The novel synthetic psychoactive ketamine analog methoxetamine is reportedly being used for recreational purposes. As ketamine use can result in urinary dysfunction, we conducted the present study to investigate how methoxetamine affects the bladder. A cystometry investigation showed that female Sprague-Dawley rats experienced increased micturition frequency bladder dysfunction after receiving a daily intraperitoneal injection of 30 mg/kg methoxetamine or ketamine for periods of 4 or 12 weeks. Histologic examinations of rat bladder tissue revealed damaged urothelium barriers, as well as evidence of inflammatory cell infiltration and matrix deposition. The drug-treated rats showed significantly upregulated levels of pro-inflammatory cytokines such as IL-1β, IL-6, CCL-2, CXCL-1, CXCL-10, NGF, and COX-2. In addition, interstitial fibrosis was confirmed by increased levels of collagen I, collagen III, fibronectin and TGF-β. Besides direct toxic effect on human urothelial cells, methoxetaminealso induced the upregulation related cytokines. Our results indicate that long term methoxetamine treatment can induce bladder dysfunction and inflammation in rats. Methoxetamine was confirmed to produce direct toxic and pro-inflammatory effects on human urothelial cells. Methoxetamine-associated bladder impairment may be similar to ketamine-induced cystitis. N-methyl-d-aspartate receptor antagonist [Methoxetamine (MXE) is a structural analog of ketamine (KET), which serves as a tagonist . MXE bintagonist . MXE hastagonist . Currenttagonist  that shotagonist . A serietagonist , abnormatagonist , mast cetagonist , cell aptagonist ,10, and tagonist ,12. Althtagonist , and thutagonist .MXE was originally designed and synthesized to function as an antidepressant , and wasp = 0.009 (p = 0.008) or KET group (p = 0.006). From Week 2 to Week 12, the changes in body weight in the three groups showed similar trends and were statistically significant , suggesting that exposure to methoxetamine could significantly delay weight gain (p < 0.05 vs. both the CON group and KET group). In contrast, ketamine showed a negligible effect on body weight (p > 0.05).After two weeks of treatment, significant differences in body weight were observed among the three groups of rats: 241.0 ± 9.0 g  vs. 224.3 ± 9.9 g (MXE group) vs. 239.1 ± 7.1 g (KET group),  = 0.009 . The meaAs shown in Urodynamic examinations were performed to gain further insight into the development of bladder dysfunction due to MXE and KET administration, and representative continuous cystometrograms are shown in Gross pathological changes in the bladder mucosa were evaluated by microscopy A. The blE-cadherin staining appeared decreased in methoxetamine- or ketamine-treated rats . Our results demonstrated a dose- and time-dependent up-regulation of these cytokines/chemokines mRNA levels .We further detected fibrotic damage in bladder tissue by Masson trichrome staining methods A, and alTo elucidate whether methoxetamine or ketamine had any direct toxic effect on bladder epithelial cells in vitro, SV-HCU-1 cells which had been stimulated with 0–8 mM ketamine or methoxetamine for 24 or 48 h were assessed for their viability with the CCK-8 assay A. The reWe selected a 0.25 mM drug concentration for use in studying the effects of KET and MXE on pro-inflammatory molecule synthesis, because at that concentration, the SV-HCU-1 cells displayed a level of viability that was ~80% that of control cells. This allowed us to exclude the effects of interference resulting from the excessive release of cytokines/chemokines due to necrocytosis. Similar to results in the in vivo experiments, the expression levels of mRNA for cytokines/chemokines in SV-HUC-1 cells appeared to increase after the cells had been incubated with methoxetamine or ketamine for 24 or 48 h, respectively B,C. ThesOur results suggest that MXE and KET cause changes in the urination patterns of rats. Cystometry investigations showed that bladder capacity, and bladder compliance decreased, while the numbers of non-voiding contractions and urination frequency increased in the drug-treated rats. Histologic studies revealed damaged urothelium barriers, reduced amounts of glycosaminoglycans, but increased levels of congestion, inflammatory cell infiltration, and fibrosis in the bladders of drug-treated rats. Moreover, the expression levels of several cytokines in bladder tissue were significantly increased following MXE or KET exposure. In addition to its cytotoxic effects, MXE also stimulated human urothelium cells to generate cytokines. When taken together, these findings explain that MXE induces cystitis and impairs bladder function. In other words, MXE may not be “bladder friendly”.This study is the first to investigate the impact of MXE on the rat bladder, and is also novel because urodynamic measurements have not been previously employed to measure the effects of MXE in animals. Various researchers have used cystometry to objectively evaluate the status of the lower urinary tract in animal models of ketamine-induced cystitis ,16,17, aKetamine was found to cause damage in the bladder mucosal, submucosal, and muscular layers. Several previous studies have reported that ketamine administration resulted in a thinner or denuded epithelium, inflammatory cell infiltration, and vascular proliferation in the submucosal layer of bladder tissue, as well as collagen deposition in the muscular layer ,18. We fGlycosaminoglycans (GAGs) are distributed on the surface of the bladder mucosal epithelium. Our alcian blue staining studies revealed lower GAG staining intensities in the MXE group and KET group as compared with GAG staining intensity in the control group. As an important component of bladder epithelium barriers, GAGs protect the vesical mucosa from bacteria, molecules, and ions present in the urine. A decreased excretion of GAGs and defects in the epithelial permeability barrier are regarded as mechanisms of bladder pain syndrome/interstitial cystitis (BPS/IC). Moreover, a previous study showed that rats with ketamine-induced cystitis had reduced levels of glycoprotein GP51 and potassium in their urine .E-cadherin is a glycoprotein found in cell-to-cell adherens junctions, and is crucial for maintaining epithelial morphology and polarity. E-cadherin expression is often repressed in patients with various bladder disorders associated with LUTS, such as BPS/IC, ketamine-induced cystitis (KC), and recurrent UTIs [E-cadherin, involved in bladder dysfunction secondary to bladder outlet obstruction. In the present study, E-cadherin content in the urothelium became significantly reduced somewhat later after MXE or KET administration. Consistent with KC [ent UTIs . Jiang eent UTIs  proposed with KC , the aboAnother important feature in the examined bladder specimens was evidence of mast cell infiltration. Activated mast cells play a dominant role in the mechanism of BPS/IC . In prevWe detected increased mRNA levels of numerous cytokines in bladder tissues obtained from the MXE group. Among those cytokines, IL-1β, IL-6, CCL-2, and NGF are widely regarded as biomarkers for BPS/IC . Aside fNumerous animal studies have proven that long term ketamine treatment leads to bladder fibrosis , which iNo previous study has focused on how MXE affects bladder epithelial cells. In our study, we found that MXE and KET produced toxic effects in SV-HUC-1 cells in a time and dose-related manner. A study by Shen et al. showed that KET was cytotoxic to human urothelial cell lines . Those fOur study has several limitations: (1) Clinical investigation and data were not included in present study. The characteristics of MXE abusers awaits further studies; (2) The occurrence and development of bladder inflammation involve various cells. Our study solely estimated the expression of pro-inflammatory mediators in urothelial cells after MXE administration. However, we consider that it is still valuable for our purpose to understand the pro-inflammatory effect of MXE; (3) Only one dose, one preparation and one administration route were tested.The potential harm resulting from MXE abuse has not been widely recognized. Lawn et al. conducted a survey of 427 MXE abusers who had once used ketamine, and found that 23% of them experienced LUTS . This waThirty-six female Sprague-Dawley rats (180–210 g) were purchased from the Laboratory Animal Center of Southern Medical University (SMU) in Guangzhou China. The animals were randomly assigned to three groups which received a single daily intraperitoneal injection of 0.9% saline (control group), 30 mg/kg methoxetamine (MXE group) or 30 mg/kg ketamine (KET group) for a period of either 4 or 12 weeks. Ketamine hydrochloride solution  and methoxetamine solid  were approved for use in this study by the Food and Drug Administration of Guangdong Province, China. The rats were weighed each week to adjust the amount of drug they received. All experimental procedures were performed in accordance with the Guidelines for Laboratory Animal Care, and the study protocol was approved by the Institutional Animal Care and Use Committee of SMU .4·5H2O for >40 min and then dehydrated at 160 °C for 30 min to promote conversion of CuSO4·5H2O to CuSO4 was stretched across the underside of the cage. Direct contact between falling urine and the paper immediately initiated hydration of the CuSO4, resulting in discoloration of the paper. Micturition frequency was determined by calculating the number of spots on the paper as determined using Photoshop software  in reversed color contrast phase. Overlapped urine spots with legible edges were considered to be a single urination incident. Micturition frequency was measured for a period of 3 h per day, for 3 consecutive days, and the mean value was determined via statistical analysis.Micturition frequency was measured as previously described . BrieflyT-branch adaptor. After emptying the bladder with a syringe, the bladder was infused with 0.9% sterile saline at a steady rate of 12 mL/h (200 μL/min). When a consistent and reproducible pattern of micturition was established, digital intravesical pressure signals were continuously recorded for 60 min or at least 5 void cycles using a MMS Solar system . The measured parameters included VCs, NVCs, bladder capacity, PVR volume , ICI, baseline pressure (lowest pressure between adjacent voids), threshold pressure , micturition pressure (peak pressure during urination), and bladder compliance .The rats were anesthetized by injection with 20% urethane (1.0 g/kg); after which, they were immobilized in the supine position, and a 1.0 mm inner diameter epidural catheter was transurethrally inserted into the bladder. The other end of the catheter was connected to a pressure transducer and a micro-pump via a E-cadherin expression. Staining for CD68 was performed to analyze macrophages aggregates present in the urinary bladders. Briefly, the tissue sections were deparaffinized and immersed in 3% H2O2 for 30 min to quench endogenous peroxidase activity. After being blocked with 2% BSA, the sections were incubated with E-cadherin , CD68 antibody , collagen I antibody , fibronectin antibody  or α-SMA antibody  overnight at 4 °C. Following incubation with the primary antibodies, the tissue sections were incubated with the appropriate biotinylated secondary antibody  for 30 min; after which, they were incubated with 3,3′-diaminobenzidine and lightly counterstained with hematoxylin. Quantitative digital image analysis was performed using Image-Pro Plus 6.0 software , and representative images were selected for presentation in this report.After completing the urodynamic investigations, the rats were sacrificed and their bladder tissues were removed. Specimens of bladder tissue for histologic staining were fixed in 4% paraformaldehyde; after which, they were embedded in paraffin and cut into 4 μm sections for analysis. Sections for histopathologic evaluation were stained with hematoxylin and eosin , alcian blue, masson’s trichrome and toluidine blue. Epithelial integrity was determined by 2 atmosphere. Prior to treatment, the cells were transferred to serum-free medium and incubated overnight to synchronize their growth; after which, they were treated with ketamine or methoxetamine solution at the indicated concentration for the designated time period.SV-HUC-1 cells  were cultured in DMEM/F12 medium containing 10% at 37 °C in a 5% COSV-HUC-1 cells (100 µL containing 5000 cells) were seeded into individual wells of a 96-well plate, and then pre-incubated for 24 h to allow their adherence. The cells were then incubated with 0.25 mM ketamine or methoxetamine for 24 or 48 h, respectively. Following incubation, 10 μL of CCK-8 solution , which serves as a tetrazolium salt colorimetric assay, was added to each well of the plate and incubated with the cells for 2 h. Next, a microplate reader  was used to measure the absorbance of each well at 450 nm.® Premix Ex TaqTM II (Takara Bio Inc.). The sequences of the primer pairs were designed by Invitrogen  (Ct) of various genes was determined, and changes in gene expression were calculated using the delta-delta Ct method. The results are expressed as a ratio relative to GAPDH. Allof the PCR experiments were repeated individually at least 3 times.Total RNA was extracted from rat bladder tissue and SV-HUC-1 cells using RNAiso Plus reagent . Complementary DNA (cDNA) was synthesized using the PrimeScript™ RT reagent Kit (Takara Bio Inc.), and genomic DNA was eliminated according to the manufacturer’s instructions. Next, qRT-PCR was performed using an Applied Biosystems 7500 Real Time PCR System and SYBRley, UK) , and prit3 test was preferred. For all analyses, two-sided p-values < 0.05 were considered statistically significant.All data were analyzed using SPSS for Windows, Version 13.0. Chicago, IL, USA: SPSS Inc., and results are expressed as the mean ± SD. Multiple group comparisons were performed using analysis of variance (ANOVA), and the LSD test was used for pair-wise comparisons . Otherwise, the Dunnett’s Methoxetamine is a novel synthetic psychoactive ketamine analog. Considering the increasing cases of ketamine-induced cystitis, it is crucial to explore the effect of MXE to urinary system. Our results of cystometry and histologic examinations indicate that long term MXE treatment can induce bladder dysfunction and inflammation in rats. In vitro, methoxetamine was confirmed to produce direct toxic and pro-inflammatory effects on human urothelial cells. MXE-associated bladder impairment may be similar to ketamine-induced cystitis. Our findings not only reveal the influence of MXE on the bladder, but also suggest that MXE-associated bladder impairment may be similar to ketamine-induced cystitis.


