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E7050 is an inhibitor of VEGFR2 with anti-tumor activity; however, its therapeutic mechanism remains incompletely understood. In the present study, we aim to evaluate the anti-angiogenic activity of E7050 in vitro and in vivo and define the underlying molecular mechanism. It was observed that treatment with E7050 markedly inhibited proliferation, migration, and capillary-like tube formation in cultured human umbilical vein endothelial cells (HUVECs). E7050 exposure in the chick embryo chorioallantoic membrane (CAM) also reduced the amount of neovessel formation in chick embryos. To understand the molecular basis, E7050 was found to suppress the phosphorylation of VEGFR2 and its downstream signaling pathway components, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. Moreover, E7050 suppressed the phosphorylation of VEGFR2, FAK, Src, Akt, JNK, and p38 MAPK in HUVECs exposed to MES-SA/Dx5 cells-derived conditioned medium (CM). The multidrug-resistant human uterine sarcoma xenograft study revealed that E7050 significantly attenuated the growth of MES-SA/Dx5 tumor xenografts, which was associated with inhibition of tumor angiogenesis. E7050 treatment also decreased the expression of CD31 and p-VEGFR2 in MES-SA/Dx5 tumor tissue sections in comparison with the vehicle control. Collectively, E7050 may serve as a potential agent for the treatment of cancer and angiogenesis-related disorders.
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Sarcoma , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Animais , Embrião de Galinha , Humanos , Inibidores da Angiogênese/uso terapêutico , Movimento Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sarcoma/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
E7050 is a potent inhibitor of c-Met receptor tyrosine kinase and has potential for cancer therapy. However, the underlying molecular mechanism involved in the anti-cancer property of E7050 has not been fully elucidated. The main objective of this study was to investigate the anti-tumor activity of E7050 in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells in vitro and in vivo, and to define its mechanisms. Our results revealed that E7050 reduced cell viability of MES-SA/Dx5 cells, which was associated with the induction of apoptosis and S phase cell cycle arrest. Additionally, E7050 treatment significantly upregulated the expression of Bax, cleaved PARP, cleaved caspase-3, p21, p53 and cyclin D1, while it downregulated the expression of survivin and cyclin A. On the other hand, the mechanistic study demonstrated that E7050 inhibited the phosphorylation of c-Met, Src, Akt and p38 in HGF-stimulated MES-SA/Dx5 cells. Further in vivo experiments showed that treatment of athymic nude mice carrying MES-SA/Dx5 xenograft tumors with E7050 remarkably suppressed tumor growth. E7050 treatment also decreased the expression of Ki-67 and p-Met, and increased the expression of cleaved caspase-3 in MES-SA/Dx5 tumor sections. Therefore, E7050 is a promising drug that can be developed for the treatment of multidrug-resistant uterine sarcoma.
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Sarcoma , Neoplasias de Tecidos Moles , Neoplasias Uterinas , Camundongos , Feminino , Animais , Humanos , Proteínas Proto-Oncogênicas c-met/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Camundongos Nus , Apoptose , Sarcoma/metabolismo , Neoplasias Uterinas/patologia , Transdução de SinaisRESUMO
BACKGROUND: Pulmonary fibrosis is a progressive diffuse parenchymal lung disorder with a high mortality rate. Studies have indicated that injured lung tissues release various pro-inflammatory factors, and produce a large amount of nitric oxide. There is also accumulation of collagen and oxidative stress-induced injury, collectively leading to pulmonary fibrosis. Antrodia cinnamomea is an endemic fungal growth in Taiwan, and its fermented extracts exert anti-inflammatory effects to alleviate liver damages. Hence, we hypothesized and tested the feasibility of using A. cinnamomea extracts for treatment of pulmonary fibrosis. METHODS: The TGF-ß1-induced human lung fibroblast cells (MRC-5) in vitro cell assay were used to evaluate the effects of A. cinnamomea extracts on the collagen production in MRC-5. Eight-week-old ICR mice were intratracheally administered bleomycin and then fed with an A. cinnamomea extract on day 3 post-administration of bleomycin. At day 21 post-bleomycin administration, the pulmonary functional test, the expression level of inflammation- and fibrosis-related genes in the lung tissue, and the histopathological change were examined. RESULTS: The A. cinnamomea extract significantly attenuated the expression level of collagen in the TGF-ß1-induced MRC-5â¯cells. In the A. cinnamome-treated bleomycin-induced lung fibrotic mice, the bodyweight increased, pulmonary functions improved, the lung tissues expression level of inflammatory factor and the fibrotic indicator were decreased, and the histopathological results showed the reduction of thickening of the inter-alveolar septa. CONCLUSIONS: The Antrodia cinnamomea extract significant protects mice against bleomycin-induced lung injuries through improvement of body weight gain and lung functions, and attenuation of expression of inflammatory and fibrotic indicators.
