Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) signaling pathway and whether CD40 signaling requires TRAF2. Methods Human B cell li...Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) signaling pathway and whether CD40 signaling requires TRAF2. Methods Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2,TRAF2-shRNA,or TRAF6-shRNA. The activation of NF-κB was detected by Western blot,kinase assay,transfactor enzyme-linked immunosorbent assay (ELISA),and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-κB activity was examined following stimulation with recombinant CD154. Results TRAF2 induced activity of IκB-kinases (IKKα,IKKi/ε),phosphorylation of IκBα,as well as nuclear translocation and phosphorylation of p65/RelA. In contrast,TRAF6 strongly induced NF-κB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA,but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However,the two TRAFs competed for CD40 binding. Conclusions These results indicate that TRAF2 can signal in human B cells,but it is not essential for CD40-mediated NF-κB activation. Moreover,TRAF2 can compete with TRAF6 for CD40 binding,and thereby limit the capacity of CD40 engagement to induce NF-κB activation.展开更多
AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of c...AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide(MTT) assay.The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay(ELISA) detection kit,flow cytometry(FCM) after propidium iodide(PI) staining and DNA agarose gel electrophoresis.The caspase activities were measured using ELISA.Reactive oxygen species(ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCFH-DA) probe labeling.Intracellular glutathione(GSH) content was measured using a glutathione assay kit.The expression of death receptor(DR)4 and DR5 proteins was analyzed by Western blotting and FCM.RESULTS:Casticin significantly inhibited the growth of human HCC(PLC/PRF/5 and Hep G2) cells in a dosedependent manner(P < 0.05).Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner.The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil(26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h.Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3,-8 and-9 in a concentration-dependent manner(P < 0.05).Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder.Casticin reduced the GSH content(P < 0.05),but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells.The thiol antioxidants,acetylcysteine(NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis.In contrast,the nonthiol antioxidants,butylated hydroxyanisole and mannitol failed to do so.In the HCC cells treated with casticin for 24 h,DR5 protein level was increased.The expression of DR5 protein induced by casticin was inhibited by NAC.Pretreatment with DR5/Fc chimera protein,a blocking antibody,effectively attenuated the induction of apoptosis by casticin.CONCLUSION:Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.展开更多
AIM:To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor(MIF)/Toll-like receptor 4(TLR4) in gastric cancer.METHODS:CD74,MIF and TLR4 expression in the paraffin-embedded...AIM:To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor(MIF)/Toll-like receptor 4(TLR4) in gastric cancer.METHODS:CD74,MIF and TLR4 expression in the paraffin-embedded sections of gastric cancer from 120 patients were detected by immunohistochemical staining.Knock down of CD74 expression in gastric cancer cell line MKN-45 was performed by lentivirus transduction and detected by Western blotting.MKN-45 cell proliferation assay under the stimulants was measured by the cell counting kit 8(CCK8) assay and MIF concentration in the culture medium was detected by enzymelinked immunosorbent assay.Surface staining of CD74 in the MKN-45 cell line under the stimulation of lipopolysaccharide(LPS) was measured by flow cytometry.MIF,CD74 and TLR4 co-localization in the MKN-45 cell line was performed by the immunoprecipitation.RESULTS:CD74,MIF and TLR4 were found to be expressed in gastric cancer and increased significantly in the advanced stage,and were also associated with lymph node metastasis.Correlation analysis revealed that CD74 was positively correlated with MIF(r = 0.2367,P < 0.01) and both proteins were also associated with TLR4(r = 0.4414,r = 0.5001,respectively,P < 0.01).LPS can significantly promote MKN-45 cell proliferation(3.027 ± 0.388 vs 4.201 ± 0.092,P < 0.05),induce MIF production(54.333 ± 2.906 pg/mL vs 29.667 ± 3.180 pg/mL,P < 0.01) and cell surface expression of CD74(75.6% ± 4.046%vs 9.4% ± 0.964%,P < 0.01) at LPS concentration of 1 μg/mL compared to medium control.Knockdown of CD74 or using antiCD74 and MIF antagonist ISO-1 significantly reduced LPS-induced MKN-45 cell proliferation(4.201 ± 0.092 vs 3.337 ± 0.087,4.534 ± 0.222 vs 3.368 ± 0.290,4.058 ± 0.292vs 2.934 ± 0.197,respectively,P < 0.01).MIF,CD74 and TLR4 could co-localize in the MKN-45 cell line.CONCLUSION:Upregulation of MIF,CD74 and TLR4 are associated with increasing clinical stage and provide an opportunity as novel gastric cancer chemoprevention and/or treatment strategy.展开更多
