RESUMEN
Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Neoplasias/inmunología , Neovascularización Patológica/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Área Bajo la Curva , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/inmunología , Glioblastoma/metabolismo , Células HCT116 , Células HT29 , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Approximately 25% of patients with colorectal cancer develop metastases to the liver, and surgery is currently the best treatment available. But there are several patients who are unresectable, and isolated hepatic perfusion (IHP) offers a different approach in helping to treat these patients. IHP is a method used for isolating the liver and delivering high doses of chemotherapeutic agents. The efficacy of IHP has been improved by combining hyperthermia not only with chemotherapeutics but with other deliverable agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we used human colorectal cancer CX-1 cells and treated them with hyperthermia and TRAIL, causing cytotoxicity. We were able to demonstrate that the numbers of live cells were significantly reduced with hyperthermia and 10 ng/ml of TRAIL combined. We also showed that the effect of hyperthermia on TRAIL in our studies was enhancement of the apoptotic pathway by the promotion of JNK and Bim(EL) activity as well as PARP cleavage. We have also used our CX-1 cells to generate tumors in Balb/c nude mice. With intratumoral injections of TRAIL combined with hyperthermia at 42 degrees C, we were able to show a delayed onset of tumor growth in our xenograft model.
Asunto(s)
Neoplasias Colorrectales/prevención & control , Hipertermia Inducida , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antracenos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Terapia Combinada , Calor , Humanos , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Carga Tumoral/efectos de los fármacosRESUMEN
Our recent findings show that astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) samples, and AEG-1 plays a central role in regulating development and progression of HCC. In the present study, we elucidate a molecular mechanism of AEG-1-induced chemoresistance, an important characteristic of aggressive cancers. AEG-1 increases the expression of multidrug resistance gene 1 (MDR1) protein, resulting in increased efflux and decreased accumulation of doxorubicin, promoting doxorubicin resistance. Suppression of MDR1 by small interfering RNA or chemical reagents, or inhibition of AEG-1 or a combination of both genes, significantly increases in vitro sensitivity to doxorubicin. In nude mice xenograft studies, a lentivirus expressing AEG-1 short hairpin RNA, in combination with doxorubicin, profoundly inhibited growth of aggressive human HCC cells compared with either agent alone. We document that although AEG-1 does not affect MDR1 gene transcription, it facilitates association of MDR1 mRNA to polysomes, resulting in increased translation, and AEG-1 also inhibits ubiquitination and subsequent proteasome-mediated degradation of MDR1 protein. This study is the first documentation of a unique aspect of AEG-1 function (i.e., translational and posttranslational regulation of proteins). Inhibition of AEG-1 might provide a means of more effectively using chemotherapy to treat HCC, which displays inherent chemoresistance with aggressive pathology.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/enzimología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Lentivirus/genética , Proteínas de la Membrana , Ratones , Ratones Desnudos , Trasplante de Neoplasias , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARNRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether treatment of docetaxel (DTX) can enhance apoptotic cell death by TRAIL against androgen-independent prostate cancer (AIPC). The cell death effect of combinations of TRAIL and docetaxel on prostate cancer cell lines (androgen-dependent LNCaP and its derived androgen-independent, metastatic C4-2B) was evaluated by synergisms of apoptosis. Western blot assay and DNA fragmentation assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL induced apoptosis in the presence of docetaxel. In addition, we investigated the in vitro anti-tumor effects of combined docetaxel and TRAIL using MAP kinase inhibitors. Docetaxel itself could not induce apoptotic cell death in 24 h even in high concentration. Apoptotic cell death, however, was drastically enhanced by pretreatment of docetaxel 20 h before TRAIL treatment. Docetaxel enhanced the PARP-1 cleavage and caspases activation by TRAIL especially in androgen-independent, metastatic C4-2B cell line, mainly by phosphorylation of Bcl-2 by JNK activation. It appears that apoptotic cell death was protected by the JNK inhibitor SP600125. The results of our study show that pretreatment of docetaxel is able to enhance the apoptosis produced by TRAIL in prostate cancer cells, especially in hormone-refractory prostate cancer (HRPC).