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2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-Glucoside (THSG) is the main active ingredient extracted from Polygonum multiflorum Thunb. (PMT), which has been reported to possess extensive pharmacological properties. Nevertheless, the exact role of THSG in pulmonary fibrosis has not been demonstrated yet. The main purpose of this study was to investigate the protective effect of THSG against bleomycin (BLM)-induced lung fibrosis in a murine model, and explore the underlying mechanisms of THSG in transforming growth factor-beta 1 (TGF-ß1)-induced fibrogenesis using MRC-5 human lung fibroblast cells. We found that THSG significantly attenuated lung injury by reducing fibrosis and extracellular matrix deposition. THSG treatment significantly downregulated the expression levels of TGF-ß1, fibronectin, α-SMA, CTGF, and TGFBR2, however, upregulated the expression levels of antioxidants (SOD-1 and catalase) and LC3B in the lungs of BLM-treated mice. THSG treatment decreased the expression levels of fibronectin, α-SMA, and CTGF in TGF-ß1-stimulated MRC-5 cells. Conversely, THSG increased the expression levels of SOD-1 and catalase. Furthermore, treatment of THSG profoundly reduced the TGF-ß1-induced generation of reactive oxygen species (ROS). In addition, THSG restored TGF-ß1-induced impaired autophagy, accompany by increasing the protein levels of LC3B-II and Beclin 1. Mechanism study indicated that THSG significantly reduced TGF-ß1-induced increase of TGFBR2 expression and phosphorylation of Smad2/3, Akt, mTOR, and ERK1/2 in MRC-5 cells. These findings suggest that THSG may be considered as an anti-fibrotic drug for the treatment of pulmonary fibrosis.
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Resveratrol, a naturally occurring dietary compound with chemopreventive properties has been reported to trigger a variety of cancer cell types to apoptosis. Whether resveratrol shows any activity on human nasopharyngeal carcinoma (NPC) cells remained to be determined. The aim of this study was to investigate the effect and mechanism of resveratrol on human NPC cells. Treatment of resveratrol resulted in significant decrease in cell viability of NPC cell lines in a dose- and time-dependent manner. A dose-dependent apoptotic cell death was also measured by flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled resveratrol treatment resulted in a significant loss of mitochondrial transmembrane potential, release of cytochrome c, enhanced expression of Fas ligand (FasL), and suppression of glucose-regulated protein 78 kDa (GRP78). These were followed by activation of caspases-9, -8, -4, and -3, subsequently leading to DNA fragmentation and cell apoptosis. Furthermore, up-regulation of proapoptotic Bax and down-regulation of antiapoptotic Bcl-2 protein were also observed. Taken together, resveratrol induces apoptosis in human NPC cells through regulation of multiple apoptotic pathways, including death receptor, mitochondria, and endoplasmic reticulum (ER) stress. Resveratrol can be developed as an effective compound for human NPC treatment.
