RESUMO
BACKGROUND: Successful immunotherapy will require alteration of the tumour microenvironment and/or decreased immune suppression. Tumour-associated macrophages (TAMs) are one major factor affecting tumour microenvironment. We hypothesised that altering TAM phenotype would augment the efficacy of immunotherapy. METHODS: We and others have reported that 5,6-Dimethylxanthenone-4-acetic-acid (DMXAA, Vadimezan) has the ability to change TAM phenotypes, inducing a tumour microenvironment conducive to antitumour immune responses. We therefore combined DMXAA with active immunotherapies, and evaluated anti-tumour efficacy, immune cell phenotypes (flow cytometry), and tumour microenvironment (RT-PCR). RESULTS: In several different murine models of immunotherapy for lung cancer, DMXAA-induced macrophage activation significantly augmented the therapeutic effects of immunotherapy. By increasing influx of neutrophils and anti-tumour (M1) macrophages to the tumour, DMXAA altered myeloid cell phenotypes, thus changing the intratumoural M2/non-M2 TAM immunoinhibitory ratio. It also altered the tumour microenvironment to be more pro-inflammatory. Modulating macrophages during immunotherapy resulted in increased numbers, activity, and antigen-specificity of intratumoural CD8(+) T cells. Macrophage depletion reduced the effect of combining immunotherapy with macrophage activation, supporting the importance of TAMs in the combined effect. CONCLUSION: Modulating intratumoural macrophages dramatically augmented the effect of immunotherapy. Our observations suggest that addition of agents that activate TAMs to immunotherapy should be considered in future trials.
Assuntos
Adenocarcinoma Bronquioloalveolar/terapia , Carcinoma Pulmonar de Lewis/terapia , Imunoterapia , Neoplasias Pulmonares/terapia , Ativação de Macrófagos/efeitos dos fármacos , Microambiente Tumoral/imunologia , Xantonas/uso terapêutico , Adenocarcinoma Bronquioloalveolar/imunologia , Adenocarcinoma Bronquioloalveolar/patologia , Animais , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Terapia Combinada , Feminino , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/imunologia , Neutrófilos/citologia , Neutrófilos/imunologiaRESUMO
BACKGROUND: Species selectivity of DMXAA (5,6-dimethylxanthenone-4-acetic acid, Vadimezan) for murine cells over human cells could explain in part the recent disappointing phase III trials clinical results when preclinical studies were so promising. To identify analogues with greater human clinical potential, we compared the activity of xanthenone-4-acetic acid (XAA) analogues in murine or human cellular models. METHODS: Analogues with a methyl group systematically substituted at different positions of the XAA backbone were evaluated for cytokine induction in cultured murine or human leukocytes; and for anti-vascular effects on endothelial cells on matrigel. In vivo antitumour activity and cytokine production by stromal or cancer cells was measured in human A375 and HCT116 xenografts. RESULTS: Mono-methyl XAA analogues with substitutions at the seventh and eighth positions were the most active in stimulating human leukocytes to produce IL-6 and IL-8; and for inhibition of tube formation by ECV304 human endothelial-like cells, while 5- and 6-substituted analogues were the most active in murine cell systems. CONCLUSION: Xanthenone-4-acetic acid analogues exhibit extreme species selectivity. Analogues that are the most active in human systems are inactive in murine models, highlighting the need for the use of appropriate in vivo animal models in selecting clinical candidates for this class of compounds.
