RESUMO
The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.
Assuntos
Apoptose/efeitos dos fármacos , Terapia Viral Oncolítica/métodos , Neoplasias Ovarianas/patologia , Vaccinia virus/fisiologia , Animais , Apoptose/fisiologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Camundongos , Camundongos Nus , Microscopia Eletrônica de Transmissão , Necrose , Neoplasias Experimentais , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Necrose Tumoral/metabolismo , Vaccinia virus/genéticaRESUMO
Adaptive immune responses may be vital in the overall efficacy of oncolytic viruses in human malignancies. However, immune responses to oncolytic adenoviruses are poorly understood because these viruses lack activity in murine cells, which precludes evaluation in immunocompetent murine cancer models. We have evaluated human adenovirus activity in murine cells. We show that a panel of murine carcinoma cells, including CMT64, MOVCAR7, and MOSEC/ID8, can readily be infected with human adenovirus. These cells also support viral gene transcription, messenger RNA (mRNA) processing, and genome replication. However, there is a profound failure of adenovirus protein synthesis, especially late structural proteins, both in vitro and in vivo, with reduced loading of late mRNA onto ribosomes. Our data also show that in trans expression of the nonstructural late protein L4-100K increases both the amount of viral mRNA on ribosomes and the synthesis of late proteins, accompanied by reduced phosphorylation of eIF2α and improved anticancer efficacy. These results suggest that murine models that support human adenovirus replication could be generated, thus allowing evaluation of human adenoviruses in immunocompetent mice.
Assuntos
Adenovírus Humanos/genética , Vírus Oncolíticos/genética , Neoplasias Ovarianas/terapia , Biossíntese de Proteínas , RNA Viral/metabolismo , Proteínas não Estruturais Virais/genética , Imunidade Adaptativa , Adenovírus Humanos/imunologia , Animais , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Expressão Gênica , Humanos , Camundongos , Vírus Oncolíticos/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ovário/efeitos dos fármacos , Ovário/imunologia , Ovário/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Ribossomos/genética , Ribossomos/metabolismo , Especificidade da Espécie , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genéticaRESUMO
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
Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Neoplasias Ovarianas/terapia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Reparo do DNA , Replicação do DNA , Feminino , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , Transplante Heterólogo , Fosfatases cdc25/metabolismoRESUMO
In this study, we established an in vitro organoid model of normal human ovarian surface epithelial (HOSE) cells. The spheroids of these normal HOSE cells resembled epithelial inclusion cysts in human ovarian cortex, which are the cells of origin of ovarian epithelial tumor. Because there are strong correlations between chronic inflammation and the incidence of ovarian cancer, we used the organoid model to test whether protumor inflammatory cytokine tumor necrosis factor alpha would induce malignant phenotype in normal HOSE cells. Prolonged treatment of tumor necrosis factor alpha induced phenotypic changes of the HOSE spheroids, which exhibited the characteristics of precancerous lesions of ovarian epithelial tumors, including reinitiation of cell proliferation, structural disorganization, epithelial stratification, loss of epithelial polarity, degradation of basement membrane, cell invasion, and overexpression of ovarian cancer markers. The result of this study provides not only an evidence supporting the link between chronic inflammation and ovarian cancer formation but also a relevant and novel in vitro model for studying of early events of ovarian cancer.