RESUMO
Most solid tumors undergo hypoxia, leading to rapid cell division, metastasis and expansion of a cell population with hallmarks of cancer stem cells (CSCs). Tumor-selective replication of oncolytic adenoviruses may be hindered by oxygen deprivation in tumors. It is desirable to develop a potent oncolytic adenovirus, retaining its antitumor activity even in a hypoxic environment. We have previously generated an Oct4-dependent oncolytic adenovirus, namely Ad9OC, driven by nine copies of the Oct4 response element (ORE) for specifically killing Oct4-overexpressing bladder tumors. Here, we developed a novel Oct4 and hypoxia dual-regulated oncolytic adenovirus, designated AdLCY, driven by both hypoxia response element (HRE) and ORE. We showed that hypoxia-inducible factor (HIF)-2α and Oct4 were frequently overexpressed in hypoxic bladder cancer cells, and HIF-2α was involved in HRE-dependent and Oct4 transactivation. AdLCY exhibited higher cytolytic activities than Ad9OC against hypoxic bladder cancer cells, while sparing normal cells. AdLCY exerted potent antitumor effects in mice bearing human bladder tumor xenografts and syngeneic bladder tumors. It could target hypoxic CD44- and CD133-positive bladder tumor cells. Therefore, AdLCY may have therapeutic potential for targeting hypoxic bladder tumors and CSCs. As Oct4 is expressed in various cancers, AdLCY may be further explored as a broad-spectrum anticancer agent.
Assuntos
Antineoplásicos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Vírus Oncolíticos/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Xenoenxertos , Humanos , CamundongosRESUMO
OBJECTIVES: Selectively targeting signalling pathways represents a promising pharmacological approach in rheumatoid arthritis (RA). Abundant levels of epidermal growth factor receptor (EGFR) are expressed in the synovial lining layers, and the anti-arthritis effect of erlotinib and lapatinib, small-molecule EGFR tyrosine kinase inhibitors (TKIs), has been demonstrated through the systemic administration on experimental arthritis models. Nevertheless, their therapeutic responses by the intra-articular (i.a.) route remain to be explored in rheumatoid joint. METHODS: The administration of an EGFR TKI (a gefitinib analogue) was explored in two in vivo models of collagen-induced arthritis (CIA) and in vitro experiments by using synovial fibroblasts (SF) from RA patients and CIA rats. RESULTS: There was a significant reduction of arthritis scores in CIA mice receiving the daily intraperitoneal injection. After the onset of arthritis in CIA rats, ankle joints receiving a single i.a. injection had significant lower articular indexes with reduced synovial inflammation, pannus formation and erosion on cartilage and bone as well as total histological scores by histopathological analyses. In CIASF or RASF, upon in vitro human EGF stimulation, there was a dose-dependent increase in cell proliferation and Akt activation with suppressed responses under the EGFR TKI treatment. CONCLUSIONS: These findings demonstrate the effect of i.a. injection of an EGFR TKI on amelioration of rheumatoid joint through the suppression of synovial inflammation, pannus formation and erosion on cartilage and bone in experimental arthritis, implicating targeting the i.a. EGFR signalling transduction as a pharmacological strategy.
