Background： Circulating tumor cells （CTCs） are often undetected through the immunomagnetic epithelial cell adhesion molecule （EpCAM）-based CellSearch~ System in breast and colorectal cancer （CRC） patients treat...Background： Circulating tumor cells （CTCs） are often undetected through the immunomagnetic epithelial cell adhesion molecule （EpCAM）-based CellSearch~ System in breast and colorectal cancer （CRC） patients treated with bevacizumab （BEV）, where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. Methods： The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch, untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch. Results： Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins （CK）, while no evidence of epithelial to mesenchymal transition （EMT） in treated cells was observed. The recovery rate of cells through CellSearch was gradually reduced in course of treatment with BEV, being 84% , 70% and 40% at l, 2 and 3 months, respectively. Conclusions： We hypothesize that BEV may prevent CellSearch from capturing CTCs through altering EpCAM isoforms.展开更多
AIM To compare the capacity of newly developed epidermal growth factor receptor(EGFR)-targeted immune magnetic liposomes(EILs) vs epithelial cell adhesion molecule(Ep CAM) immunomagnetic beads to capture colorectal ci...AIM To compare the capacity of newly developed epidermal growth factor receptor(EGFR)-targeted immune magnetic liposomes(EILs) vs epithelial cell adhesion molecule(Ep CAM) immunomagnetic beads to capture colorectal circulating tumor cells(CTCs).METHODS EILs were prepared using a two-step method, and the magnetic and surface characteristics were confirmed. The efficiency of capturing colorectal CTCs as well as the specificity were compared between EILs and Ep CAM magnetic beads. RESULTS The obtained EILs had a lipid nanoparticle structure similar to cell membrane. Improved binding with cancer cells was seen in EILs compared with the method of coupling nano/microspheres with antibody. The binding increased as the contact time extended. Compared with Ep CAM immunomagnetic beads, EILs captured more CTCs in peripheral blood from colorectal cancer patients. The captured cells showed consistency with clinical diagnosis and pathology. Mutation analysis showed same results between captured CTCs and cancer tissues. CONCLUSION EGFR antibody-coated magnetic liposomes show high efficiency and specificity in capturing colorectal CTCs.展开更多
Aim:Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesion dynamics,a process required for the tumor cell migration and invasion.Phosphorylation of the serine ...Aim:Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesion dynamics,a process required for the tumor cell migration and invasion.Phosphorylation of the serine residue 178 requires c-Jun NH2-terminal kinase(JNK)activation,which occurs downstream of epidermal growth factor receptor(EGFR)-mediated signaling and drives cell migration.In this study,we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.Methods:We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs.With those,we next performed several in vitro assays including cell proliferation,migration and focal adhesion dynamics.Finally,we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Results:Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration,which was correlated with impaired focal adhesion dynamics.Moreover,paxillinS178A attenuated lung metastasis formation in an orthotopic in vivo mammary gland tumor/metastasis model,demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression.Expression of paxillinS178A caused a decrease in EGFR expression, ;while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics.Furthermore,re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Conclusion:Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby,in EGF-driven cell migration and metastasis formation.展开更多
This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, ...This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, pancreatitis, and normal pancreas. The plasmid pcDNA3.1-Hy- gro（＋）/CADM1 was transfected into PANC-1 cells （a pancreatic cancer cell line）. The expression of CADM1 in the transfected cells was determined by RT-PCR and Western blotting. Cell growth was measured by the MTT method and cell apoptosis by flow cytometry. The results showed that CADM1 was weakly expressed in tissues of pancreatic cancer in contrast to its high expression in normal pancreatic and pancreatitis tissues. The expression level of CADM in pancreatic caner was intensely correlated with the differentiation degree, lymph node metastasis and TNM stages. The growth of CADMl-transfected PANC-1 cells was significantly suppressed in vitro by a G1 cell cycle arrest and apoptosis occurrence. It was concluded that re-expression of CADM1 inhibits the growth of pancreatic cancer cells and induces their apoptosis in vitro. As a tumor suppressor gene, CADM1 plays an important role in the occurrence, progression and metastasis of pancreatic cancer.展开更多
