RESUMO
This research describes a thermally responsive elastin-like polypeptide (ELP) for the delivery of dnMAML peptides that inhibit the Notch pathway. Exploiting passive targeting and a thermally active tumor-targeting technique available through the use of ELP, the dnMAML peptide was efficiently delivered to tumor tissue. Furthermore, this ELP-dnMAML was modified with the addition of a cell penetrating peptide (SynB1) for improved infiltration of ELP-dnMAML into the tumor cells. In this study, we verified that intravenously delivered SynB1-ELP-dnMAML was cleared from circulation under physiological conditions (37 °C) but accumulated at tumors grown in mice at sites to which an externally induced, local heat (40-41 °C) was applied, thereby resulting in greatly reduced tumor growth in animals. Additionally, in combination with Taxol, SynB1-ELP-dnMAML showed more potent tumor growth retardation.
Assuntos
Proteínas de Ligação a DNA/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias Mamárias Experimentais/patologia , Paclitaxel/administração & dosagem , Peptídeos/administração & dosagem , Receptores Notch/antagonistas & inibidores , Fatores de Transcrição/administração & dosagem , Animais , Antineoplásicos Fitogênicos , Linhagem Celular Tumoral , Peptídeos Penetradores de Células , Feminino , Humanos , Hipertermia Induzida , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The recurrent Coxsackievirus B3 (CVB3) infection is the most important cause of intractable myocarditis which often leads to chronic myocarditis and even dilated cardiomyopathy. Therefore, enhanced DNA vaccines capable of memory CD8 T cells are essential for long-lasting immunological protection against CVB3 infection. In this study, absent in melanoma 2 (AIM2) was used as an adjuvant to enhance the induction of memory CD8 T cells elicited by VP1 (viral capsid protein 1) vaccine. Mice were intramuscularly injected with 50 µg AIM2 plasmid and equal amount of VP1 plasmid (pAIM2/pVP1) vaccine 4 times at 2 week-intervals. We observed that the protection of pAIM2/pVP1 vaccine against CVB3 challenge was evidenced by significantly improved cardiac function, reduced myocardial injuries, and increased survival rate when compared with immunization with pVP1. Co-immunization with pAIM2/pVP1 robustly augmented T lymphocytes proliferation and CVB3-specific cytotoxic T lymphocyte responses. Importantly, 16 weeks after the last immunization, pAIM2/pVP1 co-immunization significantly enhanced the expression of Bcl-6, SOCS3, and Sca-1 which are critical for memory CD8 T cells as compared with pVP1 immunization. Notably, CD8 T cells that are likely vaccine-induced memory T cells were responsible for the protective efficacy of pAIM2/pVP1 vaccine by abolition of a CD8 T cell immune response following a lethal dose of CVB3 infection. Our results indicate that AIM2-adjuvanted vaccine could be a potential and promising approach to promote a long-lasting protection against CVB3-induced myocarditis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas do Capsídeo/imunologia , Infecções por Coxsackievirus/prevenção & controle , Proteínas de Ligação a DNA/imunologia , Enterovirus Humano B/imunologia , Imunização , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Animais , Antígenos Ly/metabolismo , Proteínas do Capsídeo/genética , Proliferação de Células , Infecções por Coxsackievirus/imunologia , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Células HeLa , Traumatismos Cardíacos/patologia , Humanos , Injeções Intramusculares , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/imunologia , Miocardite/prevenção & controle , Miocardite/virologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Taxa de Sobrevida , Vacinas de DNA/administração & dosagem , Vacinas Virais/imunologiaRESUMO
Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood-brain barrier penetration. The SynB1-ELP1-dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes' expression. SynB1-ELP1-dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1-ELP1-dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Proteínas de Ligação a DNA/administração & dosagem , Glioblastoma/tratamento farmacológico , Receptores Notch/antagonistas & inibidores , Fatores de Transcrição/administração & dosagem , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacologia , Proteínas de Ligação a DNA/farmacocinética , Proteínas de Ligação a DNA/farmacologia , Sistemas de Liberação de Medicamentos , Elastina/administração & dosagem , Glioblastoma/patologia , Humanos , Hipertermia Induzida/métodos , Peptídeos/administração & dosagem , Fatores de Transcrição/farmacocinética , Fatores de Transcrição/farmacologiaRESUMO