Assuntos
Antineoplásicos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Xantenos/farmacologia , Xantonas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Combinação de Medicamentos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Laminina/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos , Camundongos Nus , Proteoglicanas/metabolismo , Xantenos/químicaRESUMO
Rac1b, an alternative splice form of Rac1, has been previously shown to be upregulated in colon and breast cancer cells, suggesting an oncogenic role for Rac1b in these cancers. Our analysis of NSCLC tumor and matched normal tissue samples indicates Rac1b is upregulated in a significant fraction of lung tumors in correlation with mutational status of K-ras. To directly assess the oncogenic potential of Rac1b in vivo, we employed a mouse model of lung adenocarcinoma, in which the expression of Rac1b can be conditionally activated specifically in the lung. Although expression of Rac1b alone is insufficient to drive tumor initiation, the expression of Rac1b synergizes with an oncogenic allele of K-ras resulting in increased cellular proliferation and accelerated tumor growth. Finally, we show that in contrast to our previous findings demonstrating a requirement for Rac1 in K-ras-driven cell proliferation, Rac1b is not required in this context. Given the partially overlapping spectrum of downstream effectors regulated by Rac1 and Rac1b, our findings further delineate the signaling pathways downstream of Rac1 that are required for K-ras driven tumorigenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Transformação Celular Neoplásica/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteínas ras/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras/genéticaRESUMO
Esophageal carcinoma is the most rapidly increasing tumor in the United States and has a dismal 15% 5-year survival. Immunotherapy has been proposed to improve patient outcomes; however, no immunocompetent esophageal carcinoma model exists to date to test this approach. We developed two mouse models of esophageal cancer by inoculating immunocompetent mice with syngeneic esophageal cell lines transformed by cyclin-D1 or mutant HRAS(G12V) and loss of p53. Similar to humans, surgery and adjuvant chemotherapy (cisplatin and 5-fluorouracil) demonstrated limited efficacy. Gene-mediated cyototoxic immunotherapy (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir; AdV-tk/GCV) demonstrated high levels of in vitro transduction and efficacy. Using in vivo syngeneic esophageal carcinoma models, combining surgery, chemotherapy and AdV-tk/GCV improved survival (P=0.007) and decreased disease recurrence (P<0.001). Mechanistic studies suggested that AdV-tk/GCV mediated a direct cytotoxic effect and an increased intra-tumoral trafficking of CD8 T cells (8.15% vs 14.89%, P=0.02). These data provide the first preclinical evidence that augmenting standard of care with immunotherapy may improve outcomes in the management of esophageal carcinoma.
Assuntos
Carcinoma/terapia , Neoplasias Esofágicas/terapia , Terapia Genética/métodos , Imunoterapia/métodos , Terapia Neoadjuvante/métodos , Neoplasias Experimentais/terapia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclina D1/genética , Citotoxicidade Imunológica , Feminino , Genes ras/genética , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/prevenção & controle , Simplexvirus/genética , Timidina Quinase/genética , Resultado do Tratamento , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The eighth International Mesothelioma Interest Group (IMIG) meeting was held in Chicago, IL, United States, in 19-22 October 2006 to discuss mesothelioma - the cancer often linked to asbestos exposure. It is a very aggressive malignancy with a median survival of less than 1 year from diagnosis. Millions of people have been exposed worldwide to asbestos, especially during the second half of the twentieth century when asbestos use increased significantly. The tons of asbestos utilized in the past remain a health hazard for current and future generations because asbestos is difficult to be disposed off. This makes asbestos and mesothelioma research a public health issue in addition to a medical problem. Moreover, the very high costs of asbestos litigation have a significant impact on the whole economy. In the United States, up until 2001, defendant companies had paid 54 billion dollars in claims and estimated future liabilities ranged from 145 to 210 billion. Therefore, asbestos research is of great interest to a large audience that includes patients, millions of asbestos-exposed individuals, scientists, physicians, public health officials, politicians, unions of asbestos workers, lawyers and the public at large. During the past few years, there has been significant progress in understanding the process of mineral fiber carcinogenesis and mesothelioma pathogenesis. With improved understanding of the pathogenesis of mesothelioma, new diagnostic, preventive and therapeutic options are being developed. A total of 247 papers were presented at the IMIG: the abstracts of these presentations were published in Lung Cancer, Supplement 1, October 2006. Here, experts in different disciplines critically review some of the most exciting presentations of the IMIG meeting. The result is a comprehensive review of the research field of asbestos carcinogenesis and mesothelioma, and of the progress that has been made in recent years in both basic and clinical sciences.
