RESUMO
Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3'-epi-12ß-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Humanos , Camundongos , Apoptose , Autofagia , Linhagem Celular Tumoral , Morte Celular Imunogênica , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Microambiente TumoralRESUMO
A combination of chemotherapy and photothermal therapy has emerged as a promising method of cancer treatment since it can enhance therapeutic efficacies and reduce side effects. Herein, we fabricated doxorubicin (DOX) loaded PEG-dBSA-RuS1.7 which could be used as a synergistic therapeutic nanoplatform. The PEG-dBSA-RuS1.7/DOX nanoparticles exhibit good monodispersity, physiological stability and biocompatibility. Moreover, the prepared PEG-dBSA-RuS1.7/DOX nanoparticles can intelligently release DOX by pH- and NIR-triggered therapy. In comparison with chemotherapy or photothermal treatment alone, the combined therapy shows a better therapeutic effect. We believe that the PEG-dBSA-RuS1.7/DOX can act as an efficient multifunctional nanoplatform for chemophotothermal synergistic cancer therapy.
Assuntos
Antineoplásicos/química , Doxorrubicina/química , Sistemas de Liberação de Medicamentos , NanopartículasRESUMO
Cardenolides with special chemical structures have been considered as effective anti-cancer drugs in clinic trials. Strophalloside is a cardenolide we recently isolated from Antiaris toxicaria obtained from Hainan, China. The aim of this study was to investigate the possible anticancer effects induced by strophalloside and the underlying molecular mechanism. Gastric carcinoma SGC-7901 cells were treated with strophalloside at various concentrations for different times, and resulting cell viability was determined by the MTT assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Apoptosis were measured by Annexin V-FITC/PI and Hoechst staining. The changes of mitochondrial transmembrane potential were examined by a JC-1 kit. The expressions of pro-apoptotic protein cytochrome c, caspase-3 and caspase-9 were detected by western blotting analysis. The results showed that strophalloside was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion in a time- and dose-dependent manner. Mitochondrial membrane potential declined and the concentration of cytochrome c increased in cytoplasm and caspase-3 and caspase-9 were cleaved into activated states, suggesting that cytochrome c was released from the mitochondrion to cytoplasm and finally activated the caspase-dependent apoptosis pathway. Our results indicate that strophalloside is a potential anticancer drug.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Cardenolídeos/administração & dosagem , Neoplasias Gástricas/tratamento farmacológico , Cardenolídeos/química , Caspase 3/biossíntese , Caspase 3/metabolismo , Caspase 9/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/patologiaRESUMO
The IgE Fcε3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains α and ß subunits (the α subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fcε3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fcε3, which was used to transform Tan109 for Ag43/Fcε3 surface expression. Thereafter, Ag43/Fcε3 was investigated as an asthma vaccine in a mouse model. Ag43/Fcε3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fcε3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti-asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fcε3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self-molecules.
Assuntos
Adesinas de Escherichia coli/imunologia , Asma/prevenção & controle , Hiper-Reatividade Brônquica/prevenção & controle , Receptores de IgE/imunologia , Vacinas Sintéticas/imunologia , Adesinas de Escherichia coli/biossíntese , Adesinas de Escherichia coli/genética , Transferência Adotiva , Animais , Anticorpos Neutralizantes/sangue , Asma/sangue , Asma/imunologia , Asma/fisiopatologia , Autoanticorpos/sangue , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição , Células Cultivadas , Clonagem Molecular , Citocinas/metabolismo , Modelos Animais de Doenças , Histamina/metabolismo , Tolerância Imunológica , Imunoglobulina E/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ovalbumina/imunologia , Receptores de IgE/biossíntese , Receptores de IgE/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Tempo , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genéticaRESUMO
Various angiogenesis-related self-molecules have been considered to be therapeutic targets. However, the direct use of self-molecules as vaccines is not recommended because of the inherent ability of the host to develop immune tolerance. Antigen 43 (Ag43) is a surface protein found in E. coli and contains an α and a ß subunits, which contains multiple T epitopes in α subunit. Here we construct a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against self-molecules. The Ag43 system was constructed from an Escherichia coli strain Tan109, derived from JM109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43') expressing a partial Ag43 gene was introduced. The extracellular domain of angiogenesis-related endoglin gene was then subcloned into plasmid pETAg43', resulting in a recombinant plasmid pETAg43'/END(e) which was then used to transform Tan109 for protein expression. We found that Ag43 and endoglin chimeric protein (Ag43'/END(e) ) was expressed on the bacterial surface. The chimeric protein could be separated from the bacterial surface by heating to 60°C and yet retain activity. We used Ag43'/END(e) as a protein vaccine and found that it could disrupt immune tolerance against endoglin by inducing significant antitumor activities and inhibit angiogenesis in several tumor models without significant side effects. These data suggest that Ag43'/END(e) chimeric protein is a potential model vaccine for active tumor immunotherapy, and that Ag43 system could be an effective tool for novel vaccine preparation to break immune tolerance to other angiogenesis-related self-molecules for cancer therapy.
