RESUMO
Inflammatory bowel disease (IBD) is a poorly understood chronic immune disorder for which there is no medical cure. Milk and colostrum are rich sources of bioactives with immunomodulatory properties. Here we compared the therapeutic effects of oral delivery of bovine milk-derived iron-saturated lactoferrin (Fe-bLF), angiogenin, osteopontin (OPN), colostrum whey protein, Modulen IBD (Nestle Healthsciences, Rhodes, Australia), and cis-9,trans-11 conjugated linoleic acid (CLA)-enriched milk fat in a mouse model of dextran sulfate-induced colitis. The CLA-enriched milk fat significantly increased mouse body weights after 24d of treatment, reduced epithelium damage, and downregulated the expression of proinflammatory cytokines and nitrous oxide. Modulen IBD most effectively decreased the clinical score at d 12, and Modulen IBD and OPN most effectively lowered the inflammatory score. Myeloperoxidase activity that denotes neutrophil infiltration was significantly lower in mice fed Modulen IBD, OPN, angiogenin, and Fe-bLF. A significant decrease in the numbers of T cells, natural killer cells, dendritic cells, and a significant decrease in cytokine expression were observed in mice fed the treatment diets compared with dextran sulfate administered mice. The Fe-bLF, CLA-enriched milk fat, and Modulen IBD inhibited intestinal angiogenesis. In summary, each of the milk components attenuated IBD in mice, but with differing effectiveness against specific disease parameters.
Assuntos
Colite/tratamento farmacológico , Colostro/química , Proteínas do Leite/farmacologia , Leite/química , Animais , Austrália , Doença Crônica , Colite/induzido quimicamente , Citocinas/metabolismo , Laticínios , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Glicolipídeos/farmacologia , Glicoproteínas/farmacologia , Lactoferrina/farmacologia , Ácidos Linoleicos Conjugados/farmacologia , Gotículas Lipídicas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nitroso/metabolismo , Osteopontina/farmacologia , Ribonuclease Pancreático/farmacologia , Proteínas do Soro do Leite/farmacologiaRESUMO
BACKGROUND AND PURPOSE: The aim of this study was to determine the potency and molecular mechanism of action of YM155, a first-in-class survivin inhibitor that is currently under phase I/II clinical investigations, in various drug-resistant breast cancers including the oestrogen receptor positive (ER(+) ) tamoxifen-resistant breast cancer and the caspase-3-deficient breast cancer. EXPERIMENTAL APPROACH: The potency of YM155 in SK-BR-3, MDA-MB-231, MCF7 and its tamoxifen-resistant sublines, TamR6, TamR7, TamR8, TamC3 and TamC6, were determined by MTT assay. Western blot analysis, flow cytometric analysis, reverse transcription-PCR, fluorescent microscopy and comet assay were used to determine the molecular mechanism of action of YM155 in different breast cancer cell lines. KEY RESULTS: YM155 was equally potent towards the parental ER(+) /caspase-3-deficient MCF7 breast cancer cells and its tamoxifen-resistant sublines in vitro. The ER(-) /HER2(+) SK-BR-3 breast cancer cells and the triple-negative/caspase-3-expressing metastatic aggressive MDA-MB-231 breast cancer cells were also sensitive to YM155 with IC50 values in the low nanomolar range. Targeting survivin by YM155 modulated autophagy, induced autophagy-dependent caspase-7 activation and autophagy-dependent DNA damage in breast cancer cells. Interestingly, YM155 also induced XIAP degradation and the degradation of XIAP might play an important role in YM155-induced autophagy in breast cancer cells. CONCLUSIONS AND IMPLICATIONS: YM155 is a potent survivin inhibitor that has potential for the management of various breast cancer subtypes regardless of the expression of ER, HER2 and caspase-3. Importantly, this study provides new insights into YM155's molecular mechanism of action and therapeutic potential in the treatment of tamoxifen-resistant breast cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Dano ao DNA , Imidazóis/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Naftoquinonas/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/genética , L-Lactato Desidrogenase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , SurvivinaRESUMO
This review aims to highlight many of the difficulties encountered in trying to achieve the task of delivering proteins and peptides through oral administration. The necessity of controlled protein and peptide release, protection and stability in the gastrointestinal tract, and ability to target specific areas are only a handful of the many problems associated with trying to engineer a useful solution. Current research gives strong indication that both cyclodextrins and nanoparticles could be highly useful in the search for a suitable method for such successful oral delivery of proteins and peptides. This review focuses on the use of cyclodextrins in pharmaceuticals, aiming to discuss the use of cyclodextrins in conjunction with nanoparticles for oral delivery of proteins. Both classical applications and more advanced "nanomedical" approaches are discussed. In order to achieve a complete overview this review will include background information about cyclodextrins, nanomedicine and their role in oral delivery systems. The use of absorption enhancers like cyclodextrins, bile salts and surfactants was used to facilitate bio-availability into the system. The state-of-the-art technology and challenges in this area are discussed, with typical examples.
