The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.
Cadherins/biosynthesis ; Carcinoma, Non-Small-Cell Lung/*genetics/mortality ; DNA Methylation/*genetics ; Female ; Humans ; Lung Neoplasms/*genetics/mortality ; Male ; Middle Aged ; Neoplasm Metastasis/genetics ; Neoplasm Staging ; Promoter Regions, Genetic/*genetics ; Republic of Korea ; Tumor Markers, Biological/genetics ; Wnt Proteins/*genetics

The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.
Cadherins/biosynthesis ; Carcinoma, Non-Small-Cell Lung/*genetics/mortality ; DNA Methylation/*genetics ; Female ; Humans ; Lung Neoplasms/*genetics/mortality ; Male ; Middle Aged ; Neoplasm Metastasis/genetics ; Neoplasm Staging ; Promoter Regions, Genetic/*genetics ; Republic of Korea ; Tumor Markers, Biological/genetics ; Wnt Proteins/*genetics

OBJECTIVE: To investigate the expression of EBV-encoded latent membrane protein 2A (LMP2A) and epithelial-mesenchymal transformation(EMT) associated markers (E-cadherin and fibronectin) in nasopharyngeal carcinoma (NPC) and its clinical significance. METHOD: The expression of LMP2A, E-cad-herin and fibronectin proteins in 32 cases of chronic nasopharyngeal inflammation, 56 cases of NPC and 18 cases of NPC lymph node metastasis were examined byimmunohistochemical SP method. RESULT: (1)The positive rates of LMP2A in NPC and its lymph node metastasis were significantly higher than those of chronic nasopharyngeal inflammation (89. 3%vs 37. 5%o and 77. 8% vs 37. 5%) respectively (both P<0. 01); The normal expression rates of E-cadherin in NPC and its lymph node metastasis were significantly lower than those of chronic nasopharyngeal inflammation (33. 9% vs 90. 6% and 5. 6% vs 90. 6%) respectively (both P<0. 01); The positive rates of fibronectin in NPC and its lymph node metastasis were significantly higher than those of chronic nasopharyngeal inflammation (83. 9% vs 28. 1% and 72. 2% vs 28. 1%) respectively (both P<0. 01). (2) ZLMP2A expression were negatively correlated with normal expression of E-cadherin (r= -0. 387, P<0. 01), and were positively correlated with fibronectin (r= 0. 421, P<0. 01). (3)LMP2A, E-cadherin and fibronectin expression were significantly correlated with N stage and clinical stage (both P<0. 05), but the three proteins were not significantly correlated with M stage (both P> 0. 05). In addition, LMP2A and E-cadherin expression were significantly correlated with T stage (both P<0. 01). CONCLUSION LMP2A and fibronectin expressions were increased in NPC, but normal expression of E-cadherin were decreased. LMP2A may promote lymph node metastasis and malignant progression of NPC by induce EMT through downregulation of E-cadherin and upregulation of fibronectin.
Cadherins ; biosynthesis ; Carcinoma ; Epithelial-Mesenchymal Transition ; Fibronectins ; biosynthesis ; Humans ; Lymphatic Metastasis ; Membrane Proteins ; biosynthesis ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Nasopharynx

The effects of E-cadherin-transfected neural stem cells (NSCs) transplantation for spinal cord injury (SCI) in rats were investigated. Sixty SD rats were randomly divided into model control group, NSCs group, empty plasmid group and E-cadherin overexpression group (n=15 each). The animal SCI model was established by using the modified Allen's method. NSCs were cultured. Rats in NSCs group were subjected to NSCs transplantation. E-cadherin gene eucaryotic expression vector and pcDNA3.1-E-cadherin were respectively transfected into cultured NSCs, serving as empty plasmid group and E-cadherin overexpression group respectively. At 7th day after transplantation, neurological function of all rats was assessed by Tarlov score. After rats were sacrificed in each group, the number of BrdU and Nestin positive cells was counted by immunohistochemistry. Immumofluorescence method was used to detect the expression of neurofilament protein (NF) and glial fibrillary acidic protein (GFAP). As compared with model control group, the Tarlov score and the number of of BrdU and Nestin positive cells, and the expression of NF and GFAP in NSCs group, empty plasmid group, and E-cadherin overexpression group were increased significantly (P<0.05), and those in the E-cadherin overexpression group were increased more significantly than the other transplantation groups (P<0.05). It was suggested that E-cadherin could be conductive to nerve regeneration and repair probably by promoting the proliferation and differentiation of NSCs.
Animals ; Cadherins ; biosynthesis ; genetics ; Male ; Nerve Regeneration ; Neural Stem Cells ; metabolism ; transplantation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; therapy ; Stem Cell Transplantation ; Transfection

OBJECTIVETo investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation.

METHODSDPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction.

RESULTSThe relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P < 0.05). For in vitro tube formation assay, tubular structures were formed on the matrigel by differentiated DPC. A total of 47 miRNA were differentially expressed, in which 15 miRNA were up-regulated and 32 miRNAs down-regulated in differentiated DPC compared with the control. Of these, 4 miRNA were confirmed by qRT-PCR. The target genes of differential miRNA were predicted to associate with several biological functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway.

CONCLUSIONSThe differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

Antigens, CD ; Cadherins ; Cell Differentiation ; Collagen ; Computational Biology ; Dental Pulp ; metabolism ; Drug Combinations ; Fibroblast Growth Factor 2 ; Humans ; Laminin ; MicroRNAs ; Platelet Endothelial Cell Adhesion Molecule-1 ; biosynthesis ; Proteoglycans ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Vascular Endothelial Growth Factor A ; Wnt Signaling Pathway ; von Willebrand Factor

OBJECTIVETo obtain monoclonal antibodies (mAb) against LI-cadherin and investigate their effects on the proliferation of human hepatocellular carcinoma cells.

METHODSBalb/c mice were immunized with recombinant LI-cadherin, and hybridoma cell lines secreting monoclonal antibodies against LI-cadherin were established with routine cell fusion and subcloning approach. The specificity of these mAbs was determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of the mAbs obtained on the growth of HepG2 cells was assessed using inverted microscope and MTT assay.

RESULTSTwo hybridoma cell lines (F001 and F002) stably secreting specific mAbs were obtained. Western blot analysis showed that the two antibodies specifically recognized LI-cadherin antigen derived from human eucaryotic cells or tissue. Treatment of the HepG2 cells with the mAbs resulted in reduced viable cell number and changes in the cell morphologies, and the two mAbs inhibited the proliferation of HepG2 cells in a concentration-dependent manner (P<0.05).

CONCLUSIONThe two specific mAbs obtained can inhibit the proliferation of HepG2 cells in vitro, which facilitates further study of the relationship between LI-cadherin and tumors.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; pharmacology ; Cadherins ; immunology ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Humans ; Hybridomas ; secretion ; Liver Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C

Total saponins of Panax notoginseng (PNS) have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rg1 on renal fibrosis in rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into 3 groups: sham-operation (n=15), UUO (n=15) and UUO with ginsenoside Rg1 treatment (n=15, 50 mg per kg body weight, intraperitoneally (i.p.) injected). The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rg1 significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition. alpha-smooth muscle actin (alpha-SMA) and E-cadherin are two markers of tubular epithelial-myofibroblast transition (TEMT). Interestingly, ginsenoside Rg1 notably decreased alpha-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA (mRNA) of transforming growth factor-beta1 (TGF-beta1), a key mediator to regulate TEMT, in the obstructed kidney increased dramatically, but was found to decrease significantly after administration of ginsenoside Rg1. Further study showed that ginsenoside Rg1 considerably decreased the levels of both active TGF-beta1 and phosphorylated Smad2 (pSmad2). Moreover, ginsenoside Rg1 substantially suppressed the expression of thrombospondin-1 (TSP-1), a cytokine which can promote the transcription of TGF-beta1 mRNA and the activation of latent TGF-beta1. These results suggest that ginsenoside Rg1 inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.
Actins ; biosynthesis ; Animals ; Cadherins ; biosynthesis ; Collagen Type I ; genetics ; metabolism ; Fibronectins ; genetics ; metabolism ; Ginsenosides ; pharmacology ; Immunohistochemistry ; Male ; Nephritis, Interstitial ; drug therapy ; genetics ; metabolism ; pathology ; Panax notoginseng ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Smad2 Protein ; biosynthesis ; Thrombospondin 1 ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; biosynthesis ; genetics ; Ureteral Obstruction ; metabolism ; pathology

OBJECTIVETo determine whether human prostate cancer cell lines undergo epithelial-mesenchymal transition (EMT) and become more invasive when induced by HIF-1alpha, and to explore the underlying molecular mechanism.