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Apoptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Carcinoma , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Chaperona BiP do Retículo Endoplasmático , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Resveratrol , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
PURPOSE: Human papillomavirus (HPV) 16/18 infection is reported to be associated with nonsmoking Taiwanese female lung cancer. In this study, we attempted to further reveal the association between HPV infection with Mcl-1 and interleukin (IL)-6 expressions and to elucidate the roles of HPV infection in lung tumorigenesis. EXPERIMENTAL DESIGN: IL-6 and Mcl-1 expressions were investigated in 79 tumor tissues from lung cancer patients by immunohistochemistry. Secreting IL-6 levels and Mcl-1 expressions were examined by ELISA and Western blot, respectively, in HPV 16/18 E6- and E7-transfected A549 human lung cancer cells, as well as in the HPV16-infected TL-1 lung cancer cells established from lung cancer patients. RESULTS: Lung tumors (70.9% and 57.0%) had positive IL-6 and Mcl-1 immunostainings, respectively. Significant correlation between IL-6 and Mcl-1 expression were observed (P < 0.0001). Both IL-6 and Mcl-1 expression were significantly associated with HPV 16/18 infection (P = 0.014 and P = 0.004, respectively). IL-6 and Mcl-1 protein levels were not only elevated in HPV 16/18 E6- and E7-transfected A549 cells but also in TL-1 cells. Phosphatidylinositol-3-OH kinase pathway was the major pathway contributing to the up-regulation of Mcl-1 by IL-6 in HPV-infected lung cancer cells. CONCLUSIONS: The up-regulating effects of HPV 16/18 E6 and E7 to IL-6 and Mcl-1 expressions were observed in E6- and E7-transfected A549 cells and in HPV16-infected TL-1 cells, mainly through the phosphatidylinositol-3-OH kinase pathway. The involvement of HPV infection in lung tumorigenesis may be partly through a concomitant increased expression of autocrine and/or paracrine IL-6 and the downstream Mcl-1.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/metabolismo , Interleucina-6/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células MieloidesRESUMO
INTRODUCTION: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). Emphysema progression attributed not only to alveolar structure loss and pulmonary regeneration impairment, but also to excessive inflammatory response, proteolytic and anti-proteolytic activity imbalance, lung epithelial cells apoptosis, and abnormal lung remodeling. To ameliorate lung damage with higher efficiency in lung tissue engineering and cell therapy, pre-differentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. In this study, we aimed to evaluate the regenerative and repair ability of lung alveolar epithelium in emphysema model by using lung epithelial progenitors which pre-differentiated from amniotic fluid mesenchymal stem cells (AFMSCs). METHODS: Pre-differentiation of eGFP-expressing AFMSCs to lung epithelial progenitor-like cells (LEPLCs) was established under a modified small airway growth media (mSAGM) for 7-day induction. Pre-differentiated AFMSCs were intratracheally injected into porcine pancreatic elastase (PPE)-induced emphysema mice at day 14, and then inflammatory-, fibrotic-, and emphysema-related indices and pathological changes were assessed at 6 weeks after PPE administration. RESULTS: An optimal LEPLCs pre-differentiation condition has been achieved, which resulted in a yield of approximately 20% lung epithelial progenitors-like cells from AFMSCs in a 7-day period. In PPE-induced emphysema mice, transplantation of LEPLCs significantly improved regeneration of lung tissues through integrating into the lung alveolar structure, relieved airway inflammation, increased expression of growth factors such as vascular endothelial growth factor (VEGF), and reduced matrix metalloproteinases and lung remodeling factors when compared with mice injected with AFMSCs. Histopathologic examination observed a significant amelioration in DNA damage in alveolar cells, detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), the mean linear intercept, and the collagen deposition in the LEPLC-transplanted groups. CONCLUSION: Transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse elastase-induced pulmonary emphysema in PPE-induced pulmonary emphysema mice.
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Líquido Amniótico/citologia , Diferenciação Celular/fisiologia , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/terapia , Fibrose Pulmonar/metabolismo , Animais , Western Blotting , Diferenciação Celular/genética , Citometria de Fluxo , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Elastase Pancreática/toxicidade , Alvéolos Pulmonares/citologia , Reação em Cadeia da Polimerase em Tempo Real , Mucosa Respiratória/citologia , Suínos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We examined the effects of an Antrodia cinnamomea ethanol extract (ACEE) on lung cancer cells in vitro and tumor growth in vivo. ACEE produced dose-dependent cytotoxic effects and induced apoptosis in Lewis lung carcinoma (LLC) cells. ACEE treatment increased expression of p53 and Bax, as well as cleavage of caspase-3 and PARP, while reducing expression of survivin and Bcl-2. ACEE also reduced the levels of JAK2 and phosphorylated STAT3 in LLC cells. In a murine allograft tumor model, oral administration of ACEE significantly inhibited LLC tumor growth and metastasis without affecting serum biological parameters or body weight. ACEE increased cleavage of caspase-3 in murine tumors, while decreasing STAT3 phosphorylation. In addition, ACEE reduced the growth of human tumor xenografts in nude mice. Our findings therefore indicate that ACEE inhibits lung tumor growth and metastasis by inducing apoptosis and by inhibiting the STAT3 signaling pathway in cancer cells.