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Próstata/patología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Taxoides/farmacología , Caspasas/metabolismo , Línea Celular Tumoral , Ciclina B/metabolismo , Ciclina B1 , Docetaxel , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Masculino , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL-induced apoptosis. We observed in human colorectal cancer cell line CX-1 that TRAIL-induced apoptotic death and also that mild hyperthermia promoted TRAIL-induced apoptotic death through caspase activation and cytochrome-c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX-1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP-1 and several caspases and antiapoptotic proteins were performed. Activities of casapse-8, caspase-9, and caspase-3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non-apoptotic, more efficiently. We demonstrate here that CX-1 cells exposed to 42 degrees C and chemotherapeutic agents were sensitized and died by apoptotic and non-apoptotic cell death even in low concentration (10 ng/ml) of TRAIL.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/patología , Melfalán/farmacología , Compuestos Organoplatinos/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Temperatura , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Citocromos c/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxaliplatino , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether acetylsalicylic acid (ASA), so-called aspirin, enhances TRAIL-induced apoptosis in androgen-dependent LNCaP and androgen-independent LNCaP-derived prostate cancer cells. To evaluate the cell death effects of TRAIL in combination with ASA on tumor cells, we performed DNA fragmentation assay and immunoblot analysis for poly(ADP-ribose) polymerase-1, caspases, and anti-apoptotic proteins. We observed that ASA promoted TRAIL-induced apoptotic death in both LNCaP and its derived cells (C4, C4-2, and C4-2B). These enhancements of TRAIL's effect were related to the decrease in survivin protein expression by pretreatment with ASA. We also confirmed that knockdown in survivin expression by transfecting survivin small interfering RNA increased TRAIL-induced apoptosis. To study the mechanism of survivin down-regulation, we determined the levels of mRNA and the activities of survivin promoter in the ASA-treated and untreated cells. Reduction of the intracellular levels of survivin protein was due to a decrease in transcriptional activity. Data from electrophoretic mobility shift assay and chromatin immunoprecipitation analyses revealed that ASA inhibited the transcription factor E2F-1 binding activity to the survivin promoter region, which is known to regulate survivin gene transcription. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated by down-regulating survivin, and the down-regulation of survivin is due to inhibition of E2F-1 binding activity to the survivin promoter region.
Asunto(s)
Apoptosis/fisiología , Aspirina/farmacología , Regulación hacia Abajo/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Regulación hacia Arriba/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Survivin , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Approximately 25% of patients with colorectal cancer will develop metastatic disease exclusively or largely confined to the liver, and the vast majority of these cases are not amenable to surgical resection. These unresectable cases of liver metastatic disease can be treated with isolated hepatic perfusion (IHP), which involves a method of complete vascular isolation of the liver to allow treatment of liver tumors with toxic systemic doses of chemotherapeutic agents. To improve the efficacy of IHP, hyperthermia and biological agents have been applied along with the chemotherapeutic agents. In this study, we investigated whether hyperthermia in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) enhances mortality in human colorectal carcinoma CX-1 cells. Cells were treated with various concentrations of TRAIL (0-200 ng/ml) at various temperatures (40-46 degrees C) for 1 h and further incubated at 37 degrees C in the presence of TRAIL. We observed that hyperthermia at 42-43 degrees C effectively promoted TRAIL-induced apoptosis, as indicated by cell death, poly (ADP-ribose) polymerase (PARP) cleavage, and activation of caspase-8, -9, and -3. In contrast, hyperthermia at 45-46 degrees C suppressed TRAIL-induced apoptosis. We also observed that mild hyperthermia, but not acute hyperthermia, promoted cytochrome c release during treatment with TRAIL. Our data suggest that promotion of cytochrome c release during mild hyperthermia is responsible for the enhancement of TRAIL cytotoxicity.
Asunto(s)
Apoptosis/efectos de los fármacos , Calor , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Caspasas/metabolismo , Línea Celular Tumoral , Colágeno Tipo XI/efectos de los fármacos , Colágeno Tipo XI/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Citocromos c/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , TemperaturaRESUMEN
The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1alpha (HIF-1alpha), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. UCP is detected coincidently with HIF-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1alpha and may be a new molecular target for therapeutic intervention in human cancers.