Assuntos
Mesotelioma , Neoplasias Pleurais , Humanos , Mesotelioma/etiologia , Mesotelioma/patologia , Neoplasias Pleurais/etiologia , Neoplasias Pleurais/patologiaRESUMO
Oxidant stress may contribute to acute lung injury under some circumstances. The rapid depletion of plasma gelsolin following major trauma in patients who subsequently develop respiratory distress suggests that this actin-scavenging protein might protect against delayed pulmonary complications. The specific aim of these experiments was to explore the temporal and quantitative relationship between gelsolin levels and lung damage. Gelsolin levels were measured in three murine models of oxidant injury: immunotargeting of pulmonary endothelium with an H2O2-generating enzyme; continuous exposure to >95% O2; and single high-dose thoracic radiation. The degree of lung injury was inversely related to gelsolin levels in mice treated with glucose oxidase-conjugated antibodies against platelet endothelial cell adhesion molecule-1 (p <0.0001). By 60-72 hours of hyperoxic exposure, gelsolin levels had dropped precipitously in all mice who sustained major lung damage (p <0.0001), establishing a quantitative association between gelsolin concentration and hyperoxic lung injury (r = -0.72; 95% confidence interval: ?0.81 to ?0.59). Gelsolin levels modestly but progressively fell in irradiated mice over the 3 days following treatment (p = 0.012) despite the development of only microscopic lung damage during this timeframe. These findings are consistent with the hypothesis that gelsolin depletion is involved in the pathogenesis of acute oxidant lung injury.
Assuntos
Gelsolina/sangue , Lesão Pulmonar , Pulmão/efeitos da radiação , Oxidantes/efeitos adversos , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/etiologia , Animais , Especificidade de Anticorpos/imunologia , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Relação Dose-Resposta à Radiação , Glucose Oxidase/administração & dosagem , Glucose Oxidase/efeitos adversos , Hiperóxia/sangue , Hiperóxia/complicações , Hiperóxia/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Oxidantes/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/administração & dosagem , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos adversos , Proteínas/metabolismo , Doses de Radiação , Estatística como Assunto , Fatores de TempoRESUMO
UNLABELLED: The aim of this study was to investigate the difference in the rates of FDG uptake between malignant and inflammatory cells and processes. IN VITRO STUDIES: (18)F-FDG uptake by different tumor cell lines (human mesothelioma [REN]; rat mesothelioma [II45]; mice melanoma [B18F10]; mice mesothelioma [AB12]; human myeloma [GM1500]; and human ovarian cancer [SKOV3]) and peripheral blood mononuclear cells isolated from 8 healthy human volunteers was measured 20 and 60 min after FDG was added into growth medium. Animal studies: II45 cells were implanted into the left flank of rats (n = 5) and a focal inflammatory reaction (mechanical irritation) was generated in the right flank. PET images at 45 and 90 min after injection of FDG were obtained and standardized uptake values (SUVs) were determined. Patient studies: Seventy-six patients who had dual time FDG PET scans were retrospectively analyzed. All results were expressed as the percentage change in SUV of the later time image from that of the earlier time (mean +/- SD). IN VITRO STUDIES: Except for the SKOV3 cell line, which had only minimally increased FDG uptake (+10% +/- 26%; P > 0.3), all other tumor cell lines tested showed significantly increased FDG uptake over time (GM1500, +59% +/- 19%; B18F10, +81% +/- 15%; AB12, 93% +/- 21%; II45, +161% +/- 21%; REN, +198% +/- 48%; P < 0.01 for all). By contrast, FDG uptake in mononuclear cells was decreased in 7 of 8 donors. Animal studies: SUVs of tumors from 90-min images were significantly higher than those from 45-min images (+18% +/- 8%; P < 0.01), whereas the SUVs of inflammatory lesions decreased over time (-17% +/- 13% of the early images; P < 0.05). CLINICAL STUDIES: The SUVs of delayed images from the known malignant lesions compared with those of earlier scans increased over time (+19.18% +/- 9.58%; n = 31; P < 0.001; 95% confidence interval, 15.8%-22.6%). By contrast, the SUVs of benign lung nodules decreased slightly over time (-6.3% +/- 8.1%; n = 12; P < 0.05; 95% confidence interval, -10.9% to -1.7%). The SUV of inflammatory lesions caused by radiation therapy (+1.16% +/- 7.23%; n = 8; P > 0.05; 95% confidence interval, -3.9%-6.2%) and the lesions of painful lower limb prostheses (+4.03% +/- 11.32%; n = 25; P > 0.05; 95% confidence interval, -0.4%-8.5%) remained stable over time. CONCLUSION: These preliminary data show that dual time imaging appears to be useful in distinguishing malignant from benign lesions. Further research is necessary to confirm these results.