The interfacial adhesion of recycled carbon fiber (CF) reinforced epoxy composite heated by microwave (MW) irradiation were investigated by changing the curing state of the epoxy resin. The recycled CF was recovered from the composite, which was prepared by vacuum-assisted resin transfer molding, by thermal degradation at 500 or 600 °C. Thermogravimetric analysis showed that the heating at 600 °C caused rough damage to the CF surface, whereas recycled CF recovered at 500 °C have few defects. The interfacial shear strength (IFSS) between recycled CF and epoxy resin was measured by a single-fiber fragmentation test. The test specimen was heated by MW after mixing the epoxy resin with a curing agent or pre-curing, in order to investigate the curing effects on the matrix resin. The IFSSs of the MW-irradiated samples were significantly varied by the curing state of the epoxy resin and the surface condition of recycled CF, resulting that they were 99.5 to 131.7% of oven heated samples Furthermore, rheological measurements showed that the viscosity and shrinking behaviors of epoxy resin were affected based on the curing state of epoxy resin before MW irradiation. Carbon fiber-reinforced plastic (CFRP) is attracting attention due to its excellent mechanical and light-weight properties ,4,5,6,7.Recently, microwave (MW) processes have attracted attention as an innovative curing system for composites composed of CF and thermosetting resins ,14. In pThe main objective of this study was to clarify the effects of the curing state of epoxy resin and the surface condition of recycle CF on interfacial adhesion between various recycled CFs and epoxy resin as the thermosetting resin matrix heated by MW irradiation. Furthermore, the viscosity and the shrinkage behavior of epoxy resin at the curing temperature were investigated by rheological measurements.2. Bisphenol A-type epoxy resin (JER827), bisphenol F-type epoxy resin (JER806), and curing agents (JER cure 3080 or ST11) were purchased from Mitsubishi Chemical Co. Ltd., Tokyo, Japan. These epoxy materials are listed in f) of the prepared CFRP was measured using JIS K7075 [f was 65 vol %. The recycled CF was recovered from prepared CFRP by thermal degradation processing at 500 or 600 °C for 1 h in an air atmosphere. In the recycling process, the heating rate was adjusted to 5 °C/min. Hereafter, virgin CF, recycled CF heated at 500 °C, and recycled CF heated at 600 °C are termed vCF, rCF-500, and rCF-600, respectively.Commercially available CF fabrics  were used in this work. The textile weight of used CF fabrics is 100 g/mIS K7075 . The Vf Thermogravimetric (TG) analyses of CF and CFRP were performed using a thermal analysis instrument  from 30 °C to 900 °C under natural air of 200 mL/min at a heating rate of 5 °C/min. Furthermore, TG analyses of CFRP were carried out at temperatures of 500 and 600 °C for 3 h at a heating rate of 5 °C/min.β is the shape parameter, γ is the scale parameter, Γ(x) is the gamma function, and σ is the tensile strength.Tensile testing of a single CF was performed using an autograph apparatus  with a load cell of 10 N. Test specimens for tensile testing were prepared by fixing a single CF onto a paper with an adhesive agent. The test specimen was set in the tensile apparatus and the paper was cut into two parts before evaluation. Single-fiber tensile tests were carried out at a gauge length of 25 mm. The cross-head speed was 1.0 mm/min. The tensile strength of the CFs was calculated using the standard Weibull distribution using Equations (1) and (2) ,18. The A single CF was placed in the center of a dog-bone shaped silicon mold. The gauge length of the test specimens for fragmentation testing was fixed at 35 mm. Each end of the CF was fixed with adhesive tape. Epoxy resin JER827 was mixed with curing agent JER cure 3080 at a mass ratio of 2:1 using a hybrid deformation blender for 1 min at 2000 rpm. Epoxy resin was cast into the silicon mold. For this test, the test specimen was irradiated by MW at different times. One was MW-irradiated immediately after resin mixing, and the sample was pre-cured at 23 °C for 24 h. Then, the sample was post-cured at 80 °C for 3 h. The other sample was MW-irradiated after pre-curing at 23 °C for 24 h, and the specimen was post-cured. In this examination, the absorption of MW irradiation of the specimen could not be controlled, as the sample was a single CF. Therefore, the output powers of MW irradiation were controlled at 10, 20, and 30 W. The MW irradiation absorbed by the specimen was calculated by subtracting the reflection power from the output power.fσ is tensile strength of CF, D is diameter of CF, and cl is the critical fiber length. The critical fiber length (cl) can be determined using the average fragment length (l) by Equation (4):The IFSS between CF and epoxy resin was immediately measured without any conditioning after the preparation of test specimen by a single-fiber fragmentation test. The fragmentation test of the prepared composite was performed using a compact tensile apparatus at a displacement rate of 10 mm/s. The specimen was pulled until the fragments of fiber no more break. During this test, the specimen was observed using a microscope  with a digital camera . The fragment length within the gauge length was measured via image analysis with a calibrated measuring scale. The IFSS between the carbon fiber and epoxy resin was calculated using the following Equation (3) ,20,21,22The average fragment length is the arithmetic mean of the measured fragment lengths at the saturation stage . The IFSLε was calculated using the following the Equation (5) [ν is Poisson’s coefficient of the resin, taken to be 0.35 [h is the gap distance, and h0 represents the initial gap. Here, this relation assumes that the in-plane strain of the resin is zero.The shrinkage and rheological behavior of the epoxy resin composed of JER827 and JER cure 3080 were measured using a rheometer  Disposable aluminum parallel plates with diameters of 25 mm were used in the oscillation mode. The normal force is the amount of force applied by the top plate and is defined as negative when the epoxy resin is in compression. The test was performed in the torque- or strain-controlled mode at a constant angular velocity. Immediately after mixing, the epoxy resin was poured between the plates at room temperature and the gap was set at 1.0 mm. After pouring, the epoxy resin was maintained at 25 °C for 5 min, and heated to 80, 100, and 120 °C until its gelation point was reached. The gelation point of epoxy resin was determined based on the cross-over point of the elastic modulus and loss modulus. After gelation, the sample was heated further at the same temperature for 1 h and post-heated at 120 °C for 1 h. Finally, the sample was cooled to 25 °C. The rheometer was programmed with two segments. In segment I, the gap between the plates was kept constant and the strain was fixed at 15% with an oscillation frequency of 0.2 Hz. When the epoxy resin reached the gelation point, the rheometer was controlled according to segment II. In segment II, the torque was fixed at 0.5 mN with an oscillation frequency of 30 Hz and the normal force was kept constant at 0.1 N. The typical temperature profile, gap, and normal force of epoxy resin at a heating temperature of 100 °C are shown in tion (5) :(5)εL=[ be 0.35 ,25, h is8 and 2.10 × 104 MPa·s, respectively. The viscosity of the resin pre-heated at 23 °C, which was not gelled for 24 h, decreased greatly. However, the viscosity of the resin pre-heated at 30 °C, which was gelled for 16.5 h, only minimally decreased. Although the viscosity of the resin heated after pre-curing at 23 °C was reduced by heating, the viscosity remained higher than the initial viscosity of the resin. In MW heating, CFs act as a conductor and get abruptly heated due to ionic polarization and dipole orientation, rather than relying on heat transfer through conduction and convection [The improvement of the interfacial adhesion between recycled CF and epoxy resin by MW irradiation is now discussed. The improved IFSS is assumed to be mainly derived from the viscosity and shrinkage of the epoxy resin. For the purpose of validating whether the viscosity and the shrinkage of epoxy resin influence the interfacial adhesion, the viscosity variation and the shrinkage behavior of epoxy resin were investigated by rheological measurements. In previous studies, the temperature of CF increased with increasing output power of MW . In factnvection ,28,29,30The effect of shrinkage of the epoxy resin on interfacial adhesion is also discussed. To improve the interfacial adhesion between various recycled CFs and epoxy resin by MW irradiation, the IFSSs of composites prepared under different conditions was investigated. In this study, recycled CFs, with or without surface defects, were recovered from CFRP by thermal degradation processes at different temperatures. In the case of the composite MW-irradiated immediately after mixing, the IFSSs of both recycled CFs samples increased with increasing absorbed MW. On the other hand, the IFSS of the composite composed of recycled CF with surface defects did not improve by MW irradiation after pre-curing. We also investigated the curing behavior of epoxy resins by using a rheometer. The viscosity of the epoxy resin decreased greatly by heating immediately after mixing, whereas its reduction was suppressed by heating after pre-curing. The pre-curing of epoxy resin before gelation did not affect the shrinking behavior of epoxy resin. These results suggest that the MW process improved the interfacial adhesion between recycled CF and epoxy resin from two aspects, namely, by enhanced impregnation owing to the decrease of viscosity of the resin on the interface, and the shrinkage of epoxy resin along the fiber direction.One important consequence of the present work is that the interfacial adhesion between recycled CF and the epoxy resin is significantly affected by the curing state of the epoxy resin before MW irradiation. The interfacial adhesion between various recycled CF and epoxy resin can be improved by MW irradiation immediately after mixing the epoxy resin and curing agent.


