RESUMO
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
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
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
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
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
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
BACKGROUND: The oncolytic Newcastle disease virus (NDV) is inherently able to trigger the lysis of tumor cells and induce the immunogenic cell death (ICD) of tumor cells and is also an excellent gene-engineering vector. The macrophage inflammatory protein-3α (MIP-3α) is a specific chemokine for dendritic cells (DCs). Thus, we constructed a recombinant NDV expressing MIP-3α (NDV-MIP3α) as an in vivo DC vaccine for amplifying antitumor immunities. METHODS: The recombinant NDV-MIP3α was constructed by the insertion of MIP-3α cDNA between the P and M genes. Western blotting assay and ELISA were used to detect MIP-3α, HMGB1, IgG, and ATP in the supernatant and sera. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells (eg, DCs) were analyzed by flow cytometry. The antitumor efficiency of NDV-MIP3α was observed in B16 and CT26 tumor-bearing mice. Immunofluorescence and immunohistochemistry were applied to observe the ecto-calreticulin (CRT) and intratumoral attraction of DCs. Adoptive transfer of splenocytes and antibodies and depletion of T-cell subsets were used to evaluate the relationship between antitumor immunities and the role of the T-cell subtype. RESULTS: The findings show that NDV-MIP3α has almost the same capabilities of tumor lysis and induction of ICD as the wild-type NDV (NDV-WT). MIP-3α secreted by NDV-MIP3α could successfully attract DCs in vitro and in vivo. Both B16 and CT26 cells infected with NDV-MIP3α could strongly promote DC maturation and activation. Compared with NDV-WT, intratumoral injection of NDV-MIP3α and the adoptive transfer of T lymphocytes from mice injected with NDV-MIP3α resulted in a significant suppression of B16 and CT26 tumor growth. The NDV-MIP3α-induced production of tumor-specific cellular and humoral immune responses was dependent on CD8+ T cells and partially on CD4+ T cells. A significant reversion of tumor microenvironments was found in the mice injected with NDV-MIP3α. CONCLUSIONS: Compared with NDV-WT, the recombinant NDV-MIP3α as an in vivo DC vaccine demonstrates enhanced antitumor activities through the induction of stronger system immunities and modulation of the tumor microenvironment. This strategy may be a potential approach for the generation of an in vivo DC vaccine.
Assuntos
Quimiocina CCL20/metabolismo , Vírus da Doença de Newcastle/patogenicidade , Vírus Oncolíticos/metabolismo , Animais , Humanos , Camundongos , Microambiente TumoralRESUMO
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.
RESUMO
Multiple exopolysaccharides (EPSs) have been isolated from various organisms in extreme environments and have yielded a variety of activities. The present study evaluated the immunomodulatory capabilities of an EPS (termed PHEPS) derived from the fungus Paecilomyces lilacinus PH0016, which was isolated from a tropical and hyperhaline environment in southern China. The macrophage RAW 264.7 cell line was used to investigate the mechanism of PHEPSinduced macrophage activation. The results indicated that RAW 264.7 macrophages were activated by PHEPS, in an effect slightly inferior to lipopolysaccharide (LPS), as evidenced by secretion of interleukin (IL)1ß, tumor necrosis factor (TNF)α and nitric oxide (NO), and by significantly increased phagocytosis in the cells treated with PHEPS. Nuclear factor (NF)κB p65 was significantly translocated into the nucleus in the PHEPStreated cells. In addition, expression of inducible NO synthase (iNOS) and IκBα degradation were enhanced in PHEPStreated cells. The phosphorylation levels of p38, JNK and ERK were also significantly increased in the PHEPStreated cells. Furthermore, IL1ß and TNFα production was markedly decreased in PHEPStreated cells when the mitogenactivated protein kinase (MAPK) pathways were blocked by the inhibitor Dectin1 and by antibodies against Tolllike receptor 4 (TLR4). The present results indicated that PHEPS from Paecilomyces lilacinus possessed the capability of activating RAW 264.7 cells via the TLR4/NFκB/MAPKs signaling pathway.