Assuntos
Fluordesoxiglucose F18 , Inflamação/diagnóstico por imagem , Neoplasias/diagnóstico por imagem , Compostos Radiofarmacêuticos , Tomografia Computadorizada de Emissão , Idoso , Animais , Diagnóstico Diferencial , Feminino , Fluordesoxiglucose F18/farmacocinética , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Endogâmicos F344 , Estudos Retrospectivos , Células Tumorais Cultivadas/metabolismoRESUMO
Malignant mesothelioma remains an incurable disease for which immune-modulatory therapies, such as exogenous cytokines, have shown some promise. One such cytokine, IFN-beta, has potent antiproliferative and immunostimulatory activity in vitro, but its in vivo use has been limited by toxicity. We thus conducted studies evaluating intracavitary delivery of a replication-deficient adenoviral (Ad) vector encoding for the murine IFN-beta gene (Ad.muIFN-beta) in mouse models of malignant mesothelioma. In contrast to multiple injections of recombinant protein, a single i.p. injection of Ad.muIFN-beta into animals with established tumors elicited remarkable antitumor activity leading to long-term survival in >90% of animals bearing either AB12 or AC29 i.p. mesotheliomas. A control adenovirus vector had minimal antitumor effect in vivo. Significant therapeutic effects were also seen in animals treated with large tumor burdens. Importantly, treatment of i.p. tumor also led to reduction of growth in tumors established at a distant site (flank). A number of experiments suggested that these effects were attributable to an acquired CD8(+) T-cell-mediated response including: (a) the induction of long-lasting antitumor immunity; (b) loss of efficacy of Ad.muIFN-beta in tumor-bearing, immune-deficient (SCID, SCID/beige) mice; (c) detection of high levels of specific antitumor cytolytic activity from unstimulated splenocytes harvested from Ad.muIFN-beta-treated animals that was abolished by CD8(+) T-cell depletion; and (d) abrogation of antitumor effects of Ad.muIFN-beta in tumor-bearing CD8(+) T-cell-depleted animals. These data show that intracavitary IFN-beta gene therapy using an adenoviral vector provides strong CD8(+) T-cell-mediated antitumor effects in murine models of mesothelioma and suggest that this may be a promising strategy for the treatment of localized tumors such as mesothelioma or ovarian cancer in humans.
