RESUMEN
Pancreatic cancer is one of the intractable diseases and an effective therapeutic strategy is required to improve the prognosis. We examined possible antitumor effects of adenoviruses expressing melanoma differentiation-associated gene-7/interleukin-24 (Ad-mda-7) and a heat-shock protein 90 (Hsp90) inhibitor to human pancreatic carcinoma cells. Ad-mda-7 and an Hsp90 inhibitor, geldanamycin (GA), produced cytotoxic effects, and a combinatory use of Ad-mda-7 and GA further achieved synergistic effects. Administration of N-acetyl-L-cysteine, an inhibitor of reactive oxygen species, eliminated Ad-mda-7- and GA-mediated cytotoxicity. Ad-mda-7 augmented phosphorylated AKT levels but GA did not influence the phosphorylation. GA-treated cells showed cleavage of poly-(ADP-ribose) polymerase but not caspase-3, and upregulated Hsp70 and LC3A/B II levels, whereas Ad-mda-7-treated cells did not. GA treatments augmented ubiquitination and markedly increased melanoma differentiation-associated gene-7 (MDA-7) expression levels. These findings suggest that Ad-mda-7-mediated cytotoxicity is dependent on reactive oxygen species but independent of apoptosis or autophagy, and that GA-mediated cytotoxicity was linked with caspase-independent apoptosis and/or autophagy. A mechanism underlying the combinatory effects of Ad-mda-7 and GA remained complex and the synergism is attributable to multiple factors including increased MDA-7 protein stability by GA.
Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/genética , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Interleucinas/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Adenoviridae/genética , Benzoquinonas/farmacología , Ciclo Celular/genética , Ciclo Celular/inmunología , Línea Celular Tumoral , Sinergismo Farmacológico , Expresión Génica , Vectores Genéticos/genética , Humanos , Lactamas Macrocíclicas/farmacología , Neoplasias Pancreáticas/metabolismo , Transducción Genética , Neoplasias PancreáticasRESUMEN
The melanoma differentiation-associated gene-7 (mda-7) is a known mediator of apoptosis in cancer cells but not in normal cells. We hypothesized that MDA-7 interferes with the prosurvival signaling pathways that are commonly altered in cancer cells to induce growth arrest and apoptosis. We also identified the cell signaling pathways that are antagonized by MDA-7 leading to apoptosis. Using an adenoviral expression system, mda-7 was introduced into the breast cancer cell lines SKBr3, MCF-7 and MDA-MB-468, each with a different estrogen receptor (ER) and HER-2 receptor status. Downstream targets of MDA-7 were assessed by reverse phase protein array analysis, western blot analysis and immunofluorescence confocal microscopy. Our results show that MDA-7-induced apoptosis was mediated by caspases in all cell lines tested. However, MDA-7 modulates additional pathways in SKBr3 (HER-2 positive) and MCF-7 (ER positive) cells including downregulation of AKT-GSK3ß and upregulation of cyclin-dependent kinase inhibitors in the nucleus. This leads to cell cycle arrest in addition to apoptosis. In conclusion, MDA-7 abrogates tumor-promoting pathways including the activation of caspase-dependent signaling pathways ultimately leading to apoptosis. In addition, depending on the phenotype of the breast cancer cell, MDA-7 modulates cell cycle regulating pathways to mediate cell cycle arrest.