Objective: To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells. Methods: RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation. Results: Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (P<0.05). The cell proliferation was inhibited (P<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (P<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all P<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (P<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(r=0.478, P<0.01). Conclusion: Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/fisiopatologia , RNA Interferente Pequeno/farmacologia , Ubiquitina-Proteína Ligases/farmacologia , Adjuvantes Farmacêuticos/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Ciclina D1/efeitos dos fármacos , Ciclina D1/genética , Proteínas de Ligação a DNA/administração & dosagem , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Carcinoma de Células Escamosas do Esôfago , Fluoruracila/farmacologia , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Humanos , Metáfase/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , Transfecção , Ubiquitina-Proteína Ligases/administração & dosagemRESUMO
Nesfatin-1 is an anorexigenic peptide involved in energy homeostasis. Recently, nesfatin-1 was reported to decrease blood glucose level and improve insulin sensitivity in high-fat diet-fed rats. However, little information is known about the influence of nesfatin-1 on lipid metabolism either in physiological or diabetic condition. This study undertook whether nesfatin-1 was involved in the pathophysiology in Streptozotocin-induced type 2 diabetic mice (T2DM), which was induced by a combination of high-calorie diet and two low-doses Streptozotocin. We observed that plasma nesfatin-1 was significantly increased while expression of nesfatin-1 neurons were decreased in hypothalamus in diabetes group compared to only high-calorie diet control group; intravenous injection of nesfatin-1 decreased 0-1h, 0-2h, 0-3h cumulative food intake in T2DM, but 0-24h total food intake had no difference between groups. Body weight and plasma FFA were normalized after nesfatin-1(10 µg/Kg) administration for 6 days. These results suggested that nesfatin-1 improved lipid disorder in T2DM. It was found that blood glucose and insulin resistance coefficient decreased with treatment of nesfatin-1 (both in 1 µg/Kg and 10 µg/Kg doses) in diabetes mice. For further understanding the role of nesfatin-1 on lipid metabolism, we detected p-AMPK and p-ACC of skeletal muscle in T2DM using western blotting. The expression of p-AMPK and p-ACC increased when nesfatin-1 was given with doses 1 µg/Kg but not in doses 10 µg/Kg. Taken together, nesfatin-1 participated in the development of T2DM and stimulated free fatty acid utilization via AMPK-ACC pathway in skeletal muscle in T2DM.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Glicemia/metabolismo , Proteínas de Ligação ao Cálcio/administração & dosagem , Proteínas de Ligação ao Cálcio/sangue , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/sangue , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Ativação Enzimática , Ácidos Graxos não Esterificados/sangue , Mucosa Gástrica/metabolismo , Hipotálamo/metabolismo , Insulina/sangue , Resistência à Insulina , Masculino , Camundongos , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/sangue , Nucleobindinas , Oxirredução , Fosforilação , Estreptozocina/toxicidadeRESUMO
Mutations or deletions of p53 are relatively rare in multiple myeloma (MM), at least in newly diagnosed patients. Thus, restoration of p53 tumor suppressor function in MM by blocking the inhibitory role of murine double minute 2 (MDM2) is a promising and applicable therapeutic strategy. RITA and nutlin are two new classes of small molecule MDM2 inhibitors that prevent the p53-MDM2 interaction. Earlier reports showed p53-dependent activity of RITA in solid tumors as well as in leukemias. We and others recently described nutlin-induced apoptosis in MM cells, but it remains unclear whether RITA exerts antimyeloma activity. Here, we found that RITA activates the p53 pathway and induces apoptosis in MM cell lines and primary MM samples, preferentially killing myeloma cells. The activation of p53 induced by RITA was mediated through modulation of multiple apoptotic regulatory proteins, including upregulation of a proapoptotic protein (NOXA), downregulation of an antiapoptotic protein, Mcl-1, and activation of caspases through extrinsic pathways. Moreover, a number of key p53-mediated apoptotic target genes were identified by gene expression profiling and further validated by quantitative real-time PCR. Importantly, the combination of RITA with nutlin displayed a strong synergism on growth inhibition with the combination index ranging from 0.56 to 0.82 in MM cells. Our data support further clinical evaluation of RITA as a potential novel therapeutic intervention in MM.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Imidazóis/farmacologia , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/farmacologia , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Antineoplásicos/farmacologia , Apoptose/genética , Caspases/fisiologia , Células Cultivadas , Citotoxinas/administração & dosagem , Citotoxinas/farmacologia , Proteínas de Ligação a DNA/administração & dosagem , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Análise em Microsséries , Mieloma Múltiplo/genética , Proteínas de Neoplasias/administração & dosagem , Piperazinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
