Objective To investigate the role of cytoplasmic domain of integrin alphaⅡb in platelet signal transduction. Methods Binding capacity of integrin alphaⅡb R995A to antibody platelet activation complex-1 (PAC-1) and p...Objective To investigate the role of cytoplasmic domain of integrin alphaⅡb in platelet signal transduction. Methods Binding capacity of integrin alphaⅡb R995A to antibody platelet activation complex-1 (PAC-1) and pp125 focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting. Results Without activation, wild-type alphaⅡbbeta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alphaⅡb R995A beta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK) in wild-type alphaⅡbbeta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions. Conclusion The mutation in integrin alphaⅡb R995A alters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.展开更多
The RGD sequence generally exists in the extracellular matrix proteins and can be recognized by many integrin proteins. The binding ability of immobilized biotinylated cyclic hexapeptide [cyclo(-Arg-Gly-Asp-D-Phe-Lys-...The RGD sequence generally exists in the extracellular matrix proteins and can be recognized by many integrin proteins. The binding ability of immobilized biotinylated cyclic hexapeptide [cyclo(-Arg-Gly-Asp-D-Phe-Lys-Gly-)] containing RGD to integrin ααbβ3 was tested by the methods of ELISA and SPR. Results showed that a spacer of 1.48-2.2 nm between the peptide and the biotin residue was long enough to send the RGD sequence into the binding center embedded within αⅡbβ3, and the equilibrium dissociation constant was 1.1μm. The work provides an ideal model system for the research of cell adhesion on solid surfaces.展开更多
Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current st...Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.展开更多
characterize the activation of platelet integrin αⅡbβ3 induced by two anti human platelet tetraspanin monoclonal antibodies (mAbs), HI117 and SJ9A4, and investigate their potential mechanism of action Method...characterize the activation of platelet integrin αⅡbβ3 induced by two anti human platelet tetraspanin monoclonal antibodies (mAbs), HI117 and SJ9A4, and investigate their potential mechanism of action Methods Using 125 I labeled human fibrinogen (Fg), specific Fg binding to human platelets induced by HI117 and SJ9A4 was measured Results HI117 and SJ9A4 (10?μg/ml and 20?μg/ml) induced specific Fg binding to human platelets, suggesting that the two mAbs evoked activation of platelet integrin αⅡbβ3 Further study indicated that HI117 and SJ9A4 induced integrin αⅡbβ3 activation independent of platelet Fc receptors, and that HI117 and SJ9A4 induced integrin αⅡbβ3 activation was inhibited by pretreatment of platelets with sphingosine, aspirin, apyrase, and/or PGI 2 Conclusions Anti platelet tetraspanin (CD9) mAbs, HI117 and SJ9A4, can induce platelet integrin αⅡbβ3 activation independent of Fc receptors Three signaling pathways, namely thromboxane, secreted ADP, and cAMP pathways, may be involved in the process, with protein kinase C activation presumably being the common step of the three pathways展开更多
Objective:To examine the antiplatelet and antithrombotic activity of Rumex acetosella extract.Methods:Standard light aggregometry was used for platelet aggregation,intracellular calcium mobilization assessed using Fur...Objective:To examine the antiplatelet and antithrombotic activity of Rumex acetosella extract.Methods:Standard light aggregometry was used for platelet aggregation,intracellular calcium mobilization assessed using Fura-2/AM,granule secretion(ATP release)by luminometer,and fibrinogen binding to integrin α_(Ⅱb)β_(3) detected using flow cytometry.Western blotting is carried out to determine the phosphorylation of mitogen-activated protein kinase(MAPK)and phosphoinositide 3-kinase(PI3K)/Akt signaling.Results:Rumex acetosella displayed the ability to inhibit platelet aggregation,calcium mobilization,granule secretion,and fibrinogen binding to integrin α_(Ⅱb)β_(3).Rumex acetosella has also down-regulated MAPK and PI3K/Akt phosphorylation(all P<0.01).Conclusion:Rumex acetosella extract exhibits antiplatelet activity via modulating GPVI signaling,and it may protect against the development of platelet-related cardiovascular diseases.展开更多
文摘Objective To investigate the role of cytoplasmic domain of integrin alphaⅡb in platelet signal transduction. Methods Binding capacity of integrin alphaⅡb R995A to antibody platelet activation complex-1 (PAC-1) and pp125 focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting. Results Without activation, wild-type alphaⅡbbeta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alphaⅡb R995A beta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK) in wild-type alphaⅡbbeta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions. Conclusion The mutation in integrin alphaⅡb R995A alters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.
文摘The RGD sequence generally exists in the extracellular matrix proteins and can be recognized by many integrin proteins. The binding ability of immobilized biotinylated cyclic hexapeptide [cyclo(-Arg-Gly-Asp-D-Phe-Lys-Gly-)] containing RGD to integrin ααbβ3 was tested by the methods of ELISA and SPR. Results showed that a spacer of 1.48-2.2 nm between the peptide and the biotin residue was long enough to send the RGD sequence into the binding center embedded within αⅡbβ3, and the equilibrium dissociation constant was 1.1μm. The work provides an ideal model system for the research of cell adhesion on solid surfaces.
文摘Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.
基金grantfromtheNationalNaturalScienceFoundationofChina (No 39470 172 )
文摘characterize the activation of platelet integrin αⅡbβ3 induced by two anti human platelet tetraspanin monoclonal antibodies (mAbs), HI117 and SJ9A4, and investigate their potential mechanism of action Methods Using 125 I labeled human fibrinogen (Fg), specific Fg binding to human platelets induced by HI117 and SJ9A4 was measured Results HI117 and SJ9A4 (10?μg/ml and 20?μg/ml) induced specific Fg binding to human platelets, suggesting that the two mAbs evoked activation of platelet integrin αⅡbβ3 Further study indicated that HI117 and SJ9A4 induced integrin αⅡbβ3 activation independent of platelet Fc receptors, and that HI117 and SJ9A4 induced integrin αⅡbβ3 activation was inhibited by pretreatment of platelets with sphingosine, aspirin, apyrase, and/or PGI 2 Conclusions Anti platelet tetraspanin (CD9) mAbs, HI117 and SJ9A4, can induce platelet integrin αⅡbβ3 activation independent of Fc receptors Three signaling pathways, namely thromboxane, secreted ADP, and cAMP pathways, may be involved in the process, with protein kinase C activation presumably being the common step of the three pathways
基金Supported by a grant of National Research Foundation of Korea(No.2018R1D1A1A09083797)。
文摘Objective:To examine the antiplatelet and antithrombotic activity of Rumex acetosella extract.Methods:Standard light aggregometry was used for platelet aggregation,intracellular calcium mobilization assessed using Fura-2/AM,granule secretion(ATP release)by luminometer,and fibrinogen binding to integrin α_(Ⅱb)β_(3) detected using flow cytometry.Western blotting is carried out to determine the phosphorylation of mitogen-activated protein kinase(MAPK)and phosphoinositide 3-kinase(PI3K)/Akt signaling.Results:Rumex acetosella displayed the ability to inhibit platelet aggregation,calcium mobilization,granule secretion,and fibrinogen binding to integrin α_(Ⅱb)β_(3).Rumex acetosella has also down-regulated MAPK and PI3K/Akt phosphorylation(all P<0.01).Conclusion:Rumex acetosella extract exhibits antiplatelet activity via modulating GPVI signaling,and it may protect against the development of platelet-related cardiovascular diseases.