Assuntos
Terapia Genética/métodos , Interferon beta/genética , Interferon beta/imunologia , Mesotelioma/terapia , Neoplasias Peritoneais/terapia , Adenoviridae/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Divisão Celular/imunologia , Citotoxicidade Imunológica , Relação Dose-Resposta Imunológica , Feminino , Vetores Genéticos/genética , Injeções Intraperitoneais , Interferon beta/metabolismo , Mesotelioma/genética , Mesotelioma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/imunologia , Linfócitos T Citotóxicos/imunologia , TransfecçãoRESUMO
Replication-incompetent adenoviruses (Ad) carrying the herpes simplex thymidine kinase (HSVtk) gene have been used in a number of human cancer gene therapy trials, however transduction has generally been limited to a small minority of tumor cells. To solve this problem, replication-competent adenoviral vectors carrying transgenes such as HSVtk have been developed. However, contradictory evidence exists regarding the efficacy of these new vectors. Accordingly, we constructed and tested a replication-competent E3-deleted adenoviral vector containing the HSVtk suicide gene driven by the endogenous E3 promoter (Ad.wt.tk). This virus showed high level production of the HSVtk transgene and was more efficacious than a non-replicating virus in vitro, after injection into flank tumors, and against established intraperitoneal tumors. However, addition of ganciclovir (GCV) therapy to cells or tumor-bearing animals treated with the replicating vector containing the HSVtk suicide gene did not result in increased cell killing. Our results indicate that addition of HSVtk to a replicating Ad virus will not likely be useful in augmenting antitumor effects.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Neoplasias Pulmonares/terapia , Neoplasias Experimentais/terapia , Simplexvirus/enzimologia , Timidina Quinase/genética , Análise de Variância , Animais , Antivirais/uso terapêutico , Feminino , Ganciclovir/uso terapêutico , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Injeções Intralesionais , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Transdução Genética/métodos , Replicação ViralAssuntos
Sinalização do Cálcio , Endotélio Vascular/metabolismo , Canais Iônicos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Cálcio/metabolismo , Cátions , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Ativação do Canal Iônico , Cinética , Modelos Biológicos , Técnicas de Patch-Clamp , Fosfatidilinositóis/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Transdução de Sinais , TransfecçãoRESUMO
Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM-1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross-linking protein that facilitates anti-PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM-directed vascular immunotargeting of a reporter enzyme (beta-galactosidase, beta-Gal) in intact animals. Intravenous injection of [125I]SA-beta-Gal conjugated with either anti-PECAM or IgG led to a high 125I uptake in liver and spleen, yet beta-Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti-PECAM/[125I]SA-beta-Gal, but not IgG/[125I]SA-beta-Gal, accumulated in the lungs (36.0+/-1.3 vs. 3.9+/-0.6% injected dose/g) and induced a marked elevation of beta-Gal activity in the lung tissue persisting for up to 8 h after injection (10-fold elevation 4 h postinjection). Using histochemical detection, the beta-Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti-PECAM carrier also provided 125I uptake and beta-Gal activity in the renal glomeruli. Predominant intracellular localization of anti-PECAM/SA-beta-Gal was documented in the PECAM-expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell-selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.
Assuntos
Endotélio Vascular/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , beta-Galactosidase/genética , Animais , Genes Reporter , Humanos , Imunoglobulina G/metabolismo , Radioisótopos do Iodo/farmacocinética , Glomérulos Renais/enzimologia , Fígado/enzimologia , Pulmão/enzimologia , Mesotelioma , Camundongos , Camundongos Endogâmicos BALB C , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Circulação Pulmonar , Proteínas Recombinantes/metabolismo , Baço/enzimologia , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
HSV-1716, a replicating nonneurovirulent herpes simplex virus type 1, has shown efficacy in treating multiple types of human tumors in immunodeficient mice. Since the majority of the human population has been previously exposed to herpes simplex virus, the efficacy of HSV-based oncolytic therapy was investigated in an immunocompetent animal tumor model. EJ-6-2-Bam-6a, a tumor cell line derived from h-ras-transformed murine fibroblast, exhibit a diffuse growth pattern in the peritoneal cavity of BALB/c mice and replicate HSV-1716 to titers observed in human tumors. An established intraperitoneal (ip) tumor model of EJ-6-2-Bam-6a in naive and HSV-immunized mice was used to evaluate the efficacy of single or multiple ip administrations of HSV-1716 (4 x 10(6) pfu/treatment) or of carrier cells, which are irradiated, ex vivo virally infected EJ-6-2-Bam-6a cells that can amplify the viral load in situ. All treated groups significantly prolonged survival versus media control with an approximately 40% long-term survival rate (cure) in the multiply treated, HSV-naive animals. Prior immunization of the mice with HSV did not significantly decrease the median survival of the single or multiply treated HSV-1716 or the carrier cell-treated groups. These studies support the development of replication-selective herpes virus mutants for use in localized intraperitoneal malignancies.