Assuntos
Artrite Reumatoide , Receptores ErbB/antagonistas & inibidores , Quinazolinas/farmacologia , Membrana Sinovial , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Técnicas de Cultura de Células , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gefitinibe , Humanos , Injeções Intra-Articulares , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Resultado do TratamentoRESUMO
Knockdown of Toll-like receptors (TLRs) is a novel therapeutic strategy in treating patients with rheumatoid arthritis (RA). We examined the effects of lentiviral vector-mediated delivery of TLR7 short hairpin RNA gene (Lt.shTLR7) on collagen-induced arthritis (CIA). After being immunized on days 0 and 7, Sprague-Dawley rats received intra-articular (i.a.) injection of Lt.shTLR7 or scramble control vector on days 7 and 10. The therapeutic effects were evaluated by measuring ankle circumferences, articular index, and radiographic and histological scores on killing on day 16. Microvessel densities, vascular endothelial growth factor (VEGF) levels, pro-inflammatory cytokine concentrations and T-cell numbers within the synovial tissues were measured. Moreover, VEGF and pro-inflammatory cytokine concentrations in culture supernatants from TLR7-transfected synovial fibroblasts (SFs) stimulated with imiquimod or endogenous ligands were examined. There were significant reduction in ankle circumferences, articular indexes, and radiographic and histological scores. Microvessel densities, VEGF concentrations, interleukin (IL)-1ß and IL-6 levels and T-cell densities within synovial tissues were significantly lower. Induction of VEGF, IL-1ß and IL-6 production from stimulated SFs was significantly suppressed. Taken together, these data demonstrate the effects of i.a. lentiviral vector-mediated delivery of shTLR7 RNA gene on inhibition of CIA, and implicate the manipulation of TLR7 as a potential therapeutic strategy in RA patients.
Assuntos
Artrite Experimental/terapia , RNA Interferente Pequeno/farmacologia , Receptor 7 Toll-Like/genética , Animais , Articulação do Tornozelo/efeitos dos fármacos , Artrite Experimental/patologia , Citocinas/biossíntese , Vetores Genéticos , Injeções Intra-Articulares , Lentivirus/genética , Masculino , Microcirculação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Membrana Sinovial/metabolismo , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
Different members of the galectin family may have inhibitory or stimulatory roles in controlling immune responses and regulating inflammatory reactions in autoimmune diseases such as rheumatoid arthritis (RA). A hypothetical model of a cross talk between galectin-1 and galectin-3 has been established in the circumstance of rheumatoid joints. As galectin-3 is a positive regulator and galectin-1 is a negative regulator of inflammation and autoimmune responses, in this study we evaluated the effects of local knockdown of galectin-3 or overexpression of galectin-1 on ameliorating collagen-induced arthritis (CIA) in rats. Lentiviral vectors encoding galectin-3 small hairpin RNA (shRNA) and galectin-1, as well as two control vectors expressing luciferase shRNA and green fluorescent protein, were individually injected intra-articularly into the ankle joints of rats with CIA, and their treatment responses were monitored by measuring the clinical, radiological and histological changes. Our results show that both knockdown of galectin-3 and overexpression of galectin-1 induced higher percentages of antigen-induced T-cell death in the lymph node cells from arthritic rats. Furthermore, these treatments significantly reduced articular index scores, radiographic scores and histological scores, accompanied with decreased T-cell infiltrates and reduced microvessel density in the ankle joints. Our findings implicate galectin-3 and galectin-1 as potential therapeutic targets for the treatment of RA.
Assuntos
Artrite Experimental/terapia , Galectina 1/genética , Galectina 3/genética , Vetores Genéticos/administração & dosagem , Lentivirus/genética , RNA Interferente Pequeno/genética , Animais , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , Células Cultivadas , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Injeções Intra-Articulares , Ratos , TransfecçãoRESUMO
DNA vaccine and dendritic cells (DCs)-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4(+) T-cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen-presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-gamma production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated with pN-neu in conjunction with pFLAG. Importantly, a potent CD8(+) T-cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the vaccinated mice. Collectively, our results indicate that murine Flt3L and GM-CSF genes coexpressed by a bicistronic plasmid modulate the class of immune responses and may be superior to those codelivered by two separate monocistronic plasmids as the genetic adjuvants for HER2/neu DNA vaccine.
