Objective:To study the effects of Transient receptor potential cation channel,subfamily V,member 1(TRPV1) combined with lidocaine on status and apoptosis of U87-MG glioma cell line,and explore whether local anesthetic...Objective:To study the effects of Transient receptor potential cation channel,subfamily V,member 1(TRPV1) combined with lidocaine on status and apoptosis of U87-MG glioma cell line,and explore whether local anesthetic produces neurotoxicity by TRPVI.Methods:U87-MG cells were divided into control group,gene silencing group,empty vector group and TRPV gene up-rcgulation group.For cells in each group,flow cytometry was employed to detect the intracellular calcium ion concentration and mitochondrial membrane potential at different lime point from cellular perspective.Cell apoptosis of U87-MG was assayed by flow cytometry and MTT from a holistic perspective.Results:Calcium ion concentration increased along with time.The concentration in TRPV1 gene up-regulation group was significantly higher than those in other groups at each time point(P<0.05).After adding lidocaine.mitochondrial membrane potential in U87-MG significantly increased(P<0.05).This increasing trend in TRPV1 gene up-regulation group was more significant than other groups(P<0.05).while in TRPV1 gene silencing group,the trend significantly decreased(P<0.05).Flow cytometry result and MTT result both showed that cell apoptosis in each group significantly increased after lidocaine was added(P<0.05).This increasing trend in TRPV1 gene up-regulation group was more significant than other groups(P<0.05),while in TRPV1 gene silencing group,the trend significandy decreased(P<0.05).Moreover,apoptosis was more severe along with the increasing concentration of lidocaine(P<0.05).Conclusions:In this study,it was proved that lidocaine could dose-dependently induce the increase of intracellular calcium ion concentration,mitochondrial membrane potential and apoptosis in U87-MG glioma cell line.The up-regulation of TRPV1 enhanced cytotoxicity of lidocaine,which revealed the correlations between mem.Lidocaine might have increased intracellular calcium ion concentration by activating TRPVI gene and induced apoptosis of U87-GM glioma cell line by up-regulating mitochondrial membrane potential.展开更多
AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the ...AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the heterogenic cancer-derived NCI-N87 cell line were isolated,by stably-transducing it with the human telomerase reverse-transcriptase(h TERT) catalytic subunit gene.The clones were first characterized regarding their cell growth pattern and phenotype.For that we measured the clones' adherence properties,expression of cell-cell junctions' markers(ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance.The gastric properties of the clones,concerning expression of mucins,zymogens and glycan contents,were then evaluated by haematoxylin and eosin staining,Periodic acid Schiff(PAS) and PAS/Alcian Blue-staining,immunocytochemistry and Western blot.In addition,we assessed the usefulness of the h TERT-expressing gastric cell line for H.pylori research,by performing co-culture assays and measuring the IL-8 secretion,by ELISA,upon infection with two H.pylori strains differing in virulence.RESULTS:Compared with the parental cell line,themost promising NCI-hT ERT-derived clones(CL5 and CL6) were composed of cells with homogenous phenotype,presented higher relative telomerase activities,better adhesion properties,ability to be maintained in culture for longer periods after confluency,and were more efficient in PAS-reactive mucins secretion.Both clones were shown to produce high amounts of MUC1,MUC2 and MUC13.NCI-hT ERT-CL5 mucins were shown to be decorated with blood group H type 2(BG-H),Lewis-x(Lex),Ley and Lea and,in a less extent,with BG-A antigens,but the former two antigens were not detected in the NCI-h TERT-CL6.None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans.Entailing good gastric properties,both NCIhT ERT-clones were found to produce pepsinogen-5 and human gastric lipase.The progenitor-like phenotype of NCI-hT ERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5,supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.CONCLUSION:These traits,in addition to resistance to microaerobic conditions and good responsiveness to H.pylori co-culture,in a strain virulence-dependent manner,make the NCI-hT ERT-CL6 a promising model for future in vitro studies.展开更多