Assuntos
Ciclodextrinas/química , Portadores de Fármacos/química , Nanopartículas/química , Preparações Farmacêuticas/administração & dosagem , Proteínas/administração & dosagem , Administração Oral , Animais , Ciclodextrinas/metabolismo , Portadores de Fármacos/metabolismo , HumanosRESUMO
Multidrug ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. Recently, the mouse P-gp/Abcb1a structure has been determined and this has significantly enhanced our understanding of the structure-activity relationship (SAR) of mammalian ABC transporters. This paper highlights our current knowledge on the structural and functional properties and the SAR of human MRP1/ABCC1. Although the crystal structure of MRP1/ABCC1 has yet to be resolved, the current topological model of MRP1/ABCC1 contains two transmembrane domains (TMD1 and TMD2) each followed by a nucleotide binding domain (NBD) plus a third NH2-terminal TMD0. MRP1/ABCC1 is expressed in the liver, kidney, intestine, brain and other tissues. MRP1/ABCC1 transports a structurally diverse array of important endogenous substances (e.g. leukotrienes and estrogen conjugates) and xenobiotics and their metabolites, including various conjugates, anticancer drugs, heavy metals, organic anions and lipids. Cells that highly express MRP1/ABCC1 confer resistance to a variety of natural product anticancer drugs such as vinca alkaloids (e.g. vincristine), anthracyclines (e.g. etoposide) and epipodophyllotoxins (e.g. doxorubicin and mitoxantrone). MRP1/ABCC1 is associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. However, most compounds that efficiently reverse P-gp/ABCB1-mediated multidrug resistance have only low affinity for MRP1/ABCC1 and there are only a few effective and relatively specific MRP1/ABCC1 inhibitors available. A number of site-directed mutagenesis studies, biophysical and photolabeling studies, SAR and QSAR, molecular docking and homology modeling studies have documented the role of multiple residues in determining the substrate specificity and inhibitor selectivity of MRP1/ABCC1. Most of these residues are located in the TMs of TMD1 and TMD2, in particular TMs 4, 6, 7, 8, 10, 11, 14, 16, and 17, or in close proximity to the membrane/cytosol interface of MRP1/ABCC1. The exact transporting mechanism of MRP1/ABCC1 is unclear. MRP1/ABCC1 and other multidrug transporters are front-line mediators of drug resistance in cancers and represent important therapeutic targets in future chemotherapy. The crystal structure of human MRP1/ABCC1 is expected to be resolved in the near future and this will provide an insight into the SAR of MRP1/ABCC1 and allow for rational design of anticancer drugs and potent and selective MRP1/ABCC1 inhibitors.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Mutagênese , Estrutura Terciária de Proteína , Interferência de RNA , Relação Estrutura-Atividade , Especificidade por Substrato , Distribuição TecidualRESUMO
Apoptosis occurs via extrinsic or intrinsic signalling each triggered and regulated by many different molecular pathways. In recent years, the selective induction of apoptosis through survivin in tumour cells has been increasingly recognized as a promising approach for cancer therapy. Survivin has multiple functions including cytoprotection, inhibition of cell death, and cell-cycle regulation, especially at the mitotic process stage, all of which favour cancer survival. Many studies on clinical specimens have shown that survivin over expression is invariably up regulated in human cancers, associated with resistance to chemotherapy or radiation therapy, and linked to poor prognosis, suggesting that cancer cells survive with survivin. On the basis of these findings, survivin has been proposed as an attractive target for new anticancer interventions. Survivin inhibitors recently entered clinical trials. Recent studies suggest a possible role for survivin in regulating the function of normal adult cells. However, the expression and function of survivin in normal tissues are still not well characterized and understood. Still better understandings of survivin's role in tumour versus normal cells are needed for designing the strategies to selectively disrupt survivin in cancers. In the present review, we summarise the importance of recent survivin-targeted cancer therapy for future clinical application.