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Antineoplásicos Fitogênicos/farmacologia , Antrodia/química , Carcinoma Pulmonar de Lewis , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Extratos Vegetais/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Animais , Antineoplásicos Fitogênicos/química , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Extratos Vegetais/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal mushroom Antrodia cinnamomea has been used to treat cancer but its anti-angiogenic effects have not been studied in detail. AIM OF THE STUDY: The main objective of this study was to determine the molecular mechanism of activity underlying the anti-angiogenic effects of A. cinnamomea. MATERIALS AND METHODS: The effects of an A. cinnamomea ethanol extract (ACEE) on cell migration and microvessel formation were investigated in endothelial cells in vitro and Matrigel plugs implanted into mice in vivo. Activation of intracellular signaling pathways was examined using Western blotting. Protein expression was assessed using immunohistochemistry in a mouse model of lung metastasis. RESULTS: We show that treatment with ACEE inhibits cell migration and tube formation in human umbilical vein endothelial cells (HUVECs). ACEE suppresses phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and expression of pro-angiogenic kinases in vascular endothelial growth factor (VEGF)-treated HUVECs, in addition to reducing expression of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3). ACEE treatment inhibits VEGF-induced microvessel formation in Matrigel plugs in vivo. In addition, ACEE significantly reduces VEGFR2 expression in Lewis lung carcinoma cells and downregulates the expression of cluster of differentiation 31 (CD31) and VEGFR2 in murine lung metastases. CONCLUSION: These results indicate that A. cinnamomea produces anti-angiogenic effects by inhibiting the VEGFR2 signaling pathway.
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Inibidores da Angiogênese/farmacologia , Antrodia/química , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/isolamento & purificação , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mesenchymal stem cells (MSCs) are widely considered for treatment of pulmonary fibrosis based on the anti-inflammatory, antifibrotic, antiapoptotic, and regenerative properties of the cells. Recently, elevated levels of oncostatin M (OSM) have been reported in the bronchoalveolar lavage fluid of a pulmonary fibrosis animal model and in patients. In this work, we aimed to prolong engrafted MSC survival and to enhance the effectiveness of pulmonary fibrosis transplantation therapy by using OSM-preconditioned MSCs. OSM-preconditioned MSCs were shown to overexpress type 2 OSM receptor (gp130/OSMRß) and exhibited high susceptibility to OSM, resulting in upregulation of the paracrine factor, hepatocyte growth factor (HGF). Moreover, OSM-preconditioned MSCs enhanced cell proliferation and migration, attenuated transforming growth factor-ß1- or OSM-induced extracellular matrix production in MRC-5 fibroblasts through paracrine effects. In bleomycin-induced lung fibrotic mice, transplantation of OSM-preconditioned MSCs significantly improved pulmonary respiratory functions and downregulated expression of inflammatory factors and fibrotic factors in the lung tissues. Histopathologic examination indicated remarkable amelioration of the lung fibrosis. LacZ-tagged MSCs were detected in the lung tissues of the OSM-preconditioned MSC-treated mice 18 days after post-transplantation. Taken together, our data further demonstrated that HGF upregulation played an important role in mediating the therapeutic effects of transplanted OSM-preconditioned MSCs in alleviating lung fibrosis in the mice. Stem Cells Translational Medicine 2017;6:1006-1017.