Assuntos
Adesinas de Escherichia coli/imunologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Lewis/terapia , Tolerância Imunológica/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neovascularização Patológica/terapia , Adesinas de Escherichia coli/genética , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Endoglina , Epitopos de Linfócito T , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Patológica/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
OBJECTIVE: To explore the effects of SANPAOCAO (SPC), a compound traditional Chinese folk medicine, on chronic dermatitis/eczema in mice induced by 2, 4-dinitrochlorobenzene (DNCB). METHODS: Thirty-three Balb/c mice were randomly divided into a negative control group, a positive control group, a prednisolone treatment group, an SPC ethanol extract treatment group, a Cardiospermum halicacabum ethanol extract treatment group, a Physalis minima ethanol extract treatment group, and a Jussiaea repens ethanol extract treatment group. Mice in the six treatment groups had twenty-five microliters of 0.1% DNCB in acetone/olive oil (3: 1) applied to each side of their right ears and dorsal skin three times a week, over a 5 week period. They were treated with prednisolone or the various kinds of ethanol extract after each challenge. The weight difference between the two ears, pathological changes in the right ears, dermal inflammatory cell numbers, and total serum Ig E levels were used to assess the effects of the drugs. RESULTS: after the 5 weeks of challenges, the weight differences of the ears in the SPC group and the prednisolone group were significantly less than those in the other groups. There was evidence of significant suppression of the development of dermatitis, as determined by a histological examination and the serum Ig E levels. CONCLUSION: SPC has beneficial effects when used in the treatment of chronic dermatitis-eczema in mice.
Assuntos
Dermatite Atópica/tratamento farmacológico , Derme/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Medicina Tradicional Chinesa , Animais , Dermatite Atópica/sangue , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Derme/imunologia , Derme/patologia , Medicamentos de Ervas Chinesas/química , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The efficacy of rosuvastatin in reducing allergic inflammation has been established. However, its potential to reduce airway remodeling has yet to be explored. This study aimed to evaluate the efficacy of rosuvastatin in reducing airway inflammation and remodeling in a mouse model of chronic allergic asthma induced by sensitization and challenge with OVA. Histology of the lung tissue and the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) showed a marked decrease in airway inflammation and remodeling in mice treated with rosuvastatin, as evidenced by a decrease in goblet cell hyperplasia, collagen deposition, and smooth muscle hypertrophy. Furthermore, levels of inflammatory cytokines, angiogenesis-related factors, and OVA-specific IgE in BALF, plasma, and serum were all reduced upon treatment with rosuvastatin. Western blotting was employed to detect AMPK expression, while immunohistochemistry staining was used to observe the expression of remodeling signaling proteins such as α-SMA, TGF-ß, MMP-9, and p-AMPKα in the lungs. It was found that the activity of 5'-adenosine monophosphate-activated protein kinase alpha (AMPKα) was significantly lower in the lungs of OVA-induced asthmatic mice compared to Control mice. However, the administration of rosuvastatin increased the ratio of phosphorylated AMPK to total AMPKα, thus inhibiting the formation of new blood vessels, as indicated by CD31-positive staining mainly in the sub-epithelial region. These results indicate that rosuvastatin can effectively reduce airway inflammation and remodeling in mice with chronic allergic asthma caused by OVA, likely due to the reactivation of AMPKα and a decrease in angiogenesis.