Objective:The aim of this study was to observe and compare the antiemetic effectiveness and adverse events of magnetotherapy plus 5-hydroxytryptamine (5-HT3) receptor inhibitor granisetron hydrochloride vs granisetron...Objective:The aim of this study was to observe and compare the antiemetic effectiveness and adverse events of magnetotherapy plus 5-hydroxytryptamine (5-HT3) receptor inhibitor granisetron hydrochloride vs granisetron hydrochloride alone in patients with chemotherapy.Methods:Sixty-four patients were randomized to receive either granisetron hydrochloride alone (control group:granisetron hydrochloride 3 mg intervenous infusion before chemotherapy, from the 1st day of chemotherapy course until the day after chemotherapy course completed) or magnetotherapy plus granisetron hydrochloride (treatment group:the same granisetron hydrochloride regimen plus rotatory magnetotherapy of 1 every day after chemotherapy for 5 begin with chemotherapy).Baseline characteristics were similar in both groups.The patients' emesia was evaluated according to WHO's criteria.The density of 5-HT3 was detected by enzyme-linked immunosorbent assay (ELISA).Results:In the treatment of acute vomiting, there was no significant difference between two groups (P>0.05), but in the treatment of tardive vomiting, the effectiveness in treatment group was better than that in control group (P<0.05).The density of 5-HT3 in treatment group and control group were (225.32±57.29) ng/mL vs (213.00 ±53.29) ng/mL before chemotherapy and (273.88 ±75.42) ng/mL vs (313.17±76.36) ng/mL after chemotherapy (P<0.01); the rate of adverse events was 36.36% and 48.39% respectively in treatment group and control group (P>0.05).Conclusion:Magnetotherapy plus granisetron hydrochloride provide better effectiveness than granisetron hydrochloride alone, and both therapies have synergistic effect.The adverse events didn't raised in treatment group.展开更多
G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced...G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant （K220R） caused precocious differentiation of ceUs into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.展开更多
基金Supported by Key Projects of the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (2008-BAI59B02)
文摘Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) signaling pathway and whether CD40 signaling requires TRAF2. Methods Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2,TRAF2-shRNA,or TRAF6-shRNA. The activation of NF-κB was detected by Western blot,kinase assay,transfactor enzyme-linked immunosorbent assay (ELISA),and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-κB activity was examined following stimulation with recombinant CD154. Results TRAF2 induced activity of IκB-kinases (IKKα,IKKi/ε),phosphorylation of IκBα,as well as nuclear translocation and phosphorylation of p65/RelA. In contrast,TRAF6 strongly induced NF-κB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA,but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However,the two TRAFs competed for CD40 binding. Conclusions These results indicate that TRAF2 can signal in human B cells,but it is not essential for CD40-mediated NF-κB activation. Moreover,TRAF2 can compete with TRAF6 for CD40 binding,and thereby limit the capacity of CD40 engagement to induce NF-κB activation.
基金Supported by The Scientifi c Research Project of Hunan Provincial Administration Bureau of Traditional Chinese Medicine,No. 2010081Scientific Research Project of Hunan Provincial Health Department,No. B2010-030Major Projects of Scien-tific Research of Hunan Provincial Department of Education,No. 09A054
文摘AIM:To investigate the apoptotic activities of casticin in hepatocellular carcinoma(HCC) cells and its molecular mechanisms.METHODS:PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide(MTT) assay.The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay(ELISA) detection kit,flow cytometry(FCM) after propidium iodide(PI) staining and DNA agarose gel electrophoresis.The caspase activities were measured using ELISA.Reactive oxygen species(ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCFH-DA) probe labeling.Intracellular glutathione(GSH) content was measured using a glutathione assay kit.The expression of death receptor(DR)4 and DR5 proteins was analyzed by Western blotting and FCM.RESULTS:Casticin significantly inhibited the growth of human HCC(PLC/PRF/5 and Hep G2) cells in a dosedependent manner(P < 0.05).Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner.The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil(26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h.Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3,-8 and-9 in a concentration-dependent manner(P < 0.05).Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder.Casticin reduced the GSH content(P < 0.05),but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells.The thiol antioxidants,acetylcysteine(NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis.In contrast,the nonthiol antioxidants,butylated hydroxyanisole and mannitol failed to do so.In the HCC cells treated with casticin for 24 h,DR5 protein level was increased.The expression of DR5 protein induced by casticin was inhibited by NAC.Pretreatment with DR5/Fc chimera protein,a blocking antibody,effectively attenuated the induction of apoptosis by casticin.CONCLUSION:Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.
