OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

The aim of this study was to investigate the effect of Wnt5a on the vincristine (VCR) resistance in human ovarian carcinoma SKOV3 cells and its possible mechanism. The drug-resistant SKOV3/VCR cells were established by stepwise exposure to VCR, and then the SKOV3/VCR cells were stably transfected with specific shRNA interference plasmid vector targeting for Wnt5a. The mRNA expression level of Wnt5a was measured by RT-PCR. CCK-8 assay was used to detect the cell viability of SKOV3/VCR cells. The apoptosis was analyzed by flow cytometry. The protein expression levels of Wnt5a, MDR1, Survivin, β-catenin, Akt, p-Akt(S473), GSK3β and p-GSK3β(Ser9) were detected by Western blot. The result showed that SKOV3/VCR cells had significantly higher protein expression levels of Wnt5a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5a, compared with SKOV3 cells (P < 0.05). WNT5A gene silencing significantly increased the sensitivity of SKOV3/VCR cells to VCR, the IC of VCR being decreased from 38.412 to 9.283 mg/L (P < 0.05), synergistically enhanced VCR-induced apoptosis of SKOV3/VCR cells (P < 0.05), down-regulated the protein expression levels of MDR1, β-catenin and Survivin (P < 0.05), and inhibited phosphorylation of Akt and GSK3β (P < 0.05). Meanwhile, LY294002 (PI3K inhibitor) decreased the protein expression levels of MDR1, β-catenin and Survivin, as well as the phosphorylation levels of Akt and GSK3β in SKOV3/VCR cells (P < 0.05). These results suggest that WNT5A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.
ATP Binding Cassette Transporter, Subfamily B ; metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Gene Silencing ; Glycogen Synthase Kinase 3 beta ; metabolism ; Humans ; Ovarian Neoplasms ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Survivin ; metabolism ; Vincristine ; pharmacology ; Wnt-5a Protein ; metabolism

OBJECTIVES: To observe the effects of Yougui pill (Traditional Chinese Medicine) on the related factors of Wnt signal pathway of rats with knee osteoarthritis (KOA), and explore its protective mechanism. METHODS: Sixty SPF SD rats were randomly divided into the sham-operative group, model group, glucosamine sulfate group, high-dose, middle-dose, low-dose of Yougui pill treated group (=10). KOA model was established by modified Hulth method for six weeks. The rats in the high, middle and low-dose of Yougui pill group were treated with Yougui pills at the doses of 20,10 and 5 g/kg respectively by gastrogavage once a day for 8 weeks, while equal volume of normal saline was given to those in the sham and model control group and an equal volume of glucosamine sulfate (1.7 g/kg·d) was given to those in glucosamine sulfate group for 8 weeks. The knee joint was removed after the last dose of drug. The pathological changes of cartilaginous tissues were observed under a microscope. The mRNA levels of Dickkopf homolog 1(DKK1), Wnt induced secreted protein 1(WISP1), Wnt1, low density lipoprotein receptor related protein 5(LRP5) and beta -catenin in rats cartilaginous tissues were analyzed by using RT-PCR method, and the protein contents of DKK1, WISP1, Wnt1, LRP5 and beta-catenin in cartilaginous tissues were detected by Western blot. RESULTS: Compared with the sham group, the articular cartilage was severely damaged, the Mankin score was increased significantly (<0. 05), the mRNA and protein expression levels of DKK1 in cartilaginous tissue were markedly decreased(<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were increased significantly in model group(<0.05). Compared with model group, the articular cartilage lesions was light (<0.05), the Mankin Score was decreased significantly(<0.05), and the mRNA and protein levels of DKK1 in cartilaginous tissue were increased(<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were decreased in Yougui pill high-dose group and glucosamine sulfate group (<0.05). CONCLUSIONS Yougui pill has protective effects on the KOA by inhibiting the expressions of WISP, Wnt1, LRP5, beta-catenin and increasing the expression of DKK1 cytokine in the Wnt signaling pathway.
Animals ; CCN Intercellular Signaling Proteins ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Glucosamine ; pharmacology ; Intercellular Signaling Peptides and Proteins ; metabolism ; Osteoarthritis, Knee ; drug therapy ; Proto-Oncogene Proteins ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Wnt Signaling Pathway ; Wnt1 Protein ; metabolism ; beta Catenin ; metabolism