Assuntos
Proteínas Quinases Ativadas por AMP , Remodelação das Vias Aéreas , Asma , Modelos Animais de Doenças , Rosuvastatina Cálcica , Transdução de Sinais , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Asma/patologia , Rosuvastatina Cálcica/farmacologia , Rosuvastatina Cálcica/uso terapêutico , Proteínas Quinases Ativadas por AMP/metabolismo , Transdução de Sinais/efeitos dos fármacos , Remodelação das Vias Aéreas/efeitos dos fármacos , Camundongos , Ovalbumina , Feminino , Camundongos Endogâmicos BALB C , Líquido da Lavagem Broncoalveolar , Doença Crônica , Inflamação/tratamento farmacológico , Inflamação/patologia , Inflamação/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Imunoglobulina E/sangueRESUMO
PURPOSE: Our team identified a new cardiac glycoside, Toxicarioside H (ToxH), in a tropical plant. Previous research has indicated the potential of cardenolides in mitigating inflammation, particularly in the context of NETosis. Therefore, this study sought to examine the potential of ToxH in attenuating allergic airway inflammation by influencing the immune microenvironment. METHODS: An OVA-induced airway inflammation model was established in BALB/c mice. After the experiment was completed, serum, bronchoalveolar lavage fluid (BALF), and lung tissue samples were collected and further examined using H&E and PAS staining, flow cytometry, immunofluorescence observation, and Western blot analysis. RESULTS: Treatment with ToxH was found to be effective in reducing airway inflammation and mucus production. This was accompanied by an increase in Th1 cytokines (IFN-γ, IL-2, and TNF-ß), and the Th17 cytokine IL-17, while levels of Th2 cytokines (IL-4, IL-5, and IL-13) and Treg cytokines (IL-10 and TGF-ß1) were decreased in both the bronchoalveolar lavage fluid (BALF) and the CD45+ immune cells in the lungs. Additionally, ToxH inhibited the infiltration of inflammatory cells and decreased the number of pulmonary CD44+ memory T cells, while augmenting the numbers of Th17 and Treg cells. Furthermore, the neutrophil elastase inhibitor GW311616A was observed to suppress airway inflammation and mucus production, as well as alter the secretion of immune Th1, Th2, Th17, and Treg cytokines in the lung CD45+ immune cells. Moreover, our study also demonstrated that treatment with ToxH efficiently inhibited ROS generation, thereby rectifying the dysregulation of immune cells in the immune microenvironment in OVA-induced allergic asthma. CONCLUSIONS: Our findings indicate that ToxH could serve as a promising therapeutic intervention for allergic airway inflammation and various other inflammatory disorders. Modulating the balance of Th1/Th2 and Treg/Th17 cells within the pulmonary immune microenvironment may offer an effective strategy for controlling allergic airway inflammation.
Assuntos
Citocinas , Pulmão , Camundongos Endogâmicos BALB C , Ovalbumina , Animais , Ovalbumina/imunologia , Citocinas/metabolismo , Pulmão/imunologia , Pulmão/patologia , Pulmão/efeitos dos fármacos , Camundongos , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Modelos Animais de Doenças , Asma/imunologia , Asma/tratamento farmacológico , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Humanos , Muco/metabolismo , Muco/imunologia , Alérgenos/imunologiaRESUMO
AIMS: Penicilazaphilone C (PAC) is hypothesized to potentially serve as a therapeutic treatment for allergic airway inflammation by inhibiting the NLRP3 inflammasome and reducing oxidative stress. METHODS: An allergic asthma model was induced in female BALB/c mice of the OVA, OVA+PAC, OVA+PAC+LPS, and OVA+Dex groups by sensitizing and subsequently challenging them with OVA. The OVA+PAC and Normal+PAC groups were treated with PAC, while the OVA+PAC+LPS group also received LPS. The OVA+Dex group was given dexamethasone (Dex). Samples of serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected for histological and cytological analysis. RESULTS: Allergic mice treated with PAC or Dex showed inhibited inflammation and mucus production in the lungs. There was a decrease in the number of inflammatory cells in the BALF, lower levels of inflammatory cytokines in the serum and BALF, and a reduction in the protein expression of NLRP3, ASC, cleaved caspase-1, IL-1ß, activated gasdermin D, MPO, Ly6G, and ICAM-1. Additionally, oxidative stress was reduced, as shown by a decrease in MDA and DCF, but an increase in SOD and GSH. Treatment with PAC also resulted in a decrease in pulmonary memory CD4+ T cells and an increase in regulatory T cells. However, the positive effects seen in the PAC-treated mice were reversed when the NLRP3 inflammasome was activated by LPS, almost returning to the levels of the Sham-treated mice. SIGNIFICANCE: PAC acts in a similar way to anti-allergic inflammation as Dex, suggesting it may be a viable therapeutic option for managing allergic asthma inflammation.