A cationic photo-curable cycloaliphatic epoxy resin has been investigated as reactive monomer in blue light crosslinking process. We have demonstrated that camphorquinone is able to abstract labile hydrogen from the epoxy monomer, giving rise to the formation of carbon-centered radicals that are oxidized by the onium salt; a complete epoxy group conversion was reached after 50 s of irradiation. The presence of water up to 1 wt% was tolerated without any important detrimental effect on the kinetics of light-curing. The presence of the inorganic filler up to 65 wt% did not significantly influence the curing process. ShrinkaIn order to overcome this limitation, a strategy to reduce shrinkage is the delaying of gelation of the acrylic systems in order to reduce strain during curing . This meAmong cyclic monomers, cycloaliphatic epoxy monomers show a very little volume shrinkage upon irradiation ; therefoIn particular, cycloaliphatic epoxy monomers are of great interest since they demonstrate significantly lower shrinkage than methacrylic resins, still showing high reactivity to be cured by cationic photopolymerization in an acceptable time frame and to an adequate depth using a visible light source.A typical formulation contains camphorquinone (CQ) as visible light sensitizer, which is actually the most frequently used photoinitiator in dental materials cured by visible light, a diphenyl iodonium salt as photoinitiator and a low basic amine as electron-donor co-initiator . For catWhile it has been demonstrated that visible light cationic photopolymerization of epoxides can be initiated with success using a three component initiator system, it is important at this stage to investigate the effect of the presence of a high ceramic filler content on crosslinking reaction.In this paper, we are focusing on the investigation of the conditions to induce a blue light-curing of epoxy-based formulations for dental applications. For this reason, a silicone cycloaliphatic diepoxy resin was used as matrix. The suitable photoinitiator system composition was defined and the effect of water content and filler content was taken into account.2.2.1.Photoinitiated ring opening polymerization of epoxy resins has been the subject of extensive studies during the last two decades ,14. UV-cThe initiation of cationic UV-curing polymerization is a multistep process involving first the photoexcitation of diaryliodonium or triarylsulfonium salts, and then the decay of the resulting excited singlet state with both heterolytic and homolytic cleavages. Cations and aryl-cations generated are very reactive with monomers to give the formation of a Bronsted acid, which is the actual initiator of cationic polymerization. The ring opening polymerization takes place by protonation of the monomer, followed by the addition of further monomer molecules, thus resulting in a chain growth reaction see  15]..15].The main problem for cationic process is related to the fact that the major absorption bands of these onium salts initiators fall in the deep UV region and do not overlap with the emission band of visible light. The strategy usually adopted consists in shifting the photoinitiator light absorption towards longer wavelengths using a photosensitizer in the presence of a iodonium salt, and obtaining the promotion of photoinduced cationic polymerization by photochemical sources of free radicals.In our investigated systems, we selected CQ as photosensitizer. Usually CQ is employed together with a hydrogen donor as co-iniatitor . At this point, the onium salt can oxidize this radical. The carbon-centered monomer radical M• can interact with the onium salt to induce its reduction. The generation of an aryl radical by the decomposition of the diaryliodide free radical closes the cycle by providing a species that again can abstract a hydrogen atom from the monomer.Amines are often used as hydrogen donors. Unfortunately the basicity of the amines is a major issue when they are used as co-initiator in a cationic photopolymerization system. In fact, owing to the living character of cationic polymerization it is common to use aliphatic amines to terminate cationic chains. In this study, we wanted to polymerize under visible light an epoxy monomer; therefore besides the onium salt and the photosensitizer CQ, we needed a hydrogen donor like an amine. An aromatic amine was selected as it is characterized by lower basicity, therefore can be used, in moderate amount, even for cationic UV-curable systems.The selected epoxy resin (UV30) shows a low cytotoxicity and no mutagenity (negative Ames test) in contrast to the other commercially available dicycloaliphatic epoxy resins. This can be explained based on the hydrophobic properties of the resin .The chemical structures of the monomer and the photoinitiators  used in this work are reported in The epoxy resin in the presence of a three component initiating system made of the onium salt, the photosensitizer and the amine  and F-(2%)-containing very low refractive dental glass filler G018-163 (Schott) with a mean particle size of 1.5 μm, which was surface modified with 3-glycidoxypropyltrimethoxysilane. The glass fillers were silanized analogous to the method already reported in literature  tetramethyldisiloxane, UV30) was purchased from ABCR . Iodonium salt photoinitiator [4(1-methylethyl)phenyl][4-methylphenyl] iodonium tetrakis (pentafluorophenyl) borate, Rhodorsil 2074, was given by Rhodia . Camphoquinone and ethyl 4-(dimethylamino)benzoate (EMBO) were purchased by Sigma-Aldrich . The chemical structures of the monomer and the photoinitiators are reported in 3.2.In the case of pure cationic formulations, the defined photoinitiator system was directly added to the epoxy monomer. The filler was dispersed into the photocurable formulations using an Exakt three roll mill .in situ the extent of the reaction. Because the IR absorbance is proportional to the monomer concentration, conversion versus irradiation time profiles could be obtained. Epoxy group conversion was followed by monitoring the decrease in the absorbance due to epoxy groups in the region 760–780 cm−1.The kinetics of photopolymerization was determined by real-time Fourier Transform Infrared (FT-IR) spectroscopy, employing a Thermo-Nicolet 5700 . The formulations were coated onto a silicon wafer forming 50 μm thick films. The samples were exposed simultaneously to blue light, which induces the polymerization, and to the IR beam, which analyzes An Astralis 5  gun lamp was employed to induce photocuring; the results are the average of up to 5 runs per each formulation investigated.The Astralis 5 lamp was 75 W and 12 V and it was put at a distance of 2 cm from the sample.A Mettler-Toledo DSC  equipped with a robotic arm was used for thermal analysis. The equipment was calibrated using indium standards. Samples having masses of approximately 10 mg were cured in an open aluminum pans in nitrogen atmosphere. The DSC was used to study isothermal curing at 25 °C using the Astralis 5 gun-lamp for irradiation.4.This paper reports for the first time the possibility to use composites based on cationic systems for dental restorative material applications. Important beneficial aspects such as lowering of shrinkage and therefore lowering of residual stress are expected.An epoxy monomer cured by visible light was studied in this paper as a candidate material for dental applications. The cationic ring-opening polymerization activated via a radical-induced cationic process was investigated using a silicone containing dicycloaliphatic epoxy resin. We demonstrated that CQ is promoting the photopolymerization even in the absence of amines as hydrogen donor, thus showing to be able to abstract labile hydrogen from the epoxy monomer and give rise to the formation of carbon-centered radicals that are oxidized by the onium salt. A complete epoxy group conversion was reached after 50 s of irradiation with blue light.By adding water to the photocurable formulation, up to a content of 1 wt%, it was not observed any detrimental effect on photopolymerization rate and epoxy group conversion.In the presence of a high amount of filler (up to 65 wt% inorganic filler content), the epoxy resin showed a good reactivity. Both real-time FT-IR and photo-DSC analyses were in agreement, and a complete epoxy group conversion was achieved in all the investigated formulations.