Considering the great potential of natural products as anticancer agents, the present study was designed to explore the molecular mechanisms responsible for anticancer activities of Mesua ferrea stem bark extract against human colorectal carcinoma. Based on MTT assay results, bioactive sub-fraction (SF-3) was selected for further studies using HCT 116 cells. Repeated column chromatography resulted in isolation of less active α-amyrin from SF-3, which was identified and characterized by GC-MS and HPLC methods. α-amyrin and betulinic acid contents of SF-3 were measured by HPLC methods. Fluorescent assays revealed characteristic apoptotic features, including cell shrinkage, nuclear condensation, and marked decrease in mitochondrial membrane potential in SF-3 treated cells. In addition, increased levels of caspases-9 and -3/7 levels were also observed in SF-3 treated cells. SF-3 showed promising antimetastatic properties in multiple in vitro assays. Multi-pathway analysis revealed significant down-regulation of WNT, HIF-1α, and EGFR with simultaneous up-regulation of p53, Myc/Max, and TGF-β signalling pathways in SF-3 treated cells. In addition, promising growth inhibitory effects were observed in SF-3 treated HCT 116 tumour spheroids, which give a hint about in vivo antitumor efficacy of SF-3 phytoconstituents. In conclusion, these results demonstrated that anticancer effects of SF-3 towards colon cancer are through modulation of multiple molecular pathways.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Colorectal Neoplasms ; drug therapy ; metabolism ; pathology ; physiopathology ; ErbB Receptors ; genetics ; metabolism ; HCT116 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Magnoliopsida ; chemistry ; Neoplasm Metastasis ; prevention & control ; Plant Bark ; chemistry ; Plant Extracts ; pharmacology ; Signal Transduction ; drug effects ; Wnt Proteins ; genetics ; metabolism

OBJECTIVETo observe the effect of parathyroid hormone (PTH)(1-34) on the expression of matrix Gla protein (MGP) and Wnt/β-catenin signaling pathway and elucidate the possible molecular mechanism of PTH (1-34) in the prevention and treatment of osteoporosis.

METHODSMG63 cells treated with PTH (1-34) at 10(-9), 10(-8), and 10(-7) mol/L, alone or in combination with Wnt/β-catenin signaling pathway inhibitors DKK-1 (200 ng/ml) were examined for mRNA and protein expressions related with Wnt/β-catenin signaling with real-time PCR and Western blotting. The cell differentiation after the treatment was assessed with alkaline phosphatase (ALP) staining and cell viability assay.

RESULTSPTH (1-34) significantly increased the expression of MGP in a dose-dependent manner in MG63 cells (P<0.05 or P<0.01). PTH treatment obviously enhanced ALP activity in the cells, and this effect was suppressed by DKK-1. Combined treatment with DKK-1 partially blocked PTH-induced enhancement of ALP activity (P<0.05). PTH promoted the expression of MGP and enhanced LRP5, β-catenin, and Runx2 expressions in Wnt/β-catenin signaling pathway at both protein and mRNA levels (P<0.05 or P<0.01). DKK-1 partially blocked the effect of PTH (1-34) on Wnt/β-catenin signaling pathway (P<0.05) without affecting MGP expression.

CONCLUSIONPTH (1-34) significantly increases the expressions of MGP and proteins in the Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling pathway and MGP mediate the regulation of osteogenosis by PTH.

Alkaline Phosphatase ; metabolism ; Calcium-Binding Proteins ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Cell Survival ; Extracellular Matrix Proteins ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Osteogenesis ; Osteoporosis ; Parathyroid Hormone ; pharmacology ; Real-Time Polymerase Chain Reaction ; Wnt Signaling Pathway

OBJECTIVETo study the effects of Weipixiao (胃痞消, WPX) on Wnt pathway-associated proteins in gastric mucosal epithelial cells from rats with gastric precancerous lesions (GPL).

METHODSSprague Dawley rats were randomly divided into control, model, vitacoenzyme (0.2 g·kg(-1)·day(-1)), WPX high-dose (H-WPX, 15 g·kg(-1)·day(-1)), WPX medium-dose (M-WPX, 7.5 g·kg(-1)·day(-1)) and WPX low-dose (L-WPX, 3.75 g·kg(-1)·day(-1)) groups. After successfully establishing the GPL model, the rats were consecutively administered WPX or vitacoenzyme by gastrogavage for 10 weeks. Differential expression of Leucine-rich repeat-containing G-proteincoupled receptor 5 (Lgr5), matrix metalloproteinase-7 (MMP-7), Wnt1, Wnt3a, and β-catenin in gastric mucosal epithelial cells in all groups were immunohistochemically detected, and the images were taken and analyzed semiquantitatively by image pro plus 6.0 software.

RESULTSGastric epithelium in the model group showed significantly higher expression levels of Lgr5, MMP-7, Wnt1, Wnt3a and β-catenin than those of the control group(P<0.01). Interestingly, we also observed Lgr5+ cells, which generally located at the base of the gastric glandular unit, migrated to the luminal side of gastric epithelium with GPL. The expression levels of Lgr5, MMP-7, Wnt1, and β-catenin were all down-regulated in the L-WPX group as compared with those of both model and vitacoenzyme groups (P<0.05). A similar, but nonsignificant down-regulation in expression level of Wnt3a was noted in all WPX groups (P>0.05).

CONCLUSIONOur findings suggested that the therapeutic mechanisms of WPX in treating GPL might be related with its inhibitory effects on the expressions of Lgr5, MMP-7, Wnt1, β-catenin and the aberrant activation of Wnt/β-catenin pathway.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Gastric Mucosa ; pathology ; Immunohistochemistry ; Male ; Matrix Metalloproteinase 7 ; metabolism ; Precancerous Conditions ; drug therapy ; pathology ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; metabolism ; Staining and Labeling ; Stomach Neoplasms ; drug therapy ; pathology ; Wnt Proteins ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism.