Assuntos
Asma , Líquido da Lavagem Broncoalveolar , Inflamassomos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Feminino , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Asma/tratamento farmacológico , Asma/imunologia , Asma/induzido quimicamente , Camundongos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/metabolismo , Pulmão/imunologia , Estresse Oxidativo/efeitos dos fármacos , Ovalbumina , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Modelos Animais de Doenças , Dexametasona/farmacologia , Anti-Inflamatórios/farmacologiaRESUMO
Studies have demonstrated that prior to puberty, girls have a lower incidence and severity of asthma symptoms compared to boys. This study aimed to explore the role of progesterone (P4), a sex hormone, in reducing inflammation and altering the immune microenvironment in a mouse model of allergic asthma induced by OVA. Female BALB/c mice with or without ovariectomy to remove the influence of sex hormones were used for the investigations. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue samples were collected for analysis. The results indicated that P4 treatment was effective in decreasing inflammation and mucus secretion in the lungs of OVA-induced allergic asthma mice. P4 treatment also reduced the influx of inflammatory cells into the BALF and increased the levels of Th1 and Th17 cytokines while decreasing the levels of Th2 and Treg cytokines in both BALF and lung microenvironment CD45+ T cells. Furthermore, P4 inhibited the infiltration of inflammatory cells into the lungs, suppressed NETosis, and reduced the number of pulmonary CD4+ T cells while increasing the number of regulatory T cells. The neutrophil elastase inhibitor GW311616A also suppressed airway inflammation and mucus production and modified the secretion of immune Th1, Th2, Th17, and Treg cytokines in lung CD45+ immune cells. These changes led to an alteration of the immunological milieu with increased Th1 and Th17 cells, accompanied by decreased Th2, Treg, and CD44+ T cells, similar to the effects of P4 treatment. Treatment with P4 inhibited NETosis by suppressing the p38 pathway activation, leading to reduced reactive oxygen species production. Moreover, P4 treatment hindered the release of double-stranded DNA during NETosis, thereby influencing the immune microenvironment in the lungs. These findings suggest that P4 treatment may be beneficial in reducing inflammation associated with allergic asthma by modulating the immune microenvironment. In conclusion, this research indicates the potential of P4 as a therapeutic agent for ameliorating inflammation in OVA-induced allergic asthma mice.
Assuntos
Asma , Ovalbumina , Progesterona , Animais , Feminino , Camundongos , Asma/imunologia , Asma/tratamento farmacológico , Asma/metabolismo , Líquido da Lavagem Broncoalveolar , Microambiente Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/metabolismo , Pulmão/imunologia , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Progesterona/farmacologia , Células Th17/imunologia , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p < 0.001), as indicated by data from flow cytometry detection of membrane-permeability cell death stained with 7-aminoactinomycin, lipid peroxidation, and Fe2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.
Assuntos
Neoplasias da Mama , Ferroptose , Feminino , Humanos , Neoplasias da Mama/genética , Coenzima A Ligases/genética , Ferroptose/genética , Peroxidação de Lipídeos , Monoéster Fosfórico Hidrolases , Fosforilação , Piruvato Desidrogenase (Lipoamida)-Fosfatase/metabolismoRESUMO
OBJECTIVE: To examine the immunoprotection effect induced by MIC8 DNA vaccine co-immunized with a plasmid encoding murine IL-12 (pcIL-12) as an adjuvant in mice against the challenge of Toxoplasma gondii. METHODS: The gene sequence encoding MIC8 of T. gondii RH strain was inserted into eukaryotic expression vector pcDNA3.1 to construct the pcMIC8 expression plasmid. The recombinant plasmid was transfected into HeLa cells to test its expression and the recombinant protein was then characterized by Western blotting. Eighty Kunming mice were randomly divided into 5 groups (16 per group): 3 control groups (PBS, pcDNA3.1, and pcIL-12), pcMIC8 group, and pcMIC8 plus pcIL-12 group. Mice in the pcMIC8 plus pcIL-12 group were co-injected intramuscularly at a dosage of 100 microl each of pcMIC8 and pcIL-12 suspended in 100 microg sterile PBS. Mice in other groups were