Here we report such a galvanic corrosion can be greatly resisted via bathing the steels in Cu2+-containing solutions. This chemical bath generates Cu2−δS layers on the surfaces of MnS inclusions, invalidating the nano-galvanic cells. Our study provides a low-cost approach via an atomic scale decoration to improve the pitting corrosion resistance of stainless steels in a volume-treated manner.Stainless steels are susceptible to the localized pitting corrosion that leads to a huge loss to our society. Studies in the past decades confirmed that the pitting events generally originate from the local dissolution in MnS inclusions which are more or less ubiquitous in stainless steels. Although a recent study indicated that endogenous MnCr Stainless steels, widely used in industry and in our daily life, are in fact not “stainless”. In the presence of aggressive anionic species, they are susceptible to the localized pitting corrosion that is one of the major causes of the materials' failure, leading to a huge loss to our society. Therefore, to clarify the origin of pitting process and to improve the pitting resistance in stainless steels has been one of the major research focuses in several disciplines such as materials science, chemistry, and metallurgy12342O4 nano-octahedra embedded in the MnS medium catalyzed the local dissolution of MnS by the local nano-galvanic cells of MnCr2O4/MnS. Since then, effective solutions of restraining the cells from electrochemically working have been being tantalizingly searched.Sulphur, particularly in austenitic stainless steels, plays critical roles in tuning the material's properties. For example, manganese sulphide (MnS) as a major existence of sulphur in stainless steels taking a role of lubricant is needed for machining and extending cutting tool life7891012However, invalidating the cells in a low-cost and high-efficiency way has been unattainable with current treatments performed both in industry and laboratories. Usually, final annealing and the subsequent pickling performed on the finished products are the two most common processes to enhance the localized corrosion resistance of stainless steels. During the former processes, the improvement of corrosion resistance is ascribed to the oxidation of steel surface10172S212324252−δS on the exposure layer of MnS. Such chemical decoration leads to the inactivation of the anodic MnS inclusions and avoiding the nano-galvanic cells from electrochemically working, by which the pitting resistance of austenite stainless steels is significantly enhanced.It is known that the composition of sulphide inclusions plays a critical role in the initiation of pitting corrosion of stainless steels19A commercial hot-rolled 316F stainless steel with high sulphur content was chosen since it provides a large number of MnS inclusions for analysis. The mechanical rolling made MnS inclusions needle-shaped and parallel to the rolling direction. Dimensions of the MnS inclusions are several tens of micrometers in length and a few hundred nanometers in diameter. MnS has a NaCl-type structure with lattice parameter of 0.52 nm. TEM observations were performed under the newly developed high-angle-angular-dark-field (HAADF) technique, which provides strong contrast associated with the local variety of chemical composition (atomic number) and/or thickness contribution by detecting high-angle scattering electrons2O4, or Mn2O4 if small amount of titanium is taken into account. By means of large-angle tilting experiments and 3D tomography in the TEM, we found that each of the MnCr2O4 nano-particles has a specific geometric shape, which was identified basically to be an octahedron enclosed by eight triangles. The 3D morphology of an octahedron is displayed in 2O4 nano-particles feature a regular octahedron-shaped, as that shown in our previous study. In some cases, they are octahedron but with remarkable distortion, shown in Based on a series of EDPs and energy dispersive X-ray spectroscopy (EDS), the nanoparticles are identified to be spinel MnCr2O4 particles shown here, although they are as fine as nano-meter scale, are present in almost all the MnS inclusions. So far, little attention has been paid to the ultra-fine inclusions in stainless steels, which is based on the cognition that they do not undermine the mechanical properties of the steels. However, the nano-sized oxide particles shown above are found to play a critical chemical role in catalyzing MnS dissolution and pitting corrosion of the steels.The spinel MnCr4 solution at room temperature. The electron-probe X-ray microanalysis (EPMA) experiment clearly reveals that the cation exchange reaction has taken place during the immersion, namely, Cu ions have selectively substituted some Mn2+ in MnS inclusions after 1 hour immersion  and pitting potential (pitE) and an obvious decrease of passive current density, which indicates the chemical treatment results in an improved pitting resistance. Generally, the pitting potential data of stainless steel is scattered. To evaluate the beneficial effect of the chemical treatment in a statistic analysis, we tested many samples, including untreated and treated samples under different treating conditions. The distributions of pitE values for the samples before and after different treatment were plotted as illuminated in cumP = i/(N + 1), where cumP is the cumulative probability of measured pitting potential (pitE), i is the order in the total number N . In 4 solution with duration from 10 min to 10 h, all pitE shift to noble values by 100 mV on average. The treating duration longer than half an hour seems not to have more positive effect. It is probably because the cation exchange reaction reaches the equilibrium state in 30 minutes. We have carried out systemic electrochemical tests on the samples experienced bathing in CuSO4 solution with variant concentrations, like 0.001 mol/L, 0.005 mol/L, 0.01 mol/L, 0.05 mol/L, and 0.5 mol/L. The pitting potential of the samples treated in solution with different concentrations is shown in pit values of the samples, treated in such a wide range of solution concentrations, display similar.To evaluate the enhancement of pitting resistance after chemical treatment, the potentiodynamic polarization tests on untreated and treated samples were performed in 0.5 mol/L NaCl electrolyte. Some typical potentiodynamic polarization curves are shown in For comparison with other steels, the pitting potential of a commercial 316L stainless steel sample was determined which is about 320 mV . 2O4/MnS have been invalided by the chemical decoration.The samples undergoing different treatments were potentiodynamically polarized to the potential above In order to characterize the substantial decrease of the number of pits on the chemically modified samples, the number of pits per unit length of the inclusion was statistically counted, based on over 300 MnS inclusions in each sample . The min2O4. The novel MnCr2O4 nano-octahedra embedded in the MnS medium catalyzed the local dissolution of MnS by the local nano-galvanic cells between them122+-containing solution, the surface layer of MnS inclusions transforms into copper sulphide crystals, which is thought to be electrochemically inactive to initiate pitting corrosionThe general phenomenon of MnS dissolution is that it happens at the interface of MnS/MnCr4 solution for 20 minutes were observed, respectively. Detailed analyses of chemical compositions onto a section of a MnS inclusion  provides the thermodynamic driving force for the spontaneous ions replacement2−δS thin film which prevents the MnS from dissolution. In the galvanic system of MnS/MnCr2O4, the potential difference between MnS dissolution (EMnS) and oxygen reduction reaction (EORR) provides the thermodynamic driving force. Anode MnS tends to dissolve, while ORR takes place on the cathode MnCr2O4. Once the cation exchange occurs, the exposure layers of MnS are replaced by Cu2−δS which make the MnS be insulated from the solution. According to the E-pH diagram72−δS is much higher than that of MnS in a neutral environment and Cu2−δS can stably preserve in a wide range near EORR, Therefore, the nano-galvanic cells was effectively invalided and the pitting potential of the alloy was enhanced by such a chemical decoration.Since cubic Cuices see . In thisEver since the MnS inclusion was identified as the initiation site for pitting corrosion, several methods have been tried in engineering to remove surface MnS inclusions, such as pickling, laser and high current beam modification. These approaches are useful but have considerable deficiencies. In contrast, the chemical modification via cation exchange reaction in the present study exhibits a much more feasible methodology to improve pitting corrosion resistance of stainless steels, since it is easy to operate for variant dimensions of components. Hence, this study provides a low-cost approach via an atomic scale decoration to improve the corrosion resistance of steels in a volume-treated manner, and has a great signification in improving the integrated properties of stainless steels serving in wet environments.Commercial 316F type stainless steel with high-sulphur content (0.16%) was chosen because it can provide a large number of MnS inclusions for analysis. The steel was made by Nippon Steel and Sumikin Stainless Steel Corporation. 