RESUMO
Myofibroblasts are specialized contractile cells that participate in tissue fibrosis and remodeling, including idiopathic pulmonary fibrosis (IPF). Mechanotransduction, a process by which mechanical stimuli are converted into biochemical signals, regulates myofibroblast differentiation. Relaxin is a peptide hormone that mediates antifibrotic effects through regulation of collagen synthesis and turnover. In this study, we demonstrate enhanced myofibroblast contraction in bleomycin-induced lung fibrosis in mice and in fibroblastic foci of human subjects with IPF, using phosphorylation of the regulatory myosin light chain (MLC(20)) as a biomarker of in vivo cellular contractility. Compared with wild-type mice, relaxin knockout mice express higher lung levels of phospho-MLC(20) and develop more severe bleomycin-induced lung fibrosis. Exogenous relaxin inhibits MLC(20) phosphorylation and bleomycin-induced lung fibrosis in both relaxin knockout and wild-type mice. Ex vivo studies of IPF lung myofibroblasts demonstrate decreases in MLC(20) phosphorylation and reduced contractility in response to relaxin. Characterization of the signaling pathway reveals that relaxin regulates MLC(20) dephosphorylation and lung myofibroblast contraction by inactivating RhoA/Rho-associated protein kinase through a nitric oxide/cGMP/protein kinase G-dependent mechanism. These studies identify a novel antifibrotic role of relaxin involving the inhibition of the contractile phenotype of lung myofibroblasts and suggest that targeting myofibroblast contractility with relaxin-like peptides may be of therapeutic benefit in the treatment of fibrotic lung disease.
Assuntos
Fibrose Pulmonar Idiopática/prevenção & controle , Miofibroblastos/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Relaxina/farmacologia , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fibrose Pulmonar Idiopática/fisiopatologia , Mecanotransdução Celular , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Cadeias Leves de Miosina/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Proteínas RecombinantesRESUMO
Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin alpha(v)beta(5)-dependent latent transforming growth factor (TGF)-beta1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin alpha(v)beta(5), both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin alpha(v)beta(5) interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin alpha(v)beta(5)-dependent latent TGF-beta1 activation and TGF-beta1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-alpha(v)beta(5) interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-beta1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin alpha(v)beta(5) interactions inhibit contraction-induced latent TGF-beta1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-beta1 with integrin alpha(v)beta(5). Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.
Assuntos
Diferenciação Celular , Fibroblastos/metabolismo , Pulmão/citologia , Mioblastos/citologia , Receptores de Vitronectina/metabolismo , Antígenos Thy-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Motivos de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sistema Livre de Células , Endotelina-1/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Proteínas Imobilizadas/metabolismo , Camundongos , Contração Muscular/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Oxazóis/farmacologia , Ligação Proteica/efeitos dos fármacos , Ratos , Solubilidade/efeitos dos fármacos , Antígenos Thy-1/química , Antígenos Thy-1/isolamento & purificaçãoRESUMO
Human ovarian cancer is a highly lethal malignant neoplasm in woman with no effective treatment if conventional chemotherapy fails. In this regard, conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution to the development of CRAds was the introduction of tumor-selective viral replication to restrict amplification to the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells, killing the cells by cytolysis, leaving normal cells unaffected. However, to date, there have been limitations to the clinical application of these CRAd agents i.e. poor viral infectivity, poor tumor specificity and high toxicity. Here, we report the in vitro and in vivo comparison of four CRAd agents developed for ovarian cancer application, specifically, Ad-Delta24.F5/3, CRAd-C.F5/3, CRAd-M.F5/3 and CRAd-S.F5/3. All CRAd agents contained fiber knob chimeras of adenovirus serotype 3, which enhanced the viral infectivity at the transductional level via a non-Coxsackie-Adenovirus Receptor alternative pathway. In addition, these CRAds embodied distinct mechanisms for the achievement of replication specificity. Tumor cell killing was assessed by using an oncolytic assay and a cell viability assay (MTS) in vitro, while tumor growth was examined in a xenograft model in vivo by using a bioluminescent imaging assay. In addition, the replication rates of the CRAd agents were determined in human liver slices. Both the Ad-Delta24.F5/3 and CRAd-S.F5/3 were demonstrated to have higher tumor killing effects in tumor cells and a lower viral replication rate in human liver. These agents are thus excellent candidates for clinical trials of CRAd agents against human ovarian cancer.