METHODSThe cell line LNCaP, appropriate for the HIF-1alpha induction test, was screened out from 4 different EMT-negative prostate cell lines through vimentin gene detection by RT-PCR. The recombinant plasmid pCDNA3. 1(-)/HIF-1alpha was constructed and transfected into LNCaP with the Lipofectamine 2000 system. The control plasmid pCDNA3.1 (-) was transfected by the same method. The positive clone cells were selected by G418 and confirmed by Western blot and immunofluorescence staining. Then a Transwell polycarbonate filter, coated with 100 micol Matrigel at 1:20 dilution in the serum-free medium, was used to analyze the invasive potency. The expression of E-cadherin and vimentin was detected by Western blot.

RESULTSAmong the 4 different EMT-negative cell lines, LNCaP was the only one that expressed the vimentin gene but not protein. The expression of HIF1alpha was obviously higher in LNCaP/HIF1alpha than in LNCaP/pCDNA3. 1 (- an LNCaP. The number of the LNCaP/HIF1alpha cells that penetrated through the Transwell polycarbonate filter was significantly larger than that of the LNCaP and LNCaP/pCDNA3. 1(-) cells. Compared with the LNCaP/pCDNA3.1(-) and LNCaP cells, the expression of vimentin was up-regulated, while that of E-cadherin down-regulated, in LNCaP/HIF1alpha.

CONCLUSIONThe over-expression of HIF-1alpha could induce EMT in the human prostate carcinoma cell line LNCaP and enhance its invasiveness through E-cadherin and vimentin regulation.

Cadherins ; biosynthesis ; Cell Line, Tumor ; metabolism ; pathology ; Epithelium ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Vimentin ; biosynthesis

OBJECTIVETo explore the protective effect of the Phyllanthus Urinaria (PU) extract on the N-cadherin expression in the testicular tissues disrupted by nitrogen mustard (HN2) in vivo.

METHODSHN2 was intraperitoneally injected into male KM mice at the dose of 5 mg/kg to make reproductive toxicity models, and at the same time PU was administered for intervention at the dose of 125 mg/kg, 250 mg/kg and 500 mg/kg. N-cadherin distribution, mRNA and protein expression in the testicular tissues were detected by immunohistochemistry, RT-PCR and Western blotting.

RESULTSN-cadherin was mainly distributed in the membrane and cytoplasm of Sertoli cells at the basement of seminiferous epithelia, Leydig cells and peritubular cells, scarcely expressed in the basement of seminiferous epithelia and peritubular cells after HN2 administration. The expressions of mRNA and proteins of N-cadherin were significantly elevated with the increased dose of PU (P < 0.01). Compared with the normal control, the distribution and expression of N-cadherin showed no significant differences in either the high-dose PU group or the HN2 with high-dose PU intervention group (P > 0.05).

CONCLUSIONThe PU extract can effectively promote the N-cadherin expression in the testis tissues disrupted by HN2.

Animals ; Blotting, Western ; Cadherins ; biosynthesis ; genetics ; Immunohistochemistry ; Leydig Cells ; cytology ; drug effects ; metabolism ; Male ; Mechlorethamine ; toxicity ; Mice ; Mice, Inbred Strains ; Phyllanthus ; chemistry ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; cytology ; drug effects ; metabolism ; Testis ; cytology ; drug effects ; metabolism

OBJECTIVETo determine the effect of the transforming growth factor beta (TGF-beta) on the expression of invasion and metastasis associated proteins in the prostate cancer LNCaP cell line in vitro.

METHODSThe prostate cancer cell line LNCaP was treated with TGF-beta in vitro. Western blotting was used to detect the expression of the "invasion and metastasis" associated proteins E-Cadherin, N-Cadherin and Vimentin.

RESULTSThe expression of N-Cadherin and Vimentin of the LNCaP cells treated with TGF-beta for 12 hours was significantly upregulated, but not that of E-Cadherin.

CONCLUSIONTGF-beta may induce epithelial-mesenchymal transition (EMT) of LNCaP cells which might be of importance in promoting prostate cancer cells invading to ambient tissues and metastasizing to distant organs.

Blotting, Western ; Cadherins ; biosynthesis ; Cell Line, Tumor ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Transforming Growth Factor beta ; pharmacology ; Up-Regulation ; drug effects ; Vimentin ; biosynthesis