Assuntos
Vacinas Anticâncer/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteínas de Membrana/genética , Receptor ErbB-2/genética , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapia , Animais , Apresentação de Antígeno , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/genética , Células Dendríticas/imunologia , Terapia Genética , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos , Plasmídeos/genética , Baço/imunologia , Carga Tumoral , Neoplasias da Bexiga Urinária/patologia , Vacinação , Vacinas de DNA/genética , Vacinas de DNA/uso terapêuticoRESUMO
The murine MBT-2 bladder tumor model in syngeneic C3H/HeN mice was used to investigate the feasibility of gene therapy based on the delivery of interferon-gamma (IFN-gamma) in vivo by retroviral vectors. We constructed a recombinant retroviral vector pRUFneo/IFN-gamma, which was transfected into a retroviral packaging cell line psiCRE, to produce psiCRE/pRUFneo/IFN-gamma cells. The expressions of the neo and IFN-gamma genes were verified by reverse transcription-polymerase chain reaction and IFN-gamma was detected in the culture supernatant from psiCRE/pRUFneo/IFN-gamma cells. After receiving MBT-2 cells admixed with retroviral pRUFneoIFN-gamma supernatant, C3H/HeN mice exhibited lower tumor incidence, lower tumor mass, and higher survival rate, as well as higher antitumor responses compared to those injected with MBT-2 cells admixed with control retroviral supernatant. Moreover, the retroviral pRUFneoIFN-gamma supernatant was able to suppress the growth of rechallenged tumors in postoperated mice. Although the IFN-gamma protein secreted from psiCRE/pRUFneo/IFN-gamma cells partly contributes to the antitumor effect of retroviral pRUFneoIFN-gamma supernatant, the retroviruses carrying the IFN-gamma gene transduced MBT-2 cells in vivo, which may result in enhancing local IFN-gamma production from tumor cells. Because bladder is suitable for the intravesical instillation of therapeutic agents, in vivo administration of retroviral vectors encoding IFN-gamma may be explored for the treatment of bladder cancer.
Assuntos
Terapia Genética , Interferon gama/uso terapêutico , Linfoma de Células T/terapia , Mastocitose/terapia , Retroviridae/genética , Neoplasias da Bexiga Urinária/terapia , Animais , Divisão Celular/efeitos dos fármacos , Cromo/análise , Cromo/metabolismo , Citotoxicidade Imunológica/imunologia , Primers do DNA/química , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Técnicas In Vitro , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-2/metabolismo , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Linfoma de Células T/metabolismo , Linfoma de Células T/virologia , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Cuidados Pós-Operatórios , RNA Viral/análise , Proteínas Recombinantes/metabolismo , Retroviridae/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Transfecção , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/virologiaRESUMO
Thymic hormones have immunomodulatory effects on T cells and hence have been used clinically to restore the immunity of immunodeficient patients as well as to enhance the cellular immunity of cancer patients. Prothymosin alpha, which is a member of the thymic hormone family, has recently been suggested to act as a nuclear protein participating in the stimulation of cell proliferation. To characterize the biological activities ofprothymosin alpha in vitro, we established NIH3T3 cell transformants that constitutively express higher prothymosin alpha protein and its mRNA compared with the wild-type counterpart. Cells that overexpressed prothymosin alpha increased the proliferative activity assayed by the [3H]-thymidine incorporation or by the cell cycle analysis with the fluorescent-activated cell sorter. The results provide direct evidence that prothymosin alpha plays a role in cell proliferation by shortening the duration of the G1 phase.