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Bleomicina/toxicidade , Fator de Crescimento de Hepatócito/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Oncostatina M/farmacologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/terapia , Animais , Células Cultivadas , Feminino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal mushroom Antrodia cinnamomea possesses anticancer properties but the active compounds responsible for these effects are mostly unknown. AIM OF THE STUDY: We aimed to identify novel A. cinnamomea compounds that produce cytotoxic effects on cancer cells. MATERIALS AND METHODS: Using ethanol extraction and chromatography, we isolated the lanostanoid compound lanosta-7,9(11),24-trien-3ß,15α,21-triol (1) from cultured A. cinnamomea mycelium. Cytotoxicity and pro-apoptotic effects of compound 1 were evaluated using the MTS assay and flow cytometry analysis, respectively. RESULTS: Compound 1 produced cytotoxic effects on the nasopharyngeal carcinoma cell lines TW02 and TW04, with IC50 values of 63.3 and 115.0µM, respectively. On the other hand, no cytotoxic effects were observed on non-tumorigenic nasopharyngeal epithelial cells (NP69). In addition, compound 1 induced apoptosis in TW02 and TW04 cells as revealed by flow cytometry analysis. CONCLUSIONS: Our results demonstrate for the first time the presence of pinicolol B in A. cinnamomea mycelium and suggest that this compound may contribute to the anticancer effects of A. cinnamomea.
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Antineoplásicos/farmacologia , Antrodia , Triterpenos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Micélio , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológicoRESUMO
Hirsutella sinensis mycelium (HSM), the anamorph of Cordyceps sinensis, is a traditional Chinese medicine that has been shown to possess various pharmacological properties. We previously reported that this fungus suppresses interleukin-1ß and IL-18 secretion by inhibiting both canonical and non-canonical inflammasomes in human macrophages. However, whether HSM may be used to prevent lung fibrosis and the mechanism underlying this activity remain unclear. Our results show that pretreatment with HSM inhibits TGF-ß1-induced expression of fibronectin and α-SMA in lung fibroblasts. HSM also restores superoxide dismutase expression in TGF-ß1-treated lung fibroblasts and inhibits reactive oxygen species production in lung epithelial cells. Furthermore, HSM pretreatment markedly reduces bleomycin-induced lung injury and fibrosis in mice. Accordingly, HSM reduces inflammatory cell accumulation in bronchoalveolar lavage fluid and proinflammatory cytokines levels in lung tissues. The HSM extract also significantly reduces TGF-ß1 in lung tissues, and this effect is accompanied by decreased collagen 3α1 and α-SMA levels. Moreover, HSM reduces expression of the NLRP3 inflammasome and P2X7R in lung tissues, whereas it enhances expression of superoxide dismutase. These findings suggest that HSM may be used for the treatment of pulmonary inflammation and fibrosis.
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Ascomicetos/fisiologia , Bleomicina/toxicidade , Micélio/fisiologia , Pneumonia/induzido quimicamente , Fibrose Pulmonar/induzido quimicamente , Animais , Humanos , Camundongos , Superóxido Dismutase/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD) is a sustained blockage of the airways due to lung inflammation occurring with chronic bronchitis and/or emphysema. Progression of emphysema may be slowed by vascular endothelial growth factor A (VEGFA), which reduces apoptotic tissue depletion. Previously, authors of the present report demonstrated that cis-resveratrol (c-RSV)-induced heat-shock protein 70 (HSP70) promoter-regulated VEGFA expression promoted neovascularization of genetically modified mesenchymal stem cells (HSP-VEGFA-MSC) in a mouse model of ischemic disease. Here, this same stem cell line was evaluated for its protective capacity to alleviate elastase-induced pulmonary emphysema in mice. Results of this study showed that c-RSV-treatment of HSP-VEGFA-MSC exhibited synergy between HSP70 transcription activity and induced expression of anti-oxidant-related genes when challenged by cigarette smoke extracts. Eight weeks after jugular vein injection of HSP-VEGFA-MSC into mice with elastase-induced pulmonary emphysema followed by c-RSV treatment to induce transgene expression, significant improvement was observed in respiratory functions. Expression of VEGFA, endogenous nuclear factor erythroid 2-related factor (Nrf 2), and manganese superoxide dismutase (MnSOD) was significantly increased in the lung tissues of the c-RSV-treated mice. Histopathologic examination of treated mice revealed gradual but significant abatement of emphysema and restoration of airspace volume. In conclusion, the present investigation demonstrates that c-RSV-regulated VEGFA expression in HSP-VEGFA-MSC significantly improved the therapeutic effects on the treatment of COPD in the mouse, possibly avoiding side effects associated with constitutive VEGFA expression.