Our data suggest that increased Wnt signaling promotes excessive differentiation of melanocytes, leading to exhaustion of melanocyte stem cells and eventually canities in aged mice.Canities is an obvious sign of aging in mouse and human, shown as hair graying. Melanocytes in the hair follicle show cyclic activity with hair cycling, which transitions from anagen, catagen to telogen. How the hairs turn gray during aging is not completely uncovered. Here, by using immunostaining and LacZ staining in Dct-LacZ mice, we show that β-catenin is expressed in melanocytes during hair cycling. RT-PCR, western blot and immunostaining show that β-catenin expression is significantly increased in both anagen and telogen skin of aged mice, when compared to the anagen and telogen skin of young mice, respectively. Overexpression of Wnt10b not only accelerates hair follicle to enter anagen phase, but also promotes melanocytes differentiation in young adult mice (2-month old), with increased β-catenin expression in melanocytes at the secondary hair germ and matrix region of regenerated hair follicles. Overexpression of Wnt10b also promotes melanocyte progenitor cells differentiation  Aging is a physiological process associated with progressive structural and functional declines of tissues and organs . The haiRegenerative hair cycling process in single hair follicle consists of three consecutive phases including growth phase (anagen), regression phase (catagen) and resting phase (telogen) -7. Hair in vivo and in vitro [There is increasing evidence showing that many morphogenetic pathways play key roles in regulating melanocytes behaviors. Of these, Wnt signaling functions as an important pathway controlling the patterning of melanocytes and influencing the decisions of melanocyte stem cells differentiation to pigment the hairs , 13. Wntin vitro . Exogenoin vitro . Previouin vitro . Howeverin vivo or in vitro, through intracutaneous injection of adenovirus into the young adult skin, or by adding them into iMC23 melanocyte stem cells, respectively. Our study indicates that Wnt signaling promotes differentiation of melanocyte stem cell, exhaustion of which leads to hair graying during aging.Therefore, in this study, we first compared hair graying phenotype in young and adult mice. Since the important role of Wnt signaling in aging of other tissues, we examined periodic expression of β-catenin which is the effector of Wnt signaling pathway, in melanocyte lineage cells during hair cycling in Dct-Lac-Z CD1 transgenic mice. We also compared β-catenin expression at telogen phase and anagen phase skin in young mice and 34-month-old aged mice. To explore the function of Wnt signaling on melanocyte differentiation, we over expressed Wnt10b through adenovirus-mediated expression To study the influence of aging on hair pigmentation in mice, we first counted and compared the number of black and gray hairs in dorsal back skin of C57Bl/6 mice aged at postnatal day 23 , 35d (second anagen) and 34 months (34M) Figure . We obseWnt/β-catenin signaling is important for proliferation and differentiation of melanocytes. Previous study shows a cyclic expression pattern of β-catenin during hair cycling in C57BL/6 mouse strain . ConsistTo evaluate the expression of β-catenin in melanocyte cell lineages, we immunostained β-catenin expression in the skin of Dct-LacZ mouse, in which all melanocyte cell lineages are labeled by β-gal. This allows us to visualize LacZ+ melanocytes lineage cells, including melanoblasts, melanocyte stem cells, differentiating melanocytes, and mature melanocytes in the hair follicle. Translocation of β-catenin from cytoplasm to the nucleus indicates the activation of Wnt/β-catenin signaling pathway. We observed that nuclear β-catenin was only present in the early and mid-anagen phase of hair follicle. Close-up view reveals that nuclear β-catenin is expressed in the melanocytes located at hair matrix and secondary hair germ (Shg) region, and a few in the outer root sheath Figure . CombineSeveral lines of evidence show that aging of some tissues and organs is related to the activation of Wnt/β-catenin signal pathway -18. To eWnt10b by intradermal injection of adenovirus-mediated overexpression of Wnt10b (AdWnt10b) or control (AdGFP) into C57BL/6 mice at postnatal day 56 (P56), when the hair follicles are all at telogen phase. Consistent with previous studies done in C57BL/6 normal mouse [Wnt10b leads to increased expression of β-catenin in skin epidermis and hair follicles, when compared to the AdGFP-treated control group, which shows little β-catenin expression  with AdWnt10b or AdGFP (control), and harvested the supernatant which was then added to the cultured iMC23 cells  with AdWnt10b s Figure . Robust s Figure . RT-PCR s Figure . We alsos Figure . Moreoves Figure . These dTissue aging is characterized by decline of regenerative ability. Previous studies show that loss of function or incomplete maintenance of stem cells lead to compromised tissue regeneration during aging . Hair foHere, we found that β-catenin as the effector of Wnt signaling pathway was significantly increased in both telogen and anagen of aged mice skin, when compared to telogen and anagen of young mice skin, respectively. Particularly, β-catenin was expressed at the nucleus of some melanocytes during hair regeneration and growth, indicating the activation of Wnt signaling in these cells. Consistent with previous studies , 13, ourPrevious studies show that aging of tissues and organs is related to the classical Wnt/β-catenin signal pathway. However, it seems that Wnt signaling plays dual roles in contributing to aging among different tissues. Several studies demonstrate that Wnt signaling promotes aging of cells or tissues -18. For Wnt signaling usually maintains at a low level which is under the threshold for hair follicle stem cell activation at telogen . OverexpWnt10b not only accelerates hair follicles to enter anagen phase, but also promotes differentiation of melanocytes in vivo. Moreover, overexpression of Wnt10b in iMC23 cells promotes melanocyte stem cell differentiation, as indicated by melanogenesis and tyrosinase activity assays. Nevertheless, the exact β-catenin expression level that balances hair stem cell activation and melanocyte stem cell differentiation remains further investigation. Future study will evaluate if decrease of Wnt signaling can alleviate exhaustion of melanocyte stem cells in aged mice.Indeed, previous study suggests that Wnt/ β-catenin signaling has distinct roles of in determine melanocyte stem cell fate . Low levSince Wnt/β-catenin is also expressed in other regions of hair follicles where melanocytes were absent. For example, Wnt/β-catenin is expressed in the other regions of hair bulb as well as in the DP where melanocytes are absent. How does Wnt/β-catenin signaling coordinate the cellular behavior of other epithelial cell differentiation and melanocytes differentiation in the hair follicle? First, different regions express different Wnt receptors, which can sense different Wnt ligand to regulate cellular behaviors such as proliferation and differentiation. Second, Wnt signaling is also regulated by other signals which have differential expression in different regions of hair follicle; Third, epithelial-mesenchymal interaction also functions as a critical mechanism in regulating cellular behaviors in the hair follicle . MesenchIn summary, our results demonstrate that β-catenin expression is increased in hair follicles undergoing aging, compared to the hair follicle in young mice at the corresponding cycle stages. Increased Wnt signaling is insufficient to induce hair regeneration but may promote melanocyte stem cell differentiation in hair follicle bulge niche, leading to the exhaustion of melanocyte stem cells during aging process Figure . Our stuDct-LacZ CD1 transgenic mice were gifted from Professor Lan Jackson at the MRC Laboratory (UK). Animals were maintained in a SPF room at the Experimental Animal Center of the Third Military Medical University. For examining the expression of β-catenin, the dorsal back skins were harvested from the male mice at postnatal 4d , 8d , 18d , 23d  and 29d . Intracutaneous injection of adenovirus was performed as previously described .JB6 cells  were cultured in DMEM culture medium with 10% fetal bovine serum, in an atmosphere containing 5% CO2 in air at 37°C. Adenoviruses were generated and the supernatant of adenovirus-infected cells was harvested as previously described . Then thTotal RNA was extracted from the skin specimens per the instruction for use of Trizol reagent. RT-PCR was performed per the instruction for use of the reverse transcription kit  and PCR MIX kit . The primers for β-catenin were designed as: 5’-ATCACTGAGCCTGCCATCTG-3’ (sense); 5’-GTTGCCACGCCTTCATTCC -3’ (antisense), with a 581bp product in length. The annealing temperature was set at 63°C.Proteins were extracted from the skin specimens by using of RIPA lysis buffer . The extracts were vortexed, centrifuged at 12000×g for 10min. The concentration was determined by using a BCA protein concentration determination kit . Then the extracts were added with the loading buffer and incubated in boiling water for 5 min, followed by electrophoresis . The proteins were transferred to a PVDF membrane , which was treated with methanol for 1 min and electrotransfer buffer for 15 min before use. After wash with TBST (5min × 5), the PVDF membrane was blocked with 5% defatted milk (2 h), incubated with rabbit anti-β-catenin polyclonal antibody  at 4°C for overnight, and secondary antibody  at 37°C for 1 h, then developed with chemoluminescence.The back skins from Dct-Lac-Z CD1 transgenic mice were fixed at 4°C in 4% paraformaldehyde for 1 h, and stained with x-gal  for 24 h. After wash, the skin specimens were embedded in paraffin and sliced into 5 μm sections, which were incubated with rabbit anti-β-catenin polyclonal antibody  at 4℃ for overnight, and the secondary antibody  at 37°C for 1h. The samples were developed with DAB .RT-PCR and Western blot images were analyzed with Quantity One software to acquire the optical density of the bands. The relative expression level was normalized at the ratio of optical density of target band against that of internal control band. All experiments in this study were repeated at least three times. Data were expressed as x̄ ±S and One-way ANOVA of data using SPSS10.0 software.


IFIT2, a gene encoding interferon-induced protein with tetratricopeptide repeats. A reporter assay using plasmids containing a 5’-flanking region of the gene showed that the reporter activity was enhanced by either transduction of β-catenin siRNA or dnTCF7L2, suggesting that the region is involved in the transcriptional regulation as a downstream of the β-catenin/TCF7L2 complex. Consistent with this result, expression of IFIT2 was significantly lower in colorectal cancer tissues than that in normal tissues. Exogenous IFIT2 expression decreased cell proliferation and increased apoptosis of colorectal cancer cells. These data suggested that the down-regulation of IFIT2 by Wnt/β-catenin signaling may play a vital role in human colorectal carcinogenesis through the suppression of apoptosis.Impaired Wnt signaling pathway plays a crucial role in the development of colorectal cancer through activation of the β-catenin/TCF7L2 complex. Although genes up-regulated by Wnt/β-catenin signaling have been intensively studied, the roles of down-regulated genes are poorly understood. In this study, we explored a global gene expression of colorectal cancer cells transfected with β-catenin siRNAs or a dominant negative form of TCF7L2 (dnTCF7L2), and identified a set of genes down-regulated by Wnt/β-catenin signaling. Among the genes, we focused here on  APC or activating mutations of CTNNB1 [Wnt signaling pathway is associated with maintenance of organogenesis, growth, and homeostasis through the regulation of differentiation, proliferation, and cell fate of epithelial cells . Aberranf CTNNB1 , which rMany efforts have been made to identify direct downstream targets of β-catenin/TCF such as c-myc , cyclin The interferon-induced proteins with tetratricopeptide repeats (IFITs) consist of four members, IFIT1, IFIT2, IFIT3, and IFIT5, in humans. Induced by interferons, or through the membrane-bound Toll-like receptors (TLRs), or the cytoplasmic RIG-1-like receptors (RLRs), IFITs play a crucial role in host antiviral defense as an innate immune response. IFIT1 and IFIT2 bind to the multi-subunit eukaryotic translation initiation factor 3 (eIF3) , interfeIn this study, we identified IFIT1 and IFIT2 as genes down-regulated by Wnt signaling in colorectal cancer (CRC) cells, and clarified that inhibition of IFIT2 may play a role in the proliferation and anti-apoptotic properties of CRC cells. Our findings may be helpful for the profound understanding of colorectal carcinogenesis.To identify genes down-regulated by the β-catenin/TCF7L2 complex, we searched for those up-regulated by the suppression of β-catenin or TCF7L2. First, we performed gene expression profile analyses of SW480 and HCT116 