Assuntos
Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Neoplasias/terapia , Apoptose , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/uso terapêutico , Terapia Genética , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/metabolismo , Fragmentos de Peptídeos/uso terapêutico , SurvivinaRESUMO
beta-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 degrees C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.
Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Kluyveromyces/citologia , Lactose/metabolismo , Leite/metabolismo , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Animais , Enzimas Imobilizadas/biossíntese , Gelatina/química , Concentração de Íons de Hidrogênio , Hidrólise , Kluyveromyces/efeitos dos fármacos , Kluyveromyces/enzimologia , Kluyveromyces/metabolismo , Permeabilidade/efeitos dos fármacos , Temperatura , beta-Galactosidase/biossínteseRESUMO
This review describes the nature and applications of ribosome inactivating proteins (RIPs) from Momordica charantia (bitter melon). RIPs from the plant kingdom have received much attention in biomedical research because they target conserved host protein synthesis machinery and show specificity towards human and animal cell targets. Recent studies aimed at unravelling the enzymatic activities of the M charantia RIPs provide a structural basis for their activities. It has been reported that RIPs are member of the single chain ribosome inactivating protein (SCRIP) family which act irreversibly on ribosome by removing adenine residue from eukaryotic ribosomal RNA. Various activities of RIPs include anti-tumor, broad anti-viral, ribonuclease and deoxyribonuclease. MAP30 (Momordica Anti-HIV Protein), alpha- and beta-momorcharins inhibit HIV replication in acutely and chronically infected cells and thus are considered potential therapeutic agent in HIV infection and AIDS. Further, MAP30 improved the efficacy of anti-HIV therapy when used in combination with other anti-viral drugs. MAP30 holds therapeutic promise over other RIPs because not only it is active against infection and replication of both HSV and HIV but is non toxic to normal cells. Here we review the nature, action, structure function relationship and applications of RIPs from Momordica charantia and evaluate their potential for anti-cancer and anti-viral therapy.
Assuntos
Antivirais/farmacologia , Momordica charantia/química , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Antivirais/química , Humanos , Proteínas de Plantas/química , Proteínas Inativadoras de Ribossomos/química , Ribossomos/efeitos dos fármacosRESUMO
Engineering contributions have played an important role in the rise and evolution of cellular biology. Engineering technologies have helped biologists to explore the living organisms at cellular and molecular levels, and have created new opportunities to tackle the unsolved biological problems. There is now a growing demand to further expand the role of engineering in cellular biology research. For an engineer to play an effective role in cellular biology, the first essential step is to understand the cells and their components. However, the stumbling block of this step is to comprehend the information given in the cellular biology literature because it best suits the readers with a biological background. This paper aims to overcome this bottleneck by describing the human cell components as micro-plants that form cells as micro-bio-factories. This concept can accelerate the engineers' comprehension of the subject. In this paper, first the structure and function of different cell components are described. In addition, the engineering attempts to mimic various cell components through numerical modelling or physical implementation are highlighted. Next, the interaction of different cell components that facilitate complicated chemical processes, such as energy generation and protein synthesis, are described. These complex interactions are translated into simple flow diagrams, generally used by engineers to represent multi-component processes.
Assuntos
Engenharia/métodos , Células Eucarióticas/citologia , Células Eucarióticas/fisiologia , Biotecnologia/métodos , Metabolismo Energético/fisiologia , Humanos , Modelos Biológicos , Organelas/fisiologia , Biossíntese de Proteínas/fisiologiaRESUMO
BACKGROUND: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases. OBJECTIVE: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses. METHODS: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge. RESULTS: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective. CONCLUSION: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.
