Objectives:Cholecystokinin A receptor(CCKAR)has been linked to poor prognosis in colon cancer patients,but the role of CCKAR in colon cancer cell invasiveness and the underlying mechanisms remain elusive.This study ai...Objectives:Cholecystokinin A receptor(CCKAR)has been linked to poor prognosis in colon cancer patients,but the role of CCKAR in colon cancer cell invasiveness and the underlying mechanisms remain elusive.This study aimed to explore the effect of CCKAR on the invasive potential of colon cancer cells.Methods:Different human colon cancer cell lines were used.Gene expression was evaluated by reverse transcription polymerase chain reaction(RT-PCR)and quantitative real-time RT-PCR(qPCR),while protein expression and phosphorylation were assessed by Western blotting.Cell motility and invasiveness were examined through wound healing and invasion assays,respectively.Results:Our results showed that CCKAR expression levels varied across colon cancer cell lines,with DLD-1 and LoVo cells showing high expression.Knockdown of CCKAR significantly impaired the cell motility and invasiveness of DLD-1 and LoVo cells,downregulated integrinβ3 expression,and diminished the phosphorylation levels of focal adhesion kinase(FAK),Src,and paxillin.In addition,CCKAR knockdown modulated epithelialmesenchymal transition(EMT)markers ZO-1,E-cadherin,and vimentin and reduced urokinase-type plasminogen activator(uPA),uPA receptor(uPAR),Rho GTPase cell division control protein 42(CDC42)and RhoA,and matrix metalloproteinase-2(MMP-2).Conclusions:These findings indicate that CCKAR knockdown impairs the invasiveness of colon cancer cells,which may be attributed to modulating integrin/FAK/Rho GTPases,EMT markers,and the uPA/uPAR axis.It suggests that targeting CCKAR may represent a potential therapeutic strategy for colon cancer treatment.展开更多
Background:Integrins facilitate binding to the extracellular matrix and other cells.Their subunit β2 is exclusively expressed by leukocytes,binds to the intercellular cell adhesion molecule 1(ICAM-1),and is pivotal f...Background:Integrins facilitate binding to the extracellular matrix and other cells.Their subunit β2 is exclusively expressed by leukocytes,binds to the intercellular cell adhesion molecule 1(ICAM-1),and is pivotal for their recruitment to sites of inflammation such as the atherosclerotic plaque.Methods:To investigate β2-integrin-mediated adhesiveness,a well-established assay for human whole blood was adapted for the analysis of murine T cell subsets.Changes in avidity and affinity were assessed by incubation of murine complexes ICAM-1 in murine whole blood and consecutive stimulation with PMA and Mg^(2+)/EGTA.Underlying signaling pathways in β2-integrin-mediated adhesiveness upon chemokine stimulation with CCL-19 were identified by incubation with reducing substances,and a Ca^(2+)chelator and ROS and Ca^(2+)measurements were carried out.Results:Incubation of murine whole blood with PMA leads to 30-fold and Mg^(2+)/EGTA to 65-fold increase in β2-integrin-mediated adhesiveness of T cells.Specificity of the assay was proven by preincubation of a blocking antibody,leading to a 60%reduction in adhesion capacity.ROS species and Ca^(2+)are crucial for chemokine-mediated β2-integrin activation.In vivo relevance was proven by induction of T cell adhesiveness in whole blood of mice upon myocardial infarction.Conclusions:Our assay allows specific quantification of β2-integrin-mediated affinity and avidity of T cells in whole blood samples.In congruence to human adhesion,these mechanisms are ROS and Ca^(2+)dependent and significantly elevated after myocardial infarction.Our refined and robust assay may be of particular use in phenotyping involved mechanisms in T cell activation in atherosclerotic cardiovascular disease.展开更多
基金funded by Chung Shan Medical University Hospital(grant number CSH 2023-C-010)Buddhist Tzu Chi Medical Foundation,Dalin Tzu Chi Hospital(grant number DTCRD112(2)-I-24).
文摘Objectives:Cholecystokinin A receptor(CCKAR)has been linked to poor prognosis in colon cancer patients,but the role of CCKAR in colon cancer cell invasiveness and the underlying mechanisms remain elusive.This study aimed to explore the effect of CCKAR on the invasive potential of colon cancer cells.Methods:Different human colon cancer cell lines were used.Gene expression was evaluated by reverse transcription polymerase chain reaction(RT-PCR)and quantitative real-time RT-PCR(qPCR),while protein expression and phosphorylation were assessed by Western blotting.Cell motility and invasiveness were examined through wound healing and invasion assays,respectively.Results:Our results showed that CCKAR expression levels varied across colon cancer cell lines,with DLD-1 and LoVo cells showing high expression.Knockdown of CCKAR significantly impaired the cell motility and invasiveness of DLD-1 and LoVo cells,downregulated integrinβ3 expression,and diminished the phosphorylation levels of focal adhesion kinase(FAK),Src,and paxillin.In addition,CCKAR knockdown modulated epithelialmesenchymal transition(EMT)markers ZO-1,E-cadherin,and vimentin and reduced urokinase-type plasminogen activator(uPA),uPA receptor(uPAR),Rho GTPase cell division control protein 42(CDC42)and RhoA,and matrix metalloproteinase-2(MMP-2).Conclusions:These findings indicate that CCKAR knockdown impairs the invasiveness of colon cancer cells,which may be attributed to modulating integrin/FAK/Rho GTPases,EMT markers,and the uPA/uPAR axis.It suggests that targeting CCKAR may represent a potential therapeutic strategy for colon cancer treatment.
基金DZHK(German Centre for Cardiovascular Research)。
文摘Background:Integrins facilitate binding to the extracellular matrix and other cells.Their subunit β2 is exclusively expressed by leukocytes,binds to the intercellular cell adhesion molecule 1(ICAM-1),and is pivotal for their recruitment to sites of inflammation such as the atherosclerotic plaque.Methods:To investigate β2-integrin-mediated adhesiveness,a well-established assay for human whole blood was adapted for the analysis of murine T cell subsets.Changes in avidity and affinity were assessed by incubation of murine complexes ICAM-1 in murine whole blood and consecutive stimulation with PMA and Mg^(2+)/EGTA.Underlying signaling pathways in β2-integrin-mediated adhesiveness upon chemokine stimulation with CCL-19 were identified by incubation with reducing substances,and a Ca^(2+)chelator and ROS and Ca^(2+)measurements were carried out.Results:Incubation of murine whole blood with PMA leads to 30-fold and Mg^(2+)/EGTA to 65-fold increase in β2-integrin-mediated adhesiveness of T cells.Specificity of the assay was proven by preincubation of a blocking antibody,leading to a 60%reduction in adhesion capacity.ROS species and Ca^(2+)are crucial for chemokine-mediated β2-integrin activation.In vivo relevance was proven by induction of T cell adhesiveness in whole blood of mice upon myocardial infarction.Conclusions:Our assay allows specific quantification of β2-integrin-mediated affinity and avidity of T cells in whole blood samples.In congruence to human adhesion,these mechanisms are ROS and Ca^(2+)dependent and significantly elevated after myocardial infarction.Our refined and robust assay may be of particular use in phenotyping involved mechanisms in T cell activation in atherosclerotic cardiovascular disease.