Assuntos
Adjuvantes Imunológicos/farmacologia , Divisão Celular/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Timosina/análogos & derivados , Células 3T3 , Animais , Ciclo Celular/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Camundongos , Precursores de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timosina/genética , Timosina/farmacologiaRESUMO
The major purpose of this study was to define if the immunosuppressive effect of a transforming growth factor-beta (TGF-beta)-producing autologous tumor vaccine can be abrogated and rendered immunogenic by suppressing its TGF-beta secretion with antisense strategy. In this study, using a TGF-beta antisense gene modified MBT-2 tumor cell line [MBT-2/TGF-beta(-)#3] which we established by ourselves, we first demonstrated that the amounts of TGF-beta produced by irradiated (IR) and non-irradiated MBT-2/TGF-beta(-) #3 were both significantly decreased when detected after in vitro culture for 48 hours. The result of flow cytometry analysis reveals that decreased production of TGF-beta led to the increased expressions of MHC class I molecule and Fas on the surface of MBT-2 tumor cells. This finding may in part explain why the splenocytes obtained from day 17 tumor bearing mice (D17TBM) immunized with IRMBT-2/TGF-beta(-)#3 on day 26 expressed a higher in vitro cytotoxic activity against MBT-2 tumor cells and hence ensured a better survival of D17TBM when they were rechallenged with a two-fold higher amount of wild-type MBT-2 tumor cells, 48 hours after surgical removal of the primary tumor. Our result implies that decreasing the amount of TGF-beta secreted from the autologous tumor vaccine by antisense strategy may significantly improve its immunogenicity through up-regulation of both MHC class I and Fas expressions. Therefore, this could provide an alternative approach for future active immunotherapy.
Assuntos
Vacinas Anticâncer , Antígenos de Histocompatibilidade Classe I/biossíntese , Neoplasias Experimentais/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Fator de Crescimento Transformador beta/metabolismo , Receptor fas/biossíntese , Animais , Feminino , Imunização , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Baço/imunologia , Baço/patologia , Análise de Sobrevida , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
UNLABELLED: Our aims were to: a) elucidate whether MBT-2 cells, lethally irradiated or nonirradiated, express TGF-beta 1 mRNA and secrete TGF-beta 1 protein, and b) to investigate whether the adverse effects from IRMBT-2-secreting TGF-beta 1 in the tumor vaccine can be abrogated by exogenous addition of monoclonal anti-TGF-beta 1 antibody and/or IFN-alpha. MATERIALS AND METHODS: using the Northern hybridization analysis and the two-antibody sandwich ELISA, we demonstrate that both irradiated IRMBT-2 and nonirradiated MBT-2 cells secrete TGF-beta 1. The effect of anti-TGF-beta and/or IFN-alpha were studied by an in vitro splenocyte proliferation assay and in vivo tumor rechallenge study on day 17-TBM. RESULTS: Both IRMBT-2 and splenocytes from day 17-TBM secrete TGF-beta 1 which can express suppression of the proliferation of the splenocytes from day 17-TBM. This suppression can be partially reversed by the simultaneous addition of both anti-TGF-beta and IFN-alpha, either alone being insufficient. The result of the in vivo tumor rechallenge study on day 17-TBM reveals that a lower tumor outgrowth incidence can be obtained in groups of mice treated with postoperative vaccination with anti-TGF-beta modified tumor vaccine with or without an additional administration of IFN-alpha. CONCLUSION: Apart from TGF-beta, MBT-2 cells, both irradiated and nonirradiated, may also secrete other suppressive factors that adversely downregulate the immune response of TBM which can not then be adequately reversed by IFN-alpha.
Assuntos
Vacinas Anticâncer/imunologia , Interferon-alfa/farmacologia , Fator de Crescimento Transformador beta/fisiologia , Neoplasias da Bexiga Urinária/imunologia , Animais , Northern Blotting , Feminino , Interferon alfa-2 , Camundongos , RNA Mensageiro/análise , Proteínas Recombinantes , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Neoplasias da Bexiga Urinária/metabolismoRESUMO
INTRODUCTION AND OBJECTIVES: TGF-beta is a potent immunosuppressive cytokine produced by many tumor cells. Secretion of TGF-beta by malignant cells may be a mechanism by which tumor cells escape destruction by tumor-specific T lymphocytes. In this study, we used a TGF-beta producing C3H/He-MBT-2 murine bladder tumor model to investigate the feasibility of antisense oligonucleotide (ODN) gene therapy strategy to block the production of TGF-beta from tumor cells and evaluate its influence on both in vitro tumor growth and in vivo tumor formation. MATERIALS AND METHODS: Using a plasmid, pRUFCD, we constructed a recombinant plasmid pRUFCD/TGF-beta 1(-) containing antisense TGF-beta ODN and then transfected in into MBT-2 cells by electroporation. Three transfectant clones were successfully obtained by their resistance to 5-fluorouracil and cytosine. RESULTS: The secretion of TGF-beta from the three obtained TGF-beta antisense-blocked MBT-2 cell clones, as assessed by ELISA, were all decreased. Moreover, they all exhibited smaller colony size in the in vitro anchorage-independent soft agar colony forming assay. Tumor growths in mice injected with these three clones were all inhibited compared with those injected with parental tumor cells. CONCLUSION: This study demonstrates that after reducing the secretion of TGF-beta 1 on tumor cells by TGF-beta 1 antisense, ODN can inhibit their in vitro growth and in vivo tumor formation suggesting that this approach can be a potentially useful strategy to abolish the adverse immunosuppression effect of TGF-beta 1 producing autologous tumor vaccine and therefore to enhance host antitumor immune response.