Assuntos
Gorduras/imunologia , Hipersensibilidade/imunologia , Ácido Linoleico/imunologia , Pneumopatias Obstrutivas/imunologia , Pneumopatias Obstrutivas/patologia , Leite/imunologia , Ácidos Oleicos/imunologia , Alérgenos/imunologia , Animais , Sobrevivência Celular , Quimiocina CCL11/biossíntese , Eosinófilos/citologia , Eosinófilos/imunologia , Feminino , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Imunoglobulinas/biossíntese , Imunoglobulinas/imunologia , Interleucina-5/biossíntese , Pneumopatias Obstrutivas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The use of bacteria in the regression of tumors has long been known. Various approaches for using bacteria in cancer therapy include the use of bacteria as sensitizing agents for chemotherapy, as delivery agents for cancer drugs and as agents for gene therapy. The tumor regression stimulated by infecting microorganisms has been attributed to activation of the immune system of the host. However, recent studies indicate that when tumor-harboring mice with defective immune systems are infected with certain microorganisms, the regression of the tumor is still observed, suggesting that there are other host factors contributing to the microbial associated regression of tumors. Since the use of live or attenuated bacteria for tumor regression has associated toxic effects, studies are in progress to identify a pure microbial metabolite or any component of the microbial cell that might have anti-cancer activity. It has now been demonstrated that a redox protein from Pseudomonas aeruginosa, a cupredoxin, can enter into human cancer cells and trigger the apoptotic cell death. In vivo, this cupredoxin can lead to the regression of tumor growth in immunodeficient mice harboring xenografted melanomas and breast cancer tumors without inducing significant toxic effects, suggesting that it has potential anti-cancer activity. This bacterial protein interacts with p53 and modulates mammalian cellular activity. Hence, it could potentially be used as an anti-cancer agent for solid tumors and has translational value in tumor-targeted or in combinational-biochemotherapy strategies for cancer treatments. Here, we focus on diverse approaches to cancer biotherapy, including bacteriolytic and bacterially-derived anti-cancer agents with an emphasis on their mechanism of action and therapeutic potential.
Assuntos
Apoptose , Bactérias/imunologia , Vacinas Anticâncer/imunologia , Neoplasias/irrigação sanguínea , Neoplasias/terapia , Neovascularização Patológica , Animais , Bactérias/genética , Vacinas Anticâncer/genética , Vetores Genéticos/genética , Humanos , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.
Assuntos
Inibidores da Angiogênese/genética , DNA/administração & dosagem , Terapia Genética/métodos , Linfoma/terapia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Inibidores da Angiogênese/análise , Animais , Apoptose , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Injeções Intralesionais , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Oligonucleotídeos Antissenso/uso terapêutico , Fatores de Transcrição/análise , Proteínas Supressoras de Tumor/análise , Ubiquitina-Proteína Ligases/análise , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Members of the B7 family costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. While B7-1 and -2 are restricted to lymphoid tissues, and activate naïve T cells, recently identified members including B7-H2 and -H3 are widely expressed on nonlymphoid tissues, and regulate effector lymphocytes in the periphery. B7-H3 has properties that suggested it may display antitumor activity, including the ability to stimulate Th1 and cytotoxic T-cell responses. Here, we test this notion by determining whether intratumoral injection of an expression plasmid encoding a newly described mouse homologue of B7-H3 is able to eradicate EL-4 lymphomas. Intratumoral injection of a mouse B7-H3 pcDNA3 expression plasmid led to complete regression of 50% tumors, or otherwise significantly slowed tumor growth. Mice whose tumors completely regressed resisted a challenge with parental tumor cells, indicating systemic immunity had been generated. B7-H3-mediated antitumor immunity was mediated by CD8(+) T and NK cells, with no apparent contribution from CD4(+) T cells. In summary, the results indicate that B7-H3 interactions may play a role in regulating cell-mediated immune responses against cancer, and that B7-H3 is a potential therapeutic tool.