Asunto(s)
Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Interleucinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adenoviridae/genética , Apoptosis/genética , Western Blotting , Neoplasias de la Mama/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular Tumoral , Femenino , Humanos , Interleucinas/genética , Microscopía Confocal , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Secalonic acid D (SAD), a cleft palate-inducing teratogen, has been shown to inhibit proliferation/cell cycle progression in association with alteration in the levels of cell cycle regulators, p21 and cyclin E. These studies were conducted to test the hypotheses that p21 and cyclin E play an important functional role in normal human embryonic palatal mesenchymal (HEPM) cell cycle and that their up- and down-regulation, respectively, by SAD is functionally significant to its cell cycle block. Using small interfering RNA (siRNA) to silence p21 gene and transient transfection to overexpress cyclin E in control & SAD-treated HEPM cells, cell proliferation was assessed using a combination of cell numbers, thymidine uptake, CDK2 activity and Ki-67 expression. The results showed that silencing of p21 gene, although increased cell proliferation/numbers and CDK2 activity in normal HEPM cells, failed to counteract SAD-induced anti-proliferative effect despite inducing partial recovery of CDK2 activity. Similar effects were apparent with cyclin E overexpression. It is concluded that p21 and cyclin E are important for normal HEPM cell proliferation. However, SAD-induced deregulation of either protein, singly, may not be sufficient to induce anti-proliferative effect. Involvement of other cell cycle proteins such as cyclin D1 or of multiple proteins in SAD-induced cell cycle block needs to be examined.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Ciclina E/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Mesodermo/efectos de los fármacos , Hueso Paladar/efectos de los fármacos , Xantonas/farmacología , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Línea Celular , Ciclina E/genética , Ciclina E/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Silenciador del Gen , Humanos , Immunoblotting , Mesodermo/citología , Mesodermo/metabolismo , Hueso Paladar/citología , Hueso Paladar/embriología , Hueso Paladar/metabolismo , ARN Interferente Pequeño/farmacologíaRESUMEN
Steady-state protein levels are determined by the balance between protein synthesis and degradation. Protein half-lives are determined primarily by degradation, and the major degradation pathways involve either lysosomal destruction or an ATP-dependent process involving ubiquitination to target proteins to the proteosome. Studies have shown that multiple tumor-suppressor proteins are ubiquitinated and degraded by the 26S proteasome. In the present study, we investigated whether the tumor suppressor/cytokine melanoma differentiation-associated gene-7/interleukin-24 gene (MDA-7/IL-24) protein is ubiquitinated and its degradation controlled by the proteasome. Treatment of ovarian (2008) and lung (H1299) tumor cells with adenoviral delivery of mda-7 (Ad-mda7) or Ad-mda7 plus the proteosome inhibitor MG132 showed that MDA-7 protein expression was dependent upon proteosome activity. Western blot and immunoprecipitation analyses verified that the MDA-7 protein was ubiquitinated and that ubiquitinated-MDA-7 levels were increased in MG132-treated cells. These results were confirmed using small interfering RNA (siRNA)-mediated knockdown of ubiquitin. Furthermore, ubiquitinated MDA-7 protein was degraded by the 26S proteasome, as MDA-7 accumulation was observed only when cells were treated with MG132 but not with lysosome or protease inhibitors. Inhibition of the catalytic beta-5 subunit of the 20S proteasome using siRNA resulted in MDA-7 protein accumulation. Finally, treatment of tumor cells with Ad-mda7 plus the proteasome inhibitor bortezomib resulted in increased tumor cell killing. Our results show that MDA-7/IL-24 is ubiquitinated and degraded by the 26S proteasome. Furthermore, inhibition of MDA-7 degradation results in enhanced tumor killing, identifying a novel anticancer strategy.
Asunto(s)
Interleucinas/biosíntesis , Neoplasias Pulmonares/metabolismo , Neoplasias Ováricas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Ubiquitina/metabolismo , Ubiquitinación , Adenoviridae , Ácidos Borónicos/farmacología , Bortezomib , Dominio Catalítico/genética , Línea Celular Tumoral , Femenino , Terapia Genética , Humanos , Interleucinas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Lisosomas/genética , Lisosomas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma , Pirazinas/farmacología , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina/antagonistas & inhibidores , Ubiquitina/genética , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genéticaRESUMEN
Our previous studies demonstrated that adenovirus-mediated overexpression of melanoma differentiation-associated gene-7 (Ad-mda7) leads to rapid induction of double-stranded RNA-dependent protein kinase (PKR) and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. Here, we report that Ad-mda7 and the benzoquinone ansamycin geldanamycin (GA) interact in a highly synergistic manner to induce cell death in human lung cancer cells. Co-administration of Ad-mda7 and GA did not modify expression of MDA-7, and was not associated with further PKR induction and activation; instead the enhanced cytotoxicity of this combination was associated with inactivation of AKT by GA. By surface staining using anti-E-cadherin monoclonal antibody and flow cytometry, we found that treatment with the combination of Ad-mda7 and GA increased E-cadherin levels in these cancer cells. Ad-mda7 and GA cotreatment also inhibited lung cancer cell motility by increasing the beta-catenin/E-cadherin association. Moreover, combination of GA derivative 17-allyl-amino, 17-demethoxygeldanamycin (17AAG), with Ad-mda7 resulted in enhancement of cell death in A549 and H460 human lung cancer cells.
