Upregulation Of N-cadherin Expression Research Articles

Specific Binding of Novel SPION-Based System Bearing Anti-N-Cadherin Antibodies to Prostate Tumor Cells.

PurposeEpithelial–mesenchymal (EMT) transition plays an important role in metastasis and is accompanied by an upregulation of N-cadherin expression. A new nanoparticulate system (SPION/CCh/N-cad) based on superparamagnetic iron oxide nanoparticles, stabilized with a cationic derivative of chitosan and surface-modified with anti-N-cadherin antibody, was synthetized for the effective capture of N-cadherin expressing circulating tumor cells (CTC).MethodsThe morphology, physicochemical, and magnetic properties of the system were evaluated using dynamic light scattering (DLS), fluorescence spectroscopy, Mössbauer spectroscopy, magnetometry, and fluorescence spectroscopy. Atomic force microscopy (AFM), confocal microscopy and flow cytometry were used to study the interaction of our nanoparticulate system with N-cadherin expressed in prostate cancer cell lines (PC-3 and DU 145). A purpose-built cuvette was used in the cancer cell capture experiments.ResultsThe obtained nanoparticles were a spherical, stable colloid, and exhibited excellent magnetic properties. Biological experiments confirmed that the novel SPION/CCh/N-cad system interacts specifically with N-cadherin present on the cell surface. Preliminary studies on the magnetic capture of PC-3 cells using the obtained nanoparticles were successful. Incubation times as short as 1 minute were sufficient for the synthesized system to effectively bind to the PC-3 cells.ConclusionResults obtained for our system suggest a possibility of using it to capture CTC in the flow conditions.

Helicobacter pylori induces epithelial-mesenchymal transition in gastric carcinogenesis via the AKT/GSK3β signaling pathway.

Helicobacter pylori (H. pylori) is a main risk factor for gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is involved in the development and progression of H. pylori-associated GC. However, the exact molecular mechanism of this process remains unclear. The AKT/GSK3β signaling pathway has been demonstrated to promote EMT in several types of cancer. The present study investigated whether H. pylori infection induced EMT, and promoted the development and metastasis of cancer in the normal gastric mucosa, and whether this process was dependent on AKT activation. The expression levels of the EMT-associated proteins, including E-cadherin and N-cadherin, were determined in 165 gastric mucosal samples of different disease stages by immunohistochemical analysis. The expression levels of E-cadherin, N-cadherin, AKT, phosphorylated (p-)AKT (Ser473), GSK3β and p-GSK3β (Ser9) were further determined in H. pylori-infected Mongolian gerbil gastric tissues and cells co-cultured with H. pylori by immunohistochemical analysis and western blotting. The results indicated that the expression levels of the epithelial marker E-cadherin were decreased, whereas the expression levels of the mesenchymal marker N-cadherin were increased during gastric carcinogenesis. Their expression levels were associated with H. pylori infection. Furthermore, H. pylori infection resulted in downregulation of E-cadherin expression and upregulation of N-cadherin expression in Mongolian gerbils and GES-1 cells. In addition, an investigation of the associated mechanism of action revealed that p-AKT (Ser473) and p-GSK3β (Ser9) were activated in GES-1 cells following co-culture with H. pylori. Furthermore, following pretreatment of the cells with the AKT inhibitor VIII, the expression levels of E-cadherin, N-cadherin, p-AKT and p-GSK3β did not show significant differences between GES-1 cells that were co-cultured with or without H. pylori. The levels of p-AKT and p-GSK3β were increased in H. pylori-infected Mongolian gerbils. In conclusion, the present study demonstrated that H. pylori infection activated AKT and resulted in the phosphorylation and inactivation of GSK3β, which in turn promoted early stage EMT. These effects were AKT-dependent. This mechanism may serve as a prerequisite for GC development.

TGF-β1 induces N-cadherin expression by upregulating Sox9 expression and promoting its nuclear translocation in human oral squamous cell carcinoma cells.

