RESUMO
The pressing challenge for contemporary gene therapy is to deliver enough therapeutic genes to enough cancer cells in vivo. With the aim of improving viral distribution and tumor penetration, we explored the use of decorin to enhance viral spreading and tumor tissue penetration. We generated decorin-expressing replication-incompetent (dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK) and replication-competent (Ad-DeltaE1B-DCNG, Ad-DeltaE1B-DCNQ and Ad-DeltaE1B-DCNK) adenoviruses (Ads). Point mutants of decorin gene (DCNG), DCNK and DCNQ, have a negative and moderate binding affinity to type-I collagen fibril, respectively. In both tumor spheroids and established solid tumors in vivo, tissue penetration potency of dl-LacZ-DCNG was greatly enhanced than those of dl-LacZ, dl-LacZ-DCNQ and dl-LacZ-DCNK, and this enhanced tissue penetration effect derived from decorin-expressing Ad was dependent on the binding affinity of decorin to collagen fibril. Expression of DCNG enhanced viral spread of replicating Ad, leading to improved tumor reduction and survival benefit. Moreover, the tumoricidal effects of Ad-DeltaE1B-DCNQ and Ad-DeltaE1B-DCNK were lessened, as the binding affinity to collagen was decreased, showing that the increased cancer cell cytotoxicity was driven by the action of decorin on extracellular matrix (ECM). Furthermore, Ad-DeltaE1B-DCNG substantially decreased ECM components within the tumor tissue. Finally, intratumoral injection of Ad-DeltaE1B-DCNG in primary tumor site greatly reduced the formation of B16BL6 melanoma cell pulmonary metastases in mice. Taken together, these data show the utility of decorin as a dispersion agent and highlight its utility and potential in improving the efficacy of replicating Ad-mediated cancer gene therapy.
Assuntos
Adenoviridae/genética , Proteínas da Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Terapia Viral Oncolítica/métodos , Proteoglicanas/genética , Animais , Linhagem Celular Tumoral , Decorina , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Camundongos , Camundongos Nus , Proteoglicanas/metabolismo , Esferoides Celulares/metabolismo , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Radiation therapy, a mainstay for anti-tumor therapeutic regimens for a variety of tumor types, triggers tumor cell apoptotic pathways by either directly eliciting DNA damage or indirectly inducing the formation of oxygen radicals. In an effort to augment radiation therapy, we generated a double E1B 19 kDa- and E1B 55 kDa-deleted oncolytic adenovirus (Ad-DeltaE1B19/55). In combination with radiotherapy, greater cytotoxicity was observed for Ad-DeltaE1B19/55 than for the single E1B 55 kDa-deleted oncolytic Ad (Ad-DeltaE1B55). Consistent with this observation, higher levels of p53, phospho-p53, phospho-Chk1, phospho-Chk2, PI3K (phosphatidylinositol-3-kinase), phospho-AKT, cytochrome c, and cleavage of PARP (poly (ADP-ribose) polymerase) and caspase-3 were observed in cells treated with Ad-DeltaE1B19/55 compared with those treated with Ad-DeltaE1B55, indicating that the E1B 19 kDa present in Ad-DeltaE1B55 may partially block radiation-induced apoptosis. A significant therapeutic benefit was also observed in vivo when oncolytic Ads and radiation were combined. Tumors treated with Ad-DeltaE1B19/55 and radiation showed large areas of necrosis and apoptosis with the corresponding induction of p53. Finally, consistent with in vitro observations, the combination of Ad-DeltaE1B19/55 and radiation was more efficacious than the combination of Ad-DeltaE1B55 and radiation. Taken together, these results present a strong therapeutic rationale for combining radiation therapy with E1B 19 kDa-deleted oncolytic Ad.
Assuntos
Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Neoplasias do Colo do Útero/terapia , Animais , Apoptose/genética , Terapia Combinada , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/radioterapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although curcumin suppresses the growth of a variety of cancer cells, its poor absorption and low systemic bioavailability have limited its translation into clinics as an anticancer agent. In this study, we show that dimethoxycurcumin (DMC), a methylated, more stable analog of curcumin, is significantly more potent than curcumin in inducing cell death and reducing the clonogenicity of malignant breast cancer cells. Furthermore, DMC reduces the tumor growth of xenografted MDA-MB 435S cells more strongly than curcumin. We found that DMC induces paraptosis accompanied by excessive dilation of mitochondria and the endoplasmic reticulum (ER); this is similar to curcumin, but a much lower concentration of DMC is required to induce this process. DMC inhibits the proteasomal activity more strongly than curcumin, possibly causing severe ER stress and contributing to the observed dilation. DMC treatment upregulates the protein levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and Noxa, and the small interfering RNA-mediated suppression of CHOP, but not Noxa, markedly attenuates DMC-induced ER dilation and cell death. Interestingly, DMC does not affect the viability, proteasomal activity or CHOP protein levels of human mammary epithelial cells, suggesting that DMC effectively induces paraptosis selectively in breast cancer cells, while sparing normal cells. Taken together, these results suggest that DMC triggers a stronger proteasome inhibition and higher induction of CHOP compared with curcumin, giving it more potent anticancer effects on malignant breast cancer cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Curcumina/análogos & derivados , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Fator de Transcrição CHOP/metabolismo , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Fatores de Tempo , Fator de Transcrição CHOP/genética , Transfecção , Carga Tumoral/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Collaborator of ARF (CARF) was cloned as an ARF-interacting protein and shown to regulate the p53-p21(WAF1)-HDM2 pathway, which is central to tumor suppression via senescence and apoptosis. We had previously reported that CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis, however, the mechanisms governing this phenomenon remained unknown. Thus, we examined various cell death and survival pathways including the mitochondrial stress, ataxia telangiectasia mutated (ATM)-ATR, Ras-MAP kinase and retinoblastoma cascades. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, it protected the cells against genotoxicity; CARF knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and γH2AX, leading to induction of mitotic arrest and eventual apoptosis. We also show that the CARF-silencing-induced apoptosis in vitro translates to in vivo. In a human tumor xenograft mouse model, treatment of developing tumors with short hairpin RNA (shRNA) against CARF via an adenovirus carrier induced complete suppression of tumor growth, suggesting that CARF shRNA is a strong candidate for an anticancer reagent. We demonstrate that CARF has a vital role in genome preservation and tumor suppression and CARF siRNA is an effective novel cancer therapeutic agent.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Neoplasias/terapia , Proteínas de Ligação a RNA/metabolismo , Animais , Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Transplante Heterólogo , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVE: The purposes of the present study were to estimate the proportion of subjects with a high score on the Korean version of Eating Attitudes Test--26 (KEAT-26), which may provide preliminary data regarding the prevalence rate of eating disorders in the Korean general population, and to further examine the sociocultural hypothesis of eating disorders. METHOD: Using a multi-stage questionnaire sampling method, we surveyed 3062 subjects (1249 males, 1813 females) from 3896 Korean adults in a nationwide area. RESULTS: 8.5% (260/3062) of subjects scored above the cut-off on the KEAT-26. Their demographic correlates, eating traits, and other characteristics relating to general psychopathology were similar to those of patients with eating disorders and female Caucasian controls in Western countries. DISCUSSION: These results suggest that changes in various sociocultural aspects have increased the risk of developing eating disorders in Korea, and support the sociocultural hypothesis of eating disorders.