Asunto(s)
Adenoviridae/genética , Benzoquinonas/farmacología , Supervivencia Celular/efectos de los fármacos , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Benzoquinonas/química , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Humanos , Lactamas Macrocíclicas/químicaRESUMEN
The melanoma differentiation-associated gene-7 (mda-7) is a member of the interleukin-10 cytokine family and a novel tumor suppressor gene. Adenoviral-mediated mda-7 (Ad-mda7) gene transfer has tumor-specific growth inhibitory and proapoptotic effects in a broad spectrum of cancer cells. In breast cancer cells, adenoviral-induced mda-7 expression triggers antiproliferative effects by downregulation of survival signals, such as Bcl-2 and Akt. The anti-human epidermal growth factor receptor-2 (Her-2) monoclonal antibody, Trastuzumab (Herceptin), increases the sensitivity of Her-2/neu-overexpressing breast cancer cells to chemotherapeutic agents and radiotherapy. In this study, we evaluate the effects of treatment with Ad-mda7 and Herceptin combination therapy in a panel of Her-2/neu-overexpressing cell lines, and in established tumors in nude mice. Compared to individual treatments, the combination of Ad-mda7 and Herceptin elicits supra-additive antitumor activity in Her-2/neu-overexpressing tumor cell lines: increased cell death, cell cycle block and apoptosis. The Ad-mda7 and Herceptin interaction was shown to be synergistic by isobologram analysis. Ad-mda7 does not alter cell surface Her-2/neu levels, but the combination of Ad-mda7+Herceptin results in increased expression of cell surface E-cadherin with concomitant translocation of beta-catenin from the nucleus to the cell membrane. In vivo, the combination of Ad-mda7 and Herceptin showed significantly increased antitumor activity (P<0.003) against Her-2/neu-overexpressing tumors. These data suggest that the combination of Ad-mda7 with Herceptin may be a novel therapy for breast cancer patients whose tumors overexpress Her-2/neu. The observed synergistic effect may improve treatment options for otherwise poorly responsive, Her-2-positive, breast cancer patients.
Asunto(s)
Adenoviridae/genética , Anticuerpos Monoclonales/genética , Neoplasias de la Mama/genética , Interleucinas/genética , Animales , Anticuerpos Monoclonales Humanizados , Western Blotting , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Humanos , Ratones , Ratones Desnudos , TrastuzumabRESUMEN
Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3(+)CD8(+) but not the CD3(+)CD4(+) cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Fibrosarcoma/terapia , Interleucinas/inmunología , Adenoviridae , Animales , Apoptosis/inmunología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Citocinas/biosíntesis , Femenino , Fibrosarcoma/inmunología , Terapia Genética , Vectores Genéticos , Inmunocompetencia , Inyecciones Intralesiones , Interleucinas/administración & dosificación , Interleucinas/genética , Interleucinas/uso terapéutico , Ratones , Ratones Endogámicos C3H , Bazo/citología , Bazo/inmunología , Células TH1/inmunología , Trasplante Isogénico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cell cycle progression and thus proper cell number is essential for normal development of organs and organisms. Craniofacial tissues including the secondary palate are vulnerable to disruption of cell cycle progression and proliferation by many chemicals including mycotoxin, secalonic acid D (SAD), glucocorticoids, retinoic acid and 2,3,7,8-tetrachlorodibenzodioxin. Induction of cleft palate (CP) by SAD in mice occurs from a reduction in the size of developing palatal shelves. This is associated with an inhibition of proliferation of murine and human embryonic palatal mesenchymal (MEPM and HEPM) cells as well as a G1/S block of cell cycle. In murine embryonic palates and HEPM cells, SAD inhibited G1/S-phase-specific cyclin-dependent kinase (CDK)2 activity, reduced the level of cyclin E and increased the level of the CDK2 inhibitor, p21. These results, together with those from other laboratories, suggest that common cell cycle protein targets (biomarkers), relevant to the pathogenesis of CP by multiple chemical exposures, that can form the basis for the diagnosis and the development of preventive strategies, are likely to exist.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fisura del Paladar/metabolismo , Hueso Paladar/metabolismo , Anomalías Inducidas por Medicamentos/metabolismo , Animales , Fisura del Paladar/etiología , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/fisiología , Humanos , Hueso Paladar/anomalías , Hueso Paladar/embriología , Teratógenos/toxicidadRESUMEN