cells treated with β-catenin siRNA or control siRNA. These analyses identified 781 and 1636 entities whose expression levels were elevated greater than two-fold by the β-catenin siRNA compared with control siRNA in HCT116 and SW480 cells, respectively. In addition, we identified 772 entities whose expression was enhanced more than two-fold by a dominant-negative form of TCF7L2 (dnTCF7L2) in LS174T cells using public expression profile data (GSE46465) . Taking http://software.broadinstitute.org/gsea/msigdb/index.jsp), and the 54 down-regulated entities were analyzed , IFIT2 (7.8-fold), and IFIT3 (3.4-fold) expression, compared with control siRNA in SW480 cells . As shown in Figure MYC and CCND1 were significantly elevated in tumor tissues than those in normal tissues . On the other hand, expression levels of IFIT1 and IFIT2 were significantly down-regulated in colorectal adenocarcinoma compared with normal tissues  [IFIT1 and IFIT2 were also down-regulated in colorectal adenoma tissues compared with normal tissues  in colorectal adenocarcinoma  . In addi1x10-12)  and GSE81x10-12) ) reveale tissues . Howevera Figure , suggest. Figure . These dIFIT1 and IFIT2 by the Wnt signaling, we introduced an activating form of β-catenin lacking exon3 (pcDNA-CTNN1BΔexon3) [AXIN2 expression that is another target of the β-catenin/TCF7L2 complex. Quantitative RT-PCR analysis determined that overexpression of β-catenin increased AXIN2 expression by 16-fold and that it reduced expression of IFIT1 and IFIT2  (To clarify the negative regulation of BΔexon3)  into HeLctively) . These dIFIT1 and IFIT2 by the β-catenin/TCF7L2 complex, we carried out a reporter assay using plasmids containing their 5’-flanking regions (pIFIT1 -627/+22 and pIFIT2 -1366/+169) in SW480 cells. Dual-luciferase assay revealed that the reporter activity of pIFIT1 was augmented 1.92- and 1.88-fold by β-catenin siRNA#9 and siRNA#10, respectively, and that of pIFIT2 was augmented 2.62- and 1.98-fold by siRNA#9 and siRNA#10, respectively . Two prediction programs, JASPAR (http://jaspar.genereg.net/) and TFBIND (http://tfbind.hgc.jp/), did not predict TCF/LEF binding motif(s) in their promoter regions (data not shown).To clarify transcriptional regulation of y Figure . To examTo explore the function of IFIT1 and IFIT2 in CRC cells, we established SW480 and HCT116 cells that stably express exogenous IFIT1 and IFIT2 using the retrovirus transduction system. Exogenous expression of IFIT2, but not IFIT1, significantly suppressed the proliferation of SW480 and HCT116 cells Figure  and 3B. In this study, we discovered that IFIT1 and IFIT2 are negatively regulated by Wnt signaling in CRC cells and that suppressed expression of IFIT2 may confer pro-survival properties to cancer cells.IFIT2 or ISG54 (Interferon stimulated gene 54) is a protein of approximately 54 kDa, which forms a complex with IFIT1 and IFIT3. Stawowczyk and colleagues reported that exogenous expression of IFIT2 suppressed proliferation of HeLa cells and promoted their cell death through apoptosis . They alOf note, they additionally showed that IFIT3 but not IFIT1 counteracted the induction of apoptosis by IFIT2. Since activation of Wnt signaling in cancer cells decreased both IFIT1 and IFIT2, co-repression of these genes may confer the pro-survival properties to the cancer cells by increasing resistance to apoptotic signals. We found that the effect of aberrant Wnt signaling on the repression of IFIT3 is mild compared to that of IFIT1 and IFIT2, and that IFIT3 expression was not decreased in CRC tissues. These data may imply that IFIT1 and IFIT2 are the main targets of Wnt signaling among the human IFITs in CRC.et al. reported that IFIT2 is post-transcriptionally regulated by miR-645, a microRNA that is abundantly expressed in adenocarcinoma of gastric esophageal junction (AGEJ) [In addition, Feng n (AGEJ) . They prn (AGEJ) . These dImportantly, this is the first report showing that IFIT1 and IFIT2 are negatively regulated by Wnt signaling in colon cancer. Expression of IFITs are induced by viral and bacterial infection , type I Although IFIT1 and IFIT2 may not be direct targets of the β-catenin/TCF transcriptional complex, suppressed expression of these molecules may be involved in carcinogenesis through the activation of anti-apoptotic properties. Further studies of their regulation may contribute to a better understanding of colorectal carcinogenesis and the development of strategies to enhance apoptotic effect of anti-cancer drugs.Human CRC cell lines, HCT116, SW480, DLD-1, LS174T and human cervix cell lines, HeLa were purchased from the American Type Culture Collection . All cells were grown in appropriate media  supplemented with 10% FBS , and antibiotic/antimycotic solution .#9, #10, and #12) and control siRNA (ON-TARGETplus Non-targeting Pool #D-001810-10) were purchased from GE Dharmacon . Target sequences of the siRNAs are shown in β-catenin or control siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific). Forty-eight hours after the transfection, RNA and proteins were extracted from the cells. The silencing effect of β-catenin siRNAs was evaluated by quantitative RT-PCR and western blotting.Three β-catenin siRNAs , and RNA integrity was assessed by 2100 Bioanalyzer . Two-hundred microgram of total RNA were amplified and labeled using a Low Input QuickAmp Labeling Kit according to the manufacturer’s protocols (Agilent Technologies). Expression profiles were analyzed using SurePrint G3 Human Gene Expression 8x60K Microarray according to the supplier’s recommendations (Agilent Technologies). Subsequent data processing was performed using the GeneSpring GX13.1 (Agilent Technologies). Additionally, gene set enrichment analysis was performed using the Molecular signatures database  with gene sets derived from Canonical pathways, BioCarta, KEGG, and Reactome.Total RNA was extracted from the cells treated with IFIT1, IFIT2, and IFIT3 on StepOnePlus (Thermo Fisher Scientific). Sequences of the primers used are shown in GAPDH was used as internal control.Complementary DNA (cDNA) was synthesized from one μg of total RNA with Transcriptor First Strand cDNA Synthesis Kit . Real-time PCR was performed using qPCR Kapa SYBR Fast ABI Prism Kit  with sets of primers for Total protein was extracted from cultured cells using radioimmunoprecipitation assay (RIPA) buffer  supplemented with a Protease Inhibitor Cocktail Set III . Protein concentration was determined by BCA Protein Assay Kit . Protein (30-50 μg/lane) was separated by 10% SDS-PAGE and transferred on to a nitrocellulose membrane . After the blocking with 5% milk powder in TBS-T  for 1 hour, the membranes were incubated over night with primary antibodies including anti-β-catenin , anti-β-actin , anti-IFIT1 , anti-IFIT2 , anti-PARP , anti-cleaved PARP , anti-Bax , anti-Bak , anti-caspase-3 , and anti-caspase-8 antibodies . Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG  served as the secondary antibody for the ECL Detection System .IFIT1 (-627/+22) and IFIT2 (-1366/+169) were amplified by PCR, and the DNA fragments were cloned into pGL3-Basic vector  to generate reporter plasmids . The primer sequences are shown in β-catenin siRNA. Reporter assay was carried out using a dual-luciferase reporter assay system  according to the supplier’s recommendations.The 5′-flanking regions of IFIT1 and IFIT2 genes were constructed by the insertion of DNA fragments encoding full-length IFIT1 (NM_001548) and IFIT2 (NM_001547), respectively. The primer sequences used for the amplification are shown in The pMXs retroviral vectors carrying the SW480 and HCT116 cells stably expressing IFIT1, IFIT2, or control EGFP were fixed with 70% ethanol at -20°C. The cells were treated with RNase A (2 mg/ml) at 37°C for 30 min, and incubated with propidium iodide (20 μg/ml) in PBS at room temperature for 30 min. After filtration with nylon mesh, the cells were applied for flow cytometric analysis .Cell proliferation assay was carried out by water soluble tetrazolium salts (WST)-based colorimetric method using Cell-counting kit-8 according to the supplier’s protocol . Absorbance was measured at 450 nm using FLUOstar OPTIMA .Statistical analysis was performed by Student's t-test with Benjamini-Hochberg correction for the analysis of gene expression profiles. The unpaired Student’s t-test and Dunnett’s test were used for the statistical analysis of RT-PCR and reporter assay data. The data of cell proliferation was analyzed by two-way ANOVA.


Cadherin complexes mediate cell-cell adhesion and are crucial for embryonic development. Besides their structural function, cadherin complexes also transduce tension across the junction-actomyosin axis into proportional biochemical responses. Central to this mechanotransduction is the stretching of the cadherin-F-actin-linker α-catenin, which opens its central domain for binding to effectors such as vinculin. Mechanical unfolding of α-catenin leads to force-dependent reinforcement of cadherin-based junctions as studied in cell culture. The importance of cadherin mechanotransduction for embryonic development has not been studied yet. Here we used TALEN-mediated gene disruption to perturb endogenous αE-catenin in zebrafish development. Zygotic α-catenin mutants fail to maintain their epithelial barrier, resulting in tissue rupturing. We then specifically disrupted mechanotransduction, while maintaining cadherin adhesion, by expressing an αE-catenin construct in which the mechanosensitive domain was perturbed. Expression of either wild-type or mechano-defective α-catenin fully rescues barrier function in α-catenin mutants; however, expression of mechano-defective α-catenin also induces convergence and extension defects. Specifically, the polarization of cadherin-dependent, lamellipodia-driven cell migration of the lateral mesoderm was lost. These results indicate that cadherin mechanotransduction is crucial for proper zebrafish morphogenesis, and uncover one of the essential processes affected by its perturbation. Summary: Cadherin adhesions transduce tension across the junction-actomyosin axis into proportional biochemical responses via α-catenin. Here we show for the first time how this function of the cadherin complex is important during zebrafish morphogenesis. During embryonic development, tissue shape and organization is established by a series of complex cell migrations and rearrangements. These developmental cell-movement processes are largely dependent on dynamic intercellular adhesions . CentralXenopus , which facilitates body axis elongation . During Recent studies show that the cadherin complex not only serves as a structural anchor between cell adhesions and the actomyosin cytoskeleton, but is also an active site of mechanotransduction as increasing tensile forces on cadherin-based junctions induce biochemical changes, which among others lead to force-dependent reinforcement of the adhesion . A key eWhile the importance of the structural role of the cadherin complex has been well established, the importance of cadherin mechanotransduction during the development of a live organism has not been investigated. To address this we used zebrafish morphogenesis as a model system. We first generated αE-catenin-deficient zebrafish using TALEN gene engineering. Homozygous mutant embryos showed defects in epithelial barrier formation and subsequent lethality during somitogenesis. These defects could be fully rescued by restoring α-catenin function through mRNA injection. We then specifically disrupted cadherin mechanotransduction, while maintaining its structural function, by injecting the α-catenin-ΔVBS construct. The resulting embryos showed no barrier defects and survived somitogenesis, but now exhibited defects in convergent extension – underlying cell migrations that caused severely perturbed morphogenesis. Zooming in on one of these migration processes by live cell imaging, we observed that the dorsal migration of the lateral mesoderm cells was reduced. In particular, we found defects in the polarization of cadherin-dependent lammellipodial adhesion in these cells. Thus, α-catenin mediated cadherin mechanotransduction is essential for cadherin-dependent cell migration during zebrafish morphogenesis.ctnna1) deficient mutant. We disrupted the endogenous ctnna1 locus using TAL-effector nucleases (TALENs) , we can thus conclude that the TALEN is able to efficiently cleave the endogenous locus at the intended target site.To investigate the role of αE-catenin-dependent cadherin mechanotransduction during zebrafish morphogenesis, we first generated an αE-catenin ((TALENs)  by targe(TALENs) A. The TAanalysis B. Samplectnna1 mutations. Individual mutant fish were outcrossed to wild-type fish and the offspring checked for germline transmission of mutations. While individuals showed different germline transmission rates, of the 87 embryos screened from crosses of potential founders with wild-type, 16 embryos (18%) were found to be heterozygous for TALEN-generated ctnna1 mutant alleles. TALENs induce double-stranded breaks at the target site, which due to the error-prone non-homologous end-joining