Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect o...Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide(MTT), scratch-wound and transwell assays, respectively. Co Cl2 was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immuno?uorescence analysis,respectively. Results: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner(P<0.01). Furthermore, the expression of epithelial-mesenchymal transition(EMT) related markers were also remarkably suppressed in a dose-dependent manner(P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole(YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced Hep G2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect(P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.展开更多
Objective:To characterize the molecular mechanism underlying the antineoplastic activity of Celastrus orbiculatus Thunb.extracts(COE).Methods:The human hepatocellular carcinoma HepG2 cells with mammalian target of rap...Objective:To characterize the molecular mechanism underlying the antineoplastic activity of Celastrus orbiculatus Thunb.extracts(COE).Methods:The human hepatocellular carcinoma HepG2 cells with mammalian target of rapamycin(mTOR)knockdown expressed(HepG2/mTOR-)were constructed using molecular biological technology.In vitro,the HepG2/mTOR-cells were treated with COE at various concentrations(10,20,40,80,160 and 320μg/mL).Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays.According to the half-maximal inhibitory concentration(IC50)value(140 mg/L),the concentrations of COE in the subsequent experiment was set to alleviate cytotoxicity.The HepG2/mTOR-cells were divided into 5 groups:negative control(untreated),COE treatment groups(40,80,120 mg/L COE)and positive control group(cisplatin,DDP,2 mg/L),respectively.Wild-type HepG2 cells were used as a blank control.The effects of COE on the cell apoptosis were analyzed by flow cytometry and transmission electronic microscopy(TEM),respectively.The protein expression levels of mTOR signal pathways were determined by Western blotting.In vivo,HepG2/mTOR-cells(2×106 cell/mice)were subcutaneously injected into the right flank of nude mice.Thirty-six female nude mice were randomly assigned to 6 groups according to body weight(6 mice per group)as follows:solvent vehicle control,Banmao Capsule treated group(BM,195 mg/kg),Tegafur,Gimeracil and Oteracil Potassium Capsules(10 mg/kg)treated group,and different dosages of COE(10,20,40 mg/kg)groups.Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues.Results:COE inhibited the proliferation significantly in a concentration-dependent manner in HepG2/mTOR-cells(P<0.01).COE significantly induced the apoptosis of HepG2/mTOR-cells(P<0.01),and the apoptotic bodies can be observed under TEM.COE significantly inhibits the proteins expression of mTORrelated signal pathways.In vivo,COE significantly in展开更多
Objective To examine the immunoregulation of Celastrus orbiculatus extracts (COE), a traditional Chinese medicine, on maturation and function of dendritic cells (DCs) in vitro and in vivo. Methods In vitro, after trea...Objective To examine the immunoregulation of Celastrus orbiculatus extracts (COE), a traditional Chinese medicine, on maturation and function of dendritic cells (DCs) in vitro and in vivo. Methods In vitro, after treated with COE in different nontoxic concentrations (0, 10, 20, 40, 80, and 160 μg/mL) for 5 d, the surface immunological molecules and cytokine secretion of mice bone marrow-derived DCs in response to COE were analyzed by flow cytometric analysis (FACS) and enzyme linked immunosorbent assay (ELISA), respectively. In vivo, mouse hepatoma cells (Hepa1-6, 1 × 106) were injected sc and were treated with different dosages of COE (10, 20 or 40 mg/kg/d). Effects on tumor growth were determined by tumor volume and histology analysis after 28 d administration of COE. The relative proportions of mature DCs and CD8+ T cells were measured in mononuclear cells that had been isolated from spleen by FACS. Results COE stimulated IL-2 and IFN-γ secretion of DCs, simultaneously enhanced the maturation of DCs by enhancing immunological molecule (CD40, CD80, CD86, H-2Kb, and I-Ab) expression in a dose-dependent manner. Furthermore, the chemotactic responses of DCs were significantly higher in COE-treated than untreated DCs, in association with higher chemokine receptor 7 expression. Furthermore, COE increased DCs produce IFN-γ and IL-2 in a dose-dependent manner when the concentration of COE less than 40 μg/mL, decreased DCs produce IL-10 and IL-4 also in a dose-dependent manner. In in vivo studies, COE can not only suppress growth of malignant hepatocellular carcinomas but also stimulate maturation of DCs, associated with strongly enhanced CD8+ CTL responses. Conclusion These data provide new insight into the mechanism of action of COE and indicate that the stimulation of maturation and function of DCs by COE contributes to its immunoregulatory effects.展开更多
基金Supported by the National Natural Science Foundation of China(No.81403232 and No.81573656)Natural Science Foundation of Jiangsu Province(No.BK20171290 and No.BK2012686)Doctoral Fund of Ministry of Education of China(No.20133250120003)
文摘Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide(MTT), scratch-wound and transwell assays, respectively. Co Cl2 was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immuno?uorescence analysis,respectively. Results: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner(P<0.01). Furthermore, the expression of epithelial-mesenchymal transition(EMT) related markers were also remarkably suppressed in a dose-dependent manner(P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole(YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced Hep G2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect(P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.