Current therapies used in the treatment of breast cancer are limited by systemic toxicity, rapid drug metabolism and intrinsic and acquired drug resistance. We have previously shown that adenoviral-mediated transfer of the melanoma differentiation-associated gene-7 (mda-7) elicits growth inhibition and apoptosis in various tumor types. Here, we evaluate the effects of Ad-mda7, alone and in combination with other therapies, against a panel of nine breast tumor cell lines and their normal counterparts; we report selective Ad-mda7-mediated p53-independent growth inhibition, G2/M cell cycle arrest, and apoptosis. In vivo, Ad-mda7 induced p53-independent tumor growth inhibition (P<0.004) in multiple xenograft models. We then evaluated the combination of Ad-mda7 with agents commonly used to treat breast cancer: radiotherapy (XRT), Tamoxifen, Taxotere, Adriamycin, and Herceptin. These agents exhibit diverse modes of action, including formation of bulky adducts, inhibition of DNA replication (Adriamycin, XRT), damage to microtubules (Taxotere), nonsteroidal estrogen antagonists (Tamoxifen), or Her2/neu receptor blockade (Herceptin). Treated with conventional anticancer drugs or radiation, MDA-7-expressing cells display additive or synergistic cytotoxicity and apoptosis that correlates with decreased BCL-2 expression and BAX upregulation. In vivo, animals that received Ad-mda7 and XRT underwent significant reduction of tumor growth (P<0.002). This is the first report of the synergistic effects of Ad-mda7 combined with chemotherapy or radiotherapy on human breast carcinoma cells.
Asunto(s)
Neoplasias de la Mama/terapia , Carcinoma/terapia , Terapia Genética , Interleucinas/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Adenoviridae/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , Terapia Biológica , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/radioterapia , Carcinoma/tratamiento farmacológico , Carcinoma/radioterapia , Terapia Combinada , Femenino , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-bcl-2/análisisRESUMEN
Cyclic AMP is an important second messenger mediating the actions of many hormones and other ligands in a variety of cells. Cells of the developing organism are no exception. Once generated, it releases the catalytic subunit of protein kinase A (PKA) from the inhibitory influence of its regulatory subunit, which then migrates into the nucleus to phosphorylate and enhance the binding of relevant transcription factors to the promoter element CRE of genes involved in above cellular responses. This review summarizes the available data on the essential role of this pathway in embryonic development as well as the functionality, ontogeny and consequences of genetic and chemical disruption of this pathway in the developing orofacial structures, especially the secondary palate as influenced by the mycotoxin, secalonic acid D.
Asunto(s)
Fisura del Paladar/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico , Proteínas Nucleares/fisiología , Transducción de Señal/efectos de los fármacos , Transactivadores/fisiología , Xantonas/farmacología , Animales , Proteína de Unión a CREB , Fisura del Paladar/embriología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Humanos , RatonesRESUMEN
The protein kinase C (PKC) family of proteins mediates the action of growth factors and other ligands by activating a network of transcription factors that bind to TRE sequences in the promoters of many genes that regulate cell proliferation, differentiation, extracellular matrix synthesis, apoptosis and others in a cell type-, isozyme- and context-specific manner. The critical role of PKC in embryonic development is indicated by early death of embryos in which one or more of these isozymes are inactivated. Our studies together with others show that palatal PKC signalling is functional and may be essential for normal palate development. Although single gene knockouts have failed to exhibit the cleft palate (CP) phenotype, owing to compensation by other kinases, many chemicals including the mycotoxin, secalonic acid D, disrupt palatal PKC signalling leading to altered palatal mesenchymal gene expression. The potential relevance of such effects to chemical-induced CP is discussed.