mechanism results in indel mutations . Prior to embryo lysis, slight embryo deformations can be visible as indentations compared to the normally spherical morphology of the embryo at this stage . As shown in in vivo, we injected α-catenin-ΔVBS-GFP mRNA in the offspring of incrossed heterozygous ctnna1 mutants. As shown in ctnna1 mutants in a dose-dependent manner. This shows that both α-catenin-ΔVBS-GFP can rescue the barrier defect similar to wild-type α-catenin and that the replacement of the VBS does not impede the maintenance of a functional epithelial barrier throughout early development. Indeed both α-catenin-GFP and α-catenin-ΔVBS-GFP properly and similarly localize to cell-cell junctions in the developing embryo. This can be observed in the enveloping layer (EVL) as well as in the underlying deep cells in the embryos shown in Fig. S2. Movie 2 shows additional evidence of junctional localization of α-catenin-GFP, and also demonstrates the difference in dynamics between EVL adherens junctions that are stable and linear and deep cell adherens junctions that are constantly remodeled and short-lived, similar to the FAJs that we have characterized in 2D cultured mammalian epithelia .To investigate whether α-catenin-ΔVBS-GFP is able to support junction formation To conclude, we have generated an α-catenin-deficient zebrafish mutant as well as functional rescue constructs, which behave analogously to the mammalian α-catenin constructs previously described, and that allow us to specifically perturb mechanotransduction without interfering with the structural role of α-catenin in junctions.ctnna1 allele we generated is embryonic lethal in zygotic homozygous mutants, we could not establish an adult homozygous ctnna1 mutant line. In addition, from the offspring of an incross of heterozygous ctnna1 mutants, we could not distinguish the 25% homozygous mutants from their siblings before they started exhibiting the tissue rupture phenotypes described above. This negatively impacted our ability to analyze rescue experiments. To circumvent this problem, we opted to use anti-sense transcription-blocking ctnna1 morpholino knockdown. By titrating the right amount of morpholino , we could recapitulate the embryo lysis phenotype observed in ctnna1 zygotic mutants in almost 100% of the injected embryos. Similar to zygotic mutants, up until early somitogenesis, no severe developmental defects were obvious in the MO-injected embryos. A slight delay in development is apparent in all injected early-stage embryos, which is likely caused by the injection procedure . At a later stage during somitogenesis, ctnna1 morphants injected with morpholino undergo embryo lysis just like zygotic mutants . Most importantly, we could rescue ctnna1 morphants from embryo lysis by expressing either α-catenin-GFP . For quantification and initial characterization, we grouped body axis elongation defects in two classes: mild and severe. These two classes could be clearly distinguished in α-catenin-ΔVBS rescued embryos but not in non-injected control and only sporadically in α-catenin-GFP rescued embryos. In the mild phenotype, in addition to the shorter body axis, some embryos also showed a flatter, wider head, as well as a broader dorsal body axis . Severely affected embryos show significant retardation in anterior-posterior body axis formation, in combination with a highly enlarged mediolateral body axis  further indicating that the observed defects arose due to the loss of the vinculin binding domain in α-catenin.Although ody axis C. These ody axis . To rulectnna1 mutants . In addition, the notochords of more severely affected embryos show a wave-like morphology near the tailbud, while in the most affected embryos the complete structure of the notochord is misformed  A. Althou mutants A, ctnna1controls B. We couctnna1 morphant embryos rescued with α-catenin-ΔVBS is correlated with impaired migration of the lateral mesoderm, we tracked individual mesodermal cells at approximately 90 degrees laterally from the dorsal midline during 85-90% epiboly stages. Ctnna1 morphants expressing α-catenin-ΔVBS-GFP show a marked decrease in mesoderm cell migration compared to morphants expressing wild-type α-catenin-GFP, as shown in representative migration tracks (Movies 3 and 4). The quantification of the movement of these cells in One major cadherin-dependent cell migration process underlying convergent extension is the dorsal convergence of the lateral mesoderm during late epiboly stages . To testMovie 5). To directly investigate whether blocking cadherin mechanotransduction using α-catenin-ΔVBS also affects lamellipodia productiveness, we quantified the amount of lamellipodia that coincided with cell body displacements in either the dorsal or the ventral direction during lateral mesoderm migration at 85-90% epiboly. Similar to what has been reported before for wild-type embryos , and only 20% in the ventral direction  were predominant in the dorsal direction. A BMP-induced gradient in N-cadherin adhesion was hypothesized to cause the polarized productiveness of lamellipodia . Support embryos , ctnna1 irection A,B. In cThe cadherin complex present in adherens junctions is essential for the maintenance of epithelial tissue barriers. Cadherin complexes also regulate the rearrangements of cell-cell contacts between neighboring cells in multiple tissues to ultimately achieve proper tissue morphogenesis in the developing embryo. In this study we show that cadherin complex member αE-catenin is essential for epithelial barrier functioning and maintaining tissue shape during early morphogenesis. Furthermore, disruption of its vinculin-binding domain, which has been found to be important for force-induced junction reinforcement in cell culture, leads to altered lamellipodial polarization and defective dorsal migration of the lateral mesoderm during dorsal convergence movements. These results indicate that αE-catenin-dependent cadherin mechanotransduction is essential for the cell migration processes that underlie zebrafish morphogenesis.ctnna1 gene at exon 3, to generate a loss-of-function mutant called hu10414ctnna1 (hu10414ctnna1 mutants (data not shown) for lack of suitable antibodies to detect zebrafish α-catenin on western blot. Nevertheless, based on its essential role as an adherens junction component, the resulting epithelial barrier phenotype is fully consistent with a lack of αE-catenin protein. In analogy, knockdown of α-catenin in epithelial MDCK cells  of the Royal Netherlands Academy of Arts and Sciences.The following fish lines were used: Tupfel Longfin (TL) and DLD1 R2/7 α-catenin-deficient colon carcinoma cells  were cultured in high glucose DMEM containing L-glutamine, supplemented with 10% fetal calf serum and penicillin/streptomycin in standard 10 cm culture dishes. Cells were transfected with zebrafish α-catenin-GFP or α-catenin-ΔVBS-GFP using X-tremegene9 (Roche) as per manufacturer's instructions. Cells were tested negative for mycoplasma infection.Zebrafish α-catenin was kindly provided to us by James Nelson (Stanford University). We generated a GFP-tagged version by generating a PCR fragment containing XhoI/XbaI restriction sites at the terminal ends using the following primers: forward 5′- GACACTCGAGCCATGACGAGCATTAACACTGCTAACATC-3′ and reverse: 5′- GACATCTAGACGGAGGAGAGTAGATTAGACTTA-3′. We then digested the PCR product with XhoI and XbaI, and the pEGFP parental vector with SalI and XbaI (Clontech), after which we ligated the product, resulting in ZFα-catenin-GFP x pEGFP. We then subcloned α-catenin-GFP into pCS2 vector for mRNA transcription. To achieve this we generated a PCR product using the following primers: forward 5′- CGATTCGAATTCAAGGCCTCGCCACCATGGTGAGCAAG-3′ and reverse 5′- CGTAATACGACTCACTATAGTTTCAAATACTATCCATAGCTTTGAACTCGCTCAG-3′. The PCR product was then ligated into pCS2 digested with XhoI and XbaI using Gibson Assembly (NEB), resulting in zebrafish α-catenin-GFP in pCS2.We generated zebrafish α-catenin-ΔVBS-GFP by swapping the ΔVBS domains (which originate from chicken vinculin) with our previously described murine α-catenin-ΔVBS . We firsPlasmids were linearized with NotI (Promega) after which IVT mRNA was generated using the mMessage mMachine SP6 kit (Ambion), and purified using the RNeasy mini kit (Qiagen).For immunocytochemistry, DLD1 R2/7 cells were washed 1 time in PBS, 1 time in CSK buffer  and 1 time in PBS before fixation using 4% paraformaldehyde in PBS for 20 min. After fixation, cells were permeabilized with 0.4% Triton X-100 in PBS for 5 min and blocked in 2% BSA for 1 h. Phalloidin-415 (Promokine), vinculin mouse primary antibody  and Alexa-Fluor-594 secondary antibody (Life Technologies) were diluted in 2% BSA and incubated with the cells for 1 h. Afterwards, cells were mounted in Mowiol 4–88/DABCO solution (Sigma-Aldrich).Embryos were fixed in 2% PFA in PBS overnight at 80% epiboly stage. After washing in PBS, embryos were dechorionated and further washed 3-5 times for 5 min in PBT (PBS+0.1% Triton-X100). Embryos were blocked in PBT+10% Normal Goat Serum for 1 h, then incubated O/N with Phalloidin-Alexa-Fluor-594 (Life Technologies). Samples were then washed 4×30 min in PBT before mounting in 0.3% agarose in E3 embryo medium.ctnna1 locus were designed using the TALE-NT tool  using the guidelines specified in TTGTGTTTATTGCAGGTCACCACACTTGTGAACTCCAGCAACAAAGGTCCA-3′ (binding sites of the left and right TALEN arms are underlined) contains a HphI (NEB) restriction enzyme site (GGTGA). TALENs were generated using the Golden Gate kit (TALENs targeting the Gate kit  in combiGate kit .TALEN targeting plasmids were linearized with NotI (Promega) after which IVT mRNA was generated using the mMessage mMachine SP6 kit (Ambion), and purified using the RNeasy mini kit (Qiagen).ctnna1 were synthesized by GeneTools, LLC. The MO partially blocked the 5′UTR and ATG-start codon: 5′-TAATGCTCGTCATGTTCCAAATTGC-3′ and is described earlier (ctnna1 MO and α-catenin mRNA.Translation-blocking morpholinos (MOs) targeting zebrafish  earlier . Both MOctnna1 TALEN target site. Primers flanking the target site of ctnna1: Alphacat HphI forward, 5′- GTCTTGCCACCCCTTAATCC-3′ and Alphacat HphI reverse, 5′- CAATCTTCTCGCCTTTCTCC-3′.To isolate genomic DNA, zebrafish embryos or fin clips were incubated in 50 µl or 100 µl embryo lysis buffer respectively, for 60 min at 60°C. Proteinase K was heat-inactivated by incubating the samples for 15 min at 95°C. The genomic DNA was used as template to amplify the ctnna1 mutant alleles, the PCR amplicons were incubated with HphI (NEB) at 37°C for 2 h, resulting in the generation of 245 bp and 140 bp fragments. Successful mutagenesis was detected by loss of the restriction recognition site and the presence of an undigested band of 385 bp. Mutant alleles were sequenced using primer Alphacat HphI reverse, 5′- CAATCTTCTCGCCTTTCTCC-3′.To assess In situ hybridization experiments were performed as previously described  and stored in MeOH. escribed . Once stFixed cells were imaged using an inverted research widefield microscope  with perfect focus system, equipped with a 60×1.49 NA Apochromat total internal reflection fluorescence (oil) objective lens, a microscope cage incubator (OkoLab), and an EM charge-coupled device (CCD) camera (Andor Technology) controlled with NIS-Elements Ar 4.0 software (Nikon). All widefield images, unless specifically indicated otherwise were background subtracted by ImageJ's rolling ball (r=20) and sharpened for display by Fiji/ImageJ's unsharp mask filter .in situ hybridization were imaged using a Leica MZFLIII upright microscope.Fixed embryos were imaged on the same microscope using the Nikon C1 Confocal system. For time-lapse imaging, embryos were dechorionated at 50% epiboly stage and embedded in 0.3% agarose in E3 embryo medium. DIC images were taken every 15 min on the same microscope using a 10×0.3 NA Plan Fluor objective lens (Nikon). Embryo staging was determined by wild-type embryos imaged simultaneously with mutants or morphants. Live embryos at 12-24 hpf and fixed embryos after whole-mount Embryos were imaged from the lateral position at 12 hpf. Images were processed in Fiji (ImageJ) as follows: the center of the embryo was determined by manually fitting a circle over the yolk. Lines were drawn from the center to the most anterior position of the head, and most-posterior position of the tail. The head-tail angle was then assessed using the angle tool.Embryos were dechorionated at 50% epiboly stage and embedded in 0.3% agarose in E3 embryo medium. DIC images of the lateral mesoderm during dorsal convergence (85-90% epiboly) were taken every 45 s on a Nikon Ti microscope using a 20×0.75 NA Plan Fluor objective lens. Cells residing in the layer closest to the yolk syncytium were tracked using Fiji/imageJ with the Manual Tracking plugin. Lateral mesodermal cells from wild-type embryos overexpressing α-catenin-GFP were imaged during dorsal convergence (85-90% epiboly) using a Leica SP8 confocal microscope. Images were taken every 45 s. Images were sharpened for display by using Fiji/ImageJ's Gaussian blur filter (r=2) and unsharp mask filter .