Assuntos
Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos Antissenso/uso terapêutico , Fator de Crescimento Transformador beta/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapia , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Terapia Genética/métodos , Camundongos , Camundongos Endogâmicos C3H , Fatores de Tempo , Fator de Crescimento Transformador beta/biossínteseRESUMO
This study was conducted mainly to investigate the effect of interferon-alpha (IFN-alpha) on the antitumor immunity of a tumor bearing host (TBH) when postoperatively administrated with or without lethally irradiated autologous tumor cells. Using the C3H/He-MBT-2 murine bladder tumor model, a status of postoperative residual tumor was mimicked by rechallenging tumor cells 24 hours after resecting the day-17 tumor. Using immunohistochemical analysis we demonstrated that after treating with lethally irradiated MBT-2 tumor cells (IRMBT-2) + IL-2 cells of CD4+, CD8+, CD44+ and CD11b+ phenotypes prominently infiltrate the subcutaneous local injection sites. In contrast, only scanty immune responding cells could be seen locally if treated with IRMBT-2 + IFN-alpha 2b, albeit in the presence of interleukin-2 (IL-2). However, the spleens of D17TBM treated with IRMBT-2 + IFN-alpha 2b contained the highest percentage of CD44+ memory T cells and cells of the CD11b+ phenotype; moreover, their natural killer (NK), lymphokine activated killer (LAK) and cytotoxic T lymphocytes (CTL) activities were significantly augmented. The results of in vivo tumor rechallenge revealed that administration of IFN-alpha, either alone or combined with IRMBT-2, could both effectively suppress the outgrowth of perioperative rechallenged tumor cells as well as prolong the survival of TBH. We conclude that despite the presence of autologous tumor vaccine, postoperative administration of IFN-alpha can further enhance the antitumor immunity of TBH and therefore can be an effective adjuvant therapy to improve the therapeutic results of surgery on a tumor bearing host.
Assuntos
Antineoplásicos/farmacologia , Vacinas Anticâncer/uso terapêutico , Carcinoma de Células de Transição/imunologia , Imunoterapia/métodos , Interferon-alfa/farmacologia , Interleucina-2/farmacologia , Neoplasias da Bexiga Urinária/imunologia , Animais , Antineoplásicos/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Carcinoma de Células de Transição/terapia , Terapia Combinada , Feminino , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Interleucina-2/administração & dosagem , Células Matadoras Ativadas por Linfocina/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Proteínas Recombinantes , Linfócitos T Citotóxicos/efeitos dos fármacos , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/terapiaRESUMO
Tc-99m-HL91 is a hypoxia imaging biomarker. The aim of this study was to investigate the value of Tc-99m-HL91 imaging for hypoxia-induced cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy in a murine lung tumor model. C57BL/6 mice were implanted with Lewis lung carcinoma cells transduced with the hypoxia-inducible promoter-driven CD gene (LL2/CD) or luciferase gene (LL2/Luc) serving as the control. When tumor volumes reached 100 mm(3), pretreatment images were acquired after injection of Tc-99m-HL91. The mice were divided into low and high hypoxic groups based on the tumor-to-non-tumor ratio of Tc-99m-HL91. They were injected daily with 5-FC (500 mg kg(-1)) or the vehicle for 1 week. When tumor volumes reached 1000 mm(3), autoradiography and histological examinations were performed. Treatment with 5-FC delayed tumor growth and enhanced the survival of mice bearing high hypoxic LL2/CD tumors. The therapeutic effect of hypoxia-induced CD/5-FC gene therapy was more pronounced in high hypoxic tumors than in low hypoxic tumors. This study provides the first evidence that Tc-99m-HL91 can serve as an imaging biomarker for predicting the treatment responses of hypoxia-regulated CD/5-FC gene therapy in animal tumor models. Our results suggest that hypoxia imaging using Tc-99m-HL91 has the predictive value for the success of hypoxia-directed treatment regimens.