316L type stainless steel was made by Baosteel Corporation in Shanghai, China.The hot-rolled steel rod was cut into sheets with the same size along the rolling direction. The samples were carefully polished with standard SiC grinding papers. The samples were then lightly polished using 1 μm diamond paste until there was no visible scratch on the surfaces. After exposure in air for 12 hours, the samples were immersed in a Cu-containing solution at room temperature for different periods. Those treated samples were cleaned by distilled water and dried for the subsequent electrochemical tests and other structural characterization .The samples cut from the steel rod were ground using variant grit silicon carbide papers, polished with diamond paste, and finally thinned by ion-milling. After the first-round TEM observation, some of the specimens were plasma-cleaned and then immersed into 1 mol/L NaCl solution for immersion-corrosion test. The TEM specimens after immersion were quickly cleaned in distilled water and methanol, dried, and transferred into the TEM for further investigation.2−δS were prepared by ion-milling and treated in 0.05 mol/L CuSO4 solution at room temperature after first-round TEM observation.The plan-view TEM samples for analyzing the structural and chemical information of Cu4 solution for 1 hour. The treated surfaces of two samples were bonded face-to-face and then thinned by grinding, dimpling and ion-milling.The cross-sectional sample, used for determining the thickness of reaction layer, was prepared by the conventional methods. Two pieces of 316F stainless steel samples with polished surfaces were immersed in 0.05 mol/L CuSO2 F30 transmission electron microscope, equipped with HAADF detector and X-ray dispersive spectrometer systems, was used at 300 kV for diffraction, HAADF imaging, composition analysis, and 3D tomography. Microstructure characterizations of Cu2−δS were performed using a Titan G2 60-300 TEM equipped with double spherical aberration (Cs) correctors. A SUPRA35 field emission scanning electron microscope was used to investigate the morphology of MnS inclusions. The surface composition of the bulk materials was analyzed using an electron probe X-ray microanalyzer (EPMA: Shimazu Seisakusho EPMA-1610).A Tecnai G2. The tested solution of 0.5 mol/L NaCl electrolytes was maintained at 28°C with electric-heated thermostatic water bath.A traditional three-electrode system was used in electrochemical experiment. The working electrode is stainless steel specimen, Pt counter electrode and SCE (saturated with KCl) reference electrode. AUTOLAB PGSTAT302N electrochemical workstation was used in potentiodynamic polarization curve measurements with a scan rate 1 mV/s. All the tests were stopped when the anodic current density reached the value of 1 mA/cmEpit).The potentiodynamic polarization curve includes the cathodic and anodic branches. In the anodic branch, there exists a well-defined inflexion, above which anodic current density sharply increase with potential, meaning stable pitting corrosion occurs. The potential value corresponding to that inflexion is identified as pitting potential (This project was conceived by X.L.M.; TEM experiments were performed by Y.T.Z., B.Z. and S.J.Z.; electrochemical experiments were carried out by B.Z., Y.T.Z., J.W. and X.Y.S.; all the authors participated in discussion, interpretation of the data, and producing the final version of this paper.


Platinum drugs, including cisplatin, are a frontline therapeutic in ovarian cancer treatment and acquired resistance to these agents is a major contributor to ovarian cancer morbidity and mortality. In this study a novel glycosylation-dependent mechanism for cisplatin resistance is described. Specifically, cisplatin-induced cell death is blocked by the activity of the ST6Gal-I sialyltransferase. ST6Gal-I modifies specific receptors by adding a negatively charged sialic acid sugar which influences diverse receptor functions. Overexpression of ST6Gal-I is a hallmark of ovarian and other cancers and its expression has been correlated to metastasis and poor prognosis.Tumor cell viability and apoptotic induction were determined in cell lines with ST6Gal-I overexpression and knockdown. In addition, cell populations with acquired resistance to cisplatin were assayed for endogenous ST6Gal-I expression.We show that forced expression of ST6Gal-I in OV4 ovarian cancer cells that lack endogenous ST6Gal-I causes reduced activation of caspase 3 and increased cell viability following cisplatin treatment. Conversely, forced ST6Gal-I knockdown in Pa-1 cells with high endogenous ST6Gal-I increases cisplatin-induced caspase activation and cell death. A2780 ovarian cancer cells selected for stable cisplatin resistance display upregulated endogenous ST6Gal-I when compared with parental, cisplatin-sensitive, A2780 cells. Similarly, extended low dose cisplatin treatment of a Pa-1 polyclonal ST6Gal-I shRNA knockdown population led to selection for subclones with elevated ST6Gal-I expression.Receptor sialylation by ST6Gal-I confers a survival advantage for tumor cells in the presence of cisplatin. These collective findings support a role for ST6Gal-I in chemoresistance and highlight ST6Gal-I as a potential therapeutic target for platinum resistant tumors. N-linked glycans on selected cell surface or secreted proteins as they transit through the Golgi. ST6Gal-I elaborates an α2-6 linkage of sialic acid to galactose, and this enzyme appears to be the primary sialyltransferase responsible for this modification in most tissues [The β-galactoside α2-6-sialyltransferase ST6Gal-I catalyzes the addition of the negatively-charged sugar, sialic acid, to the termini of  tissues ,2. Depen tissues ). Anothe tissues . Galecti tissues . Given t tissues -7, ST6Ga tissues , albeit ST6Gal-I is overexpressed in many different types of cancers including ovarian, breast, and colon carcinoma , and In view of ST6Gal-I’s upregulation in cancer, as well as its emerging role as an inhibitor of cell death pathways, we investigated whether ST6Gal-I activity could influence the sensitivity of tumor cells to cisplatin. Cisplatin is the parent compound of the platinum family of chemotherapeutics commonly used in frontline ovarian cancer treatment. Cisplatin and other platinum derivatives  function by forming inter- and intra-strand crosslinks in DNA, leading to an apoptotic cell death. Resistance to platinum drugs represents a major treatment challenge in ovarian and other cancers. The vast majority of ovarian cancer patients have an initial response to platinum compounds, however up to 75% of patients will relapse, with most exhibiting drug resistant disease . The molThe Pa-1 ovarian cancer cell line was purchased commercially through ATCC . Pa-1 cells were cultured and grown in Dulbecco’s eagle's minimal essential medium (DMEM) with 4.5 g glucose supplemented with 10% fetal bovine serum (FBS)(Hyclone) and 1% antibacterial/antimycotic solution containing penicillin, streptomycin, and amphotericin B (Invitrogen). Pa-1 cells were previously found to express high endogenous levels of ST6Gal-I . To examCells were seeded onto 4-well chamber microscope slides (Beckin Dickinson) and allowed to adhere overnight. Cells were washed and fixed in 4% paraformaldehyde for 10 minutes followed by permeabilization in 5% Triton X-100 (in PBS) for 5 minutes. Cells were then incubated overnight at 4°C or 3 hr at room temperature with the ST6Gal-I antibody  and 3 hr at room temperature with anti-Golgi Matrix-130 (GM-130) . Following incubation with primary antibody, cells were washed and incubated with anti-goat Alexa-Fluor 594-conjugated or anti-mouse Alexa-Fluor 488 secondary antibody (Molecular Probes) for 30 minutes. Chambers were removed and DAPI-containing mounting solution, Vectamount (Vector Labs), was placed onto each well. Coverslips were added and the slides visualized under a Nikon Eclipe 80i fluorescence microscope fitted with a Photometrics CoolSNAP camera (Roper Scientific). Images were analyzed on NIS elements software. ST6Gal-I co-localization with GM-130 was imaged by confocal microscopy.5 cells per well. Cells were allowed to adhere overnight and then treated with cisplatin. Cells were lysed on ice in 50 mM Tris-HCl (pH 7.4) containing 1% Triton X-100, and a protease inhibitor cocktail (Roche Applied Bioscience). Cell lysate was kept on ice for 40 minutes vortexing regularly or lysates were sonicated using a sonicator model C-18 (Fisher). Protein concentrations of the lysates were determined using a modified Bradford Assay (Sigma). Proteins were then resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBST). Primary antibodies against ST6Gal-I (R&D Systems) or cleaved caspase-3  were added to the membrane. Membranes were then washed and incubated with horseradish peroxidase-coupled secondary antibody (Amersham) and visualized with Immobilon enhanced chemiluminscence reagent (Millipore). Protein loading was evaluated by immunoblotting for either β-tubulin or β-actin .Prior to lysis cells were grown in puromycin free media for at least one day, passaged normally, and plated onto 6 well tissue culture plates (Fisher) at a density of 7.5 × 104 cells per well in 75 μl of media and allowed to adhere overnight. Cisplatin stock solutions were made by dissolving solid cisplatin in distilled water to a concentration of 2.5 mM and stored at 4°C with new solutions made monthly. On the day of each experiment, stock solutions were used to dilute cisplatin into media to obtain the desired concentrations, and then cells were grown in the cisplatin-containing