The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced int...The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection in vitro. The expression of CH50 polypeptide was detected by using immunohistochemical S-P method. The expression of the transfected gene was identified by RT-PCR. Cell invasion assay kit was applied to detect the effect of CH50 polypeptide on the invasion ability of BIU-87. The results showed that the BIU-87 cells transfected with pCH510 could express the CH50 polypeptide, while in the control group, no CH50 polypeptide was detectable. In the transfection group, the invasion ability of BIU-87 in vitro was lower than in control group (P<0.05). It was concluded that CH50 polypeptide was successfully expressed in BIU-87 cells by gene transfection, by which the in vitro invasion ability of BIU-87 was inhibited.展开更多
The expression of different vascular endothelial growth factor (VEGF) genes was studied in glioma U87 cells with endoplasmic reticulum–nuclei-1 (ERN1) loss of function and its regulation by hypoxia and glutamine or g...The expression of different vascular endothelial growth factor (VEGF) genes was studied in glioma U87 cells with endoplasmic reticulum–nuclei-1 (ERN1) loss of function and its regulation by hypoxia and glutamine or glucose deprivation conditions as model of ischemia. The blockade of function of the ERN1 enzyme, which is a major sensor of endoplasmic reticulum stress, leads to a decrease of the VEGFA, VEGFB and VEGFC mRNA expression level. The level of VEGFA proteins also decreases at this experimental condition in the cytosolic fraction, but increases in the nuclear fraction. Hypoxia does not affect VEGFC and increases the expression level of VEGFA and VEGFB mRNA in both used cell types, however, the change was much less profound in cells with suppressed function of ERN1. The expression level of VEGFC mRNA decreases in both used cell types in glutamine deprivation condition, however, the change was more profound in control glioma cells. At the same time, the expression level of VEGFA mRNA increases and VEGFB—decreases in gluta-mine deprivation condition in control glioma cells only. Exposure of glioma cells to glucose deprivation condition increases VEGFB mRNA expression level in both used cell types;however, VEGFA—in control glioma cells only and VEGFC—in cells with ERN1 signaling enzyme loss of function only. Thus, the results of this study clearly demonstrated the down-regulation of the expression of all three VEGF genes in glioma cells with ERN1 loss of function which correlates to the suppressed angiogenesis and proliferation rate of these cells. Moreover, the effect of hy-poxia and glutamine or glucose deprivation condition on the expression level of all VEGF genes is different and mainly depends on ERN1 signaling enzyme function.展开更多
We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was sho...We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase?2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment.?Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.展开更多
基金supported by the National Natural Science Foundation(NO.81260176)
文摘Objective:To study the effects of Transient receptor potential cation channel,subfamily V,member 1(TRPV1) combined with lidocaine on status and apoptosis of U87-MG glioma cell line,and explore whether local anesthetic produces neurotoxicity by TRPVI.Methods:U87-MG cells were divided into control group,gene silencing group,empty vector group and TRPV gene up-rcgulation group.For cells in each group,flow cytometry was employed to detect the intracellular calcium ion concentration and mitochondrial membrane potential at different lime point from cellular perspective.Cell apoptosis of U87-MG was assayed by flow cytometry and MTT from a holistic perspective.Results:Calcium ion concentration increased along with time.The concentration in TRPV1 gene up-regulation group was significantly higher than those in other groups at each time point(P<0.05).After adding lidocaine.mitochondrial membrane potential in U87-MG significantly increased(P<0.05).This increasing trend in TRPV1 gene up-regulation group was more significant than other groups(P<0.05).while in TRPV1 gene silencing group,the trend significantly decreased(P<0.05).Flow cytometry result and MTT result both showed that cell apoptosis in each group significantly increased after lidocaine was added(P<0.05).This increasing trend in TRPV1 gene up-regulation group was more significant than other groups(P<0.05),while in TRPV1 gene silencing group,the trend significandy decreased(P<0.05).Moreover,apoptosis was more severe along with the increasing concentration of lidocaine(P<0.05).Conclusions:In this study,it was proved that lidocaine could dose-dependently induce the increase of intracellular calcium ion concentration,mitochondrial membrane potential and apoptosis in U87-MG glioma cell line.The up-regulation of TRPV1 enhanced cytotoxicity of lidocaine,which revealed the correlations between mem.Lidocaine might have increased intracellular calcium ion concentration by activating TRPVI gene and induced apoptosis of U87-GM glioma cell line by up-regulating mitochondrial membrane potential.