Assuntos
Polissacarídeos Fúngicos/imunologia , Sistema de Sinalização das MAP Quinases , Ativação de Macrófagos , NF-kappa B/imunologia , Paecilomyces/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Transdução de SinaisRESUMO
Purpose: Lattice corneal dystrophy (LCD) is related to the denaturation of transforming growth factor-ß-induced protein (TGFBIp). Autophagic degradation of the denatured proteins by macrophages is one pathway to remove the denatured proteins. Thus, we investigated the role of autophagy in the degradation of mutant (MU) TGFBIp in macrophages. Methods: Corneas from participants were observed by slit-lamp photography and subjected to histopathologic and genetic analysis. Wild-type (WT) and MU TGFBIp were recombined and expressed. Macrophages from MU participants were isolated and cocultured with the recombinant TGFBIp. Colocalization of the two molecules was observed by immunofluorescent microscopy. Enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to detect changes in molecule expression related to the phenotype and autophagy process. Results: Fourteen members from a family of 25 were identified as LCD sufferers. Significant TGFBIp aggregates and macrophage infiltration were found only in the corneas of LCD sufferers. Marker accumulation of TGFBIp was found in macrophages exposed to MU TGFBIp even at 5 hours after MU TGFBIp was withdrawn. High expressions of CD68 and CD36 were found in macrophages exposed to WT TGFBIp, but not to MU TGFBIp. Impaired autophagic flux due to defective autophagosome fusion to lysosomes was found in macrophages exposed to MU TGFBIp. Blockage of the autophagic process suppressed the expression of CD68 and CD36 in macrophages exposed to WT TGFBIp to levels similar to those found in macrophages exposed to MU TGFBIp. Conclusions: Our results suggested that reversion of the defective autophagic process in macrophages may be a therapeutic strategy for patients with LCD.
Assuntos
Autofagia , Distrofias Hereditárias da Córnea/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Western Blotting , Antígenos CD36/metabolismo , Criança , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/genética , Ensaio de Imunoadsorção Enzimática , Proteínas da Matriz Extracelular/genética , Feminino , Citometria de Fluxo , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Linhagem , Fagocitose , Fator de Crescimento Transformador beta/genéticaRESUMO
Nano-agent-mediated photothermal therapy (PTT) combined with chemotherapy has been proposed as an effective strategy against cancer. However, chemotherapeutic agents often cause serious side effects. Herein, a novel PTT nanoagent (Cy5.5-MSA-G250) with unanticipated intrinsic tumor-selective cytotoxicity is developed. The Cy5.5-MSA-G250 nanoparticles (NPs) are created by mixing mouse serum albumin (MSA) and coomassie brilliant blue (G250) and then conjugated with cyanine 5.5 (Cy5.5). As expected, Cy5.5-MSA-G250 NPs can efficiently kill cancer cells in vitro and in vivo by PTT. Meanwhile, we accidentally discover that Cy5.5-MSA-G250 have intrinsic specific cytotoxicity against tumor cells but not against normal cells. Moreover, the tumor-specific cytotoxicity of Cy5.5-MSA-G250 is much stronger than that of cytarabine, an FDA-approved anticancer drug. In vivo experiments also prove that Cy5.5-MSA-G250 NPs can effectively eliminate residual tumor cells and prevent metastasis. Further study indicates that selective induction of G1 cell cycle arrest and inhibition of DNA duplication in tumor cells may be the possible mechanism of the tumor cell-selective cytotoxicity of Cy5.5-MSA-G250 NPs. In addition, direct visualization, low systematic toxicity, good biodegradation, and efficient body excretion further make Cy5.5-MSA-G250 NPs attractive for in vivo applications. Taken together, Cy5.5-MSA-G250 NPs are proven to be a promising platform for combined photothermal chemotherapy.
Assuntos
Neoplasias , Animais , Antineoplásicos , Camundongos , NanopartículasRESUMO
Immunogenic cell death (ICD) is a specific kind of cell death that stimulates the immune system to combat cancer cells. Ultrasound (US)-controlled targeted release of drugs by liposome-microbubble complexes is a promising approach due to its non-invasive nature and visibility through ultrasound imaging. However, it is not known whether this approach can enhance ICD induced by drugs, such as doxorubicin. Herein, we prepared a doxorubicin-liposome-microbubble complex (MbDox), and the resultant MbDox was then characterized and tested for US-controlled release of Dox (MbDox+US treatment) to enhance the induction of ICD in LL/2 and CT26 cancer cells and in syngeneic murine models. We found that MbDox+US treatment caused more cellular uptake and nuclear accumulation of Dox in tumor cells, and more accumulation of Dox in tumor tissues. Enhanced induction of ICD occurred both in vitro and in vivo. MbDox+US treatment induced more apoptosis, stronger membrane exposure and the release of ER stress proteins and DAMPs in tumor cells, and increased DC maturation in vitro. In addition, MbDox+US treatment also resulted in stronger therapeutic effects in immunocompetent mice than in immunodeficient mice. Moreover, MbDox+US enhancement of ICD was also evidenced by a higher proportion of activated CD8+ T-lymphocytes but lower Treg in tumor tissues. Taken together, our results demonstrate that US-controlled release of ICD inducers into nuclei using liposome-microbubble complexes may be an effective approach to enhance the induction of ICD for tumor treatment.