media for 21 hours (Pa-1) or 24 hours (OV4). Cell viability was evaluated by determining ATP content using the CellGlo ATP quantification kit (Promega) following the manufacturer’s protocol. Luminescence was measured on a Synergy 2 plate reader (Biotek).Cells were plated in opaque-sided 96-well plates (Corning) at a density of 10The polyclonal Pa-1 sh.ST6 population contains stable clones with varying levels of ST6Gal-I knockdown. Two cell flasks were grown in parallel; one with DMEM containing 10% FBS and 1% antibiotic/antimycotic solution (control), and the other with this media supplemented with 1 μM cisplatin. After a 3-week interval, greater than 90% of the cells grown in cisplatin-containing media had died, whereas the control population proliferated over this interval. At the end of the 3 week incubation in cisplatin, the remaining viable cells were resuspended in 10% FBS/DMEM lacking cisplatin, and cultures were expanded to gain a sufficient number of cells for lysis and western blot analysis.In order to evaluate the effects of receptor α2-6 sialylation on tumor cell sensitivity to cisplatin, we utilized cell models with engineered ST6Gal-I expression. OV4 ovarian cancer cells have no detectable endogenous ST6Gal-I ; we therPar, EV and ST6 cells were treated with increasing doses of cisplatin and evaluated for cell viability by measuring ATP content. The ST6 cells maintained cell viability at higher cisplatin doses as compared with Par or EV cells Figure A, indicaTo further establish a role for ST6Gal-I in cisplatin sensitivity, ST6Gal-I expression was repressed by shRNA in Pa-1 ovarian cancer cells, a cell line with high endogenous levels of ST6Gal-I . EffectiWhile results shown in Figures Given that ST6Gal-I is upregulated in many types of cancer, including ovarian carcinoma , we hypoIf the response of differentially-sialylated tumor cells to cisplatin treatment is indicative of a general protective effect of ST6Gal-I in the presence of cisplatin, then it would be expected that other cell models of cisplatin resistance may exhibit elevated ST6Gal-I expression. Thus, we immunoblotted for ST6Gal-I in the A2780 ovarian carcinoma cell line and its stably cisplatin-resistant derivative, A2780cp20 [Despite the clear clinical significance of chemotherapy resistance, a single mechanism of resistance has not yet been established for all cases. The multifactorial nature of tumor cell resistance to cisplatin leaves open the possibility of novel mechanisms that remain undiscovered. In this study we show that forced expression of ST6Gal-I confers resistance to cisplatin, whereas ST6Gal-I knockdown conversely sensitizes cells to cisplatin. Furthermore, cells selected for resistance to cisplatin exhibit an upregulation in endogenous ST6Gal-I protein, suggesting that increased receptor α2-6 sialylation may provide tumor cells with a survival advantage. These findings illuminate a new mechanism for chemoresistance, and underscore the importance of the cellular glycosylation machinery in drug response. An aberrant glycan profile was one of the earliest identified characteristics of a cancer cell, and selective enrichment in α2-6 sialylation , is a common feature of transformed cells ,30, and One predominant surface receptor known to modulate cisplatin sensitivity is the Fas death receptor. Fas is activated by binding to FasL, which in turn causes receptor internalization, formation of the Death Inducing Signaling Complex (DISC), followed by activation of apoptotic caspases. Caspase activation is also a critical downstream event following cisplatin-induced DNA damage, and cisplatin-resistant cells exhibit attenuated activation of caspases 3, 8 and 9 . CisplatN-glycosylation defects were linked to reduced levels of two glycosyltransferases: (i) N-acetylglucosamine-1-phosphate transferase, gamma subunit (GNPTG) and (ii) mannosyl -glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase (MGAT5) [N-glycans are also known to be crucial for the stability of the ABCG2 transporter in the endoplasmic reticulum [Another potential mechanism for ST6Gal-I-mediated cisplatin-resistance may involve the differential sialylation of one or more drug transporters. Many cisplatin-resistant cell lines show reduced accumulation of cisplatin , pointin (MGAT5) . N-glycaeticulum ,50. ThesAlthough the mechanisms underlying the effects of ST6Gal-I activity on cisplatin sensitivity are not yet understood, the current study adds to the body of literature implicating this enzyme as a major contributor to tumor cell survival. In addition to conferring cisplatin resistance, ST6Gal-I-mediated receptor sialylation blocks apoptotic signaling by the Fas  and TNFRWe demonstrate that ST6Gal-I expression in ovarian tumor cells confers resistance to cisplatin-mediated cell death and that cell lines selected for resistance to cisplatin strongly express ST6Gal-I. Hence, tumor cell expression of ST6Gal-I possibly contributes to chemotherapy resistance in a clinical setting. This finding points to the potential of targeting ST6Gal-I in ovarian cancer treatment and identifies ST6Gal-I as a novel contributor to cisplatin resistance.The authors have no conflicts to disclose.MJS developed the methodology, acquired and interpreted data, and drafted the manuscript. AFS and JWW acquired data. ESS acquired data and aided in study design. CNL conceived and designed the study. SLB conceived and designed the study, developed the methodology, interpreted data, edited the manuscript, and oversaw the study. All authors have read and approved the final manuscript.


DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of β-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.The Wnt/β-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since  Multiple myeloma (MM), one of the most common hematological malignancies in adults, is characterized by a clonal expansion of malignant plasma cells in the bone marrow, associated with suppression of normal hematopoiesis, renal failure, and osteolytic bone lesions Wnt genes encode a family of 19 secreted glycoproteins, which promiscuously interact with several Frizzled (FRZ) receptors and the low-density lipoprotein receptor-related protein 5/6 (LRP5/6). The key event in the Wnt signaling pathway is the stabilization of β-catenin. Signaling by Wnt proteins results in inhibition of glycogen synthase kinase-3β (GSK3β) activity and dissociation of the adenomatous polyposis coli (APC)/axin complex, resulting in accumulation of β-catenin, which translocates to the nucleus. Here, β-catenin interacts with T cell factor (TCF) transcription factors to drive transcription of target genes The canonical Wnt/β-catenin signaling pathway plays a central role in modulating the delicate balance between stemness and differentiation in several adult stem cell niches, including the intestinal crypt and the hematopoietic stem cell niche in the bone marrow DKK1 promoter and unleashes β-catenin/TCF mediated transcription.Recent studies indicate that the Wnt signaling plays at least two distinct roles in the pathogenesis of MM. On the one hand, studies by our own laboratory The study involving human biopsy samples was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committee of The University of Amsterdam, AIEC (Algemene Instellingsgebonden Ethische Commissie). Patients gave written informed consent for the sample collection.A panel of BM biopsy specimen from 41 MM and 7 MGUS patients, obtained at clinical diagnosis, was selected from the files of the Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. All patients were staged according to the Salmon–Durie system. For statistical analysis patients at stage I and II disease were grouped together (n = 16) and classified as early MM, whereas patients with stage III disease (n = 25) were classified as having advanced MM.http://www.ncbi.nlm.nih.gov/geo/ were used. These concerned the U133 Plus2.0 affymetrix oligonucleotide microarray data from 559 newly diagnosed MM patients included in total therapy 2/3 , provided by the University of Arkansas for Medical Sciences, GSE2658 For the analysis of expression of Wnt family members in MM patients, expression data publically available and deposited in the NIH Gene Expression Omnibus  pH 9.0 at 100°C. After blocking with serum free blocker , the slides were either incubated for one hour at room temperature with anti-CD138  or overnight at 4°C with anti-DKK1 , or anti-β-catenin . For CD138 and β-catenin, binding of the antibody was visualized using the PowerVision plus detection system  and 3, 3-diaminobenzidine . Whereas binding of the DKK1-antibody was visualized with a biotinylated rabbit anti-goat antibody, followed by horseradish peroxidase (HRP)-conjugated streptavidin (DAKO) and DAB+ (DAKO). The sections were counterstained with hematoxylin , washed and subsequently dehydrated through graded alcohol, cleared in xylene, and coverslipped. Immunocytochemical staining was performed on formalin-fixed cells. Briefly, slides were incubated for 30 minutes with PBS 0.3% Triton X-100 followed by blocking with serum free blocker , the slides were then incubated overnight at 4°C with anti-DKK1 . Binding of the DKK1-antibody was visualized with a biotinylated rabbit anti-goat antibody, followed by horseradish peroxidase (HRP)-conjugated streptavidin (DAKO) and 3-amino-9-ethyl carbazole (AEC).Immunohistochemical staining was performed on formalin-fixed, plastic-embedded bone marrow sections as described previously i.e., low (0–25%), intermediate (25–75%) and high (75–100%), with the percentages indicating the number of DKK1 positive MM plasma cells. For β-catenin expression the intensity of nuclear staining was scored as negative/low, intermediate or high. For statistical analysis, the cases scored as negative/low and intermediate were grouped together as low β-catenin.The biopsies were analyzed for DKK1 and β-catenin expression by two independent observers (RWJG and STP). DKK1 expression was scored in three semi-quantitative categories, DKK1 gene was assessed using both methylation specific PCR (MSP) and bisulfate sequencing, as described by Aquilera et al. Genomic DNA was extracted and purified from MM bone marrow suspensions and cell lines using DNAzol  according to the manufacturer's protocol. The extracted DNA was modified by treatment with sodium bisulfite using the EpiTect Bisulfite Kit . The methylation status of the MM cell lines L363, UM-1, OPM-1, RPMI 8226, and PC-3 were cultured in RPMI medium 1640 (Invitrogen Life technologies) containing 10% clone I serum (HyClone), 100 units/ml penicillin, and 100 µg/ml streptomycin. XG-1 and LME-1 cell lines were cultured in IMDM medium (Invitrogen Life technologies) supplemented with transferrin (20 µg/ml) and β-mercaptoethanol (50 µM). For XG-1, medium was additionally supplemented with IL-6 (500 pg/m). 