Assuntos
Anticorpos Antivirais/imunologia , Terapia Genética , Herpesvirus Humano 1/fisiologia , Neoplasias Peritoneais/terapia , Replicação Viral/fisiologia , Animais , Feminino , Vetores Genéticos , Humanos , Imunidade , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Peritoneais/virologia , Taxa de SobrevidaRESUMO
Replication-restricted herpes simplex virus-1 (HSV-1) strains lacking ICP34.5 are emerging as powerful anticancer agents against several solid tumors including epithelial ovarian cancer (EOC). Although chemotherapy-resistant tumors would be likely candidates for treatment with HSV-1 mutants lacking ICP34.5, the efficacy of these mutants on such tumors is unknown. In the present study, we investigated whether chemotherapy resistance affects the response of ovarian cancer cells to HSV-R3616, an ICP34.5-deficient, replication-restricted HSV-1. Primary EOC cultures obtained from patients who varied in their responses to platinum/paclitaxel induction chemotherapy displayed similar sensitivity to HSV-R3616. Similarly, chemotherapy-sensitive ovarian cancer cells A2780 and PA-1, possessing wild-type p53, and their respective chemotherapy-resistant clones A2780/200CP, lacking p53 function, and PA-1/E6, permanently expressing the HPV E6 gene, were equally sensitive to HSV oncolysis. Because wild-type HSV can kill cells by apoptosis and nonapoptotic mechanisms, we investigated the involvement of apoptosis and the role of the p53 tumor suppressor gene in oncolysis induced by HSV-R3616. Infection of ovarian cancer cell lines by HSV-R3616 was followed by cell death via apoptosis or nonapoptotic mechanisms as noted by morphology, cell cycle analysis, and in situ TUNEL assay. p53 protein levels remained unchanged, and Bax protein levels decreased in cells possessing intact p53 and that mainly underwent HSV-induced apoptosis. Loss of p53 function did not affect the frequency or rate of apoptosis or the sensitivity of EOC cells to the oncolytic effect of HSV-R3616. These results suggest that recombinant HSV-1 lacking ICP34.5 is capable of killing ovarian cancer cells that lack p53 function, resist apoptosis, and/or are chemotherapy resistant. These data support the hypothesis that HSV-based oncolytic therapy may be efficacious in chemotherapy-resistant tumors, including tumors that are deficient in p53.
Assuntos
Herpesvirus Humano 1/fisiologia , Neoplasias Ovarianas/terapia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Supressora de Tumor p53/fisiologia , Proteínas Virais/fisiologia , Antineoplásicos/farmacologia , Apoptose/fisiologia , Morte Celular/fisiologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/virologia , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/genética , Replicação Viral , Proteína X Associada a bcl-2RESUMO
Previous studies with a mycobacterial heat shock protein (hsp-65) have demonstrated some efficacy using cationic liposome-mediated gene transfer in murine i.p. sarcoma models. To further analyze the efficacy of hsp-65 immunotherapy in clinically relevant models of localized cancer, immunocompetent mice bearing i.p. murine mesothelioma were treated with four i.p. doses of a cationic lipid complexed with plasmid DNA (pDNA) containing hsp65, LacZ, or a null plasmid. We observed >90% long-term survival (median survival, 150 days versus approximately 25 days, treated versus saline control, respectively) in a syngeneic, i.p. murine mesothelioma model (AC29). Long-term survivors were observed in all groups treated with lipid complexed with any pDNA. Lipid alone or DNA alone provided no demonstrable survival advantage. In a more aggressive i.p. model of mesothelioma (AB12), we observed >40% long-term survival in groups treated with lipid:pDNA complexes, again irrespective of the transgene. To ask whether these antitumor effects had led to an adaptive immune response against the tumor cell, we rechallenged long-term survivors in both murine models s.c. with the parental tumor cell line. Specific, long-lasting systemic immunity against the tumor was readily demonstrated in both models (AB12 and AC29). Consistent with these results, splenocytes from long-term survivors specifically lysed the parental tumor cell lines. Depleting the CD8+ T-cells from the splenocyte pool eliminated this lytic activity. Lipid:pDNA treatment of athymic, SCID, and SCID/Beige mice bearing a murine i.p. mesothelioma (AC29) resulted in only a slight survival advantage, but there were no long-term survivors. Treatment of immunocompetent mice depleted of specific immune effector cells demonstrated roles for CD8+ and natural killer cells. Although the exact mechanism(s) responsible for these antitumor effects is unclear, the results are consistent with roles for both innate and adaptive immune responses. An initial tumor cell killing stimulated by cationic lipid:pDNA complexes appears to be translated into long-term, systemic immunity against the tumor cell. These results are the first to demonstrate that adaptive immunity against a tumor cell can be induced by the administration of lipid:pDNA complexes. Multiple administrations of cationic lipid complexed with pDNA lacking an expressed transgene could provide a promising generalized immune-mediated modality for treating cancer.