Asunto(s)
Anomalías Inducidas por Medicamentos/etiología , Fisura del Paladar/inducido químicamente , Proteína Quinasa C/fisiología , Xantonas/toxicidad , Cara/embriología , Humanos , Hueso Paladar/embriología , Elementos de Respuesta , Transducción de SeñalRESUMEN
Secalonic acid-D (SAD) is a teratogenic mycotoxin inducing cleft palate (CP) in the offspring of the exposed mice by reducing palatal shelf size secondary to reduced proliferation of the palatal mesenchymal (PM) cells. Co-administration of dimethylsulfoxide (DMSO) reversed the CP-inducing effect of SAD. Although SAD has been shown to affect both protein kinases A (PKA) and C (PKC) pathways, the relevance of each of these pathways to its CP induction is unknown. The present studies were designed to test the hypothesis that the protective effect of DMSO is mediated by its specific reversal of the effect(s) of SAD on one of these two pathways using ELISA-based activity assays, Western blot analysis, electrophoretic mobility shift assays (EMSA), and murine embryonic PM (MEPM) cell growth in culture. Within the PKA pathway, SAD inhibited the activity of the catalytic subunit of PKA and its migration into the nucleus, elevated phosphorylated cyclic AMP (cAMP) response element (CRE)-binding protein (pCREB) level, and reduced the binding of CREB to CRE. In the PKC pathway, SAD reduced the activity of PKC and the binding of transcription factors (TF) to 12-O-tetradecanoate-13 phorbol acetate-response element (TRE). SAD also inhibited MEPM cell growth and the expression of the CRE- and TRE-containing gene, proliferating cell nuclear antigen (PCNA). Reversal, by DMSO, of the effects of SAD on MEPM cell growth, on PCNA expression and on all components of the PKA, but not of PKC, pathway suggests that the perturbation of the PKA pathway by SAD is relevant to its induction of CP in mice.
Asunto(s)
Fisura del Paladar/inducido químicamente , Fisura del Paladar/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Hueso Paladar/enzimología , Teratógenos/toxicidad , Xantonas/toxicidad , Animales , Western Blotting , Catálisis , División Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Fisura del Paladar/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Dimetilsulfóxido/farmacología , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Inhibidores Enzimáticos/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Ratones , Hueso Paladar/citología , Fosforilación , Embarazo , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Elementos de Respuesta/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Xantonas/antagonistas & inhibidores , Xantonas/metabolismoRESUMEN
The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers.
Asunto(s)
Adenoviridae/genética , Neoplasias Colorrectales/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Monoéster Fosfórico Hidrolasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis/genética , Western Blotting/métodos , Caspasas/metabolismo , Ciclo Celular/genética , División Celular/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/genética , Activación Enzimática , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead , Expresión Génica , Vectores Genéticos/genética , Glucógeno Sintasa Quinasa 3/genética , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fosfohidrolasa PTEN , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Factores de Transcripción/genética , Trasplante HeterólogoRESUMEN
Regulation of gene expression via the protein kinase A (PKA) pathway is mediated through Ser133 phosphorylation of the transcription factor (TF), cAMP response element (CRE) binding protein (CREB). Secalonic acid D (SAD), a mycotoxin causing cleft palate (CP), induces phosphorylation of palatal CREB in vivo. SAD-induced increase in phosphoCREB (pCREB), however, is associated with decreased binding of TF to CRE in vivo. Mechanism(s) involved in these two effects of SAD were studied using palatal nuclear extracts (PNE). Stimulation of CREB phosphorylation by SAD was confirmed in vitro in both cell culture and cell-free systems, and this phosphorylation was not altered by currently known CREB kinase (PKA, CaMK, MEK, p38MAPK, PKC) or phosphatase inhibitors. SAD-induced increase in pCREB, however, was associated with decreased TF binding to CRE in vitro. Two-dimensional gel analysis ruled out additional inhibitory phosphorylations. Addition of SAD to PNE following an increase in PKA-phosphorylated CREB resulted in reduced TF binding to CRE. Further, SAD was shown to bind directly to phosphorylated nuclear proteins (pCREB) with greater affinity. In addition, the inhibitory effect of SAD occurred with CRE of proliferating cell nuclear antigen (PCNA) gene. These studies confirm that stimulation of CREB phosphorylation by SAD does not involve sites other than Ser133 and is mediated by a novel kinase. They also indicate that SAD directly binds to CREB to inhibit its binding to CRE of genes such as PCNA. This effect could lead to reduced palatal mesenchymal cell number, smaller palatal shelf, and thus CP.