Assuntos
Adenoviridae/genética , Proliferação de Células , Terapia Viral Oncolítica , Neoplasias Ovarianas/terapia , Replicação Viral , Animais , Feminino , Terapia Genética , Vetores Genéticos , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/genética , Transdução Genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Hec1 (Highly Expressed in Cancer gene 1) has recently been shown to play an important role in the proper segregation of chromosomes during mitosis. Recently, an adenovirus delivery system carrying RNA interference (RNAi) of Hec1 has been reported in a cervical adenocarcinoma model. Adenoviral delivery systems, however, have the main limitation of poor viral infectivity due to lack of the native receptor, Coxsackie-Adenovirus Receptor (CAR), on the surface of tumor cells. We hypothesize that the viral infectivity of the adenovirus vector would be enhanced via a CAR-independent pathway by altering the targeting tropism, thus increasing the knockdown effect of Hec1 expression in ovarian carcinoma cells. METHODS: Two adenoviruses (Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3), along with a negative control (Ad-siRNA-GAPDH.F5/3), were created using homologous recombination. HEY and SKOV3.ip1 cell lines were used to perform experiments. The following assays were then used to determine RNAi knockdown efficiency: (1) quantitative PCR (QPCR), (2) Western blot, (3) MTS assay, (4) Annexin V-FITC FACS, (5) crystal violet staining. In all experiments, a negative control served as a baseline measure. RESULTS: QPCR demonstrated a 2-log viral infectivity enhancement with Ad-siRNA-Hec1.F5/3 over Ad-siRNA-Hec1. QPCR at 72 h revealed mRNA knockdown induced by Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cells, respectively (71%/60%, and 32%/78% mRNA knockdown compared to negative control). Western blot revealed translational inhibition induced by both Hec1 Ads with the least knockdown seen with Ad-siRNA-GAPDH.F5/3. FACS analysis revealed increased annexin V positivity in RNAi-infected cells, suggesting a higher rate of apoptosis. MTS assay indicated increased cell death 8 days post-infection with Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cell lines, respectively (75% vs. 35% and 43% vs. 12% viable cells). Crystal violet staining revealed increased cell death with Ad-siRNA-Hec1.F5/3 in all tested cell lines. CONCLUSIONS: RNAi against Hec1 results in gene expression knockdown and apoptosis in vitro. The infectivity-enhanced adenovirus as delivery mechanism shows potential application in future gene therapy models of RNAi in ovarian cancer.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Proteínas Nucleares/genética , Neoplasias Ovarianas/terapia , Interferência de RNA , RNA Interferente Pequeno/genética , Adenoviridae/patogenicidade , Apoptose/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , Feminino , Vetores Genéticos/genética , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/virologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/biossínteseRESUMO
Conventional cancer treatments are not adequate for the majority of most patients stricken with squamous cell carcinomas of the head and neck (SCCHN). Conditionally replicating adenoviruses (CRAds) represent a promising new modality for treating of neoplastic diseases, including SCCHN. Specifically, CRAd agents infect tumor cells and selectively replicate within them, thus causing their death while sparing surrounding normal cells in the host. Oncolysis results from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for propagation of infection and resultant lysis of neighboring cancer cells, sparing normal host cells. However, to date there have been two main limitations to successful clinical application of these CRAd agents: poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.F5/3, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter, and the viral infectivity is enhanced by a fiber modification, F5/3, containing an Ad3 knob chimeric fiber protein. As expected, this agent improved both of the viral infectivity and tumor specificity as evaluated in established SCCHN tumor cell lines and in primary tumor tissues from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as described previously. Based on these data, the CRAd-CXCR4.F5/3 is a promising novel CRAd agent for SCCHN targeting with low host toxicity.