Assuntos
Proteínas de Bactérias , DNA Bacteriano/genética , Vetores Genéticos , Imunoterapia Adotiva , Lipídeos/genética , Mesotelioma/terapia , Animais , Linfócitos T CD8-Positivos/fisiologia , Chaperonina 60 , Chaperoninas/genética , Ilhas de CpG , Intervalo Livre de Doença , Feminino , Técnicas de Transferência de Genes , Células Matadoras Naturais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos SCID , Plasmídeos , Baço/efeitos dos fármacos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Gene therapy, the treatment of any disorder or pathophysiologic state on the basis of the transfer of genetic information, was a high-priority goal in the 1990s. The lung is a major target of gene therapy for genetic disorders, such as cystic fibrosis and alpha1-antitrypsin deficiency, and for other diseases, including lung cancer, malignant mesothelioma, pulmonary inflammation, surfactant deficiency, and pulmonary hypertension. This paper examines general concepts in gene therapy, summarizes the results of published clinical trials, and highlights areas of research aimed at overcoming challenges in the field. Although progress has been slower than anticipated, gene transfer has been safely achieved in patients with lung diseases. Recent advancements in understanding of the molecular basis of lung disease and the development of improved vector systems make it likely that gene therapy will be an important tool for the 21st-century clinician.
Assuntos
Terapia Genética , Pneumopatias/terapia , Animais , Ensaios Clínicos como Assunto , Terapia Combinada , Fibrose Cística/terapia , Técnicas de Transferência de Genes , Genes p53 , Terapia Genética/métodos , Vetores Genéticos , Humanos , Imunoterapia , Pneumopatias/genética , Neoplasias Pulmonares/terapia , Doenças Vasculares Periféricas/terapia , Deficiência de alfa 1-Antitripsina/terapiaRESUMO
Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti-PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H(2)O(2) generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H(2)O(2,) and caused endothelial injury manifested by a fourfold elevation of angiotensin-converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 +/- 9 vs. 2.4 +/- 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF(2alpha)-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo.
Assuntos
Endotélio Vascular/enzimologia , Marcação de Genes , Glucose Oxidase/genética , Estresse Oxidativo , Circulação Pulmonar , Doenças Vasculares/induzido quimicamente , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Anticorpos/farmacologia , Glucose Oxidase/imunologia , Glucose Oxidase/metabolismo , Técnicas In Vitro , Pneumopatias/induzido quimicamente , Pneumopatias/patologia , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Ratos , Distribuição Tecidual , Doenças Vasculares/patologiaRESUMO
Recombinant strains of herpes simplex virus-1 (HSV-1) harboring mutations in the infected cell product (ICP)34.5 region lose their neurovirulence and replicate more efficiently in dividing tumor cells than stationary cells, becoming replication-selective oncolytic agents. Additional mutation of the ICP6 gene, which encodes ribonucleotide reductase, further impairs the ability of HSV-1 mutants to replicate in normal cells, enhancing tumor selectivity. The present study investigated the effect of HSV-G207, a recombinant HSV-1 lacking ICP34.5 and ICP6, against epithelial ovarian cancer (EOC) in vitro and in vivo in a mouse xenograft model. To assess the selectivity of multimutated HSV-G207 against malignant cells, HSV-G207 and wild-type HSV-F were comparatively tested against normal human peritoneal mesothelial cells and EOC cells in vitro. HSV-G207 infected both EOC cells and mesothelial cells; however, unlike EOC cells, mesothelial cells provided a poor substrate for replication of HSV-G207. In contrast to wild-type HSV-F, HSV-G207 exerted a potent oncolytic effect on EOC cells but spared normal mesothelial cells in vitro. Primary EOC cells were more sensitive to the virus than established EOC cell lines. A single intraperitoneal injection of HSV-G207 resulted in a significant reduction in tumor volume and tumor spread in vivo. HSV-G207 was shown to penetrate deeply within tumor nodules and caused no apparent intraperitoneal toxicity. Oncolytic therapy with multimutated replication-restricted HSV may offer a novel approach in the treatment of EOC.