Asunto(s)
Neoplasias/patología , Desacopladores/toxicidad , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/patología , Secuencia de Bases , División Celular , Hipoxia de la Célula , Línea Celular Tumoral , Vectores Genéticos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hígado/fisiología , Metástasis de la Neoplasia , Proteínas Recombinantes/metabolismo , Ubiquitina/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
The pituitary tumor transforming (PTTG) gene family comprises PTTG1, 2, and 3. Forced expression of PTTG1 (securin) induces cellular transformation and promotes tumor development in animal models. PTTG1 is overexpressed in various human cancers. However, the expression and pathogenic implications of the PTTG gene family in hepatocellular carcinoma are largely unknown. Gene silencing using short interfering RNA (siRNA) has become an efficient means to study the functions of genes and has been increasingly used for cancer gene therapy approaches. We report that PTTG1, but not PTTG2 and 3, was highly and frequently expressed in liver cancer tissues from patients and highly in SH-J1, SK-Hep1, and Huh-7 hepatoma cell lines. Adenoviral vector encoding siRNA against PTTG1 (Ad.PTTG1-siRNA) depleted PTTG1 specifically and efficiently in SH-J1 hepatoma cells, which resulted in activation of p53 that led to increased p21 expression and induction of apoptosis. The depletion of PTTG1 in HCT116 colorectal cancer cells exhibited a cytotoxic effect in a p53-dependent manner. Ad.PTTG1-siRNA-mediated cytotoxic effect was dependent on expression levels of PTTG1 and p53 in hepatoma cell lines. Huh-7 hepatoma cells, once transduced with Ad.PTTG1-siRNA, displayed markedly attenuated growth potential in nude mice. Intra-tumor delivery of Ad.PTTG1-siRNA led to significant inhibition of tumor growth in SH-J1 tumor xenograft established in nude mice. In conclusion, PTTG1 overexpressed in hepatoma cell lines negatively regulates the ability of p53 to induce apoptosis. PTIG1 gene silencing using siRNA may be an effective modality to treat liver cancer, in which PTTG1 is abundantly expressed. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/ suppmat/index.html).
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/genética , Transactivadores/genética , Adenoviridae , Animales , Apoptosis , Carcinoma Hepatocelular/genética , Proliferación Celular , Femenino , Humanos , Neoplasias Hepáticas/genética , Ratones , Ratones Endogámicos BALB C , Securina , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
PURPOSE: This study investigated whether hyperthermia can enhance TRAIL-induced apoptotic death. METHODS: Human prostate adenocarcinoma DU-145, human pancreatic carcinoma MIA PaCa-2 and BxPC-3, human colon fibroblast CCD-33Co and rat prostate endothelial YPEN-1 cells were treated with various concentrations of TRAIL (0-200 ngml(-1)) with hyperthermia (40-42 degrees C). RESULTS: It was observed in human cancer cells, but not in normal cells, that TRAIL induced apoptotic death and also that hyperthermia (40-42 degrees C) promoted TRAIL-induced apoptotic death. Enhancement of TRAIL-mediated apoptosis by hyperthermia was detected by an increase in PARP cleavage, the hallmark feature of apoptosis, as well as by activation of caspases. There were no significant changes in the intra-cellular levels of death receptors (DRs), decoy receptors (DcRs) and anti-apoptotic proteins. Interestingly, data from in vitro enzyme kinetics assay demonstrated that hyperthermia promoted caspase enzyme activity. CONCLUSIONS: These data suggest that cancer cells are more susceptible to TRAIL in the condition of hyperthermia (40-42 degrees C). The promotion of caspase enzyme activity by hyperthermia may be responsible for enhancement of TRAIL-induced apoptotic death.