AIM: To study the effect of chimeric E7 protein of human papillomavirus type 18 on activation of adaptive immunity in absence of adjuvant. MATERIALS AND METHODS: Chimeric protein was genetically engineered and represents the protein molecule consisting of full-size E7 oncoprotein and heat-shock protein 70 (HSP70) of Mycobacterium tuberculosis in one polypeptide chain. Antibody titers as well as isotypes and subisotypes of immunoglobulins were measured by ELISA in sera of immunized animals. RESULTS: It was shown that studied construction E7 (HPV-18)-HSP70 significantly increases titers of antibodies to E7 protein of HPV type 18 and have cross-reactive antigenic activity with E7 protein of HPV type 16. Immunization with chimeric protein resulted in increase of IgG1 and IgG2b levels and decrease of IgG2a and IgM levels. CONCLUSION: . Oncoprotein E7 included in chimeric construction with HSP70 could be used for further studies on development of therapeutic vaccine for treatment of cervical cancer and precancerous lesions. Skew of immune response to Th2 type after intraperitoneal administration of the studied construction points to necessity for control of immunity during such studies.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Ligação a DNA/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Proteínas Recombinantes de Fusão/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , Proteínas de Choque Térmico HSP70/administração & dosagem , Proteínas de Choque Térmico HSP70/genética , Humanos , Imunização , Esquemas de Imunização , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Injeções Intraperitoneais , Masculino , Camundongos , Proteínas Oncogênicas Virais/administração & dosagem , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/sangue , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/virologiaRESUMO
The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.
Assuntos
Regulação do Apetite/fisiologia , Comportamento Alimentar/fisiologia , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Resposta de Saciedade/fisiologia , Animais , Anorexia/induzido quimicamente , Anorexia/metabolismo , Anorexia/prevenção & controle , Regulação do Apetite/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/administração & dosagem , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Relação Dose-Resposta a Droga , Comportamento Alimentar/efeitos dos fármacos , Perfilação da Expressão Gênica , Injeções Intraventriculares , Leptina/metabolismo , Leptina/farmacologia , Masculino , Camundongos , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/farmacologia , Nucleobindinas , Obesidade/metabolismo , Ratos , Ratos Wistar , Ratos Zucker , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores para Leptina , Receptores de Melanocortina/metabolismo , Resposta de Saciedade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , alfa-MSH/metabolismoRESUMO
We previously purified a 55 kDa protein that preferentially expands anti-DNA antibody production both in vitro and in vivo across the H-2 barrier from culture supernatants of KML1-7 cells, cloned from a lupus-prone MRL/lpr mouse. By using the purified protein, termed nucleobindin (Nuc), we cloned cDNA and produced recombinant(r) Nuc in Escherichia coli. To elucidate the function of rNuc in vivo, we initially injected intraperitoneally 5 micrograms of rNuc without adjuvant into female MRL/n mice at 8 weeks of age and continued injection twice a week. As early as 5 weeks after administration, all mice treated showed an increase in IgG anti-double stranded (ds) DNA antibodies accompanied by IgG hypergammaglobulinemia (HG). Of particular interest was that these mice also produced anti-U1RNP antibodies and rheumatoid factor (RF) of IgG class, but not anti-Sm antibodies. Histopathologically, hypercellularity with occasional crescents in the glomeruli was observed, but evidence for lupus nephritis was lacking, indicating that some factors other than Nuc are necessary for the development of a lupus syndrome observed in MRL/lpr mice. Similar administration of lipopolysaccharide into MRL/n mice failed to induce autoantibodies except for a slight increase in serum IgG, suggesting that these autoimmune responses are not due simply to polyclonal B-cell activation. The presence of rNuc will give us a clue for further understanding of autoimmunity.