Assuntos
Adenoviridae/fisiologia , Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeça e Pescoço/terapia , Terapia Viral Oncolítica/métodos , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Linhagem Celular Tumoral , Citomegalovirus/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Regiões Promotoras Genéticas , Receptores CXCR4/genética , Replicação ViralRESUMO
Conventional treatments are not adequate for the majority of lung cancer patients. Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of neoplastic diseases, including non-small cell lung cancer. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect a new population of surrounding target cells, replicate again and eradicate the infected tumor cells while leaving normal cells unaffected. However, to date, there have been two main limitations to successful clinical application of these CRAd agents; i.e. poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.RGD, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter and the viral infectivity is enhanced by a capsid modification, RGD4C. This agent CRAd-CXCR4.RGD, as expected, improved both of the viral infectivity and tumor specificity as evaluated in an established lung tumor cell line and in primary tumor tissue from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as compared to three other promoters regularly used for targeting tumors. In addition, this agent has the potential of targeting multiple other tumor cell types. From these data, the CRAd-CXCR4.RGD appears to be a promising novel CRAd agent for lung cancer targeting with low host toxicity.
Assuntos
Adenoviridae/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Terapia Viral Oncolítica/métodos , Regiões Promotoras Genéticas , Receptores CXCR4/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Vetores Genéticos , Humanos , Fígado/metabolismo , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas , Replicação ViralRESUMO
Cholangiocarcinoma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to the clinical application of these CRAd agents, i.e. poor viral infectivity and tumor specificity. Here we report the construction of three new CRAd agents, CRAd-S.RGD, CRAd-S.F5/3 and CRAd-S.pk7, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD, F5/3 or pk7) in the adenovirus fiber region. These CRAd agents effectively target cholangiocarcinoma cells, induce strong cytoxicity in these cells in vitro and inhibit tumor growth in a murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting cholangiocarcinoma with low host toxicity. Such results should provide important insights into the identification of novel therapeutic strategies for cholangiocarcinoma.
Assuntos
Adenoviridae/genética , Neoplasias dos Ductos Biliares/terapia , Colangiocarcinoma/terapia , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Terapia Viral Oncolítica , Replicação Viral/genética , Animais , Neoplasias dos Ductos Biliares/genética , Capsídeo/metabolismo , Células Cultivadas , Colangiocarcinoma/genética , Citomegalovirus/genética , Feminino , Humanos , Proteínas Inibidoras de Apoptose , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas/genética , Survivina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECT: Malignant brain tumors have been proved to be resistant to standard treatments and therefore require new therapeutic strategies. Survivin, a recently described member of the inhibitor of apoptosis protein family, is overexpressed in several human brain tumors, primarily gliomas, but is downregulated in normal tissues. The authors hypothesized that the expression of tumor-specific survivin could be exploited for treatment of gliomas by targeting the tumors with gene therapy vectors. METHODS: Following confirmation of survivin expression in glioma cell lines, an adenoviral vector containing the survivin promoter and the reporter gene luciferase was tested in established and primary glioma cells, normal astrocytic cells, and normal human brain tissues. High levels of reporter gene expression were observed in established tumor and primary tumor cell lines and low levels of expression in astrocytes and normal human brain tissue. To test oncolytic potency, the authors constructed survivin promoter-based conditionally replicative adenoviruses (CRAds), composed of survivin promoter-regulated E1 gene expression and an RGD-4C capsid modification. These CRAds could efficiently replicate within and kill a variety of established glioma tumor cells, but were inactive in a normal human liver organ culture. Finally, survivin promoter-based CRAds significantly inhibited the growth of glioma xenografts in vivo. CONCLUSIONS: Together these data indicate that the survivin promoter is a promising tumor-specific promoter for transcriptional targeting of adenovirus-based vectors and CRAds for malignant gliomas. The strategy of using survivin-CRAds may thus translate into an experimental therapeutic approach that can be used in human clinical trials.