Assuntos
Carcinoma/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Mutação/genética , Neoplasias Ovarianas/virologia , Animais , Carcinoma/patologia , Carcinoma/terapia , Transformação Celular Viral/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , DNA Recombinante/uso terapêutico , DNA Viral/genética , DNA Viral/metabolismo , DNA Viral/uso terapêutico , Epitélio/virologia , Feminino , Herpesvirus Humano 1/fisiologia , Humanos , Infusões Parenterais , Camundongos , Camundongos SCID , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Células Tumorais Cultivadas , Virulência , Replicação Viral/genéticaRESUMO
A method using reversed-phase liquid chromatography coupled with electrospray ionization and selected reaction monitoring mass spectrometry has been developed for the quantitative analysis of ganciclovir in rat plasma. Acyclovir, a structurally related analog of ganciclovir, was used as the internal standard. A small volume of plasma (50 microL) was spiked with the internal standard and plasma proteins were precipitated by methanol. The supernatant was dried under nitrogen, and then reconstituted in water. The use of liquid chromatography/selected reaction monitoring/mass spectrometry effectively eliminated potential interference from endogenous constituents in the plasma. This highly selective and sensitive method made it possible to analyze plasma ganciclovir with a lower limit of quantitation of 10 ng/mL. The assay was reproducible and linear in the range 10-10,000 ng/mL. The precision and accuracy values were in the range 2.0-6.9% and 89.0-109.6%, respectively. The analyte recovery was greater than 88%. This method was successfully used to monitor the pharmacokinetic profile of ganciclovir in normal rats following intraperitoneal administration of the drug.
Assuntos
Antivirais/sangue , Cromatografia Líquida/métodos , Ganciclovir/sangue , Animais , Calibragem , Espectrometria de Massas , Ratos , Ratos Endogâmicos F344 , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
PECAM-1/CD31, a vascular cell adhesion/signaling molecule that has been implicated in a number of vascular functions (including angiogenesis and the transmigration of leukocytes through endothelium) is highly enriched at the cell-cell borders of adjacent endothelial cells. To identify the mechanisms responsible for this localization, a series of PECAM-1 mutants and chimeric PECAM-1 molecules were transfected into non-PECAM-expressing cells and the ability of the constructs to move to cell-cell borders of adjacent cells was determined using immunohistochemistry and confocal microscopy. Although neither the extracellular domain, by itself, nor the cytoplasmic domain, by itself, was sufficient to direct cell-cell localization, the combination of the extracellular and transmembrane domains with a small group of highly charged amino acids in a membrane proximal region of the cytoplasmic domain was sufficient to direct efficient localization of the molecule to cell-cell borders. Importantly, only constructs that supported PECAM-1 mediated adhesion localized to cell-cell borders. Our data are consistent with a 'diffusion trapping' model in which movement of PECAM-1 in the cell membrane occurs relatively freely until the 'stablized' extracellular domain of the molecule encounters its ligand on an adjacent cell. When this occurs, the complex is 'captured' at the cell-cell interface leading to localization at cell-cell borders.