tumor suppressor gene PTEN, and mutations of PIK3CA, contribute to constitutive activation of the phosphatidylinositol 3-kinase (PI3K) pathway. We investigated the potential antitumor activity of NVP-BEZ235, which is a novel dual PI3K/mammalian target of rapamycin (mTOR) inhibitor in gliomas. The compound suppressed glioma cell proliferation with IC(50) values in the low nanomolar range by specifically inhibiting the activity of target proteins including Akt, S6K1, S6, and 4EBP1 in the PI3K/Akt/mTOR signaling pathway. NVP-BEZ235 treatment of glioma cell lines led to G(1) cell cycle arrest and induced autophagy. Furthermore, expression of the vascular endothelial growth factor (VEGF), which is an important angiogenic modulator in glioma cells, was significantly decreased, suggesting that NVP-BEZ235 may also exert an antiangiogenic effect. Preclinical testing of the therapeutic efficacy of NVP-BEZ235 showed that it significantly prolonged the survival of tumor-bearing animals without causing any obvious toxicity. Tumor extracts harvested from animals after treatment showed that the compound inhibited the activity of target proteins in the PI3K/Akt/mTOR cascade. Immunohistochemical analyses also showed a significant reduction in staining for VEGF von Willebrand factor (factor VIII) in NVP-BEZ235-treated tumor sections compared with controls, further confirming that NVP-BEZ235 has an antiangiogenic effect in vivo. We conclude from these findings that NVP-BEZ235 antagonizes PI3K and mTOR signaling and induces cell cycle arrest, down-regulation of VEGF, and autophagy. These results warrant further development of NVP-BEZ235 for clinical trials for human gliomas or other advanced cancers with altered PI3K/Akt/mTOR signaling.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glioma/tratamiento farmacológico , Glioma/enzimología , Imidazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Ciclo Celular , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Serina-Treonina Quinasas TORRESUMEN
c-Jun, a major transcription factor in the activating protein 1 (AP-1) family of regulatory proteins, is activated by many physiologic and pathologic stimuli. However, whether c-jun is regulated by epigenetic modification of chromatin structure is not clear. We showed here that c-jun was transcriptionally repressed in response to osmotic stress via a truncated HDAC3 generated by caspase-7-dependent cleavage at aspartic acid 391. The activation of caspase-7, which is independent of cytochrome c release and activation of caspase-9 and caspase-12, depends on activation of caspase-8, which in turn requires MEK2 activity and secretion of FAS ligand. The cell apoptosis induced by the truncated HDAC3 or enhanced by c-Jun deficiency during osmotic stress was suppressed by exogenous expression of c-Jun, indicating that the downregulation of c-Jun by HDAC3-dependent transcriptional repression plays a role in regulating cell survival and apoptosis.
Asunto(s)
Apoptosis/fisiología , Histona Desacetilasas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ácido Aspártico/química , Secuencia de Bases , Caspasa 7/metabolismo , Caspasa 8/metabolismo , Línea Celular , ADN Complementario/genética , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática , Proteína Ligando Fas/metabolismo , Histona Desacetilasas/química , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Ratones , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Modelos Biológicos , Células 3T3 NIH , Presión Osmótica , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Regiones Promotoras Genéticas , Sorbitol/farmacologíaRESUMEN
Global protein analysis of treated and untreated glioblastoma cell lines was performed. Proteomic analysis revealed the identity of proteins that were significantly modulated by the treatment with wild-type TP53 and the cytotoxic chemotherapy SN38. In particular, galectin-1 was found to be negatively regulated by transfection with TP53 and further down-regulated by SN38. Expression level changes were confirmed by Western blot. Subsequent analysis of several high-grade glioma cell lines demonstrated very high levels of galectin-1, regardless if the cell lines contained mutant or wild-type TP53. High expression of galectin-1 in a human orthotopic murine tumor model was also detected by immunohistochemistry and revealed a consistent pattern of preferential expression in peripheral or leading tumor edges. Further examination of galectin-1 expression through microarray analysis in tumor materials from patients confirmed galectin-1 as a valuable biomarker and possible therapeutic target. These results demonstrate the utility of using proteomic approaches to interrogate and identify potential useful targets for cancer therapy by evaluating specific tumor responses, either positive or negative, to various therapies.