Assuntos
Antígeno B7-1/genética , DNA/administração & dosagem , Terapia Genética/métodos , Linfoma/terapia , Neoplasias do Timo/terapia , Sequência de Aminoácidos , Animais , Antígenos B7 , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Feminino , Injeções Intralesionais , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Transplante de Neoplasias , Neoplasias do Timo/imunologiaRESUMO
In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E-deficient (apoE(-/-)) mice. The apoE(-/-) mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE(-/-) mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE(+/+)) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3(+) T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.
Assuntos
Apolipoproteínas E/deficiência , Artérias/metabolismo , Artérias/patologia , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Chaperonina 60/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Western Blotting , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Imuno-Histoquímica , Macrófagos/metabolismo , Camundongos , Camundongos Mutantes , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Linfócitos T/metabolismo , Regulação para CimaRESUMO
Tumors must develop an adequate vascular network to meet their increasing demands for nutrition and oxygen. Angiostatin, a multiple kringle (1-4)-containing fragment of plasminogen, is an effective natural inhibitor of tumor angiogenesis. Here we show that gene transfer of angiostatin into small (0.1 cm in diameter) solid EL-4 lymphomas established in syngeneic C57BL/6 mice led to reduced tumor angiogenesis and weak inhibition of tumor growth. In contrast, when angiostatin gene therapy was preceded by in situ gene transfer of the T-cell costimulator B7.1, large (0.4 cm in diameter) tumors were rapidly and completely eradicated, whereas B7.1 and angiostatin monotherapies were ineffective. Combined gene transfer of B7.1 and angiostatin generated potent systemic antitumor immunity that was effective in eradicating a systemic challenge of 10(7) EL-4 cells. Gene transfer of angiostatin expression plasmids led to overexpression of angiostatin in tumors, increased apoptosis of tumor cells, and decreased density of tumor blood vessels, which may allow the immune system to overcome tumor immune resistance. The latter effects were not the result of a decrease in vascular endothelial growth factor expression, as tumoral vascular endothelial growth factor expression increased slightly after angiostatin gene transfer, presumably in response to increasing hypoxia. These results suggest that combining immunogene therapy with a vascular attack by angiostatin is a particularly effective approach for eliciting antitumor immunity.
Assuntos
Antígeno B7-1/genética , Fatores de Crescimento Endotelial/metabolismo , Terapia Genética/métodos , Imunoterapia/métodos , Linfocinas/metabolismo , Fragmentos de Peptídeos/genética , Plasminogênio/genética , Neoplasias do Timo/terapia , Angiostatinas , Animais , Western Blotting , Terapia Combinada , Primers do DNA/química , Técnicas de Transferência de Genes , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/prevenção & controle , Fragmentos de Peptídeos/metabolismo , Plasminogênio/metabolismo , Reação em Cadeia da Polimerase/métodos , Linfócitos T/imunologia , Neoplasias do Timo/irrigação sanguínea , Neoplasias do Timo/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Leukocytes infiltrate the pancreatic islets of nonobese diabetic mice, causing beta-cell destruction and autoimmune Type I diabetes. Here, we completely blocked adoptive transfer of diabetes and reduced spontaneous disease incidence from 71% to 17% by simultaneously administering a combination of antibodies directed against alpha4, beta2, and beta7 integrins and their ligands VCAM-1, MAdCAM-1, and ICAM-1 for 52 and 28 days, respectively. CD4 and CD8 T cells and macrophages were excluded from islets and remained entrapped in a peri-islet location as inactive exiles, no longer expressing normal levels of interferon-gamma, interleukin-4, and iNOS. Only IL-10 expression was retained, which could aid immunosuppression. Infiltrating leukocytes retained a peri-islet location, even 215 days following suspension of antibody treatment, potentially forming a barrier to the entry of active, autoantigen-reactive T cells. Combination treatment was effective against spontaneous disease when administered from 7 days of age but ineffective when initiated late in the prediabetic period (day 40 or 70). Nevertheless, anti-alpha4 subunit mAb monotherapy alone was very effective, reducing insulitis to levels similar to those obtained with combinational antibody treatment, suggesting that alpha4 integrins are major receptors contributing to leukocyte infiltration. Treatment with anti-alpha4 integrin antibody retained some therapeutic benefit when administered from days 7, 40, or 70 of age. The results have implications for the treatment of diabetes and provide a unique insight into the fate of disease-forming leukocytes following anti-CAM therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Moléculas de Adesão Celular/antagonistas & inibidores , Quimiotaxia de Leucócito/efeitos dos fármacos , Diabetes Mellitus Tipo 1/terapia , Integrinas/antagonistas & inibidores , Ilhotas Pancreáticas , Transferência Adotiva , Fatores Etários , Animais , Antígenos CD/imunologia , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/imunologia , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/patologia , Feminino , Inflamação/patologia , Inflamação/prevenção & controle , Integrina alfa4 , Integrinas/imunologia , Ilhotas Pancreáticas/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Baço/citologia , Fatores de TempoRESUMO