Assuntos
Antimetabólitos/uso terapêutico , Carcinoma Pulmonar de Lewis/terapia , Citosina Desaminase/genética , Flucitosina/uso terapêutico , Compostos de Organotecnécio , Oximas , Compostos Radiofarmacêuticos , Animais , Antimetabólitos/toxicidade , Peso Corporal , Carcinoma Pulmonar de Lewis/diagnóstico por imagem , Carcinoma Pulmonar de Lewis/genética , Hipóxia Celular , Linhagem Celular , Citosina Desaminase/metabolismo , Flucitosina/toxicidade , Terapia Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cintilografia , Carga Tumoral/genéticaRESUMO
The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.
Assuntos
Interleucina-12/genética , Vírus da Leucemia Murina de Moloney/fisiologia , Terapia Viral Oncolítica/métodos , Receptor ErbB-2/genética , Neoplasias da Bexiga Urinária/terapia , Neoplasias da Bexiga Urinária/virologia , Animais , Apoptose/genética , Linhagem Celular Tumoral , Terapia Genética/métodos , Vetores Genéticos , Humanos , Interleucina-12/metabolismo , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/metabolismo , Receptor ErbB-2/metabolismo , Transdução Genética , Transfecção , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Tropismo ViralRESUMO
Cervical cancer is the second most common type of malignant tumor among women worldwide. When the disease is confined locally, it can be controlled with surgical resection and radiotherapy. However, patients with recurrent or metastatic disease often have a poor prognosis. Measurement of serum levels of squamous cell carcinoma (SCC) antigens has been widely used as serological markers for SCC of uterine cervix. Recently, it has been demonstrated that cervical cancer patients with elevated squamous cell carcinoma antigen-2 (SCCA2) expression in tumor cells carry a poor prognosis. Here, by using a luciferase reporter assay, we show that SCCA2 promoter was active in SCCA2-producing human cervical cancer cell lines, including Cx, Cxwj, SiHa and HeLa cells, but relatively quiescent in normal cervical epithelial cells. We then developed a conditionally replicating adenovirus, designated Ad-KFH, under the transcriptional control of the SCCA2 promoter. This E1B-55 kDa-deleted oncolytic adenovirus replicated specifically in and lysed SCCA2-producing cervical cancer cells. Furthermore, in a peritoneal metastatic tumor model, Ad-KFH retarded Cxwj tumor growth in NOD/severe combined immunodeficient mice and prolonged survival of tumor-bearing mice, especially when combined with cisplatin. These results suggest that Ad-KFH may provide a new strategy of gene therapy for advanced or recurrent uterine cervical cancer.