基金Supported by Grants from the Fundao para a Ciência e a Tecnologia(FCT,Portugal),No.PPCDT/SAL-IMI/57297/2004 and No.PTDC/BIM-MEC/1051/2012The Swedish Cancer foundation+2 种基金The Swedish Research Council,No.K2010-79X-21372-01-3Forska utan djurfrsk,Animal Free ResearchResearch fellowship 2011 from the Sociedade Portuguesa de Gastrenterologia(Portugal)
文摘AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the heterogenic cancer-derived NCI-N87 cell line were isolated,by stably-transducing it with the human telomerase reverse-transcriptase(h TERT) catalytic subunit gene.The clones were first characterized regarding their cell growth pattern and phenotype.For that we measured the clones' adherence properties,expression of cell-cell junctions' markers(ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance.The gastric properties of the clones,concerning expression of mucins,zymogens and glycan contents,were then evaluated by haematoxylin and eosin staining,Periodic acid Schiff(PAS) and PAS/Alcian Blue-staining,immunocytochemistry and Western blot.In addition,we assessed the usefulness of the h TERT-expressing gastric cell line for H.pylori research,by performing co-culture assays and measuring the IL-8 secretion,by ELISA,upon infection with two H.pylori strains differing in virulence.RESULTS:Compared with the parental cell line,themost promising NCI-hT ERT-derived clones(CL5 and CL6) were composed of cells with homogenous phenotype,presented higher relative telomerase activities,better adhesion properties,ability to be maintained in culture for longer periods after confluency,and were more efficient in PAS-reactive mucins secretion.Both clones were shown to produce high amounts of MUC1,MUC2 and MUC13.NCI-hT ERT-CL5 mucins were shown to be decorated with blood group H type 2(BG-H),Lewis-x(Lex),Ley and Lea and,in a less extent,with BG-A antigens,but the former two antigens were not detected in the NCI-h TERT-CL6.None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans.Entailing good gastric properties,both NCIhT ERT-clones were found to produce pepsinogen-5 and human gastric lipase.The progenitor-like phenotype of NCI-hT ERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5,supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.CONCLUSION:These traits,in addition to resistance to microaerobic conditions and good responsiveness to H.pylori co-culture,in a strain virulence-dependent manner,make the NCI-hT ERT-CL6 a promising model for future in vitro studies.
文摘The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection in vitro. The expression of CH50 polypeptide was detected by using immunohistochemical S-P method. The expression of the transfected gene was identified by RT-PCR. Cell invasion assay kit was applied to detect the effect of CH50 polypeptide on the invasion ability of BIU-87. The results showed that the BIU-87 cells transfected with pCH510 could express the CH50 polypeptide, while in the control group, no CH50 polypeptide was detectable. In the transfection group, the invasion ability of BIU-87 in vitro was lower than in control group (P<0.05). It was concluded that CH50 polypeptide was successfully expressed in BIU-87 cells by gene transfection, by which the in vitro invasion ability of BIU-87 was inhibited.
文摘The expression of different vascular endothelial growth factor (VEGF) genes was studied in glioma U87 cells with endoplasmic reticulum–nuclei-1 (ERN1) loss of function and its regulation by hypoxia and glutamine or glucose deprivation conditions as model of ischemia. The blockade of function of the ERN1 enzyme, which is a major sensor of endoplasmic reticulum stress, leads to a decrease of the VEGFA, VEGFB and VEGFC mRNA expression level. The level of VEGFA proteins also decreases at this experimental condition in the cytosolic fraction, but increases in the nuclear fraction. Hypoxia does not affect VEGFC and increases the expression level of VEGFA and VEGFB mRNA in both used cell types, however, the change was much less profound in cells with suppressed function of ERN1. The expression level of VEGFC mRNA decreases in both used cell types in glutamine deprivation condition, however, the change was more profound in control glioma cells. At the same time, the expression level of VEGFA mRNA increases and VEGFB—decreases in gluta-mine deprivation condition in control glioma cells only. Exposure of glioma cells to glucose deprivation condition increases VEGFB mRNA expression level in both used cell types;however, VEGFA—in control glioma cells only and VEGFC—in cells with ERN1 signaling enzyme loss of function only. Thus, the results of this study clearly demonstrated the down-regulation of the expression of all three VEGF genes in glioma cells with ERN1 loss of function which correlates to the suppressed angiogenesis and proliferation rate of these cells. Moreover, the effect of hy-poxia and glutamine or glucose deprivation condition on the expression level of all VEGF genes is different and mainly depends on ERN1 signaling enzyme function.
文摘We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase?2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment.?Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.
