RESUMO
Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-α (TNF-α). We investigated whether inhibition of TNF-α augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-α and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-α reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-α antibody infliximab. In vivo, stable knockdown of TNF-α in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-α using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-α acts as a survival factor in adenovirus-infected cells. Combining TNF-α inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.
Assuntos
Adenoviridae , Proteínas Inibidoras de Apoptose/metabolismo , Vírus Oncolíticos , Neoplasias Ovarianas , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Adenoviridae/efeitos dos fármacos , Adenoviridae/imunologia , Adenoviridae/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Infliximab , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia Viral Oncolítica , Vírus Oncolíticos/efeitos dos fármacos , Vírus Oncolíticos/imunologia , Vírus Oncolíticos/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Neoplasias Ovarianas/virologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Fator de Necrose Tumoral alfa/farmacologia , Replicação Viral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The oncolytic adenovirus dl922-947 replicates selectively within and lyses cells with a dysregulated Rb pathway, a finding seen in > 90% human cancers. dl922-947 is more potent than wild type adenovirus and the E1B-deletion mutant dl1520 (Onyx-015). We wished to determine which host cell factors influence cytotoxicity. SV40 large T-transformed MRC5-VA cells are 3-logs more sensitive to dl922-947 than isogenic parental MRC5 cells, confirming that an abnormal G1/S checkpoint increases viral efficacy. The sensitivity of ovarian cancer cells to dl922-947 varied widely: IC50 values ranged from 51 (SKOV3ip1) to 0.03 pfu/cell (TOV21G). Cells sensitive to dl922-947 had higher S phase populations and supported earlier E1A expression. Cytotoxicity correlated poorly with both infectivity and replication, but well with expression of p21 by microarray and western blot analyses. Matched p21+/+ and -/- Hct116 cells confirmed that p21 influences dl922-947 activity in vitro and in vivo. siRNA-mediated p21 knockdown in sensitive TOV21G cells decreases E1A expression and viral cytotoxicity, whilst expression of p21 in resistant A2780CP cells increases virus activity in vitro and in intraperitoneal xenografts. These results highlight that host cell factors beyond simple infectivity can influence the efficacy of oncolytic adenoviruses. p21 expression may be an important biomarker of response in clinical trials.
Assuntos
Adenoviridae/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Concentração de Íons de Hidrogênio , Terapia Viral Oncolítica , Neoplasias Ovarianas/virologia , Proteínas E1A de Adenovirus/metabolismo , Ciclina D/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Fase SRESUMO
Constitutive production of inflammatory cytokines is a characteristic of many human malignant cell lines; however, the in vitro and in vivo interdependence of these cytokines, and their significance to the human cancer microenvironment, are both poorly understood. Here, we describe for the first time how three key cytokine/chemokine mediators of cancer-related inflammation, TNF, CXCL12, and interleukin 6, are involved in an autocrine cytokine network, the "TNF network," in human ovarian cancer. We show that this network has paracrine actions on angiogenesis, infiltration of myeloid cells, and NOTCH signaling in both murine xenografts and human ovarian tumor biopsies. Neutralizing antibodies or siRNA to individual members of this TNF network reduced angiogenesis, myeloid cell infiltration, and experimental peritoneal ovarian tumor growth. The dependency of network genes on TNF was shown by their downregulation in tumor cells from patients with advanced ovarian cancer following the infusion of anti-TNF antibodies. Together, the findings define a network of inflammatory cytokine interactions that are crucial to tumor growth and validate this network as a key therapeutic target in ovarian cancer.
Assuntos
Citocinas/metabolismo , Neoplasias Ovarianas/metabolismo , Animais , Biópsia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Virus infection may induce host cell death by apoptosis, but some DNA viruses are capable of preventing this process. RNA viruses were thought not to display anti-apoptotic activities, as their spread appears to benefit from a rapid induction of cell death. Here, we report an antiapoptotic activity in the Picornavirus Coxsackievirus B4 (CVB4). CVB4 infection of HeLa cells induced negligible apoptosis over a period of 10 h. However, infected cells developed resistance to drug-induced apoptosis using staurosporine and actinomycin D and to death receptor-induced apoptosis using tumor necrosis factor-related apoptosis-inducing ligand. Despite this resistance, the apoptotic machinery was nonetheless fully activated in these drug-treated infected cells because the levels of pro-caspase-3 processing to its active form were similar to control cells. However, the DEVDase (Asp-Glu-Val-Asp protease) activity of the processed caspase was significantly inhibited in the virus-infected staurosporine-treated cells compared with drug treatment alone. Likewise, extracts of CVB4-infected cells suppressed recombinant caspase-3 activity in vitro. Immunoprecipitation of activated caspase-3 from radiolabeled virus-infected cells revealed the co-precipitation of a 48-kDa protein that was tentatively identified as viral protein 2BC. Recombinant caspase-3 was found to co-precipitate with virus protein 2BC. Finally, when protein 2BC was expressed in HeLa cells, both staurosporine-induced apoptosis and in vitro caspase-3 DEVDase activity were significantly reduced. Taken together these data imply that CVB4 infection suppresses apoptosis through virus protein 2BC associating with caspase-3 and inhibiting its function. Thus, 2BC is the first reported RNA virus inhibitor of apoptosis protein.
Assuntos
Apoptose , Inibidores de Caspase , Enterovirus , Inibidores Enzimáticos/farmacologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3 , Caspases/metabolismo , Dactinomicina/farmacologia , Citometria de Fluxo , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Previous studies have identified virus proteins that traffic to mitochondria and may affect mitochondrial function. Here, it is reported that Human herpesvirus 1 (HHV-1, herpes simplex virus 1) and influenza virus reduced mitochondrial respiration, whilst Measles virus, cytomegalovirus, coxsackievirus B4 and Feline calicivirus did not. The inhibition of total cellular respiration was caused by a block in the mitochondrial electron-transport chain. This effect occurred during beta-phase protein synthesis and the inhibition of mitochondrial respiration could be reproduced by ectopic expression of the beta-phase protein US3. An HHV-1 mutant lacking this protein failed to inhibit oxygen consumption in infected cells relative to controls. It was concluded that US3 was mediating the suppression of mitochondrial respiration following HHV-1 infection. The integrity of the electron-transport chain in HHV-1-infected cells was analysed further and the site of the block in electron transport was located between complexes II and III, a site previously shown to be affected by Poliovirus.