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Enfisema/tratamento farmacológico , Proteínas de Choque Térmico HSP70/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Elastase Pancreática/farmacologia , Estilbenos/farmacologia , Estilbenos/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Enfisema/metabolismo , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Resveratrol , Fumaça/efeitos adversos , Nicotiana/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active ß-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.
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Apoptose/efeitos dos fármacos , Apoptose/genética , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Inibidores de Proteassoma/farmacologia , Via de Sinalização Wnt , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Humanos , Modelos BiológicosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia cinnamomea--a medicinal fungus that is indigenous to Taiwan--has been used as a health tonic by aboriginal tribes and the Asian population. Recent studies indicate that Antrodia cinnamomea extracts exhibit hepato-protective, anti-hypertensive, anti-oxidative, anti-inflammatory, immuno-modulatory, and anti-cancer effects on cultured cells and laboratory animals. This study aims to explore the anti-inflammatory activity of an Antrodia cinnamomea ethanol extract (ACEE) and elucidate its underlying mechanisms of action using lipopolysaccharide (LPS)-primed, ATP-stimulated human THP-1 macrophages. MATERIALS AND METHODS: The effects of ACEE on cell viability were studied using the MTT assay. The expressions of genes, proteins, and pro-inflammatory cytokines were measured by quantitative real-time RT-PCR, Western blotting and ELISA, respectively. The ACEE was further investigated for its effects on reactive oxygen species (ROS) production using ROS detection kit. RESULTS: Our results showed that ACEE significantly inhibits ATP-induced secretion of interleukin-1ß (IL-1ß), interleukin-18 (IL-18) and tumor necrosis factor-α (TNF-α) by LPS-primed macrophages. ACEE also suppresses the transcription and activation of caspase-1, which is responsible for the cleavage and activation of IL-1ß and IL-18. Of note, ACEE not only reduces expression of the inflammasome component NLRP3 and the purinergic receptor P2X7R but also inhibits ATP-induced ROS production and caspase-1 activation. Furthermore, the anti-inflammatory properties of ACEE correlate with reduced activation of the MAPK and NF-κB pathways. CONCLUSION: The results of the present study indicate that Antrodia cinnamomea suppresses the secretion of IL-1ß and IL-18 associated with inhibition of the NLRP3 inflammasome in macrophages. These findings suggest that ACEE may have therapeutic potential for the treatment of inflammatory diseases.
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Anti-Inflamatórios/farmacologia , Antrodia/química , Inflamassomos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Anti-Inflamatórios/isolamento & purificação , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Medicina Tradicional do Leste Asiático/métodos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cancer stem cells (CSCs) represent a unique sub-population of tumor cells with the ability to initiate tumor growth and sustain self-renewal. Although CSC biomarkers have been described for various tumors, only a few markers have been identified for nasopharyngeal carcinoma (NPC). In this study, we show that CD24+ cells isolated from human NPC cell lines express stem cell genes (Sox2, Oct4, Nanog, Bmi-1, and Rex-1), and show activation of the Wnt/ß-catenin signaling pathway. CD24+ cells possess typical CSC characteristics that include enhanced cell proliferation, increased colony and sphere formation, maintenance of cell differentiation potential in prolonged culture, and enhanced resistance to chemotherapeutic drugs. Notably, CD24+ cells produce tumors following inoculation of as few as 500 cells in immunodeficient NOD/SCID mice. CD24+ cells further show increased invasion ability in vitro, which correlates with enhanced expression of matrix metalloproteinase 2 and 9. In summary, our results suggest that CD24 represents a novel CSC biomarker in NPC.