293T cells were cultured in DMEM medium containing 10% clone I serum (HyClone), 100 units/ml penicillin, and 100 µg/ml streptomycin. The PC-3 cell line was kindly provided by Christopher Hall. L-cells and Wnt3a-producing L-cells  were cultured in DMEM medium (Invitrogen Life technologies) containing 10% clone I serum (HyClone), 100 units/ml penicillin, and 100 µg/ml streptomycin. Conditioned medium was harvested from 95% confluent flasks every 72 h and stored at 4°C. 5-aza-2′-deoxycytidine (5-aza-CdR Sigma-Aldrich) treatment was performed for 72 h at a final concentration of 5 µM, after which they were harvested and lysed in Trizol (Invitrogen Life technologies).2, 0.2 µmol/L of each primer, and 1 U platinum Taq polymerase (Invitrogen Life technologies). PCR conditions were: denaturing at 95°C for 5 minutes, followed by 30 cycles of 30 s at 95°C, 30 s at 65°C (DKK1), 55°C (β-actin) and 30 s at 72°C. The reaction was completed for 10 minutes at 72°C. Primers used were: DKK1 forward (5′–GATCATAGCACCTTGGATGGG–3′) DKK1 reverse (5′–CAGTCTGATGACCGG–3′) β-actin forward (5′–GGATGCAGAAGGAGATCACTG–3′) β-actin reverse (5′–TCCACACGGAGTACTTG–3′). All primers were manufactured by Sigma-Aldrich .Total RNA was isolated using Trizol according to the manufacturer's protocol (Invitrogen Life technologies). The RNA was further purified using iso-propanol precipitation and was concentrated using the RNeasy MinElute Cleanup kit (Qiagen). The quantity of total RNA was measured using a NanoDrop ND-1000 Spectrophotometer . 5 µg of total RNA was used for cDNA synthesis as described previously. The PCR mixture contained: 100 ng of cDNA, 1× PCR Rxn buffer (Invitrogen Life technologies), 0.2 mmol/L dNTP, 2 mmol/L MgClR@D) was detected by a horseradish peroxidase-conjugated swine anti-goat antibody, followed by detection using Lumi-Light PLUS western blotting substrate (Roche). Protein was harvested from MM cell lines, fractionated using a nuclear/cytosol fractionation kit (BioVision), separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently blotted. The following antibodies were used: β-catenin , β-tubulin (SantaCruz Biotechnologies), and histone H2B (Imgenex). Primary antibodies were detected by HRP-conjugated secondary antibodies, followed by detection using Lumi-Light PLUS western blotting substrate (Roche).Conditioned medium was directly lysed in sample buffer, separated by SDS/10% PAGE, and blotted. Primary goat anti-DKK1 antibody (DKK1 and eGFP gene was generated by inserting the DKK1 gene from pcDNA3.1 into the BamHI and XhoI sites of LZRS-pBMN-IRES-eGFP vector (S-001-AB provided by Dr. G. Nolan). The amphotropic Phoenix packaging cell line was transfected either with LZRS-pBMN-IRES-eGFP or LZRS-pBMN-DKK1-IRES-eGFP by the use of Fugene (Roche). The selection of transfected cells was performed with puromycine. Virus was harvested from the packaging cell lines when 95% of the cells were eGFP-positive as analyzed by fluorescence activated cell sorting (FACS). Transduction of MM cell lines was done by 16 h of incubation with virus supernatant in the presence of 10 µg/ml retronectin . 2 days after the transduction eGFP positive cells were analyzed and sorted by FACS. The transduced MM cells were expanded and examined regularly for eGFP and DKK1 expression. The expression of DKK1 protein was determined in conditioned medium by western blot analysis.The LZRS-pBMN-DKK1-IRES-eGFP retroviral vector encoding Renilla luciferase activity were measured using the dual luciferase assay kit (Promega) following the manufacturers instruction. Renilla luciferase activity served as an internal control for transfection efficiency.MM cells (10 mln) were transfected with TOPFLASH reporter construct (5 ug) and pRL-TK (1 ug) in 500 ul serum-free medium. Treatment with Wnt3a conditioned medium or L-cell conditioned medium was initiated 24 h upon transfection and subsequently maintained for 24 h. Next, MM cells were lysed in 100 ul passive lysis buffer (Promega) and firefly luciferase activity and 2) test was used to test the correlation between the DKK1 expression and the stage of disease, the relation between expression of nuclear β-catenin and the stage of disease and the correlation between expression of DKK1 and nuclear β-catenin.The chi-square (χhttp://www.ncbi.nlm.nih.gov/geo/ accession number GSE2658) revealed frequent (co-)expression of various Frizzleds  and the co-receptor LRP6 (LRP5 could not be studied because of defective probe sets), as well as various Wnts . Moreover, 51% of the primary MMs co-express the co-receptor LRP6 with at least one of the Frizzled genes and one of the Wnt genes  of the biopsies. However, in a significant proportion expression was either restricted to a subfraction of the malignant plasma cells or was entirely lost (DKK1 in a subset of patients with advanced stage MM (p<0.05), and an inverse relation to nuclear β-catenin expression.Since stimulation by (autocrine and/or paracrine) Wnt ligands appears to underly Wnt pathway activation in MM, loss of secreted Wnt pathway antagonists like DKKs and sFRPs could have a major impact on the pathogenesis of MM. In particular DKK1, which binds to LRP5/6 thereby preventing activation of the pathway by Wnt ligands, might act as a potent negative regulator as it has been shown to be overexpressed by MM cells ely lost . In contely lost . Furtherely lost . Direct ely lost . Our resDKK1 may unleash activation of the canonical Wnt pathway by Wnt ligands, leading to increased levels of nuclear β-catenin in MM cells of patients with advanced disease. To explore whether DKK1 expression by MM cells can indeed control the response of MM cells to ligand-induced Wnt pathway activation, we restored the DKK1 expression in the MM cell lines OPM-1 and UM-1 by retroviral transduction , or weakly expressed  in comparison to the DKK1 expression in the (positive control) prostate cancer cell line PC-3  and a bisulphate-sequencing PCR (BSP) (Since it has been reported that CR (BSP) . DNA isoCR (BSP) .i.e., L363, LME-1, UM-1, and OPM-1, showed hypermethylation of the DKK1 promoter, while XG-1 and RMPI8226 were unmethylated  of patients with MM and compared this to BM-MNC from healthy donors. All 12 patients had stage III disease. The DKK1 promoter was hypermethylated in 33% of the primary MM specimens studied, as determined by BSP (DKK1 promoter (data not shown).Since d by BSP  and MSP d by BSP . In accoDKK1 promoter methylation indeed plays a causative role in the transcriptional silencing of DKK1 in MM, we investigated whether DKK1 expression could be restored or enhanced by treatment with the demethylating agent 5-aza-2-deoxycytidine. Analysis by BSP of the genomic DNA isolated from OPM-1 and UM-1 cells, which combine a lack of DKK1 expression with hypermethylation of its promoter  of aberrant Wnt pathway activation in MM has not yet been established, the absence of detectable Wnt pathway mutations DKK1 gene itself is a direct target of β-catenin/TCF-mediated transcription DKK1 silencing during tumor progression has been reported in several types of cancer DKK1 may contribute to activation of the canonical Wnt pathway during MM progression.Interestingly, low or undetectable of DKK1 protein expression in BM samples of MM patients was correlated with an increased nuclear expression of β-catenin, a hallmark of canonical Wnt signaling . DKK1 isDKK1 promoter hypermethylation as a mechanism underlying the absence of DKK1 expression in MM . Prostate cancer cell line (PC-3) was used as positive control for the DKK1 staining.(TIF)Click here for additional data file.Figure S2Bisulfite genomic sequencing of the DKK1 promoter in MM bone marrow samples. Bisulfite sequencing analysis was performed on DNA isolated from total bone marrow samples of twelve MM patients (P1–P12). For individual patient 5 clones of the DKK1 promoter region are presented. Open circles indicate unmethylated CpG sites; closed circles represent methylated CpG sites.(TIF)Click here for additional data file.Table S1Expression of Wnt family members in 345 MM patients from total therapy 2 (TT2) patients set.(DOC)Click here for additional data file.