Assuntos
Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Antígenos de Neoplasias/genética , Terapia Genética , Regiões Promotoras Genéticas , Serpinas/genética , Neoplasias do Colo do Útero/terapia , Replicação Viral , Adenoviridae/fisiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Squamous cell carcinoma antigen (SCCA) is a tumor marker for patients with squamous cell carcinoma of uterine cervix, lung, and esophagus. It was encoded by two highly homologous genes, SCCA1 and SCCA2. However, the relevance of SCCA genes to squamous cell carcinogenesis and patient outcome remains far from clear. In this study, by using laser microdissection and real-time quantitative polymerase chain reaction procedures, the messenger RNA (mRNA) expression of the SCCA1 and SCCA2 genes in normal, dysplastic, and malignant squamous epithelia from uterine cervical tissues were analyzed and correlated with outcome of cancer patients. We found that the SCCA2/A1 mRNA ratios were progressively increased from normal, dysplastic, to cancer cells, and the mean ratio was significantly higher in cancer tissues than that in normal epithelium (P= 0.02). The SCCA2/A1 mRNA ratios were not significantly associated with types of human papillomavirus infection (P > 0.05). High SCCA2/SCCA1 mRNA ratios (ratio >1) were an independent predictor of disease recurrence (relative risk: 3.58; P= 0.003). Of the 38 patients with cervical cancer, 12 patients with high SCCA2/SCCA1 mRNA ratios had a significant lower 2-year disease-free survival of only 50%, while it was 92% in those with low SCCA2/SCCA1 mRNA ratios (P < 0.001). In conclusion, our study indicated that the ratios of SCCA2 to SCCA1 RNA were increased during the process of cervical carcinogenesis, and patients with elevated SCCA2/A1 ratio carried a higher risk for recurrence in early-stage uterine cervical cancer.
Assuntos
Antígenos de Neoplasias/biossíntese , Biomarcadores Tumorais/biossíntese , Serpinas/biossíntese , Neoplasias do Colo do Útero/imunologia , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/genética , Neoplasias do Colo do Útero/genéticaRESUMO
Fusion of peptide epitopes to the core antigen (HBcAg) of hepatitis B virus (HBV) enhances their immunogenicity, both quantitatively and qualitatively. In a number of vaccine-induced mutants of HBV, glycine145 of the surface antigen S polypeptide (HBsAg) has been replaced by arginine, resulting in loss of cross-reactivity with antibodies to normal (wild-type) HBsAg. HBcAg fusion proteins carrying the immunodominant epitope of HBsAg, in which glycine145 was replaced by arginine, glutamic acid, or lysine, were produced in Escherichia coli and formed particles that displayed HBc antigenicity and immunogenicity similar to that of HBcAg itself. The fusion proteins also elicited T-cell proliferative responsiveness to HBcAg and HBsAg. Fusions carrying either wild-type or mutated epitopes of HBsAG showed HBs antigenicity in immunoblot analysis and antigen-capture immunoradiometric assay, but both mutant and wild-type derivatives induced antibodies that cross-reacted with wild-type HBsAG. The results emphasise the potential for HBcAg fusion proteins in vaccines by broadening the antibody response in a way that could confer protection against both wild-type and variant form of HBV.
Assuntos
Epitopos/imunologia , Anticorpos Anti-Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Ativação Linfocitária , Proteínas Recombinantes de Fusão/imunologia , Animais , Formação de Anticorpos , Reações Antígeno-Anticorpo , Arginina , Células Cultivadas , Clonagem Molecular , Reações Cruzadas , Escherichia coli , Glicina , Anticorpos Anti-Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Masculino , Mutagênese Sítio-Dirigida , Mutação Puntual , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Fatores de TempoRESUMO
The extracellular slime produced by Staphylococcus epidermidis has been shown to interfere with several human neutrophil functions in vitro, such as chemotaxis, degranulation and phagocytosis. Slime production has been suggested as a useful marker for clinically significant infections with coagulase-negative Staphylococcus. Since the main role of macrophages in defense mechanisms is phagocytosis, the effect of slime on the phagocytic activity of macrophages was investigated. The phagocytic activity of murine peritoneal macrophages treated with slime in vitro decreased in a dose-dependent fashion. A similar decrease was also observed in macrophages isolated from mice that had previously received intraperitoneal injection of slime. To investigate whether interferon also plays a role in this process, mice were treated with interferon or an interferon inducer, polyinosinic-polycytidylic acid (poly I:C), together with slime before macrophage isolation. The slime-suppressed phagocytic activity of macrophages was partially relieved by both agents, and the recovery effect of poly I:C in slime-suppressed phagocytosis of macrophages in vivo might be attributed to the increased interferon level in peritoneal fluid and sera. However, when slime was given to poly I:C-pretreated mice, the phagocytic activity remained suppressed. Thus, it appears that slime is able to suppress the phagocytic activity of macrophages regardless of the state of macrophage activation by poly I:C. The results suggest that the inhibition of phagocytosis by S. epidermidis slime may be independent from the activation of interferon.