Assuntos
Neoplasias Encefálicas/genética , Marcação de Genes , Terapia Genética/métodos , Glioma/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Adenoviridae , Animais , Apoptose/genética , Linhagem Celular Tumoral , Feminino , Vetores Genéticos , Humanos , Proteínas Inibidoras de Apoptose , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Survivina , Transplante Heterólogo , Replicação Viral/genéticaRESUMO
Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculum. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect surrounding target cells, replicate and eradicate the infected tumor cells, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; i.e., both infectivity and tumor specificity are poor. Survivin protein is a novel member of the inhibitor of apoptosis (IAP) protein family, which plays an important role in the survival of cancer cells and progression of malignancies. Previous data have shown the survivin promoter has high activities in multiple cancer cells with a low activity in mouse liver. In this study, we propose an improved CRAd agent to circumvent the obstacles. We constructed a novel CRAd agent, CRAd-Survivin-RGD, which contains both the survivin promoter (either the short version, S-S, or the long version, S-L) to selectively drive E1 gene expression in tumor cells and a capsid modification and RGD4C to specifically enhance the tumor infectivity of CRAd agents. Both CRAd agents (S-S and S-L) showed high replication rates in the breast cancer cell line, MDA-MB-361, and low promoter activity in both normal mouse and human liver, thus signifying the CRAd agents have the phenotype of 'tumor on/liver off'. In cytocidal experiments, the CRAd agents demonstrated a high cytocidal effect on multiple cancer cell lines, including the breast cancer cell line, MDA-MB-231; the glioma cell line, D65, the melanoma cell line, MEL-28; and mesothelioma, Meso2374. The results also showed the tumor growth was dramatically inhibited by intertumoral administration of the CRAd agents in a breast cancer (MDA-MB-361) xenograft animal model. These data clearly demonstrate that CRAd-Survivin-RGD is a potential novel therapeutic agent for treatment in many, but not all, human cancers.
Assuntos
Adenoviridae/genética , Antineoplásicos/farmacologia , Terapia Genética/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Oligopeptídeos/genética , Regiões Promotoras Genéticas , Proteínas E1 de Adenovirus/genética , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Feminino , Vetores Genéticos , Humanos , Técnicas In Vitro , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Modelos Genéticos , Família Multigênica , Transplante de Neoplasias , Fenótipo , Reação em Cadeia da Polimerase , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Survivina , Fatores de TempoRESUMO
Metastatic renal cell carcinoma (RCC) is often resistant to standard treatment, thereby requiring new therapeutic strategies. In this regard, tumor cell migration and metastasis have recently been shown to be regulated by chemokines and their respective receptors (e.g., SDF-1alpha/CXCR4). In the context of RCC, up-regulation of CXCR4 expression is closely related to the development of invasive cancer. Thus, we hypothesized that the CXCR4 pathway could be exploited for RCC targeting with gene therapy vectors. In this regard, targeting adenoviral vectors to tumor cells is critically dependent on tumor-specific gene expression. Toward the end of RCC tumor targeting, we evaluated the utility of the CXCR4 promoter in an adenoviral context. First, overexpression of CXCR4 was confirmed in several RCC cell lines. Next, an adenoviral vector was constructed, whereby the human CXCR4 promoter drives the expression of a reporter gene. We tested the activity of the CXCR4 promoter in vitro and in vivo in relevant models. Our data indicate that the human CXCR4 promoter is highly active in RCC cells but not in normal human cells. Finally, biodistribution studies in mice demonstrated dramatic repression of the CXCR4 promoter in the liver but not in the kidney. In conclusion, the unique activity of the CXCR4 promoter in RCC lines and its repression in normal human cells and in the murine liver underscore its potential utility as a novel candidate for transcriptional targeting of RCC.
Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Regiões Promotoras Genéticas/genética , Receptores CXCR4/genética , Transcrição Gênica/genética , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Genes Reporter/genética , Terapia Genética , Vetores Genéticos/genética , Humanos , Fígado/metabolismo , CamundongosRESUMO
It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. We employed a recombinant adenoviral vector (reAdGL3BSurvivin) in which a tumor-specific survivin promoter and a luciferase reporter gene were inserted into the E1-deleted region of adenovirus vector. Luciferase activity was measured in both multiple tumor cell lines and two primary melanoma cells infected with reAdGL3BSurvivin. Human fibroblast and mammary epithelial cell lines were used as negative controls. A reAdGL3CMV, containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity generated by the survivin promoter. Our data revealed that the survivin promoter showed high activity in both established tumor cell lines and the primary melanoma cells. In contrast, the in vivo studies indicated that the activities of survivin promoter were extremely low in the major mouse organs. The survivin promoter appears to be a promising tumor-specific promoter exhibiting a "tumor on" and "liver off" profile, and therefore, it may prove to be a good candidate for transcriptional targeting of cancer gene therapy in a wide variety of tumors.
