RESUMO
Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.
Assuntos
Anti-Infecciosos/farmacologia , Neoplasias da Mama/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Receptor de Endotelina A/efeitos dos fármacos , Sulfisoxazol/farmacologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Biogênese de Organelas , Receptor de Endotelina A/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. METHODS: In this study, we identified biomarkers for diagnosing CC through proteomic analysis of small-EVs from CC cell lines. These small-EVs were characterized by western blot analysis, nanoparticle tracking analysis, and transmission electron microscopy and analyzed using mass spectrometry. RESULTS: Five selected proteins were found to be upregulated in CC by western blot analysis. Among the candidate proteins, tetraspanin 1 (TSPAN1) was found to be upregulated in plasma EVs from CC patients compared to those from healthy controls (HCs) with 75.7% sensitivity. CONCLUSIONS: These results suggest that TSPAN1 is a potent non-invasive biomarker for CC detection. Our experimental strategy provides useful insights into the identification of cancer-specific non-invasive biomarkers.
Assuntos
Biomarcadores Tumorais , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/metabolismo , Vesículas Extracelulares/metabolismo , Proteômica , Adulto , Idoso , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromatografia Líquida , Neoplasias do Colo/sangue , Neoplasias do Colo/genética , Feminino , Expressão Gênica , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteoma , Proteômica/métodos , Curva ROCRESUMO
Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Vesículas Extracelulares/metabolismo , Hepatite Alcoólica/diagnóstico , Fígado/metabolismo , Adulto , Animais , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Feminino , Células Hep G2 , Hepatite Alcoólica/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , ProteômicaRESUMO
Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1ß, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/ß phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway.
RESUMO
Eisenia bicyclis is edible brown algae recognized as a rich source of bioactive derivatives mainly phlorotannins reported for their anti-oxidant properties. Of all phlorotannins identified so far, dieckol has shown the most potent effect in anti-inflammatory, radical scavenging and neuroprotective functions. However, whether dieckol up-regulates hemeoxygenase 1 (HO-1) and this mediates its anti-inflammatory effect in murine macrophages remains poorly understood. Dieckol (12.5-50 µM) inhibited nitric oxide production and attenuated inducible nitric oxide synthase, phospho (p)-PI-3K, p-Akt, p-IKK-α/ß, p-IκB-α and nuclear p-NF-κBp65 protein expressions, and NF-κB transcriptional activity in LPS (0.1 µg/ml) stimulated murine macrophages. On the other hand, dieckol up-regulated HO-1 which partly mediated its anti-inflammatory effect in murine macrophages. Thus, dieckol appeared to be a potential therapeutic agent against inflammation through HO-1 up-regulation.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Benzofuranos/farmacologia , Heme Oxigenase-1/metabolismo , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Phaeophyceae/imunologia , Animais , Linhagem Celular , Heme Oxigenase-1/genética , Heme Oxigenase-1/imunologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Despite a multitude of reports on anti-inflammatory properties of ginseng extracts or individual ginsenosides, data on antiarthritic effect of ginseng saponin preparation with mixed ginsenosides is limited. On the other hand, a combined therapy of safe and inexpensive plant-derived natural products such as ginsenosides can be considered as an alternative to treat arthritis. Our previous in vitro data displayed a strong anti-inflammatory action of red ginseng saponin fraction-A (RGSF-A). We, herein, report a marked antiarthritic property of RGSF-A rich in ginsenoside Rb1, Rc, and Rb2. Collagen-induced arthritic (CIA) mice were treated with RGSF-A or methotrexate (MTX) for 5 weeks. Joint pathology, serum antibody production and leukocye activation, cytokine production in the circulation, lymph nodes, and joints were examined. RGSF-A markedly reduced severity of arthritis, cellular infiltration, and cartilage damage. It suppressed CD3(+)/CD69(+), CD4(+)/CD25(+), CD8(+) T-cell, CD19(+), B220/CD23(+) B-cell, MHCII(+)/CD11c(+), and Gr-1(+)/CD11b(+) cell activations. It further suppressed anti-CII- or anti-RF-IgG/IgM, TNF-α, IL-1ß, IL-17, and IL-6 secretions but stimulated IL-10 levels in the serum, joint, or splenocyte. RGSF-A attenuated arthritis severity, modified leukocyte activations, and restored cytokine imbalances, suggesting that it can be considered as an antiarthritic agent with the capacity to ameliorate the immune and inflammatory responses in CIA mice.
Assuntos
Artrite Experimental/tratamento farmacológico , Ginsenosídeos/química , Panax/química , Extratos Vegetais/uso terapêutico , Saponinas/uso terapêutico , Animais , Artrite Experimental/sangue , Cromatografia Líquida de Alta Pressão , Interleucina-10/sangue , Interleucina-17/sangue , Interleucina-6/sangue , Masculino , Camundongos , Fator de Necrose Tumoral alfa/sangueRESUMO
Regulator of G-protein signaling 5 (RGS5), an inhibitor of Gα(q) and Gα(i) activation, has been reported to have antiatherosclerosis. Previous studies showed antiatherosclerotic effect of Korean red ginseng water extract (KRGE) via multiple signaling pathways. However, potential protective effect of KRGE through RGS5 expression has not been elucidated. Here, we investigated the antiatherosclerotic effect of KRGE in vivo and in vitro and its role on RGS5 mRNA expression. Elevated levels of total cholesterol, lactate dehydrogenase (LDH), and triglyceride (TG) in western diet groups of low-density lipoprotein receptor deficient LDLr(-/-) mice were reversed by oral administration of KRGE. KRGE suppressed transcriptional activity of tumor necrotic factor alpha (TNF-α), interleukin-6 (IL-6), and leptin in adipose tissue. It also potently repressed western diet-induced atheroma formation in aortic sinus. While KRGE showed reduced mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1ß, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells, it enhanced mRNA expression of RGS5. Moreover, RGS5 siRNA transfection of microglia cells pretreated with KRGE reversed its inhibitory effect on the expression of iNOS, COX-2, and IL-1ß mRNA. In conclusion, KRGE showed antiatherosclerotic and anti-inflammatory effects in western diet fed LDLr(-/-) mice and this effect could partly be mediated by RGS5 expression.
RESUMO
A number of reagents that prevent thrombosis have been developed but were found to have serious side effects. Therefore, we sought to identify complementary and alternative medicinal materials that are safe and have long-term efficacy. In the present studies, we have assessed the ability of chlorine e6 (CE6) to inhibit ADP-induced aggregation of rat platelets and elucidated the underlying mechanism. CE6 inhibited platelet aggregation induced by 10 µM ADP in a concentration-dependent manner and decreased intracellular calcium mobilization and granule secretion (i.e., ATP and serotonin release). Western blotting revealed that CE6 strongly inhibited the phosphorylations of PI3K, Akt, c-Jun N-terminal kinase (JNK), and different mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (ERK1/2) as well as p38-MAPK. Our study also demonstrated that CE6 significantly elevated intracellular cAMP levels and decreased thromboxane A2 formation in a concentration-dependent manner. Furthermore, we determined that CE6 initiated the activation of PKA, an effector of cAMP. Taken together, our findings indicate that CE6 may inhibit ADP-induced platelet activation by elevating cAMP levels and suppressing PI3K/Akt activity. Finally, these results suggest that CE6 could be developed as therapeutic agent that helps prevent thrombosis and ischemia.