Assuntos
Biomarcadores Tumorais/metabolismo , Antígeno CD24/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Células-Tronco Neoplásicas/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Carcinoma , Contagem de Células , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Células Clonais , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/genética , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Esferoides Celulares/patologia , Via de Sinalização WntRESUMO
Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Perforina/metabolismo , Reishi/química , Animais , Anticorpos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , RNA/biossíntese , RNA/isolamento & purificação , RNA Interferente Pequeno/farmacologia , Receptores de Superfície Celular/biossíntese , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Although the mechanisms underlying the cytotoxic effect of NK cells on tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell-surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Bacteriólise , Células Matadoras Naturais/imunologia , Mycobacterium kansasii , Mycobacterium tuberculosis , Perforina/metabolismo , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/farmacologia , Bacteriólise/efeitos dos fármacos , Bacteriólise/fisiologia , Linhagem Celular Tumoral , Parede Celular/efeitos dos fármacos , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/ultraestrutura , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Nanotubos , Receptor 2 Desencadeador da Citotoxicidade Natural/antagonistas & inibidores , Receptor 2 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 2 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/antagonistas & inibidores , Receptor 3 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Perforina/biossíntese , Perforina/genética , Perforina/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Multidrug resistance in cancer cells arises from altered drug permeability of the cell. We previously reported activation of the Wnt pathway in ABCB1-overexpressed human uterus sarcoma drug-resistant MES-SA/Dx5 cells through active ß-catenin and associated transactivation activities, and upregulation of Wnt-targeting genes. In this study, Wnt5A was found to be significantly upregulated in MES-SA/Dx5 and MCF7/ADR2 cells, suggesting an important role for the Wnt5A signaling pathway in cancer drug resistance. Higher cAMP response elements and Tcf/Lef transcription activities were shown in the drug-resistant cancer cells. However, expression of Wnt target genes and CRE activities was downregulated in Wnt5A shRNA stably-transfected MES-SA/Dx5 cells. Cell viability of the drug-resistant cancer cells was also reduced by doxorubicin treatment and Wnt5A shRNA transfection, or by Wnt5A depletion. The in vitro data were supported by immunohistochemical analysis of 24 paired breast cancer biopsies obtained pre- and post-chemotherapeutic treatment. Wnt5A, VEGF and/or ABCB1 were significantly overexpressed after treatment, consistent with clinical chemoresistance. Taken together, the Wnt5A signaling pathway was shown to contribute to regulating the drug-resistance protein ABCB1 and ß-catenin-related genes in antagonizing the toxic effects of doxorubicin in the MDR cell lines and in clinical breast cancer samples.
Assuntos
Neoplasias da Mama/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Animais , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Transfecção , Proteína Wnt-5a , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
Resveratrol, a natural phenolic compound found in red grapes and wine, exists as cis and trans isomers. Recent studies have shown that trans-resveratrol possesses anti-inflammatory, anti-oxidant, anti-carcinogenic, anti-tumor and immunomodulatory properties. However, it remains unclear whether cis-resveratrol may exhibit similar activities. The objective of the present study was to examine the effects of cis- and trans-resveratrol on the production of pro-inflammatory cytokines and mediators in human macrophages. We examined the possibility that cis- and trans-resveratrol may affect cytokine secretion by modulating inflammasomes, intracellular multi-protein complexes, the assembly of which leads to caspase-1 activation and secretion of active IL-1ß by macrophages. Our results show that pre-treatment of macrophages with cis-resveratrol not only reduces pro-IL-1ß production and IL-1ß secretion, but also suppresses ATP-induced transcription and activation of caspase-1 and caspase-4. Notably, cis-resveratrol inhibits the expression of the purinergic receptor, P2X(7)R, and the endoplasmic reticulum stress marker, Glc-regulated protein 78, but also reduces reactive oxygen species production. Moreover, cis-resveratrol attenuates cyclooxygenase-2 expression and prostaglandin E2 production. cis-Resveratrol also decreases the phosphorylation of p38 MAPK and expression of the c-Jun protein. These results indicate that cis-resveratrol produces anti-inflammatory effects by inhibiting both the canonical and non-canonical inflammasomes, and associated pathways in human macrophages.