BACKGROUND: Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is detectable in most types of cancer, and its presence is associated with a poor prognosis. We determined the effects of gene-based therapies that inhibit survivin function in a mouse tumor model. METHODS: Using five to six mice per treatment group, we injected tumors derived from mouse EL-4 thymic lymphoma cells with plasmids encoding antisense survivin, a dominant-negative mutant survivin, and the T-cell costimulator B7-1. Expression of endogenous survivin and the proteins encoded by the injected plasmids were examined by immunohistochemical staining of tumor sections and by western blot and flow cytometry analyses of isolated tumor cells. Tumor growth, the generation of antitumor cytotoxic T-lymphocyte (CTL) activity, apoptosis, and the contribution of leukocyte subsets to antitumor activity were measured. All statistical tests were two-sided. RESULTS: Large (1.0-cm diameter) tumors had approximately 10-fold more survivin than small (0.2-cm diameter) tumors. At 28 days after injection, antisense and dominant-negative mutant survivin plasmids statistically significantly inhibited the growth of both small (P =.006 and P =.0018, respectively) and large (P<.001 for both plasmids) EL-4 tumors compared with tumors injected with empty plasmid. The growth of large tumors was further inhibited by intratumoral injection with antisense survivin and B7-1 (P =.004); thus, inhibition of survivin expression renders large tumors susceptible to B7-1-mediated immunotherapy. Mice whose tumors were completely eradicated by injection of B7-1 remained tumor free for 26 days after re-injection with EL-4 cells (when the experiment ended). Compared with tumors injected with empty plasmid, tumors injected with survivin-based plasmids had increased apoptosis, and animals bearing such tumors generated more antitumor CTLs. CONCLUSION: Intratumoral injection of plasmids that block survivin expression and stimulate the generation of tumor-specific CTLs may be beneficial for the treatment of large lymphomas.
Assuntos
Antígeno B7-1/uso terapêutico , Proteínas Cromossômicas não Histona/fisiologia , DNA Antissenso/uso terapêutico , Terapia Genética , Imunoterapia , Linfoma não Hodgkin/tratamento farmacológico , Proteínas Associadas aos Microtúbulos , Proteínas de Neoplasias/fisiologia , Neoplasias do Timo/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose , Antígeno B7-1/administração & dosagem , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/biossíntese , Proteínas Cromossômicas não Histona/genética , Terapia Combinada , DNA Antissenso/administração & dosagem , DNA Antissenso/genética , Progressão da Doença , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Marcação de Genes , Genes Dominantes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/uso terapêutico , Rejeição de Enxerto/imunologia , Proteínas Inibidoras de Apoptose , Injeções Intralesionais , Depleção Linfocítica , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/patologia , Linfócitos do Interstício Tumoral , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/patologia , Linfoma não Hodgkin/terapia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Survivina , Linfócitos T Citotóxicos/imunologia , Neoplasias do Timo/imunologia , Neoplasias do Timo/patologiaRESUMO
Solid tumors meet their demands for nascent blood vessels and increased glycolysis, to combat hypoxia, by activating multiple genes involved in angiogenesis and glucose metabolism. Hypoxia inducible factor-1 (HIF-1) is a constitutively expressed basic helix-loop-helix transcription factor, formed by the assembly of HIF-1alpha and HIF-1beta (Arnt), that is stablized in response to hypoxia, and rapidly degraded under normoxic conditions. It activates the transcription of genes important for maintaining oxygen homeostasis. Here, we demonstrate that engineered down-regulation of HIF-1alpha by intratumoral gene transfer of an antisense HIF-1alpha plasmid leads to the down-regulation of VEGF, and decreased tumor microvessel density. Antisense HIF-1alpha monotherapy resulted in the complete and permanent rejection of small (0.1 cm in diameter) EL-4 tumors, which is unusual for an anti-angiogenic agent where transient suppression of tumor growth is the norm. It induced NK cell-dependent rejection of tumors, but failed to stimulate systemic T cell-mediated anti-tumor immunity, and synergized with B7-1-mediated immunotherapy to cause the NK cell and CD8 T cell-dependent rejection of larger EL-4 tumors (0.4 cm in diameter) that were refractory to monotherapies. Mice cured of their tumors by combination therapy resisted a rechallenge with parental tumor cells, indicating systemic antitumor immunity had been achieved. In summary, whilst intensive investigations are in progress to target the many HIF-1 effectors, the results herein indicate that blocking hypoxia-inducible pathways and enhancing NK-mediated antitumor immunity by targeting HIF-1 itself may be advantageous, especially when combined with cancer immunotherapy.