inoculated with PBS, pcDNA3.1, pcIL-12, and pcMIC8 respectively following the same protocol. All the mice received three immunizations at 2-week intervals. Serum samples were collected on day 0, 13, 27, 41, and 55 before each inoculation for determining antibody IgG, IgG subclass IgG2a. Four weeks after the final immunization, IFN-gamma and IL-4 levels in splenocytes cultures from immunized mice were detected by ELISA. The mice were challenged with 10(3) tachyzoites of the virulent T. gondii RH strain three weeks after the last immunization to observe the survival time. RESULTS: Western blotting showed that the protein extracts in HeLa cells upon transfection with pcMIC8 were effectively expressed in cells. The levels of IgG (0.51 +/- 0.028) and IgG2a (0.261 +/- 0.04)(on day 55) in mice immunized with pcMIC8 plus pcIL-12 were higher than pcMIC8 group (497.65 +/- 98.15) and control groups (PBS 47.18 +/- 2.73, pcDNA3.1 50.08 +/- 4.62, pcIL-12 118.15 +/- 12.73) (P < 0.05). There was no significant difference in the level of IgG 1 and IL-4 among the five groups (P > 0.05). After a lethal challenge of T. gondii RH strain, the survival time in mice immunized with pcMIC8 plus pcIL-12 (15d) was prolonged in comparison to that of pcMIC8 (10d) and control groups (PBS 5 d, pcDNA3.1 6d, pcIL-12 8 d) (P < 0.05). CONCLUSION: The immune responses induced by the combined use of the recombinant plasmid encoding MIC8 of T. gondii with murine IL-2 gene adjuvant can be enhanced.
Assuntos
Adjuvantes Imunológicos , Moléculas de Adesão Celular/imunologia , Interleucina-12/imunologia , Proteínas de Protozoários/imunologia , Toxoplasmose/prevenção & controle , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/genética , Animais , Moléculas de Adesão Celular/genética , Células HeLa , Humanos , Imunização , Interleucina-12/genética , Camundongos , Camundongos Endogâmicos , Plasmídeos , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Toxoplasma , Transfecção , Vacinas de DNA/genética , Vacinas de DNA/uso terapêuticoRESUMO
OBJECTIVE: To observe the immune response induced by complex gene vaccine pcSAG1-ROP5 of Toxoplasma gondii in mice. METHODS: The recombinant eukaryotic expression plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were constructed and identified by PCR, restriction enzyme digestion, and sequencing. The three recombinant plasmids were transfected into HeLa cells to express in vitro and identified by Western blotting analysis. Seventy Kunming mice were randomly divided into 5 groups with 14 each, i.e. pcSAG1 group, pcROP5 group, pcSAG1-ROP5 group, blank plasmid group and PBS control group. The mice were immunized intramuscularly with pcSAG1, pcROP5, pcSAG1-ROP5, pcDNA3.1, and PBS, respectively, every two weeks for three times. Sera were collected before each injection and 2 weeks after the last immunization. The titer of mice serum in pcSAG1-ROP5 group combined with recombinant protein SAG1, ROP5 and SAG1-ROP5 and the level of IgG against T. gondii in 5 groups were determined by ELISA. Three weeks after the last immunization, ten mice of each group were challenged with 10(3) tachyzoites of the virulent T. gondii RH strain to observe the survival time. One week later, the rest four mice in each group were sacrificed and the supernatant of cultured splenocytes was collected for the detection of IFN-gamma and IL-4. RESULTS: Western blotting showed that the recombinant plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were expressed in HeLa cells with M(r) 31 000, 57 000, and 88 000, respectively. The serum titer in pcSAG1-ROP5 group combined with SAG1, ROP5 and SAG1-ROP5 was 1:320, 1:160, and 1:2560, respectively. The IgG level kept rising in pcSAG1, pcROP5 and pcSAG1-ROP5 groups. Two weeks after the last immunization, the IgG level in pcSAG1-ROP5 group was higher than those in other groups (P<0.05). After a lethal challenge of T. gondii RH strain, the survival time of the mice in pcSAG1-ROP5 group was (288 +/- 7) h, which was 48 h and 96h longer than the groups of pcSAG1 and pcROP5, respectively (P< 0.05). Four weeks after the last immunization, IFN-gamma in splenocyte culture of pcSAG1-ROP5 group [(908.52 +/- 6.31) pg/ml] was higher than other groups (P<0.05), with no significant difference in IL-4 (P>0.05). CONCLUSION: Compared with the single gene vaccines pcSAG1 and pcROP5, higher levels of IgG and IFN-gamma and longer survival time are observed in mice immunized with pcSAG1-ROP5.