We demonstrate in five human GEJ adenocarcinoma cell lines that nuclear β-catenin expression indeed correlates with enhanced TCF-mediated transcription of a reporter gene. In several tumour types, TCF/β-catenin activation is caused by mutations in either adenomatous polyposis coli (APC), β-catenin exon 3, AXIN1, AXIN2 or β-transducin repeat-containing protein (β-TrCP). In GEJ adenocarcinomas, very few APC and β-catenin mutations have been found. Therefore, the mechanism of Wnt pathway activation remains unclear. In the present study, we did not find AXIN1 gene mutations in 17 GEJ tumours with nuclear β-catenin expression (without β-catenin exon 3 mutations). Six intragenic single nucleotide polymorphisms (SNPs) were identified. One of these, the AXIN1 gene T1942C SNP, has a frequency of 21% but is only very recently described despite numerous AXIN1 gene mutational studies. We provide evidence why this SNP was missed in single strand conformation polymorphism analyses. The AXIN1 gene G2063A variation was previously described as a gene mutation but we demonstrate that this is a polymorphism. With these six SNPs loss of heterozygosity (LOH) was found in 11 of 15 (73%) informative tumours. To investigate a possible AXIN1 gene dosage effect in GEJ tumours expressing nuclear β-catenin, AXIN1 locus LOH was determined in 20 tumours expressing membranous and no nuclear β-catenin. LOH was found in 10 of 13 (77%) informative cases. AXIN1 protein immunohistochemistry revealed cytoplasmic expression in all tumours irrespective of the presence of AXIN1 locus LOH. These data indicate that nuclear β-catenin expression is indicative for activated Wnt signalling and that neither AXIN1 gene mutations nor AXIN1 locus LOH are involved in Wnt pathway activation in GEJ adenocarcinomas.Up to 60% of gastro-oesophageal junction (GEJ) adenocarcinomas show nuclear  The incidence of adenocarcinoma of the gastro-oesophageal junction (GEJ), that is, distal oesophagus and gastric cardia, is rising in the Western world , glycogen synthase kinase 3β (GSK-3β) and AXIN1 or AXIN2. In this complex, β-catenin is phosphorylated by GSK-3β and then targeted by β-transducin repeat-containing protein (β-TrCP) to proteasomal degradation. Activated Wnt signalling inhibits the phosphorylation of β-catenin, thereby preventing its degradation. The impaired β-catenin degradation leads to an increase in cytoplasmic β-catenin and its translocation to the nucleus. Nuclear β-catenin forms heterodimers with members of the T-cell factor (TCF) family of transcription factors and activates genes containing TCP-binding sites  analysis. The entire coding region including the exon–intron boundaries of the AXIN1 gene was analysed for genetic alterations by single strand conformation polymorphism (SSCP) analysis. The presence of six intragenic single nucleotide polymorphisms (SNPs) was used to detect LOH. These six SNPs were also used to perform AXIN1 gene LOH analysis in 20 tumours with strong membranous β-catenin expression. In addition, AXIN1 protein expression was investigated by immunohistochemistry in all 37 tumour samples.Several studies have reported nuclear β-catenin protein complexes was determined by transient transfection of the cell lines with either the pTOPGLOW or pFOPGLOW reporter constructs as described previously . Transfection efficiencies were determined by cotransfection of a pRL-TK reporter construct  that contained the Renilla luciferase gene under control of the herpes simplex virus thymidine kinase promoter. Cells were harvested 24 h after transfection. Activity of both luciferases was measured sequentially in each sample using the Dual-Luciferase Reporter Assay System . TCF-mediated gene transcription was defined by the ratio of pTOPGLOW to pFOPGLOW luciferase activities. The luciferase activity of the internal control reporter was used to correct for differences in transfection efficiency.All cell lines, JROECL19, JROEL33, SKGT-4, TE-7, OACP4C and SW480, were cultured in RPMI 1640 supplemented with 10% fetal calf serum and antibiotics. Transcriptional activation mediated by TCF/in vitro cell lines were investigated for β-catenin expression and β-catenin exon 3 mutations , 20 pmol of each primer and 0.3 U AmpliTaq Gold polymerase . AmpliTaq Gold polymerase was used because this enzyme is superior to other DNA polymerases with regard to amplification of DNA retrieved from routine formalin-fixed and paraffin-embedded tissues. To the PCRs with primer sets 4, 10, 12, 13, 14, 15, 18, 21 and 23, DMSO (5%) was added to increase the amplification efficiency. The PCRs were performed with a hotstart of 95°C for 5min followed by 35 cycles of 95°C for 30 s, 55°C  for 45 s and 72°C for 45s and ended up with an extension step of 72°C for 10 min. The PCR products were diluted 1 : 4 with loading buffer , heated for 5 min, cooled on ice and electrophoresed in 6% polyacrylamide gels containing 10% glycerol at 7 W overnight at room temperature, in 1 × TBE running buffer. Gels were dried and exposed to X-ray films at −80°C. Results were evaluated by visual inspection. With the used SSCP conditions, the PCR products from primer sets 18, 19 and 21 resulted in simple banding patterns, inefficient for the detection of aberrations. To increase the DNA aberration detection efficiency, these PCR products were electrophoresed without glycerol at 4°C for 6 h resulting in more complex banding patterns. For each variant SSCP pattern identified by SSCP analysis, the genomic DNA samples were reamplified for bidirectional direct sequencing with the amplification primers. In 20 DNA samples from tumours with strong membranous β-catenin expression, LOH was determined by SSCP analysis of six detected SNPs. These SNPs are at positions (according to GenBank accession no. AF009674) A94C, C874T, intron 4+17 G → A (nucleotide position identified from exon–intron boundary), G1396A, T1942C and G2063A. Amplification of these SNPs was performed with primer sets 1, 7, 11, 12 and 17, respectively.The 17 pairs of tumour and normal DNA from tumours with nuclear β-catenin and AXIN1 was performed on 5 μm paraffin sections with a mouse anti-human β-catenin monoclonal antibody   and diaminobenzidine hydrochloride . As negative controls, normal mouse immunoglobulins and normal rabbit serum were used.In all 37 tumour samples, immunostaining for β-catenin reporter gene assay was performed with the pTOPGLOW and pFOPGLOW constructs. Cell lines JROECL19 and JROECL33, both with nuclear β-catenin expression  cases (pression . Also inAXIN1 gene mutation studies (M Tris-HCl (pH 8.0), 50 mM KCl and 1.5 mM MgCl2 and the Promega buffer consists of 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2 and 0.1% Triton X-100. As demonstrated in Figure 4The T1942C and G2063A SNPs were found to have population frequencies of 21 and 3%, respectively  and 2. Tβ-catenin expression and 20 cases with normal membranous reactivity (β-catenin expression and 10 of 13 (77%) informative cases with membranous β-catenin immunoreactivity showed loss of the AXIN1 gene locus. In all tumour samples AXIN1 protein expression was confined to the cytoplasm of the tumour cells exclusively  in exon 6 with an allele frequency of 18 and 21% in GEJ adenocarcinoma patients and healthy controls, respectively. This frequent SNP was only recently described, with comparable allele frequencies, in two independent studies  clear LOH was observed. This frequent loss of the AXIN1 gene could point to a dosage effect where the presence of 50% of the AXIN1 protein is insufficient for proper β-catenin degradation and subsequently leads to Wnt activation. To gather more information about this possibility, we investigated AXIN1 gene LOH with the detected SNPs in a series of 20 tumours from our previous study with strong, normal membranous β-catenin expression. These included three cell lines  without enhanced TCF-mediated transcriptional activity. In this series, the T1942C and G2063A SNPs were detected