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Nucleares/metabolismo , Teratógenos/farmacología , Transactivadores/metabolismo , Xantenos/farmacología , Xantonas , Animales , Proteína de Unión a CREB , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Endogámicos , Proteínas Nucleares/genética , Hueso Paladar/anomalías , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica/efectos de los fármacos , Transducción de Señal/genética , Teratógenos/toxicidad , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xantenos/toxicidadRESUMEN
P53-based immunization is an attractive approach to cancer immunotherapy due to the accumulation of p53 protein in tumor, but not in normal cells. However, it was not known whether immune response against self-protein (p53) could be generated in vivo. Mouse dendritic cells (DCs) were transduced with adenoviral construct containing murine full-length wild-type p53 (Ad-p53). Repeated immunizations with these cells protected 60% of mice against challenge with MethA sarcoma cells bearing point mutations in p53 gene. Activation of DCs via ligation of CD40 significantly improved the results of immunization: all mice were protected against MethA sarcoma. The treatment of MethA tumor-bearing mice with activated Ad-p53-transduced DCs showed complete tumor rejection in four out of six mice. The specificity of antitumor immune response was confirmed by CTL assay. The analysis of phenotype and function of DCs demonstrated that the effect of CD40 ligation on these cells was enhanced by their infection with Ad-p53. The level of neutralizing anti-adenovirus antibody was moderately elevated in these mice. No signs of autoimmune reaction were evident during detailed pathological evaluation of treated mice. These data demonstrate that activated Ad-p53-infected DCs are able to break tolerance to this protein and can be used in immunotherapy of cancer.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/metabolismo , Genes p53 , Terapia Genética/métodos , Sarcoma Experimental/terapia , Adenoviridae/genética , Animales , Pruebas Inmunológicas de Citotoxicidad , Femenino , Vectores Genéticos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Sarcoma Experimental/inmunología , Transducción GenéticaRESUMEN
Secalonic acid D (SAD), a teratogenic mycotoxin, induces cleft palate (CP) in the offspring of exposed mice by inhibiting palatal shelf growth. Since reduced proliferation, increased apoptosis, and/or decreased extracellular matrix (ECM) synthesis of palatal mesenchymal cells (PMC) can all contribute to smaller shelf size, the hypothesis that teratogenically relevant concentrations (0 to 120 microg/ml) of SAD will have adverse effects on one or more of these cellular processes was tested, using primary murine PMC cultures. Exposure to SAD resulted in significant and dose-dependent decreases in mesenchymal cell number, uptake of (3)H-thymidine, and expression of proliferating cell nuclear antigen (PCNA). Trypan blue dye exclusion assay, however, revealed significant cell death only at higher doses, suggesting that the decrease in cell number at lower (more realistic) doses is likely a consequence of reduced cell proliferation and not cell death. Further, negative results in the DNA fragmentation analysis following SAD exposure suggested that cell death caused by higher levels of SAD was unrelated to apoptosis. Similarly, results of (3)H-glucosamine uptake assay indicated inhibitory effect of SAD on accumulation of hyaluronic acid (HA) or sulfated glycosaminoglycans (sGAG) only at the highest dose tested. Also, SAD affected neither extracellular nor cell-associated fibronectin expression at any dose tested. Taken together, these data suggest that the pathogenesis of CP by SAD is likely a result of a reduction in the size of the palatal shelf caused by SAD-induced inhibition of mesenchymal cell proliferation.
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Mesodermo/efectos de los fármacos , Micotoxinas/toxicidad , Xantenos/toxicidad , Xantonas , Animales , Apoptosis/efectos de los fármacos , Western Blotting , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Femenino , Fibronectinas/biosíntesis , Glicosaminoglicanos/biosíntesis , Ácido Hialurónico/biosíntesis , Masculino , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Micotoxinas/administración & dosificación , Hueso Paladar/citología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Timidina/metabolismo , Tritio , Xantenos/administración & dosificaciónRESUMEN
Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.