In this study, β-catenin and WIF1 expression were analyzed by immunohistochemistry for 196 patients with CC, 39 with cervical intraepithelial neoplasia (CIN), and 41 with normal cervical epithelium (NCE). Significant overexpression of β-catenin was detected in CC (67.9%) when compared to CIN (43.6%) or NCE (34.1%), p < 0.01, while low WIF1 expression was detected in CC (24.0%) when compared to CIN (59.0%) or NCE (58.5%), p < 0.001. Negative correlation was shown between β-catenin and WIF1 expression . In addition, multivariate analysis revealed that both lymph node metastasis and β-catenin expression were the independent prognostic factors not only for disease-free survival , but also for overall survival . Our findings indicate that, besides lymph node metastasis, β-catenin expression may also be a poor prognostic factor for CC while WIF1 could be a potential drug target for treatment of advanced CC.Aberrant activation of the canonical Wnt pathway plays a significant role in cervical cancer (CC). However, limited data show the correlation between the cancer clinicopathological characteristics and the key molecules such as  Cervical cancer (CC) is the fourth most common cancer in women. According to existing data, there were 528,000 registered new cases and 266,000 deaths only in year 2012 [β-catenin  is a very common pathway in human CC [β-catenin expression and leads to the activation of β-catenin target genes inside the nucleus [WIF1) is an upstream secreted Wnt antagonist, first identified as highly conserved gene in the human retina [ WIF1 main function is to bind the extracellular Wnt ligands [β-catenin degradation, therefore inhibiting the canonical Wnt pathway. Currently, there are dozens of studies regarding the correlation with β-catenin and various types of cancer [β-catenin have been strongly correlated with poor cancer prognosis. However, so far, there have been limited reports focusing on the association between β-catenin, WIF1, and the clinicopathological characteristics in CC.Aberrant activation of the Wingless-type (Wnt)/human CC . Recent human CC , while thuman CC , 5, 6. H nucleus , 8. Furtn retina . WIF1 ma ligands , disturb ligands  and consf cancer , 11. In β-catenin as the hallmark [ WIF1, and clinicopathological features of 196 patients with CC and analyzed their prognostic value in CC.In the present study, we investigated the association between the canonical Wnt pathway , WIF1, Between December 2002 and October 2007, 196 patients from the Department of Gynecologic Oncology , diagnosed with CC (stages IA–IIB) and submitted to curative surgical resection, were recruited for this study. Cases were restricted to those who did not receive any tumor related treatment before surgery. The clinicopathological characteristics were summarized in β-catenin antibody  and anti-WIF1 antibody-N-terminal . Human breast tissues were used as positive controls; negative controls were obtained by replacing the primary antibodies with phosphate buffered saline. The staining of β-catenin was evaluated according to Maruyama's method [β-catenin-positive expression. By contrast, membrane staining was only classified as β-catenin-negative expression; for WIF1 protein expression, nuclear staining was considered to be negative [According to the previously described method , 15, IHCs method . If morenegative . Finallyβ-catenin expression were examined using Pearson's χ2 test. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate survival analyses were performed using the Cox regression model for DFS and OS. A forward stepwise procedure was used to identify independent variables in the multivariate analysis. p < 0.05 indicated statistical significance.All the statistical analysis was conducted using SPSS software, version 13.0 . Associations between the clinicopathological characteristics and the pattern of WIF1 and β-catenin and nuclear staining of WIF1 were observed in most cancer and noncancerous samples; however, both staining patterns were considered to be negatively expressed. No significant difference was observed between β-catenin-positive expression ratios (cytoplasmic/nuclear staining) in CIN  compared to NCE . In addition, positive β-catenin expression was found in  cervical cancer samples (both with p < 0.01). Furthermore, low expression of WIF1 was significant in CC samples compared with CIN and NCE (p < 0.001). Briefly, from 149 WIF1 negative CC samples, 126 (84.6%) were β-catenin-positive. On the other hand, from 47 WIF1 positive CC samples, 40 (85.1%) were β-catenin-negative (p < 0.001), β-catenin expression had negative correlation compared to WIF1 expression .As shown in β-catenin staining for representative CC cases were shown in β-catenin was observed. Specifically, the expression of β-catenin was usually absent in patients who had the most intense cytoplasmic WIF1 staining while the staining of β-catenin was intense in those who had no cytoplasmic expression of WIF1.Images of WIF1 and WIF1 was usually found to be moderate-to-strong nuclear staining but without cytoplasmic staining in this study. However, as shown in β-catenin-positive expression was associated with a higher rate of lymphovascular space invasion (p = 0.017). Furthermore, WIF1 positive staining was associated with less cervical stromal invasion (p = 0.002) and a lower rate of lymphovascular space invasion (p = 0.035).As shown in β-catenin expression had poorer 5-year DFS , 46/196 (23.5%) patients underwent CC recurrence. Among those patients, 41 (88.1%) died from cancer progression and 5 patients with recurrent vaginal or lymph node CC survived after surgery and adjuvant concurrent chemoradiotherapy. The 5-year disease-free survival (DFS) and overall survival (OS) rates were 76.5% and 79.1%, respectively. CC patients with positive β-catenin expression were correlated with poorer 5-year DFS rate. In addition, lymph node metastasis  = 5.029; 95% CI: 2.623–9.645) and β-catenin expression  emerged as independent predictors of 5-year DFS in multivariate analysis, β-catenin expression were included in the multivariate analysis. Lymph node metastasis  and β-catenin expression  emerged as independent predictors of 5-year OS, The univariate analysis showed that advanced FIGO stage, parametrial invasion, positive surgical margin, lymph node metastasis, larger tumor size (>4 cm), and  Wnt pathway is involved in different diseases, including cancer. β-Catenin, the key factor of canonical Wnt pathway, conducts Wnt signals to the nucleus and upregulates oncogenes during tumorigenesis [β-catenin can be upregulated in various cancers, including CC [β-catenin accumulation inside the cytoplasm was found to be significantly increased in CC when compared with CIN and NCE in this study. In addition, by Cox regression analysis, it was shown that β-catenin-positive expression was significantly correlated with poor prognosis in 5-year DFS and 5-year OS. In the present study, a negative correlation between cytoplasmic/nuclear β-catenin accumulation and cytoplasmic WIF1 immunostaining (positive expression) was found. In addition, decreased WIF1 expression in CC was consistent with some previous studies which were based on human tumor study, such as gastrointestinal tract, kidney, glioblastoma, osteosarcoma, lung, pituitary, bladder, and oral cavity [ Wnt pathway was activated, whereas the Wnt antagonist WIF1 was inhibited, by the multistep gene process in CC.Dysregulation ofigenesis , 8. It huding CC , 5, 18. uding CC –21, β-cal cavity , 22–28. β-catenin-positive expression  and lymph node metastasis (14.8%) than in those with no surgical margin involvement (24.3%) or no lymph node metastasis  was opposite to most other CC cells in this study, and the reason needs to be explored in the future.Our results have demonstrated the upregulation of


The translocation of β-catenin/CTNNB1 to the nucleus activates Wnt signaling and cell proliferation; however, the precise mechanism underlying this phenomenon remains unknown. Previous reports have provided evidence that NOTCH1 is involved in the Wnt signaling pathway. Therefore, we sought to determine the mechanism by which NOTCH1 influences the Wnt/β-catenin pathway. We constructed a vector expressing the NOTCH1 intracellular domain (NICD1) and transfected the vector into HCT116 which has low expression of NICD1. Furthermore, inhibition of NOTCH signal pathway in SW480 which has abundant NICD1 expression, was performed by transfection of siNICD1 or DAPT, gamma secretase inhibitor, treatment. In addition, we evaluated NICD1 and β-catenin localization in colon cancer cell lines and in 189 colon cancer tissue samples and analyzed the correlation between the nuclear localization of NICD1 and the clinicopathological features of colon cancer patients.Immunohistochemical assays demonstrated that NICD1 and β-catenin exhibited a similar localization pattern in colon cancer tissues. In addition, we found that NICD1 induced the translocation of β-catenin to the nucleus and that NICD1 and β-catenin co-localized in the nucleus. Overexpression of NICD1 increased luciferase activity of Wnt signal pathway. On the other hand, reduction of NICD1 reduced luciferase activity of Wnt signaling pathway. In the 189 analyzed colon cancer cases, multivariate COX regression analysis demonstrated the independent prognostic impact of nuclear localization of NICD1(p=0.0376).NOTCH1 plays a key role in the Wnt pathway and activation of NOTCH1 is associated with the translocation of β-catenin to the nucleus. Recent progress in cancer research has revealed that β-catenin/CTNNB1, which functions in cell-to-cell adhesion and Wnt signaling, is a key contributor to carcinogenesis in various tissues, including the colon, liver, ovary, and skin . CellulaThe NOTCH signaling pathway plays a critical role in tissue development and homeostasis by regulating cell fate, proliferation, differentiation, and apoptosis , 18. NOTIn this study, we evaluated the involvement of NOTCH1 in the Wnt/CTNNB1 pathway using a vector expressing the NOTCH1 intracellular domain (NICD1). Several reports have provided evidence suggesting that NOTCH1 functions as a negative regulator of the Wnt signaling pathway . HoweverStudies investigating whether NOTCH1 negatively or positively regulates the Wnt signaling pathway have presented conflicting results. Here, we demonstrated that NOTCH1 acts as an oncogene in colon cancer by activating Wnt signaling.First, we examined the distribution of β-catenin and NOTCH1 in colon cancer tissue samples using co-immunohistochemistry. Interestingly, the distribution of the 2 proteins was similar in colon cancer cells Figures . In the We examined the localization of NOTCH1 in 189 colon cancer tissue samples using immunohistochemistry. In approximately 50% of the colon cancer samples, NOTCH1 was strongly expressed in the nucleus Figure . IntraceIn addition, we observed a statistically significant association between nuclear NICD1 expression and TNM staging  (data not shown). The prognostic value of various clinicopathological factors was evaluated using univariate Cox regression analysis  were