in four and two samples, respectively. A total of 13 cases were informative and in 10 (77%) clear LOH was observed.The AXIN1 locus loss. It is known that with the semiquantitative immunohistochemical method a two-fold reduction in protein expression cannot be detected. In oesophageal squamous cell carcinoma, reduced protein and RNA expression has recently been described by AXIN1 gene silencing by promotor methylation, in addition to allelic losses, as a mechanism for AXIN1 downregulation. Our immunohistochemical results indicate that AXIN1 gene silencing does not occur in GEJ adenocarcinomas.By AXIN1 immunohistochemistry, no differences were observed between tumours with and without AXIN1 gene haplo-insufficiency is not sufficient for Wnt activation. This is in accordance with studies that demonstrated biallelic inactivation of the AXIN1 gene in several tumour types (AXIN1 gene inactivation has also been described (AXIN1 locus LOH in our study could point to a gene dosage effect independent of Wnt pathway activation. Furthermore, it cannot be excluded that an as yet to be defined tumour suppressor gene on 16p13.3 is the actual target for LOH in GEJ adenocarcinomas.All the above-mentioned results indicate that APC, β-catenin and AXIN1 do not play a major role in the frequent TCF/β-catenin activation in GEJ adenocarcinomas, other components should be considered. AXIN2 gene mutations have been described in 11 colorectal cancers (β-catenin expression indicating activated Wnt signalling. The AXIN2 gene mutations appear to be present exclusively in tumours with a microsatellite instable phenotype since all AXIN2 mutant colorectal carcinomas and the ovarian endometrioid adencarcinoma were microsatellite instable (AXIN2 gene is an improbable candidate for Wnt activation in GEJ adenocarcinomas because microsatellite instability has been described in only less than 6% in these tumours (β-TrCP, involved in β-catenin degradation. Recently, β-TrCP mutations have been described in two prostate cancer samples, of which one had nuclear β-catenin expression (Since mutations in α has recently been demonstrated to induce TCF/β-catenin-mediated transcription in a GEJ adenocarcinoma cell line (Activated Wnt signalling can also be the result of activation of the canonical Wnt pathway by secreted Wnts or by expression of the Wnt receptors frizzled (β-catenin expression in GEJ adenocarcinoma cell lines correlates with TCF-mediated transcription activation and so with activated Wnt signalling. The frequent nuclear localisation of β-catenin in GEJ adenocarcinomas cannot be attributed by AXIN1 gene mutations. The mechanism of Wnt activation in these tumours remains to be established. In addition, the role of the frequent LOH of the AXIN1 locus in GEJ adenocarcinomas deserves further investigation.In summary, our results demonstrate that nuclear


Determining potential β-catenin signaling inhibitors would be helpful in ameliorating drug resistance in advanced or metastatic RCC. Screening for β-catenin signaling inhibitors involved in silico inquiry of the PubChem Bioactivity database followed by TCF/LEF reporter assay. The biological effects of ovatodiolide were evaluated in 4 RCC cell lines in vitro and 2 RCC cell lines in a mouse xenograft model. The synergistic effects of ovatodiolide and sorafenib or sunitinib were examined in 2 TKI-resistant RCC cell lines. Ovatodiolide, a pure compound of Anisomeles indica, inhibited β-catenin signaling and reduced RCC cell viability, survival, migration/invasion, and in vitro cell or in vivo mouse tumorigenicity. Cytotoxicity was significantly reduced in a normal kidney epithelial cell line with the treatment. Ovatodiolide reduced phosphorylated β-catenin (S552) that inhibited β-catenin nuclear translocation. Moreover, ovatodiolide decreased β-catenin stability and impaired the association of β-catenin and transcription factor 4. Ovatodiolide combined with sorafenib or sunitinib overcame drug resistance in TKI-resistant RCC cells. Ovatodiolide may be a potent β-catenin signaling inhibitor, with synergistic effects with sorafenib or sunitinib, and therefore, a useful candidate for improving RCC therapy.Dysregulated  Renal cell carcinoma (RCC) is the most lethal genitourinary cancer, and the worldwide incidence and mortality rates of RCC have increased annually. In 2008, the incidence was 4/100,000 and the mortality 1.6/100,000 people worldwide. The incidence is 3.2/100,000 and mortality 1.7/100,000 people in Taiwan ) and its downstream genes  and other WNT molecules  receptor-related proteins 5 and 6 (LRP5/6), active LRP5/6 (p-LRP5/6), Axin1, and dishevelled). For apoptotic effects, we compared caspase 3, 8, 9, poly (ADP-ribose) polymerase [PARP] and their cleaved forms, apoptotic proteins Bax, Bid, and PUMA, and antiapoptotic proteins Bcl-2, Bcl-xL, and survivin. For effects on cell invasion, we compared matrix metalloproteinase 2 (MMP-2) and MMP-9. For analyzing β-catenin stability, we compared active β-catenin (pβ--catenin [S552]), inactive β-catenin , active AKT (p-AKT [S473]), and inactive GSK3β (p-GSK3 [S9]). For synergistic effects, we compared TKI's target RAS/RAF/MEK1/ERK1 axial molecules and active STAT3 (p-STAT3 [Y705]). The immunoreactive bands were revealed by the use of enhanced chemiluminescence (Millipore) then developed and quantified by the use of the UVP BioSpectrum Imaging System (Ultra-Violet Products Ltd.).Treated cells were washed twice with phosphate buffered saline (PBS), then lysed in 200 μm sections of xenografted tumors for immunohistochemistry. After blocking with 10% goat serum for 1 hr and incubation with β-catenin, Ki-67, and survivin antibodies (1 : 200 each) for 2 hr at room temperature, sections were washed in triplicate with 1xTBST  for 10 min; slides were processed by the use of the UltraVision Quanto Detection System (Thermo Scientific) and counterstained with hematoxylin. For immunocytochemistry, about 2 × 104 cells were seeded on 18 × 18 mm cover glass. After treatment, cells were washed with 1x PBS  twice, fixed in acetone/methanol (1 : 1) at −20°C for 30 min, and permeabilized with 0.1% Triton X-100 in 1x PBS at room temperature for 10 min. Cells were then washed 3 times with 1xTBST and blocked in 10% goat serum for 1 hr. After incubation with the same antibodies (1 : 200) for 2 hr at room temperature, cells were washed 3 times with 1xTBST for 10 min, stained by the use of the UltraVision Quanto Detection System (Thermo Scientific), and counterstained with hematoxylin. Immunohistochemical and immunocytochemical results of each marker were quantified with Aperio ImageScope and Spectrum Software ver. 10.0.We used 4 μM) and controls were washed twice with 1x PBS and subjected to 3--2,5-diphenyltetrazolium bromide (MTT) assay for detection of cell viability. In brief, 20 μL of 5 mg/mL MTT reagent was added to each well and incubated at 37°C for 3.5 hr before absorbance was read at 570 nm at 0, 24, 48, and 72 hr. Each condition involved 6 repeats. The IC50 value for each cell line was determined by the use of CalcuSyn v1.1.1 (Biosoft).Cells treated with concentrations of ovatodiolide .Subconfluent cells were trypsinized, washed with 1x PBS, and adjusted to 2 × 10μM ovatodiolide and scraped with a p200 tip (time 0). Before imaging, suspended cells were washed off. The distance of migrating cells was measured from images (5 fields) at 24 and 48 hr after ovatodiolide treatment. The results of wound healing assay were normalized as ratio of wound repaired area to the nontreated control set to 100%.For wound healing assay, cells were plated in 6-well plates and cultured to 90% confluence. Each RCC cell was treated with 10, 20, or 40 μm inserts (BD Biosciences). In all, 1 × 104 cells were loaded into upper wells, and both upper (200 μL) and lower (1 mL) chambers were filled with complete medium ) containing 20 μM ovatodiolide or 0.1% DMSO. For invasion assay, each insert was coated with 1 mg/mL Matrigel at 37°C