Squamous cell carcinoma (SCC) is the most frequent cancer that develops in the oral cavity. Epithelial-mesenchymal transition (EMT) is known to play an important role in the process of metastasis of SCC cells. In our previous study, we demonstrated that TGF-β1 induced EMT in the human oral SCC (hOSCC) cell line HSC-4. We also found that Slug plays an important role in suppressing E-cadherin expression and promotion of the migratory activity of HSC-4 cells. However, we also demonstrated that Slug does not participate in upregulation of N-cadherin expression, suggesting that EMT-related transcription factors other than Slug also play an important role in the process. In the present study, we aimed to elucidate how the transcription factor Sox9 affects the TGF-β1-induced upregulation of N-cadherin expression in HSC-4 cells. We found that TGF-β1 upregulated Sox9 expression in HSC-4 cells. In addition, Sox9 siRNA significantly abrogated the TGF-β1-induced upregulation of N-cadherin expression and inhibited the TGF-β1-promoted migratory activity in HSC-4 cells. We also demonstrated that TGF-β1 upregulated the phosphorylation status of Sox9 and then promoted nuclear translocation of Sox9 from the cytoplasm, possibly resulting in an increase in N-cadherin expression. The cyclic AMP-dependent protein kinase A inhibitor H-89, which is known to suppress phosphorylation of Sox9, significantly abrogated the TGF-β1-induced upregulation of N-cadherin expression. These results suggested that TGF-β1 induced N-cadherin expression by upregulating Sox9 expression and promoting its nuclear translocation, which results in EMT progression in hOSCC cells.

Histone deacetylase inhibitors induce invasion of human melanoma cells in vitro via differential regulation of N-cadherin expression and RhoA activity.

BackgroundHistone deacetylase inhibitors (HDACi) exert multiple cytotoxic actions on cancer cells. Currently, different synthetic HDACi are in clinical use or clinical trials; nevertheless, since both pro-invasive and anti-invasive activities have been described, there is some controversy about the effect of HDACi on melanoma cells.MethodsMatrigel and Collagen invasion assays were performed to evaluate the effect of several HDACi (Butyrate, Trichostatin A, Valproic acid and Vorinostat) on two human melanoma cell line invasion (A375 and HT-144). The expression of N- and E-Cadherin and the activity of the RhoA GTPase were analyzed to elucidate the mechanisms involved in the HDACi activity.ResultsHDACi showed a pro-invasive effect on melanoma cells in vitro. This effect was accompanied by an up-regulation of N-cadherin expression and an inhibition of RhoA activity. Moreover, the down-regulation of N-cadherin through blocking antibodies or siRNA abrogated the pro-invasive effect of the HDACi and, additionally, the inhibition of the Rho/ROCK pathway led to an increase of melanoma cell invasion similar to that observed with the HDACi treatments.ConclusionThese results suggest a role of N-cadherin and RhoA in HDACi induced invasion and call into question the suitability of some HDACi as antitumor agents for melanoma patients.

Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway

C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in MatrigelTM with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.

Alterations of the myovesical plexus of the human overactive detrusor.

Objectives. The human bladder shows spontaneous autonomous activity. Detrusor overactivity could be seen as a consequence of exaggerated autonomous activity. Interstitial cells (ICs) play a potential role in coordination of autonomous activity. As it is suggested that changes in ICs coexist with detrusor overactivity (DO), we investigated possible alterations to human bladder ICs. Methods. Biopsies were obtained from 23 patients and were categorized into four groups: genuine stress incontinence (without DO) (n = 5), neurogenic disease with DO (n = 6), bladder outlet obstruction with DO (n = 6), or idiopathic DO (n = 6). Specimens were processed to investigate expression of N-cadherin and PGP9.5. N-cadherin expression was semiquantitatively analyzed and correlated to PG9.5 expression and bladder wall morphology. Results. The population of cells expressing N-cadherin is altered in the overactive detrusor, making no difference between the sources of DO. Punctate distribution of morphological changes was found and downregulation of PGP9.5 expression seemed to coexist with upregulation of N-cadherin expression in the detrusor layer. Conclusions. The population of N-cadherin+ cells of the interstitial compartment of the human bladder has the ability to proliferate. As this proliferation seems to coexist with denervation, it could be possible that a highly developed network of interstitial cells replaces the loss of innervation in overactive detrusor.

The expressions of N-cadherin and β-catenin in hepatoceilular carcinoma and its correlation with Wnt signaling pathway

Objective To investigate whether N-cadherin modulates expression of β-catenin and activation of the Wnt signaling pathway in hepatocellular carcinoma ( HCC ). MethodsThe expressions of N-cadherin and β-catenin in 64 HCC tissues were measured by immunohistochemistry and the relationship between them was studied. Furthermore, N-cadherin expression in HCC cell lines, HCCLM3 and SMMC-7721 cells, was regulated and its effect on expression of β-catenin and activity of Wnt singnaling pathway was evaluated. Results Among the 64 HCCs examined,34 HCCs showed reduced N-cadherin expression; β-catenin staining was observed on cell membranes but not in cell nuclei and 13 HCCs showed reduced membranous staining;the reduced expression of β-catenin on cell membranes was correlated with reduced N-cadherin expression(P ＜ 0. 05 ). Down-regulation of N-cadherin in HCCLM3 cells resulted in reduced membranous β-catenin expression but not activation of the Wnt signaling pathway. In contrast, up-regulation of N-cadherin expression in SMMC-7721 cells led to increased expression of membranous β-catenin but had no effect on Wnt signaling pathway. Conclusion In HCC,the level of N-cadherin was positively correlated with membranous β-catenin expression:however, N-cadherin was not involved in regulation of the Wnt signaling pathway. Key words: Carcinoma,hepatocellular; N-cadherin; β-catenin; Wnt signal pathway