METHODSThe multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively.

RESULTSThe 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P < 0.05 and r = -0.473, P < 0.05), while the culture medium without 17-AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P < 0.05); At same time of culture, the more high of 17-AAG concentration, the more high of cell ratio in G1 phase (P < 0.05), at same concentration of 17-AAG, the more long time of culture, the more high of cell ratio in G1 phase (P < 0.05).

CONCLUSIONThe HSP90 inhibitory 17-AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.

Apoptosis ; Benzoquinones ; pharmacology ; Cell Cycle ; Cell Division ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Multiple Myeloma ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo explore the effect of decitabine on Dickkopf-1 (DKK1) gene expression level and its downstream Wnt signaling pathway in acute myeloid leukemia (AML) cell line HL-60.

METHODSFlow cytometry and DNA ladder analysis were performed to detect apoptosis in HL-60 cell treated with different concentration of decitabine. Methylation-specific polymerase chain reaction (MS-PCR) was used to examine the methylation status of DKK1 gene. The expressions of mRNA and protein were determined by qRT-PCR and Western blot, respectively.

RESULTSFlow cytometric detection showed that after treating HL-60 cell line with decitabine of different concentrations for 48 h, the early apoptosis of HL-60 cells increased significantly as compared with control group (P < 0.05). DNA ladder analysis showed that the DNA ladder and demethylation of DKK1 gene appeared. RT-PCR and Western blot showed that the expressions of mRNA and protein increased. The protein expressions of β-catenin and C-MYC decreased.

CONCLUSIONThe decitabine can promote the apoptosis of HL-60 cells throngh demethylation of DDK1 gene and inhibition of Wnt signalling pathway.

Apoptosis ; Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; Gene Expression Regulation, Bacterial ; Genes, myc ; HL-60 Cells ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myeloid, Acute ; pathology ; RNA, Messenger ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo investigate the effect of inhibiting and activating Wnt signalling pathway on monocyte differentiation of HL-60 cells induced with a new steroidal drug NSC67657 and its possible mechamism.

METHODSThe HL-60 cells were treated with 5, 10 and 20 µmol/L XAV-939 (inhibitor of Wnt signalling pathway) for 3 days, and with 10, 20 and 30 mmol/L LiCl (activator of Wnt signalling pathway) for 1 day; the expression levels of down-stream genes and proteins of Wnt signolling pathway were detected by RT-PCR and Western blot, respectively; the expression of cell surface differentiation antigen CD14 and early apoptosis of HL-60 cells was detected by flow cytometry, moreover the most suitable concentration of Wnt inhibitor and activator for HL-60 cells was determined. Then the HL-60 cells with inhibited and activated Wnt pathway were treated with NSC67657 of 10 µmol/L for 3 days; the expression levels of CD14 and down-stream target proteins of Wnt signalling pathway in blank control (culture mediam) group, simple NSC67657-treated group, NSC67657 combined with inhibitor group and NSC67657 combined activator group were compared and analyzed.

RESULTS20 µmol/L XAV-939 and 20 mmol/L LiCl could effectively inhibit and activate Wnt signalling pathway of HL-60 cells respectively, could significantly down- and up-regulate the expression of cyclinD1, TCF1 and c-Jun genes (P < 0.05) and proteins (P < 0.05); moreover, the number of CD10(+) HL-60 cells in these conditions was below 1%, no early apoptosis of HL-60 cells was found. In the simple NSC67657-treated groups, the expression of cyclinD1, TCF1 and c-Jun proteins was down-regulated (P < 0.05), and the percentage of CD14(+) HL-60 cells accounted for 62.13 ± 9.44; after the HL-60 cells were treated with XAV-939, the NSC67657 could more significantly down-regulate the expression of cyclinD1, TCF1 and c-Jun proteins and the percentage of CD14(+) HL-60 cell accounted for 84.17 ± 5.39%, as compared with simple NSC67657-treated group; as compared with blank controls group, the expression of cyclinD1, TCF1 and c-Jun proteins was more obviously down-regulated and the percentage of CD14(+) HL-60 cells decreased to 33.99 ± 8.37% in NSC67657 combined LiC1 streated group, but which were higher than those in simple NSC67657-treated group (P < 0.05).

CONCLUSION20 µmol/L XAV-939 and 20 mmol/L LiCl as effective inhabitor and activator of Wnt signalling pathway respectively can significantly down- and up-regulate the expression of Wnt down-stream pathway target genes and proteins. The influence of XAV-939 and LiC1 on differentiation of HL-60 cells induced by NSC67657 suggests that Wnt signalling pathway plays a key role in monocyte differentiction of HL-60 cells induced by NSC67657.

Apoptosis ; Cell Differentiation ; Cyclin D1 ; metabolism ; Flow Cytometry ; HL-60 Cells ; Hepatocyte Nuclear Factor 1-alpha ; metabolism ; Humans ; Lipopolysaccharide Receptors ; metabolism ; Mesylates ; pharmacology ; Monocytes ; cytology ; Proto-Oncogene Proteins c-jun ; metabolism ; Steroids ; pharmacology ; Wnt Signaling Pathway