for 5 hr. An amount of 1 × 104 cells was loaded into a coated insert, and both upper (200 μL) and lower (1 mL) chambers were filled with complete medium containing 20 μM ovatodiolide or 0.1% DMSO. The migration and invasion chambers were incubated in a humidified 5% CO2 incubator at 37°C for 24 hr. Cells were fixed with 500 μL methanol/acetone (1 : 1) for 15 min; the inner surface of the upper chambers was wiped with a cotton swab to remove the unmigrated cells for migration assay or Matrigel was scraped off for invasion assay. The chambers were washed with 500 μL 1x PBS and stained with 500 μL hematoxylin for 1 min at room temperature. After washing with 1x PBS, the transwell membranes were torn off and placed on slides. The stained cells were analyzed by the use of ImageJ software and 5 random fields were counted at 100x magnification. All data represent the mean of triple independent transwell assays.Transwell assay of each RCC cell was assessed by use of 8 β-mercaptoethanol or DTT and not boiled before loading. An amount of 50 μg conditioned media underwent SDS-PAGE with 0.1% gel. After electrophoresis, gels were washed twice with 1x TBST then incubated with 2% Triton X-100 for 30 min at room temperature to remove SDS, then in 30 mL reaction buffer  for 24 hr at 37°C. Before scanning, gels were stained with Coomassie Brilliant Blue R-250 for 30 min and destained with destaining solution .The enzymatic activities of MMP-2 and MMP-9 were determined by gelatin zymography. In brief, conditioned media were prepared with standard SDS gel-loading buffer containing 0.01% SDS without 6 of each RCC cell line was treated with DMSO or ovatodiolide  for 24 hr, and cytoplasmic and nuclear extracts were separated with the use of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). In all, 20 μg nuclear and cytoplasmic extracts were used for SDS-PAGE and Western blot analysis to compare β-catenin nuclear and cytoplasmic distribution.An amount of 2 × 10β-Catenin degradation was well documented with the 26S proteasome pathway. We compared the ovatodiolide effects on β-catenin stability in RCC cells by treating cells with 40 μM ovatodiolide and translation inhibitor cyclohexamine  or 26S proteasome inhibitor MG-132 (10 or 20 μM) for 48 hr, and 20 μg total cell lysates were assessed for SDS-PAGE and western blot analysis.6 of Caki-1, 786-O, ACHN, A498, and HEK293T cells were lysed in 0.5% NP-40 protein lysis buffer  NP-40, 5 mM EDTA, and 2x proteinase inhibitors) and centrifuged at 16,000 ×g for 10 min at 4°C. The supernatant was incubated with the indicated primary antibody with protein A plus agarose (Invitrogen) for 4hr incubation at 4°C. The beads were washed 6 times with 0.5% NP-40 protein lysis buffer and resuspended in 30 μL SDS loading buffer, boiled, and used for SDS-PAGE and western blot analysis.In all, 5 × 10In vitro tumorigenicity was evaluated by colony-forming assay. In brief, 2 mL of 0.5% agarose in complete RPMI-1640 was used as bottom agar in a 6-cm dish, and 2 × 104 cells were mixed with 0.3% agarose in complete RPMI-1640 containing 20 μM ovatodiolide or 0.1% DMSO. Cells were maintained in a humidified 5% CO2 incubator at 37°C for 15 days with fresh medium replaced every 3 days. At the 15th day, cells were stained with crystal violet for 1 min and destained with tap water for 15 min. Colonies in each dish were counted by the use of ImageJ with triplicated repeats for each condition.7 786-O or ACHN cells in 100 μL of 1x PBS with ~10 mg/mL Matrigel (BD Biosciences) were implanted into the right flank of each mouse. RCC cells at passage 8 were used for xenografting. Before xenografting, cells were tested for mycoplasma by the use of the e-Myco Mycoplasma PCR Detection Kit (Intron). 786-O cells were xenografted into 25 nude mice, and 18 mice showed ~50 mm3 tumors after 7 days. ACHN cells were xenografted into 25 nude mice, and 20 mice revealed ~50 mm3 tumors after 7 days. We chose 18 mice from each group and randomly divided them into 3 groups (6 mice/group) for systematic treatment: DMSO, 50 mg/kg ovatodiolide, or 100 mg/kg ovatodiolide in 60 μL PBS with 0.5% DMSO by intraperitoneal injection daily. Control mice were intraperitoneally injected with 60 μL PBS and 0.5% DMSO daily. Tumor size was measured every 2 days with the use of calipers and calculated by (length × width2)/2. Tumors were removed at 22 days for 786-O cells and 30 days for ACHN cells, because the body weight of some 786-O xenografted mice was lower than the regulation of The Laboratory Animal Center of National Defense Medical Center and mice should be sacrificed. After measuring tumor weight, a small part of each tumor was flash-frozen in liquid nitrogen for western blot analysis and other parts were fixed with formalin for immunohistochemistry.Female Balb/c nude mice (6 weeks old) were purchased from the National Laboratory Animal Center (Taiwan) and acclimated for 1 week. In brief, 1 × 10t-test. Statistical analyses involved the use of SPSS v16.0 and Microsoft Excel 2007. All statistical tests and P values were two sided. P < 0.05 was considered statistically significant.Real-time PCR data and cell numbers from transwell assay were recorded as continuous data and analyzed by Student's β-catenin signaling activity, we performed a two-step screening of 21 pure compounds of C. reticulata Blanco, 16 compounds of H. syriacus L., and 23 compounds of A. indica L. The first step consists of in silico drug screening involving the PubChem Compound database to search for human tumor cell line growth inhibition/antiproliferative activity, in vivo antitumor/anticancer activity, induction of apoptosis, or cytotoxicity (summarized in Table S1). In all, 11 compounds were selected, including 5 pure compounds of C. reticulata Blanco, 4 compounds of H. syriacus L., and 2 compounds of A. indica L. In the second step, these 11 compounds were used to examine β-catenin signaling activity by the use of the TCF/LEF reporter assay with 786-O and Caki-1 RCC cell lines, which feature high endogenous β-catenin signaling. Psoralen, a pure compound of Psoralea corylifolia L., was used as a β-catenin signaling inductive control [μM ovatodiolide . Ovatodiolide, 40 µM, cotreated with rhWNT3a or LiCl significantly decreased rhWNT3a or LiCl-induced β-catenin signaling, respectively, with TOP/FOP ratios reduced ~80% in both cell lines, respectively .To identify potential compounds suppressing  control . Ovatodiβ-catenin signaling, we compared the ovatodiolide effects with NF-AT, CRE, and NFκB luciferase reporter assays, with their agonistic compounds used as inductive controls. Ovatodiolide specifically inhibited the luciferase activity of TOP-flash but had no effect in NF-AT, CRE, and NFκB reporters . In 4 RCC cell lines , ovatodiolide reduced levels of active β-catenin (Ser552 phosphorylation on β-catenin) and its downstream genes  but not other WNT molecules  . With 48 hr treatment, ovatodiolide significantly increased the sub-G1 cell population by ~5- to 6-fold in RCC cells than in controls  induced cell apoptosis, a sub- IC50 concentration (15 μM) was also examined in Caki-1 and 786-O for 24 h and 48 h. As in Figure S2E, 15 μM ovatodiolide also reduced levels of active β-catenin (p-Ser552-β-catenin) and its downstream genes  but not other WNT molecules (TCF4), LRP5/6 and its active phosphorylated form, Axin1, and dishevelled.To evaluate the cytotoxicity of ovatodiolide in RCC and normal kidney cell lines, we analyzed cell viability. Ovatodiolide had a significantly higher cytotoxic effect in 4 RCC cell lines  but less effect in HK-2 cells . Similar inhibitory effects were observed by transwell assay . However, β-catenin nuclear translocation was dose-dependently decreased after 24 hr ovatodiolide treatment . Consequently, RCC cells were cotreated with ovatodiolide, the translation inhibitor CHX, and 26S proteosome inhibitor MG-132 to confirm the suppression of β-catenin stability. Cotreatment with CHX decreased most of