Phosphoinositide-3 Kinase–Rac1–c-Jun NH2-terminal Kinase Signaling Mediates Collagen I–induced Cell Scattering and Up-Regulation of N-Cadherin Expression in Mouse Mammary Epithelial Cells

During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH2-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I-induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I-induced N-cadherin up-regulation. These data implicate PI3K-Rac1-JNK signaling in collagen I-induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I-induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I-induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K-Rac1-JNK signaling.

N-Cadherin andβ-Catenin involvement in BMP-2 induction of mesenchymal chondrogenesis

Controlled upregulation of N-cadherin expression and function has been shown to be essential for bone morphogenetic protein-2 (BMP-2)-induced chondrogenic differentiation of high-density micromass cultures of the murine multipotential mesenchymal cell line, C3H10T1/2. In this report, we have examined the nature of the N-cadherin-related mechanisms involved in BMP-2-mediated chondrogenesis. BMP-2 treatment altered the expression of catenins, the cytoplasmic components of the adherens junction, and their interactions with N-cadherin. Within 24 h of BMP-2 treatment, immunoprecipitation analysis showed that the association of catenins with N-cadherin was significantly lower. With long-term (9 and 13 days) BMP-2 treatment, β-catenin accumulated in a nuclear localization, with reduced association with the adenomatous polyposis tumor suppressor protein (APC), a factor known to direct β-catenin degradation via ubiquitination. The functional importance of N-cadherin-β-catenin interaction was further investigated by the effect of transfection-mediated over-expression of two forms of β-catenin: wild-type β-catenin inhibited, while an amino terminal truncated (adhesive deficient) β-catenin enhanced BMP-2-induced chondrogenesis. These data indicate that BMP-2 induction of chondrogenesis in the mesenchymal cell line C3H10T1/2 depends on functional alterations in specific cell adhesion and signaling pathways involving N-cadherin-catenin interaction.

Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-β (TGF-β) superfamily, is characterized by its ability to induce cartilage and bone formation. We have recently demonstrated that the multipotential, murine embryonic mesenchymal cell line, C3H10T1/2, when cultured at high density, is induced by BMP-2 or TGF-β1 to undergo chondrogenic differentiation. The high-cell-density requirement suggests that specific cell-cell interactions, such as those mediated by cell adhesion molecules, are important in the chondrogenic response. In view of our recent finding that N-cadherin, a Ca 2+-dependent cell adhesion molecule, is functionally required in normal embryonic limb mesenchyme cellular condensation and chondrogenesis, we examine here whether N-cadherin is also involved in BMP-2 induction of chondrogenesis in C3H10T1/2 cells. BMP-2 stimulation of chondrogenesis in high-density micromass cultures of C3H10T1/2 cells was evidenced by Alcian blue staining, elevated [ 35S]sulfate incorporation, and expression of the cartilage matrix markers, collagen type II and cartilage proteoglycan link protein. With BMP-2 treatment, N-cadherin mRNA expression was stimulated 4-fold within 24 h, and by day 5, protein levels were stimulated 8-fold. An N-cadherin peptidomimic containing the His-Ala-Val sequence to abrogate homotypic N-cadherin interactions inhibited chondrogenesis in a concentration-dependent manner. To analyze the functional role of N-cadherin further, C3H10T1/2 cells were stably transfected with expression constructs of either full-length N-cadherin or a dominant negative, N-terminal deletion mutant of N-cadherin. Moderate (2-fold) overexpression of full-length N-cadherin augmented, whereas higher (4-fold) overexpression inhibited the BMP-2-chondrogenic effect. On the other hand, expression of the dominant negative N-cadherin mutant dramatically inhibited BMP-2 stimulated chondrogenesis. These data strongly suggest that upregulation of N-cadherin expression, at defined critical levels, is a candidate mechanistic component of BMP-2 stimulation of mesenchymal chondrogenesis.