基金Supported by the National Natural Science Foundation of China(No.81403232)the National Natural Science Foundation of Jiangsu Province(No.BK20171290,BK2012686)the Doctoral Fund of the Ministry of Education of China(No.20133250120003)
文摘Objective:To characterize the molecular mechanism underlying the antineoplastic activity of Celastrus orbiculatus Thunb.extracts(COE).Methods:The human hepatocellular carcinoma HepG2 cells with mammalian target of rapamycin(mTOR)knockdown expressed(HepG2/mTOR-)were constructed using molecular biological technology.In vitro,the HepG2/mTOR-cells were treated with COE at various concentrations(10,20,40,80,160 and 320μg/mL).Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays.According to the half-maximal inhibitory concentration(IC50)value(140 mg/L),the concentrations of COE in the subsequent experiment was set to alleviate cytotoxicity.The HepG2/mTOR-cells were divided into 5 groups:negative control(untreated),COE treatment groups(40,80,120 mg/L COE)and positive control group(cisplatin,DDP,2 mg/L),respectively.Wild-type HepG2 cells were used as a blank control.The effects of COE on the cell apoptosis were analyzed by flow cytometry and transmission electronic microscopy(TEM),respectively.The protein expression levels of mTOR signal pathways were determined by Western blotting.In vivo,HepG2/mTOR-cells(2×106 cell/mice)were subcutaneously injected into the right flank of nude mice.Thirty-six female nude mice were randomly assigned to 6 groups according to body weight(6 mice per group)as follows:solvent vehicle control,Banmao Capsule treated group(BM,195 mg/kg),Tegafur,Gimeracil and Oteracil Potassium Capsules(10 mg/kg)treated group,and different dosages of COE(10,20,40 mg/kg)groups.Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues.Results:COE inhibited the proliferation significantly in a concentration-dependent manner in HepG2/mTOR-cells(P<0.01).COE significantly induced the apoptosis of HepG2/mTOR-cells(P<0.01),and the apoptotic bodies can be observed under TEM.COE significantly inhibits the proteins expression of mTORrelated signal pathways.In vivo,COE significantly in
基金Plans of Colleges and Universities in Jiangsu Province to Postgraduate Research and Innovation (CX09B-321Z)State Administration of Traditional Chinese Medicine of P.R.China (04-05ZP35)
文摘Objective To examine the immunoregulation of Celastrus orbiculatus extracts (COE), a traditional Chinese medicine, on maturation and function of dendritic cells (DCs) in vitro and in vivo. Methods In vitro, after treated with COE in different nontoxic concentrations (0, 10, 20, 40, 80, and 160 μg/mL) for 5 d, the surface immunological molecules and cytokine secretion of mice bone marrow-derived DCs in response to COE were analyzed by flow cytometric analysis (FACS) and enzyme linked immunosorbent assay (ELISA), respectively. In vivo, mouse hepatoma cells (Hepa1-6, 1 × 106) were injected sc and were treated with different dosages of COE (10, 20 or 40 mg/kg/d). Effects on tumor growth were determined by tumor volume and histology analysis after 28 d administration of COE. The relative proportions of mature DCs and CD8+ T cells were measured in mononuclear cells that had been isolated from spleen by FACS. Results COE stimulated IL-2 and IFN-γ secretion of DCs, simultaneously enhanced the maturation of DCs by enhancing immunological molecule (CD40, CD80, CD86, H-2Kb, and I-Ab) expression in a dose-dependent manner. Furthermore, the chemotactic responses of DCs were significantly higher in COE-treated than untreated DCs, in association with higher chemokine receptor 7 expression. Furthermore, COE increased DCs produce IFN-γ and IL-2 in a dose-dependent manner when the concentration of COE less than 40 μg/mL, decreased DCs produce IL-10 and IL-4 also in a dose-dependent manner. In in vivo studies, COE can not only suppress growth of malignant hepatocellular carcinomas but also stimulate maturation of DCs, associated with strongly enhanced CD8+ CTL responses. Conclusion These data provide new insight into the mechanism of action of COE and indicate that the stimulation of maturation and function of DCs by COE contributes to its immunoregulatory effects.