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Antígenos de Neoplasias/genética , Apoptosis/genética , Cromosomas Humanos Par 1/genética , Diterpenos , Genes , Sustancias de Crecimiento/genética , Interleucinas , Proteínas de Neoplasias/genética , Neoplasias/genética , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/aislamiento & purificación , Secuencia de Bases , Carcinoma/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , División Celular/genética , Clonación Molecular , Dimetilsulfóxido/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor , Glioblastoma/patología , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/aislamiento & purificación , Células HL-60/metabolismo , Células HL-60/patología , Humanos , Interferón Tipo I/farmacología , Células K562/metabolismo , Células K562/patología , Masculino , Melanocitos/metabolismo , Melanoma/química , Melanoma/genética , Melanoma/patología , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/aislamiento & purificación , Especificidad de Órganos , Osteosarcoma/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Proteínas Recombinantes de Fusión/fisiología , Proteínas Recombinantes , Terpenos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas/patologíaRESUMEN
The melanoma differentiation associated gene-7 (mda-7) has a potential inhibitory role in melanoma progression, although the mechanisms underlying this effect are still unknown. mda-7 mRNA has been found to be present at higher levels in cultured normal melanocytes compared with metastatic melanoma cell lines. Furthermore, levels of mda-7 message have shown an inverse correlation with melanoma progression in human tumor samples, suggesting that mda-7 may be a novel tumor suppressor gene. We have designed this study to investigate MDA-7 protein expression in different stages of melanoma progression and to examine its antiproliferative effects in vitro. Our data demonstrate that MDA-7 protein can be found in normal melanocytes and early stage melanomas. It is also observed in smooth muscle cells in the skin. However, in keeping with a possible role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated antitumor effects of overexpressed MDA-7 on human melanoma cells in vitro. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes.
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Regulación Neoplásica de la Expresión Génica , Sustancias de Crecimiento/genética , Interleucinas , Melanocitos/química , Melanoma/metabolismo , Apoptosis , Diferenciación Celular , División Celular , Citoplasma/química , Regulación hacia Abajo , Fase G2 , Genes Supresores de Tumor , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/fisiología , Humanos , Melanoma/patología , Melanoma/secundario , Mitosis , Células Tumorales CultivadasRESUMEN
BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.
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Terapia Genética/métodos , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Interleucinas , Neoplasias/terapia , Oxazinas , Xantenos , Adenoviridae/genética , Anexina A5/metabolismo , Western Blotting , División Celular/efectos de los fármacos , Línea Celular , Separación Celular , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Colorantes/farmacología , Relación Dosis-Respuesta a Droga , Exones , Citometría de Flujo , Genes Supresores de Tumor/genética , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Confocal , Microscopía Fluorescente , Propidio/farmacología , Timidina/metabolismo , Factores de Tiempo , Transducción Genética , Azul de Tripano/farmacología , Células Tumorales CultivadasRESUMEN
Accumulation of wild-type or mutant p53 protein occurs in approximately 50% of human malignancies. This overexpression may generate antigenic epitopes recognized by CTLs. Because normal cells have undetectable levels of p53, these CTLs are likely to be tumor specific. Here, for the first time, we test the hypothesis that full-length wild-type p53 protein can be used for generation of an immune response against tumor cells with p53 overexpression. T cells obtained from nine HLA-A2-positive cancer patients and three HLA-A2-positive healthy individuals were stimulated twice with dendritic cells (DCs) transduced with an adenovirus wild-type p53 (Ad-p53) construct. Significant cytotoxicity was detected against HLA-A2-positive tumor cells with accumulation of mutant or wild-type p53 but not against HLA-A2-positive tumor cells with normal (undetectable) levels of p53 or against HLA-A2-negative tumor cells. This response was specific and mediated by CD8+ CTLs. These CTLs recognized HLA-A2-positive tumor cells expressing normal levels of p53 protein after their transduction with Ad-p53 but not with control adenovirus. Stimulation of T cells with Ad-p53-transduced DCs resulted in generation of CTLs specific for p53-derived peptide. These data demonstrate that DCs transduced with the wild-type p53 gene were able to induce a specific antitumor immune response. This offers a new promising approach to immunotherapy of cancer.