Cell-cell and cell-matrix signaling and communication between adhesion sites involve mechanisms which are required for cellular functions during normal development and homeostasis; however these cellular functions and...Cell-cell and cell-matrix signaling and communication between adhesion sites involve mechanisms which are required for cellular functions during normal development and homeostasis; however these cellular functions and mechanisms are often deregulated in cancer. Aberrant signaling at cell-cell and cell-matrix adhesion sites often involves downstream mediators including Rho GTPases and tyrosine kinases. This review discusses these molecules as putative mediators of cellular crosstalk between cell-cell and cell-matrix adhesion sites, in addition to their attractiveness as therapeutic targets in cancer. Interestingly, inter-junctional crosstalk mechanisms are frequently typified by the way in which bacterial and viral pathogens opportunistically infect or intoxicate mammalian cells. This review therefore also discusses the concept of learning from pathogen-host interaction studies to better understand coordinated communication between cell-cell and cell-matrix adhesion sites, in addition to highlighting the potential therapeutic usefulness of exploiting pathogens or their products to tap into inter-junctional crosstalk. Taken together, we feel that increased knowledge around mechanisms of cell-cell and cell-matrix adhesion site crosstalk and consequently a greater understanding of their therapeutic targeting offers a unique opportunity to contribute to the emerging molecular revolution in cancer biology.展开更多
背景与目的:EB病毒编码的潜伏膜蛋白1(latent membrane protein 1,LMP1)在鼻咽癌的浸润和转移过程中起重要的作用。本实验目的在于研究EB病毒LMP1对人鼻咽癌细胞转移能力的影响和探讨其中的相关机制。方法:应用免疫细胞化学RT-PCR法和We...背景与目的:EB病毒编码的潜伏膜蛋白1(latent membrane protein 1,LMP1)在鼻咽癌的浸润和转移过程中起重要的作用。本实验目的在于研究EB病毒LMP1对人鼻咽癌细胞转移能力的影响和探讨其中的相关机制。方法:应用免疫细胞化学RT-PCR法和Western blot检测LMP1、E-cadherin和intercellular adhesion molecule-1 (ICAM-1)在人高分化鼻咽癌细胞系CNE1和转染了LMP1基因的CNE1-GL细胞中的表达。应用细胞-细胞粘附实验、细胞-基质粘附实验、划痕实验和细胞运动实验观察LMP1对鼻咽癌细胞粘附和运动能力的影响。结果:免疫细胞化学结果显示:LMP1在CNE1和CNE1-GL细胞中的阳性率分别为0和(96.60±3.03)%(P<0.01);E-cadherin蛋白的阳性率分别为(37.47±1.50)%和(19.53±1.92)%(P<0.01);ICAM-1蛋白的阳性率分别为(5.27±1.45)%和(93.33±4.23)%(P<0.01)。RT-PCR和Westernblot结果表明,与CNE1细胞相比较,CNE1-GL细胞中E-cadherin的mRNA和蛋白表达明显降低(P<0.01),而ICAM-1的mRNA和蛋白表达则明显升高(P<0.01)。肿瘤细胞同质粘附实验显示,CNE1-GL细胞的同质粘附能力较CNE1细胞弱(P<0.05)。肿瘤细胞-基质粘附实验得出CNE1-GL细胞的异质粘附能力(A值=0.60±0.03)较CNE1细胞(A值=0.46±0.01)强(P<0.01)。肿瘤细胞运动实验显示,穿过PVPF膜的CNE1-GL细胞数多于CNE1细胞(119.3±6.0 vs 46.3±7.0,P<0.05)。结论:LMP1通过抑制E-cadherin表达、促进ICAM-1表达从而抑制肿瘤细胞的同质粘附能力、增强其异质粘附能力和运动能力来参与鼻咽癌的侵袭和转移。展开更多
文摘Background： Circulating tumor cells （CTCs） are often undetected through the immunomagnetic epithelial cell adhesion molecule （EpCAM）-based CellSearch~ System in breast and colorectal cancer （CRC） patients treated with bevacizumab （BEV）, where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. Methods： The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch, untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch. Results： Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins （CK）, while no evidence of epithelial to mesenchymal transition （EMT） in treated cells was observed. The recovery rate of cells through CellSearch was gradually reduced in course of treatment with BEV, being 84% , 70% and 40% at l, 2 and 3 months, respectively. Conclusions： We hypothesize that BEV may prevent CellSearch from capturing CTCs through altering EpCAM isoforms.
文摘AIM To compare the capacity of newly developed epidermal growth factor receptor(EGFR)-targeted immune magnetic liposomes(EILs) vs epithelial cell adhesion molecule(Ep CAM) immunomagnetic beads to capture colorectal circulating tumor cells(CTCs).METHODS EILs were prepared using a two-step method, and the magnetic and surface characteristics were confirmed. The efficiency of capturing colorectal CTCs as well as the specificity were compared between EILs and Ep CAM magnetic beads. RESULTS The obtained EILs had a lipid nanoparticle structure similar to cell membrane. Improved binding with cancer cells was seen in EILs compared with the method of coupling nano/microspheres with antibody. The binding increased as the contact time extended. Compared with Ep CAM immunomagnetic beads, EILs captured more CTCs in peripheral blood from colorectal cancer patients. The captured cells showed consistency with clinical diagnosis and pathology. Mutation analysis showed same results between captured CTCs and cancer tissues. CONCLUSION EGFR antibody-coated magnetic liposomes show high efficiency and specificity in capturing colorectal CTCs.
基金the EU FP7 Metafight project(HEALTH-F2-2007-201862)Dutch Cancer Society(KWF-UL2007-3860)NWO grant(911-02-022).
文摘Aim:Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesion dynamics,a process required for the tumor cell migration and invasion.Phosphorylation of the serine residue 178 requires c-Jun NH2-terminal kinase(JNK)activation,which occurs downstream of epidermal growth factor receptor(EGFR)-mediated signaling and drives cell migration.In this study,we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.Methods:We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs.With those,we next performed several in vitro assays including cell proliferation,migration and focal adhesion dynamics.Finally,we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Results:Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration,which was correlated with impaired focal adhesion dynamics.Moreover,paxillinS178A attenuated lung metastasis formation in an orthotopic in vivo mammary gland tumor/metastasis model,demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression.Expression of paxillinS178A caused a decrease in EGFR expression, ;while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics.Furthermore,re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Conclusion:Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby,in EGF-driven cell migration and metastasis formation.