Assuntos
Biofilmes , Interferons/farmacologia , Macrófagos Peritoneais/imunologia , Fagocitose , Staphylococcus epidermidis/patogenicidade , Animais , Interferons/imunologia , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos CBA , Poli I-C/farmacologia , Staphylococcus epidermidis/fisiologiaRESUMO
The core antigen of hepatitis B virus (HBcAg) made in Escherichia coli yields particles that closely resemble the viral nucleocapsid. Extensive modifications can be made to the primary structure of HBcAg without impairing particle assembly. This enables other peptide sequences, including very long sequences, to be added, substituted, or inserted into the nucleocapsid subunit while retaining the ability to form highly immunogenic particles. These also retain the T cell epitopes of HBcAg and constitute powerful delivery systems for a diverse range of immunogenic epitopes and have significant potential for development of multicomponent vaccines.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Peptídeos/imunologia , Vacinas Sintéticas/imunologia , Animais , Humanos , Proteínas Recombinantes de Fusão/imunologiaRESUMO
To explore the potential use of prothymosin alpha(ProT), a putative thymic hormone, in gene therapy for bladder cancer, we generated a replication-defective recombinant retroviral vector encoding ProT and tested its antitumor effect on the MBT-2 murine bladder cancer. C3H/HeN mice injected with MBT-2 cells in conjunction with retroviruses encoding ProT exhibited smaller tumor mass, lower tumor incidence and higher survival rate, as well as higher antitumor cytotoxic activities compared with those injected with control viruses. However, such effects were not observed in severe combined immunodeficiency mice, suggesting that ProT exerts antitumor effects through its immunomodulatory activities. Cell growth in monolayer culture and colony formation in soft agar were enhanced in ProT gene-modified MBT-2 clones, and such growth-promoting activities of ProT could be reversed if its nuclear localization signal (NLS) was deleted. To circumvent the proliferation-promoting effect of ProT on tumor cells, a retroviral vector encoding ProT lacking NLS was constructed. Our results showed that retroviruses encoding NLS-deleted ProT was more efficacious than those encoding wild-type ProT in prolonging survival of tumor-bearing mice. This is the first report indicating that ProT, in particular NLS-deleted ProT, delivered by retroviral vectors may be further explored for the treatment of bladder cancer.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Precursores de Proteínas/genética , Retroviridae/genética , Timosina/análogos & derivados , Timosina/genética , Neoplasias da Bexiga Urinária/terapia , Animais , Divisão Celular , Feminino , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Transplante de Neoplasias , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Attenuated intracellular bacteria, such as Salmonella and Shigella, have been exploited to act as gene delivery vectors. In this study, we report that nonpathogenic, live Escherichia coli can be used for the delivery of DNA vaccines in vivo, leading to generation of immune responses against plasmid-encoded foreign antigens. The pseudorabies virus (PrV) DNA vaccine carrying the glycoprotein D (gD) gene delivered by E. coli was able to induce protective immune responses in mice against a lethal PrV challenge. Co-delivery of E. coli carrying plasmid DNA encoding prothymosin alpha enhanced cellular immune responses to the PrV DNA vaccine delivered by E. coli. Our results suggest that nonpathogenic E. coli may be used as a vector for DNA vaccines in veterinary uses.