Asunto(s)
Apoptosis/fisiología , Hipertermia Inducida , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Animales , Terapia Biológica , Caspasa 1/metabolismo , Línea Celular Tumoral , Terapia Combinada/métodos , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiologíaRESUMEN
BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno-associated virus (rAAV) vectors have a number of advantages including in vivo long-term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector. METHODS: A plasmid and rAAV2 vectors were constructed encoding sTRAIL composed of a leader sequence, the isoleucine zipper, and the active domain of TRAIL (aa 95-281). The functionality of sTRAIL was validated by cell viability, FACS analysis, caspase-3 activity, and TUNEL staining. rAAV-sTRAIL was injected intratumorally to nude mice bearing human A549 lung tumor cells. Nude mice received A549 tumor cells after intravenous delivery of rAAV-sTRAIL. The antitumor effect was then evaluated by measuring tumor regression and occurrence in the experimental animal. RESULTS: sTRAIL was released from cells transfected with the sTRAIL expression construct or transduced with rAAV-sTRAIL, and induced apoptosis in cancer cells, but spared normal fibroblast cells. Secreted sTRAIL formed oligomers including trimers with intersubunit disulfide. Purified sTRAIL exerted much lower cytotoxicity on primary human hepatocytes compared to recombinant TRAIL. Intratumoral delivery of rAAV-sTRAIL significantly inhibited growth of A549 tumors established in nude mice. A number of apoptotic tumor cells were detected by TUNEL staining in mice treated with rAAV-sTRAIL. Systemic pretreatment with rAAV-sTRAIL significantly inhibited tumor formation in nude mice. CONCLUSION: The results suggest that rAAV-sTRAIL may be useful for local or systemic cancer gene therapy for treating TRAIL-sensitive tumors.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Dependovirus , Terapia Genética , Vectores Genéticos , Neoplasias Pulmonares/terapia , Glicoproteínas de Membrana/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Cistina/metabolismo , Femenino , Hepatocitos/metabolismo , Humanos , Neoplasias Pulmonares/prevención & control , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Desnudos , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Therapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12), a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD) and 5-fluorocytosine (5-FC) has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca) tumors. METHODS: Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-gamma production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-gamma production. RESULTS: The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-gamma production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time. CONCLUSION: The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers.
Asunto(s)
Adenoviridae/genética , Antineoplásicos/farmacología , Citosina Desaminasa/farmacología , Fluorouracilo/farmacología , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Interleucina-12/genética , Neoplasias/terapia , Animales , Western Blotting , Carcinoma de Células Renales/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Femenino , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Neoplasias Renales/metabolismo , Células Asesinas Naturales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Profármacos/farmacología , Bazo/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Solid tumors contain normoxic and hypoxic regions depending on the distance from the capillary. Normal cells may also be exposed to hypoxia under certain physiological conditions. Tumor hypoxia has been shown to associate strongly with tumor propagation and malignant progression. Hypoxia-inducible factor (HIF)-1alpha is stable under hypoxia and induces transcription of target genes by binding to the hypoxia-response element (HRE). Here we investigated the oncolytic effects of a novel adenovirus mutant with a deleted E1B55 gene (Ad.Delta55.HRE), in which the expression of E1A, which is essential for adenoviral replication, is regulated under the control of an HRE-expression system. Ad.Delta55.HRE expressed E1A under normoxia and more E1A under hypoxia and exhibited oncolytic effects on various cultured tumor cells, but its cytotoxic effect is relatively attenuated in normal fibroblast cells under normoxic and hypoxic conditions. Ad.Delta55.HRE lysed Huh-7 hepatoma cells stably expressing HIF-1alpha more effectively compared to parental cells. Ad.Delta55.HRE treatment exhibited significant antitumor activity in PC-3 prostate- and MDA-MB-435 breast tumor-bearing nude mice in which HIF-1alpha protein was immunohistochemically detected. The E1A and hexon proteins of adenovirus were immunostained in MDA-MB-435 xenografts after Ad.Delta55.HRE treatment, suggestive of viral replication. Our results suggest that Ad.Delta55.HRE may be useful for the treatment of solid tumors.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Neoplasias/terapia , Proteínas E1A de Adenovirus/análisis , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Animales , Proteínas de la Cápside/análisis , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular , Eliminación de Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Trasplante de Neoplasias , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Factores de Transcripción/análisis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Replicación Viral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5'-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32-), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32- produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32- compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32- contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18.