Assuntos
Terapia Genética/métodos , Proteínas Associadas aos Microtúbulos/genética , Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2 , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter/genética , Vetores Genéticos/genética , Humanos , Proteínas Inibidoras de Apoptose , Isoenzimas/genética , Luciferases/análise , Luciferases/genética , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias , Neoplasias/terapia , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/análise , Survivina , Transcrição Gênica/genéticaRESUMO
OBJECTIVES: Current virotherapy strategies for ovarian cancer have been hampered by limitations in target cell infectivity and nonspecific tissue replication. In an effort to circumvent these limitations, we evaluated various CRAds modified to incorporate novel capsid targeting motifs (RGD and chimeric Ad5/3) with a novel tissue-specific promoter (CXCR4). METHODS: Two novel CRAds (Ad5-CXCR4-F5/3 and Ad5-CXCR4-RGD) were constructed via homologous recombination and verified by PCR and DNA sequencing. The infectivity and viral replication rates of these two CRAds were analyzed via quantitative real-time PCR (QRT-PCR) in cell line experiments using three ovarian cancer cell lines (SKOV3.ip1, Hey, and OV4) and compared to that achieved with a clinical grade CRAd (delta24-RGD) to be evaluated in a Phase I trial. Cytocidal effects were determined by crystal violet staining in these same cell lines infected with different concentrations of viral particles per cell (0, 0.1, 1, 10, 100, and 500). Additionally, viral replication was evaluated by QRT-PCR in primary ovarian cancer tissue slices from multiple patients with ovarian cancer as well as in primary human normal liver tissue slices in order to establish CRAd selectivity. All experiments incorporated appropriate controls and repeated in triplicate. RESULTS: Compared to RGD-capsid CRAds (delta24-RGD and CXCR4-RGD), the F5/3-capsid CRAd (CXCR4-F5/3) demonstrated significant improvements in infection rates (p=0.025, 0.006, and 0.006) in all ovarian cancer cell lines tested (SKOV3.ip1, Hey, and OV4, respectively). In addition to improved transduction of virus into the cells, the TSP CXCR4-based CRAds demonstrated improved viral replication. Specifically, CXCR4-F5/3 further enhanced viral replication 89-fold (p=0.009, 0.010, 0.003) in the same cancer cell lines. Furthermore, CXCR4-F5/3 showed a 4-log improvement in oncolytic potential over delta24-RGD. In the ex vivo primary ovarian tissue slices, CXCR4-F5/3 showed a 58-fold improvement in viral replication (p=0.005) compared to the clinical grade delta24-RGD. Both CXCR4-F5/3 and CXCR4-RGD demonstrated significant reduction of viral replication in normal liver slices (p=0.001). CONCLUSIONS: These data suggest that a dual targeted approach is feasible for the combined enhancement of infectivity and replication in ovarian cancer with a specificity that was attenuated in normal liver tissues. In fact, CXCR4-F5/3 outperformed our best CRAd agent to date nearly 60-fold in our most stringent ex vivo model of primary ovarian cancer tissue slices and suggests that this novel agent could be useful for the treatment of ovarian cancer.
Assuntos
Adenovírus Humanos/fisiologia , Terapia Viral Oncolítica/métodos , Neoplasias Ovarianas/terapia , Neoplasias Ovarianas/virologia , Replicação Viral/fisiologia , Adenovírus Humanos/genética , Adenovírus Humanos/patogenicidade , Capsídeo/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Fígado/virologia , Oligopeptídeos/genética , Receptores CXCR4/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/genéticaRESUMO
Mesothelioma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard is the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; viral infectivity and tumor specificity have been poor. Herein we report on two CRAd agents, CRAd-S.RGD and CRAd-S.F5/3, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD or F5/3) in the adenovirus fiber region. These CRAd agents effectively target human mesothelioma cell lines, induce strong cytoxicity in these cells in vitro, and viral replication in a H226 murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting mesothelioma with low host toxicity. These agents should provide important insights into the identification of novel therapeutic strategies for mesothelioma.