Asunto(s)
Antineoplásicos/toxicidad , Galectina 1/genética , Glioblastoma/genética , Proteómica/métodos , Proteína p53 Supresora de Tumor/genética , Animales , Western Blotting , División Celular , Línea Celular Tumoral , Supervivencia Celular , Electroforesis en Gel Bidimensional , Galectina 1/aislamiento & purificación , Terapia Genética , Glioblastoma/patología , Humanos , Espectrometría de Masas , Trasplante de Neoplasias , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/aislamiento & purificaciónRESUMEN
BACKGROUND: Laboratory and clinical data suggest that the anti-angiogenic agent, thalidomide, if combined with cytotoxic agents, may be effective against recurrent glioblastoma multiforme (GBM). OBJECTIVES: To determine 6-month progression-free survival (6PFS) and toxicity of temozolomide plus thalidomide in adults with recurrent GBM. PATIENTS AND METHODS: Eligible patients had recurrent GBM after surgery, radiotherapy, and/or adjuvant chemotherapy. Temozolomide was given at 150-200 mg/m(2)/day on days 1-5 of each 28-day cycle. Thalidomide was given orally at 400 mg at bedtime (days 1-28) and increased to 1,200 mg as tolerated. Patients were evaluated with magnetic resonance imaging scans every 56 days. The study was designed to detect an increase of the historical 6PFS for GBM from 10 to 30%. RESULTS: Forty-four patients were enrolled, 43 were evaluable for efficacy and safety. The study population included 15 women, 29 men; median age was 53 years (range 32-84); median Karnofsky performance status was 80% (range 60-100%). Thirty-six (82%) patients were chemotherapy-naïve. There were 57 reports of toxicity of grade 3 or greater. Non-fatal grade 3-4 granulocytopenia occurred in 15 patients (34%). The objective response rate was 7%. The estimated probability of being progression-free at 6 months with this therapy is 24% [95% confidence interval (C.I.) 12-38%]. The median time to progression is 15 weeks (95% C.I. 10-20 weeks). There was no observed correlation between serum levels of vascular endothelial growth factor, basic fibroblast growth factor, and IL-8 and the 6PFS outcome. CONCLUSION: This drug combination was reasonably safe, but with little indication of improvement compared to temozolomide alone.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/mortalidad , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Supervivencia sin Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Glioblastoma/mortalidad , Humanos , Masculino , Persona de Mediana Edad , América del Norte , Análisis de Supervivencia , Temozolomida , Talidomida/uso terapéuticoRESUMEN
Mutations of PTEN, a tumor suppressor gene located on chromosome 10, which encodes a protein-tyrosine and lipid-phosphatase, are prevalent in various human cancers, including glioblastoma. Despite extensive characterization of PTEN mutations in human cancers and a relatively good understanding of the molecular roles of PTEN in the control of cellular processes, little is known about modes of PTEN regulation. To understand the regulation of expression of the tumor suppressor gene PTEN, we isolated a 2212 bp fragment from the human BAC clone 46B12 DNA. The 3' end of this fragment starts at the Not I site of -745 relative to the first translation codon ATG (+1) and ends at the Sal I site of -2957 at the 5' end. Using classical 5'RACE and primer extension techniques, nine start sites were observed between -817 and -984 upstream of the ATG start site. We located a 137 bp fragment (-958/-821) as the minimum promoter region using promoter deletion and luciferase assays. A 704 bp fragment (-33/-737) downstream of the 2212 bp fragment was also cloned. As indicated by luciferase assays, the data show that this region possesses no promoter function. Interestingly, a p53 binding sequence is located within the 599 bp fragment (-1344/-745), although p53 expression had a minimal effect on PTEN, demonstrating its insignificant role in PTEN gene expression.