文摘This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, pancreatitis, and normal pancreas. The plasmid pcDNA3.1-Hy- gro（＋）/CADM1 was transfected into PANC-1 cells （a pancreatic cancer cell line）. The expression of CADM1 in the transfected cells was determined by RT-PCR and Western blotting. Cell growth was measured by the MTT method and cell apoptosis by flow cytometry. The results showed that CADM1 was weakly expressed in tissues of pancreatic cancer in contrast to its high expression in normal pancreatic and pancreatitis tissues. The expression level of CADM in pancreatic caner was intensely correlated with the differentiation degree, lymph node metastasis and TNM stages. The growth of CADMl-transfected PANC-1 cells was significantly suppressed in vitro by a G1 cell cycle arrest and apoptosis occurrence. It was concluded that re-expression of CADM1 inhibits the growth of pancreatic cancer cells and induces their apoptosis in vitro. As a tumor suppressor gene, CADM1 plays an important role in the occurrence, progression and metastasis of pancreatic cancer.
基金Supported by Past and present funding in the senior author’s laboratory as follows--Health Research Board of Ireland(HRA--POR-2014-545HRA/2009/49+6 种基金RP/2006/95,to Hopkins AM)Science Foundation Ireland(13/IA/19942008/RFP/NSC14272008/RFP/NSC1427 TIDA Feasibility 10,to Hopkins AM)Cancer Research Ireland,Breast Cancer IrelandBrazil Science Without Borders(CAPES-13306-13-8)the Beaumont Hospital Cancer Research and Development Trust
文摘Cell-cell and cell-matrix signaling and communication between adhesion sites involve mechanisms which are required for cellular functions during normal development and homeostasis; however these cellular functions and mechanisms are often deregulated in cancer. Aberrant signaling at cell-cell and cell-matrix adhesion sites often involves downstream mediators including Rho GTPases and tyrosine kinases. This review discusses these molecules as putative mediators of cellular crosstalk between cell-cell and cell-matrix adhesion sites, in addition to their attractiveness as therapeutic targets in cancer. Interestingly, inter-junctional crosstalk mechanisms are frequently typified by the way in which bacterial and viral pathogens opportunistically infect or intoxicate mammalian cells. This review therefore also discusses the concept of learning from pathogen-host interaction studies to better understand coordinated communication between cell-cell and cell-matrix adhesion sites, in addition to highlighting the potential therapeutic usefulness of exploiting pathogens or their products to tap into inter-junctional crosstalk. Taken together, we feel that increased knowledge around mechanisms of cell-cell and cell-matrix adhesion site crosstalk and consequently a greater understanding of their therapeutic targeting offers a unique opportunity to contribute to the emerging molecular revolution in cancer biology.
文摘背景与目的:EB病毒编码的潜伏膜蛋白1(latent membrane protein 1,LMP1)在鼻咽癌的浸润和转移过程中起重要的作用。本实验目的在于研究EB病毒LMP1对人鼻咽癌细胞转移能力的影响和探讨其中的相关机制。方法:应用免疫细胞化学RT-PCR法和Western blot检测LMP1、E-cadherin和intercellular adhesion molecule-1 (ICAM-1)在人高分化鼻咽癌细胞系CNE1和转染了LMP1基因的CNE1-GL细胞中的表达。应用细胞-细胞粘附实验、细胞-基质粘附实验、划痕实验和细胞运动实验观察LMP1对鼻咽癌细胞粘附和运动能力的影响。结果:免疫细胞化学结果显示:LMP1在CNE1和CNE1-GL细胞中的阳性率分别为0和(96.60±3.03)%(P<0.01);E-cadherin蛋白的阳性率分别为(37.47±1.50)%和(19.53±1.92)%(P<0.01);ICAM-1蛋白的阳性率分别为(5.27±1.45)%和(93.33±4.23)%(P<0.01)。RT-PCR和Westernblot结果表明,与CNE1细胞相比较,CNE1-GL细胞中E-cadherin的mRNA和蛋白表达明显降低(P<0.01),而ICAM-1的mRNA和蛋白表达则明显升高(P<0.01)。肿瘤细胞同质粘附实验显示,CNE1-GL细胞的同质粘附能力较CNE1细胞弱(P<0.05)。肿瘤细胞-基质粘附实验得出CNE1-GL细胞的异质粘附能力(A值=0.60±0.03)较CNE1细胞(A值=0.46±0.01)强(P<0.01)。肿瘤细胞运动实验显示,穿过PVPF膜的CNE1-GL细胞数多于CNE1细胞(119.3±6.0 vs 46.3±7.0,P<0.05)。结论:LMP1通过抑制E-cadherin表达、促进ICAM-1表达从而抑制肿瘤细胞的同质粘附能力、增强其异质粘附能力和运动能力来参与鼻咽癌的侵袭和转移。