RESUMO
Rationale: Cardenolides have potential as anticancer drugs. 3'-epi-12ß-hydroxyfroside (HyFS) is a new cardenolide structure isolated by our research group, but its molecular mechanisms remain poorly understood. This study investigates the relationship between its antitumor activities and autophagy in lung cancer cells. Methods: Cell growth and proliferation were detected by MTT, lactate dehydrogenase (LDH) release, 5-ethynyl-20-deoxyuridine (EDU) and colony formation assays. Cell apoptosis was detected by flow cytometry. Autophagic and signal proteins were detected by Western blotting. Markers of autophagy and autophagy flux were also detected by immunofluorescence, transmission electron microscopy and acridine orange staining. Real time RT-PCR was used to analyze the gene expression of Hsp90. Hsp90 ubiquitination was detected by coimmunoprecipitation. The antitumore activities of HyFS were observed in nude mice. Results: HyFS treatment inhibited cell proliferation and induced autophagy in A549 and H460 lung cancer cells, but stronger inhibition of cell proliferation and induction of cell apoptosis were shown when HyFS-mediated autophagy was blocked. The Hsp90/Akt/mTOR axis was found to be involved in the activation of HyFS-mediated autophagy. Evidence of direct interaction between Hsp90 and Akt was observed. HyFS treatment resulted in decreased levels of heat shock protein 90 (Hsp90) and phosphorylated Akt, overexpression of Hsp90 increased activation of autophagy, and inhibition of Hsp90 expression decreased autophagy. In addition, ubiquitin-mediated degradation of Hsp90 and subsequent dephosphorylation of its client protein Akt were also found in HyFS-treated lung cancer cells. Moreover, combination treatment with HyFS and chloroquine showed remarkably increased tumor inhibition in both A549- and H460-bearing mice. Conclusion: Our results demonstrate that HyFS induced cytoprotective autophagy through ubiquitin-mediated degradation of Hsp90, which further blocked the Akt/mTOR pathway in lung cancer cells. Thus, a combination of a HyFS-like cardenolide and an autophagic inhibitor is a potential alternative approach for the treatment of lung cancer.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Cardenolídeos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Western Blotting , Cardenolídeos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Formazans/análise , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica de Transmissão , Transplante de Neoplasias , Proteína Oncogênica v-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Coloração e Rotulagem , Serina-Treonina Quinases TOR/metabolismo , Sais de Tetrazólio/análise , Resultado do TratamentoRESUMO
Metastatic invasion is the primary cause of treatment failure for GBM. EMT is one of the most important events in the invasion of GBM; therefore, understanding the molecular mechanisms of EMT is crucial for the treatment of GBM. In this study, high expression of DRR1 was identified to correlate with a shorter median overall and relapse-free survival. Loss-of-function assays using shDRR1 weakened the invasive potential of the GBM cell lines through regulation of EMT-markers. The expressions of p-AKT were significantly decreased after DRR-depletion in SHG44 and U373â¯cells. Moreover, the invasion was inhibited by the AKT inhibitor, MK-2206. The expression of Vimentin, N-cadherin, MMP-7, snail and slug was significantly inhibited by MK-2206, while the expression of E-cadherin was upregulated. Our results provide the first evidence that DRR1 is involved in GBM invasion and progression possibly through the induction of EMT activation by phosphorylation of AKT.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Glioblastoma/genética , Humanos , Masculino , Invasividade Neoplásica , Fosforilação , Prognóstico , Análise de Sobrevida , Regulação para CimaRESUMO
OBJECTIVES: Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti-cancer agent. However, the molecular mechanisms involved remain poorly understood. MATERIALS AND METHODS: Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA-ß-gal assay, western blotting and immunofluorescence were performed to determine CGN-induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN-induced senescence and autophagy. The anti-tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models. RESULTS: We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell-cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN-mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti-tumour effects in vivo. CONCLUSIONS: Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN-mediated anti-cancer therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Cardenolídeos/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Animais , Calotropis/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quimioterapia Combinada , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
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.