基金Supported by Shanghai Municipal Natural Science Foundation, No.09DZ1907203 and No.10411950400National Natural Science Foundation of China,No.81072009
文摘AIM:To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor(MIF)/Toll-like receptor 4(TLR4) in gastric cancer.METHODS:CD74,MIF and TLR4 expression in the paraffin-embedded sections of gastric cancer from 120 patients were detected by immunohistochemical staining.Knock down of CD74 expression in gastric cancer cell line MKN-45 was performed by lentivirus transduction and detected by Western blotting.MKN-45 cell proliferation assay under the stimulants was measured by the cell counting kit 8(CCK8) assay and MIF concentration in the culture medium was detected by enzymelinked immunosorbent assay.Surface staining of CD74 in the MKN-45 cell line under the stimulation of lipopolysaccharide(LPS) was measured by flow cytometry.MIF,CD74 and TLR4 co-localization in the MKN-45 cell line was performed by the immunoprecipitation.RESULTS:CD74,MIF and TLR4 were found to be expressed in gastric cancer and increased significantly in the advanced stage,and were also associated with lymph node metastasis.Correlation analysis revealed that CD74 was positively correlated with MIF(r = 0.2367,P < 0.01) and both proteins were also associated with TLR4(r = 0.4414,r = 0.5001,respectively,P < 0.01).LPS can significantly promote MKN-45 cell proliferation(3.027 ± 0.388 vs 4.201 ± 0.092,P < 0.05),induce MIF production(54.333 ± 2.906 pg/mL vs 29.667 ± 3.180 pg/mL,P < 0.01) and cell surface expression of CD74(75.6% ± 4.046%vs 9.4% ± 0.964%,P < 0.01) at LPS concentration of 1 μg/mL compared to medium control.Knockdown of CD74 or using antiCD74 and MIF antagonist ISO-1 significantly reduced LPS-induced MKN-45 cell proliferation(4.201 ± 0.092 vs 3.337 ± 0.087,4.534 ± 0.222 vs 3.368 ± 0.290,4.058 ± 0.292vs 2.934 ± 0.197,respectively,P < 0.01).MIF,CD74 and TLR4 could co-localize in the MKN-45 cell line.CONCLUSION:Upregulation of MIF,CD74 and TLR4 are associated with increasing clinical stage and provide an opportunity as novel gastric cancer chemoprevention and/or treatment strategy.
基金Supported by a grant from the Government Science and Technology Agency of Zhejiang Province (No.2006C23018)
文摘Objective:The aim of this study was to observe and compare the antiemetic effectiveness and adverse events of magnetotherapy plus 5-hydroxytryptamine (5-HT3) receptor inhibitor granisetron hydrochloride vs granisetron hydrochloride alone in patients with chemotherapy.Methods:Sixty-four patients were randomized to receive either granisetron hydrochloride alone (control group:granisetron hydrochloride 3 mg intervenous infusion before chemotherapy, from the 1st day of chemotherapy course until the day after chemotherapy course completed) or magnetotherapy plus granisetron hydrochloride (treatment group:the same granisetron hydrochloride regimen plus rotatory magnetotherapy of 1 every day after chemotherapy for 5 begin with chemotherapy).Baseline characteristics were similar in both groups.The patients' emesia was evaluated according to WHO's criteria.The density of 5-HT3 was detected by enzyme-linked immunosorbent assay (ELISA).Results:In the treatment of acute vomiting, there was no significant difference between two groups (P>0.05), but in the treatment of tardive vomiting, the effectiveness in treatment group was better than that in control group (P<0.05).The density of 5-HT3 in treatment group and control group were (225.32±57.29) ng/mL vs (213.00 ±53.29) ng/mL before chemotherapy and (273.88 ±75.42) ng/mL vs (313.17±76.36) ng/mL after chemotherapy (P<0.01); the rate of adverse events was 36.36% and 48.39% respectively in treatment group and control group (P>0.05).Conclusion:Magnetotherapy plus granisetron hydrochloride provide better effectiveness than granisetron hydrochloride alone, and both therapies have synergistic effect.The adverse events didn't raised in treatment group.
文摘G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant （K220R） caused precocious differentiation of ceUs into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.