Assuntos
Vacinas Protozoárias/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Feminino , Células HeLa , Humanos , Imunização , Imunoglobulina G/sangue , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos , Plasmídeos , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/genética , Toxoplasma/genética , Toxoplasmose Animal/prevenção & controle , TransfecçãoRESUMO
Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.
Assuntos
Ferroptose , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fibroblastos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genéticaRESUMO
Electro-acupuncture (EA) at Fenglong acupoint (ST40) can lower the levels of serum cholesterol and triacylglycerols. To study the hepatic genes responsible for the cholesterol-lowering effect of EA, suppression subtractive hybridization combined with the switch mechanism at the 5'-end of RNA template cDNA synthesis and long-distance PCR were employed using hepatic tissues from hypercholesterolemia and EA-treated mice. 68 % of the identified genes are involved in metabolism, immune response, and signal transduction pathways. Real-time PCR and western blot indicate that EA at ST40 induces the expression of nNOS and Mt1, two genes involved in NO signal transduction. EA treatment for hypercholesterolemia thus involves the modulation of several biological pathways and provides a physiological link between NO signal transduction and the cholesterol-lowering effect of EA.
Assuntos
Acupuntura/métodos , Colesterol/sangue , Regulação da Expressão Gênica , Hipercolesterolemia/terapia , Óxido Nítrico/metabolismo , Transdução de Sinais , Animais , Biópsia , Western Blotting , Perfilação da Expressão Gênica , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Triglicerídeos/sangueRESUMO
OBJECTIVE: To study the antitumor efficacy of an immunoconjugate composed of adriamycin (ADM) and downsized Fab fragment of mouse anti-Endoglin monoclonal antibody. METHODS: The Fab fragment of mouse anti-Endoglin monoclonal antibody was downsized and conjugated with ADM by m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The antitumor effect of the conjugate was tested in mice bearing subcutaneous injection of H22 tumor in vivo. RESULTS: The molecular ratio of Fab:ADM in conjugate was approximately 1:2. The Fab-ADM conjugate inhibited the growth of H22 by 91.94% on day 14 after injection at the dose of 0.4 mg/ kg, much higher the inhibition rate of 25.00% by the equivalent dose of free ADM. The median survival time of the mice treated with the conjugate was longer than those treated with free ADM. The Fab-ADM conjugate was significantly more effective than free ADM in tumor suppression and life span prolongation. CONCLUSION: Fab -ADM displayed more significant antitumor efficacy than free ADM in vivo and might be a novel candidate for cancer treatment.
Assuntos
Anticorpos Monoclonais/farmacologia , Doxorrubicina/farmacologia , Imunotoxinas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Animais , Antibióticos Antineoplásicos/farmacologia , Endoglina , Masculino , Camundongos , Neoplasias Experimentais/tratamento farmacológicoRESUMO
BACKGROUND: Several agents for oncolytic immunotherapy have been approved for clinical use, but monotherapy is modest for most oncolytic agents. The combination of several therapeutic strategies through recombinant and nanotechnology to engineer multifunctional oncolytic viruses for oncolytic immunotherapy is a promising strategy. METHODS: An endothelium-targeting iRGD-liposome encapsulating a recombinant Newcastle disease virus (NDV), which expresses the dendritic cell (DC) chemokine MIP-3α (iNDV3α-LP), and three control liposomes were constructed. MIP-3α, HMGB1, IgG, and ATP were detected by western blotting or ELISA. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells were analyzed by flow cytometry. The antitumor efficiency was investigated in B16 and 4T1 tumor-bearing mice. Immunofluorescence and immunohistochemistry were used to observe the localization of liposomes, molecular expression and angiogenesis. Synergistic index was calculated using the data of tumor volume, tumor angiogenesis and tumor-infiltrating lymphocytes. RESULTS: Compared with NDV-LP, treatment with iNDV3α-LP and NDV3α-LP induced stronger virus replication and cell lysis in B16 and 4T1 tumor cells and human umbilical vein endothelial cells (HUVECs) with the best response observed following iNDV3α-LP treatment. B16 and 4T1 cells treated with iNDV3α-LP produced more damage-associated molecular pattern molecules, including secreted HMGB1, ATP, and calreticulin. Moreover, iNDV3α-LP specifically bound to αvß3-expressing 4T1 cells and HUVECs and to tumor neovasculature. Tumor growth was significantly suppressed, and survival was longer in iNDV3α-LP-treated B16-bearing and 4T1-bearing mice. A mechanism study showed that iNDV3α-LP treatment initiated the strongest tumor-specific cellular and humoral immune response. Moreover, iNDV3α-LP treatment could significantly suppress tumor angiogenesis and reverse the tumor immune suppressive microenvironment in both B16-bearing and 4T1-bearing mice. CONCLUSIONS: In this study, iNDV3α-LP had several functions, such as tumor and vessel lysis, MIP-3α immunotherapy, and binding to αvß3-expressing tumor and its neovasculature. iNDV3α-LP treatment significantly suppressed tumor angiogenesis and reversed the tumor immunosuppressive microenvironment. These findings offer a strong rationale for further clinical investigation into a combination strategy for oncolytic immunotherapy, such as the formulation iNDV3α-LP in this study.