identified as significant and independent prognostic indicators (p=0.0376) of survival in postoperative colon cancer patients.The hazard ratio for reduced survival associated with positive nuclear NICD1 expression compared with negative nuclear NICD1 expression was 2.181  -transfected HCT116 cells compared with cells transfected with the control vector Figure . FurtherGiemsa staining demonstrated that cell proliferation was enhanced in HCT116 colon cancer cells compared with the control cells Figure . MoreoveNext, to inhibit the translocation of NICD1 to the nucleus in SW480 colon cancer cells with abundant nuclear NICD1, we used DAPT which is a gamma-secretase inhibitor. DAPT inhibits the cutting of the site between NICD1 and NECD (NOTCH1 extracellular domain). The results of Western blotting using extracted nuclear protein revealed that nuclear β-catenin and Cyclin D1 levels were decreased in DAPT-treated SW480 cells compared with cells transfected with DMSO as a control Figure . Cell prNOTCH1 activation is mediated by gamma secretase-mediated cleavage of the NOTCH S3 site . S3 cleaThe expression of NOTCH1 has been reported to be up-regulated in colon cancer tissue , 26. FurFurther investigation into the role of NOTCH1 in colon cancer might help to elucidate its role in the Wnt signaling pathway.There are many reports describing the crosstalk between the Wnt/β-catenin and NOTCH signaling pathways , 44–47. Membrane-bound NOTCH1 binds to and inhibits unphosphorylated (active) β-catenin . HoweverTransfecting colon cancer cells with the NICD1 expression vector promoted the translocation of β-catenin into the nucleus and induced cell proliferation. As β-catenin lacks a nuclear localization signal (NLS), it might translocate to the nucleus by interacting with NICD1, which contains an NLS. In fact, luciferase assay using TOPFLASH indicated that NICD1 induced TCF/β-catenin activity. Thus, we hypothesize that NICD1 promotes Wnt signaling by mediating the translocation of β-catenin to the nucleus and that NICD1 functions as an oncogene in colon cancer.Interestingly, the accumulation of NICD1 in the nucleus induces the transformation of rat kidney cells (RKE cells) by up-regulating cyclin D1 expression . SeveralOverexpression of components of the NOTCH signaling pathway is often associated with poor outcomes or tumor metastasis . TogetheOur data indicate that the activation of the NOTCH pathway promotes colon cancer. Therefore, we hypothesize that NOTCH1 acts as an oncogene in colon cancer.NOTCH1 mutations have been observed in various cancers , 56. TheThe association between NOTCH1 and colon cancer was confirmed by the results of experiments using SW480 human colon cancer cells, which express high levels of β-catenin. siRNA-mediated NICD1 depletion in SW480 cells reduced Wnt signaling activity mediated by the β-catenin-Tcf/Lef complex, thereby significantly reducing cyclin D1 expression. Together, the results of our experiments using the NICD1 expression vector strongly suggest that NOTCH1 plays an important role in the β-catenin signaling pathway.Upon the activation of gamma secretase, NOTCH1 is cleaved at its S3 site, resulting in the release of activated NICD1. In our experiments, activated NOTCH1 (the cleaved NICD1) interacted with β-catenin and translocated to the nucleus. Moreover, cell proliferation was induced in pcDNA3.1 NICD1-transfected HCT116 colorectal cancer cells expressing low levels of β-catenin. However, additional molecular functions of NOTCH1 in colon cancer have yet to be characterized. We subsequently investigated the association of NOTCH1 expression in resected tumor tissues samples with the clinicopathological features and prognosis of colon cancer patients.Our findings indicate that the role of NOTCH1 in colon cancer progression merits further investigation. Although the precise molecular mechanisms underlying the up-regulation of NOTCH1 expression and the activation of NICD1 have yet to be elucidated, our data suggest that NOTCH1 is a candidate prognostic molecular marker and a promising molecular target for the development of effective therapeutic options for patients with colon cancer.The HCT116, SW480, LoVo and WiDr human colon cancer cell lines were obtained from American Type Culture Collection . SW480 cells, LoVo, WiDr and HCT116 cells were cultured in L15, Ham F12, EMEM and RPMI medium, respectively, supplemented with 5-10% fetal bovine serum and a 1% antibiotic/antimycotic solution  at 37°C in a humidified atmosphere. An antibody ab8925  which actually recognizes the active form of Notch1 receptor, exposed after cleavage by γ-secretase in the methods, was obtained as previously described .Anti-β-catenin was purchased from BD Transduction Laboratories (Japan) and anti-GAPDH was purchased from Cell Signaling Technology, Inc. .Colorectal cancer tissue samples were obtained from 189 patients who had undergone surgery at the Nagoya City University Hospital  between January 1998 and December 2007. The resected specimens were staged by pathological evaluation according to the UICC guidelines for clinical and pathological studies of colon cancer , 2% SDS, 10% glycerol, and 1 Mini Protease Inhibitor Cocktail Tablet . Cytoplasmic and nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents  according to the manufacturer's instructions. Equal amounts of protein were separated by electrophoresis on 10% Tris-glycine gels. Western blot analyses were performed as previously described , and theThe NICD1 cDNA expression vector pcDNA3.1-NICD1 was constructed to express the region of NOTCH1 between the codon encoding Ser-1748 and the stop codon. To examine the effects of NICD1 on Wnt transcriptional activity, NICD1-expressing cells were plated in 6-well plates and transiently transfected with 3 μg of the pcDNA3.1-NICD1 plasmid with 9 μL of FuGENE 6  according to the manufacturer's protocol. The cells were harvested 72 h after transfection, and the localization of NICD1 was analyzed using immunohistochemistry and western blot assays.For inhibition of Notch1 in HCT116 cells and SW480, Notch1-siRNA and negative control-siRNA and Lipofectamine® RNAiMAX transfection Reagent were purchased from Thermo Fisher Scientific Inc. . Cells were plated in 10cm or 6cm dish and transiently transfected with Lipofectamine® RNAiMAX transfection Reagent according to the manufacturer's protocol. The cells were incubated with transfection mixtures containing 20 nM of Notch1-siRNA or negative control-siRNA for 72 hours.Cells were treated with 25 μmol/L DAPT  and 0.1% dimethyl sulfoxide (DMSO)  as a control. After treatment for 72 h, all cells were collected for protein extraction.We transfected HCT116 and SW480 cells with the pcDNA3.1-NICD1 expression vector. The transiently transfected cell lines were plated on chamber slides and fixed with 4% paraformaldehyde in PBS. The cells were subsequently permeabilized using 0.1% Triton X-100 in PBS for 3 min at 4°C and incubated with blocking solution (2% BSA in PBS) for 30 min at room temperature to block nonspecific antibody-binding sites. Then, the cells were incubated with a mouse antibody against NOTCH1 (diluted 1:100 in blocking solution). The primary antibodies were detected using a goat anti-rabbit secondary antibody conjugated to rhodamine, and the stained cells were imaged using a laser scanning confocal imaging system, BZX-700. We confirmed that the transfected cells expressed the NICD protein using western blotting as previously described [Equal amounts of total cell lysate solubilized in Laemmli's sample buffer were separated using SDS-PAGE and transferred to Immobilon-P filters . The filters were incubated with anti-NOTCH1 and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies [anti-mouse IgG and anti-rabbit IgG  and anti-goat IgG ]. The reactions were visualized using an enhanced chemiluminescence system, Amersham Imager 600 .Colon cancer sections were obtained from Nagoya City University Hospital . For antigen retrieval, deparaffinized sections were boiled in citrate buffer  prior to incubation with the primary antibodies. NOTCH1 and β-catenin protein levels were examined using rabbit polyclonal antibodies (ab8925) and mouse monoclonal antibodies . Nuclear CyclinD1 and HistonH3 protein levels were examined using rabbit monoclonal antibodies Antibody staining was conducted using the peroxidase-based DAKO EnVision System.6 cells/dish) for 24 h and subsequently transfected with pcDNA3.1-NICD1 or pcDNA3.1-Mock plasmid using 10 μg of plasmid DNA and 30ul of FuGENE6® Transfection Reagent . After the cells were transfected for 24 h, they were diluted 1:8 and cultured for 14 days in the presence of 800 μg/ml geneticin (G418).Cell proliferation was analyzed in the HCT116 and SW480 cancer cell lines using colony formation assays. The cells were plated in 10 cm dishes . The cells were washed once with phosphate-buffered saline, fixed with 4% paraformaldehyde for 15 min, and permeabilized using 0.2% Triton X-100 on ice for 5 min. Cells were then incubated with blocking solution (3% BSA in PBS) for 60 min at room temperature. The cells were subsequently incubated for overnight at 4°C with rabbit anti-NOTCH1 (ab8925) (1:400 dilution) in Tris-buffered saline with 3% bovine serum albumin. Goat anti-rabbit Cy3® (IgG H&L)-preadsorbed ab6939 (Abcam) was visualized in the red channel, and goat anti-mouse Alexa Fluor® 488 (IgG H&L) (Abcam) was visualized in the green channel. Microscopy analysis and image acquisition were conducted using a laser scanning confocal imaging system, BZX-700.Cells were seeded onto 4-chamber slides before being fixed and permeabilized. The fixed cells were incubated with anti-NOTCH1and anti-β-catenin  for 1 h at room temperature. The cells were subsequently incubated with the Cy3-conjugated goat anti-rat and FITC-conjugated goat anti-rabbit secondary antibodies diluted 1:100 for 30 min at room temperature. The cell nuclei were stained with ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Protein expression and localization were examined using a confocal microscopy system .4) in 24well plates, and the activities of both firefly and Renilla luciferase reporters was determined at 72 hours after transfection using a Dual Luciferase Assay Kit  according to the manufacturer's instructions. The TOP-FLASH reporter activity is presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. All experiments were performed three times in triplicate.TOP-Flash reporter and pTK-RL plasmids were transiently co-transfected into colon cancer cells . Student's t-test was employed to determine the optimal cut-off value for comparing gene expression levels between 2 groups. The associations between various clinicopathological characteristics and the localization of NOTCH1 and β-catenin were evaluated using Fisher's exact test. Kaplan-Meier estimates of overall survival were compared via the log-rank test. Cox regression analysis of potential prognostic indicators of survival was used to identify independent factors that significantly affect survival. All tests were two-tailed, and p<0.05 was considered statistically significant.