Assuntos
Proteínas de Ligação a DNA/genética , Terapia Genética/métodos , Linfoma de Células T/terapia , Neovascularização Patológica/terapia , Proteínas Nucleares/genética , Animais , Elementos Antissenso (Genética)/genética , Antígeno B7-1/uso terapêutico , Terapia Combinada , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Técnicas de Transferência de Genes , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Células Matadoras Naturais/imunologia , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Plasmídeos , Subpopulações de Linfócitos T/imunologia , Fatores de Transcrição/genéticaRESUMO
The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60-180 microg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2-6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 10(7) parental EL-4 tumor cells but not a challenge of 1 x 10(4) Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 10(8) splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 microg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients' immune responses to their own tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Antígeno B7-1/imunologia , Imunoterapia/métodos , Neovascularização Patológica/terapia , Xantonas , Animais , Antígeno B7-1/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Terapia Combinada , Feminino , Flavonoides/administração & dosagem , Dosagem de Genes , Terapia Genética , Vetores Genéticos/genética , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Linfoma/imunologia , Linfoma/patologia , Linfoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia , Neoplasias do Timo/irrigação sanguínea , Neoplasias do Timo/imunologia , Neoplasias do Timo/patologia , Neoplasias do Timo/terapia , Xantenos/administração & dosagemRESUMO
Heat shock proteins (hsps) chaperone cytosolic peptides, forming complexes that stimulate antitumor immunity. Hsps facilitate signal 1 in the two-signal model of T-cell costimulation, whereas cell adhesion molecules such as B7.1 provide secondary (signal 2) costimulatory signals. B7.1 gene transfer into tumors in situ has been shown to eradicate small (<0.3 cm in diameter) tumors in mice, and induce systemic antitumor immunity, but is ineffective against larger tumors. We examine whether mammalian hsps, as facilitators of T-cell costimulation, also exhibit this ability, and whether simultaneously stimulating both signal 1 (hsp-facilitated antigen presentation) and signal 2 (B7.1-mediated costimulation) enhances antitumor immunity compared to that achieved with either monotherapy. Prophylactic vaccination of mice with an hsp preparation from an EL-4 lymphoma weakly retarded tumor growth, to the same extent as that achieved with a single EL-4-derived peptide (AQHPNAELL), previously shown to induce antitumor immunity establishing that a preparation of EL-4 hsp-peptide complexes has antitumor activity. Here we show that injection of rat hsp70.1 into mouse tumors in situ causes the complete eradication of tumors, and generates potent systemic antitumor immunity mediated by CD4+ and CD8+ T cells. Unexpectedly, simultaneous gene transfer of hsp70.1 and B7.1 compromised the efficacy of hsp-mediated tumor rejection--a problem which could be partially overcome by the timed delivery of hsp70.1 and B7.1. Thus, gene transfer of hsp70 into tumors can be employed to generate potent systemic antitumor immunity, but further consideration is required if this approach is to be successfully combined with immunotherapies employing other T-cell costimulators.