Assuntos
Proteína HMGB1 , Neoplasias , Terapia Viral Oncolítica , Trifosfato de Adenosina/metabolismo , Animais , Células Endoteliais , Endotélio , Proteína HMGB1/metabolismo , Humanos , Fatores Imunológicos , Imunoterapia , Lipossomos/metabolismo , Camundongos , Neoplasias/terapia , Vírus da Doença de Newcastle , Microambiente TumoralRESUMO
Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and adjuvant CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8+-induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.
Assuntos
Burkholderia pseudomallei , Vacinas Anticâncer , Neoplasias , Células Dendríticas , Humanos , Porosidade , Microambiente TumoralRESUMO
Virus is a nanosized pathogen and mainly composed of viral protein and nucleic acids. Under the pressure of long-term selection, mammals have gradually evolved effective immune mechanisms to defend themselves against viruses. In addition to recognizing viral proteins, immune system can also respond to viral sequence-specific nucleic acids, including CpG ODN, single- and double- strand RNA, and thereby enhancing the ability to remove infected viruses. Inspired by these immune mechanisms, we have attempted to develop a tracing virus-mimicking nanovaccine for tumor immunotherapy. This nanovaccine mainly consists of nucleic acids (CpG ODN), proteins (including tumor-associated antigen, and neutravidin (nAvidin) as skeleton materials for constructing nanovaccine and carriers for loading tumor-associated antigen and CpG ODN), and the dye molecules for assembling nAvidin to form nanoparticles comparable in size to viruses and tracing the vaccine in vitro and in vivo. The as-prepared nanovaccine efficiently induces the maturation of dendritic cell, the enhancement of antigen cross-presentation ability, and amplification of cytokine production in vitro. Furthermore, in vivo analysis clearly shows that it targets lymph nodes, successfully presents antigens to generate tumor-antigen-specific CD8+ T cells and induces a Th1-biased immune response. Most notably, this virus-mimicking nanovaccine significantly inhibits the growth of antigen-expressed tumor and prolongs the survival time of the antigen-expressed tumor bearing mice.
Assuntos
Vacinas Anticâncer , Nanopartículas , Vírus , Animais , Avidina , Biotina , Linfócitos T CD8-Positivos , Células Dendríticas , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Toxicarioside O (TCO), a natural product derived from Antiaris toxicaria, has been identified to be a promising anticancer agent. In this study, we aimed to investigate the effect of TCO on the proliferation and epithelial-mesenchymal transition (EMT) of lung cancer cells and its molecular mechanisms. Here, we indicated that TCO inhibits the proliferation of lung cancer cells both in vitro and in vivo. Our results demonstrated that TCO induces apoptosis in lung cancer cells. Moreover, we found that TCO suppresses EMT program and inhibits cell migration in vitro. Mechanistically, TCO decreases the expression of trophoblast cell surface antigen 2 (Trop2), resulting in inhibition of the PI3K/Akt pathway and EMT program. Overexpression of Trop2 rescues TCO-induced inhibition of cell proliferation and EMT. Our findings demonstrate that TCO markedly inhibits cell proliferation and EMT in lung cancer cells and provides guidance for its drug development.