Assuntos
Adenoviridae , Mesotelioma/terapia , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Terapia Viral Oncolítica , Neoplasias Pleurais/terapia , Replicação Viral , Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Animais , Proteínas do Capsídeo/genética , Feminino , Vetores Genéticos , Humanos , Proteínas Inibidoras de Apoptose , Fígado/metabolismo , Mesotelioma/genética , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Cultura de Órgãos , Neoplasias Pleurais/genética , Regiões Promotoras Genéticas , Recombinação Genética , Survivina , Transdução Genética , Células Tumorais CultivadasRESUMO
OBJECTIVE: Icodextrin, a novel glucose polymer solution utilized for peritoneal dialysis, has been demonstrated to have prolonged intraperitoneal (IP) instillation volumes in comparison to standard PBS solutions. In an animal model of ovarian cancer, we explored whether a survival advantage exists utilizing icodextrin rather than PBS as a delivery solution for an infectivity enhanced virotherapy approach. METHODS: Initial experiments evaluated whether icodextrin would adversely affect replication of a clinical grade infectivity enhanced conditionally replicative adenovirus (Delta24-RGD). Virus was added to prepared blinded solutions of PBS or icodextrin (20%) and then evaluated in vitro in various human ovarian cancer cell lines (SKOV3.ip1, PA-1, and Hey) and in vivo in a SKOV3.ip1 human ovarian cancer IP murine model. Viral replication was measured by detecting adenovirus E4 gene levels utilizing QRT-PCR. Survival was subsequently evaluated in a separate SKOV3.ip1 ovarian cancer IP murine model. Cohorts of mice were treated in blinded fashion with PBS alone, icodextrin alone, PBS+Delta24-RGD, or icodextrin+Delta24-RGD. Survival data were plotted on Kaplan-Meier curve and statistical calculations performed using the log-rank test. RESULTS: There was no adverse affect of icodextrin on vector replication in the ovarian cancer cell lines nor murine model tumor samples evaluated. Median survival in the IP treated animal cohorts was 23 days for the PBS group, 40 days for the icodextrin group, 65 days for the PBS+Delta24-RGD group, and 105 days for icodextrin+Delta24-RGD (p=0.023). Of note, 5 of the 10 mice in the icodextrin+Delta24-RGD group were alive at the end of the study period, all without evidence of tumor (120 days). CONCLUSIONS: These experiments suggest that the use of dialysates such as icodextrin may further enhance the therapeutic effects of novel IP virotherapy and other gene therapy strategies for ovarian cancer. Phase I studies utilizing icodextrin-based virotherapy for ovarian cancer are currently in development.
Assuntos
Soluções para Diálise/uso terapêutico , Terapia Genética , Glucanos/uso terapêutico , Glucose/uso terapêutico , Neoplasias Ovarianas/terapia , Animais , Soluções para Diálise/administração & dosagem , Modelos Animais de Doenças , Feminino , Glucanos/administração & dosagem , Glucose/administração & dosagem , Icodextrina , Infusões Parenterais , Camundongos , Neoplasias Ovarianas/patologiaRESUMO
Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers such as the epithelium or endothelium. We report here the construction of an adenoviral vector that has the capability to be transported across polarized epithelial monolayers of Caco-2 cells (a colon carcinoma cell line) by transcytosis. This transcytosis is transferrin receptor (TfR)-mediated with use of a bifunctional adaptor, soluble coxsackie adenovirus receptor (sCAR)-Tf, and is both temperature and iron dependent. Under experimental conditions, the adenoviral transcytosis was inhibited by pretreatment of Caco-2 cells with colchicine, an inhibitor of transcytosis, and was not enhanced by pretreatment with Brefeldin A (BFA), an enhancer of transcytosis. In these Caco-2 cells, the transcytosis rate was 0.3 +/- 1.3% (SD). The transcytosed adenoviruses remain biologically functional. These data suggest the potential clinical benefit under conditions where drug delivery is a challenge, such as within the airway epithelium, at the bladder lumen urothelial cell interface, and across the blood-brain barrier for clinical treatment of lung, urogenital, and brain disorders, respectively, by adenoviral transcytosis of transgene delivery.