the β-catenin protein level, and MG-132 treatment abrogated this inhibitory effect of ovatodiolide .To explore the ovatodiolide inhibition of odiolide . Ovatodiβ-catenin, TCF4, and β-catenin was further compared by coimmunoprecipitation. TCF4-β-catenin interaction but not E-cadherin-β-catenin interaction was remarkably reduced in each cell . Despite the Caki-1, 786-O, and A498 being so-called E-cadherin-negative cells, the faintly immunoprecipitated E-cadherins were unchanged after ovatodiolide treatment. β-Catenins phosphorylated by GSK3β at residues T41, S37, and S33 are recognized by the β-TrCP E3 ubiquitin-ligase complex, ubiquitinylated, and ultimately degraded by the 26S proteosome [β (S9) phosphorylated by active AKT  inhibits GSK3β kinase activity [β-catenin phosphorylated at S552 by active AKT enhances β-catenin protein levels and nuclear signaling [β-catenin phosphorylation status with ovatodiolide treatment. Ovatodiolide dose- and time-dependently reduced both phosphorylated AKT (S473) and GSK3β (S9) levels (β-catenin S552 forms were decreased but phosphorylated S33/37/T41 forms were increased. Treatment with the AKT inhibitor VIII (5 μM) induced similar effects, and constitutively active AKT abrogated the ovatodiolide-induced inhibition of β-catenin signaling . The effect of constitutively active Akt also partially rescued the OVA-induced cell death . Besides, ovatodiolide treatment did not modify other downstream molecules of AKT, including p-Foxo3a (T32), p-mTOR (S2448), and p-p70S6K (T389) levels . Therefore, ovatodiolide inhibited β-catenin signaling by reducing β-catenin activity and stability. With ovatodiolide treatment of xenografted mice, levels of phosphorylated β-catenin, cell cycle markers Ki-67 and cyclin D1, and survival marker survivin were decreased as compared with controls  were decreased  and β-catenin (PDB: 1QZ7). As in Figure S6B, the ovatodiolide inserted into the β-catenin molecule enclosing by the AKT phosphorylation site, Ser-552 residue, and may result in a stereochemical change to reduce its activation. However, there is no proper 3D structure including N-terminus of β-catenin and it is uneasy to evaluate whether ovatodiolide also bound to the GSK3β targeting Ser33, Ser37, or Thr41 residues.The interaction between E-cadherin, oteosome . GSK3β  levels . Therefo Figures  and leveecreased . Thus, oμM sorafenib or sunitinib for 48 hr, all drug-resistant 786-O and ACHN cells showed at least 2.6-fold significantly greater IC50 than their parental cells . STAT3 (Y775) is another target of sorafenib or sunitinib [β-catenin signaling without affecting RAS-RAF-MEK-ERK signaling or STAT3 status. Ovatodiolide conferred a synergistic effect that resensitized sorafenib- or sunitinib-resistant cells towards these chemotherapeutic agents.Assessment of the synergistic activity of 20 c method  for drugHN cells . Combineunitinib . Phosphoβ-catenin signaling compound, at least in RCC, as evidenced by its ability to reduce β-catenin stability and suppress β-catenin activation in vitro and in vivo. More importantly, when combined with sorafenib or sunitinib, ovatodiolide could enhance the treatment response and overcome drug resistance. We successfully used compound screening with the PubChem BioActivity database combined with TOP/FOP reporter assays to target β-catenin signaling in RCC with ovatodiolide. And this procedure should be easily and quickly possessed for screening of specific signaling inhibitors among most purified compounds or synthetic chemicals.This study demonstrated that ovatodiolide is an anti-A. indica L. It can reduce lipopolysaccharide-induced nitric oxide and cytokine levels in macrophages [A. indica. Ovatodiolide also has cytotoxic effects in some human cancer cell lines by inducing apoptotic pathways [κB-MMP-9 axis activation [Ovatodiolide is a cemsbrane-type diterpenoid and one of the major components of rophages  and bloorophages  and is rpathways , 47 and tivation .β-catenin signaling [β-catenin stability [β-catenin-TCF interaction [β-catenin-CREB binding protein interaction [β (S9) and β-catenin (S552) in RCC cell lines. Reduced phosphorylation of GSK3β (S9) prolongs GSK3β activation [β-catenin protein stability. Reduced phosphorylation of β-catenin (S552) inhibits β-catenin activation and nuclear signaling [in vitro and in vivo. As in Figure S5C, schematic diagram depicts the mechanism of inactivation of β-catenin by ovatodiolide in RCC cells. Survivin knockdown has been associated with G2/M arrest [β-catenin signaling inhibitors induced G2/M arrest [Several drugs and synthetic or natural compounds have been reported to inhibit and/or modulate ignaling , 50; howtability , blockineraction , 53 or βeraction , stabilieraction , preventeraction , or othetivation  and decrignaling , 41. We M arrest , 37, 57,M arrest , althougM arrest .γ-related angiostatic chemokines [Combined treatment with ovatodiolide and sorafenib or sunitinib overcame drug resistance in TKI-resistant RCC cells. Sorafenib and sunitinib are approved for treatment of advanced RCC by the US Food and Drug Administration. Both have been reported as multikinase inhibitors of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, RAF, and several different tyrosine kinases and therefore are used in treating several cancers . RCC patemokines . Currentemokines .β-catenin signaling dysregulation involved in the drug resistance of RCC cells [β-catenin signaling inhibitors and TKIs may be useful to enhance the treatment response of highly chemorefractory, advanced RCC. For evaluation of bioavailability in human, the human equivalent dose (HED) [We also found that RAS-RAF-MEK1-ERK1 signaling is reversibly expressed in sorafenib- or sunitinib-resistant 786-O and ACHN cells. In addition, we found reversed phosphorylated STAT3 status, another target of these TKIs in different cancer types . After cCC cells  and the se (HED)  was calcβ-catenin signaling and therefore inhibits cell viability, migration, invasion, and both in vitro and in vivo tumorigenicity of RCC but induces less cytotoxicity in normal kidney cells. Ovatodiolide had synergistic effects with sorafenib or sunitinib and enhanced the combined treatment response. Ovatodiolide may be a promising candidate for RCC treatment.In conclusion, ovatodiolide is a potent inhibitor of βcatenin signaling and therefore reduces TOP/FOP ratio , βcatenin activation and downstream gene expression . Its purity was also examined . Ovatodiolide inhibits RCC cell viability and induces apoptosis were confirmed in all four RCC cells and normal HK-2 cell lines . And sub-IC50 dose also resulted in similarly inhibitory effects . Ovatodiolide inhibitory abilities on cell migration, invasion and tumorigenicity were also confirmed in all four RCC cells . Ovatodiolide reduces βcatenin stability and activation on a 26S proteasome dependent manner . Ovatodiolide significantly suppresses in vivo tumorigenicity without recognizably systemic toxicity . AKT inhibitor treatment induced effects similar to that of ovatodiolide and constitutively active AKT abrogated the ovatodiolide-induced inhibition of WNT/β-catenin signaling . According to molecular docking simulation, ovatodiolide inserted into β-catenin enclosing Ser-552 residue, the AKT phosphorylation site, and may result in a stereochemical change to reduce its activation.Ovatodiolide specifically inhibits WNT/Click here for additional data file.


Cell adhesion to the extracellular matrix is a key event in cell migration and invasion and endocytic trafficking of adhesion receptors and signaling proteins plays a major role in regulating these processes. Beta2-adaptin is a subunit of the AP-2 complex and is involved in clathrin-mediated endocytosis. Herein, β2-adaptin is shown to bind to the focal adhesion protein actopaxin and localize to focal adhesions during cells spreading in an actopaxin dependent manner. Furthermore, β2-adaptin is enriched in adhesions at the leading edge of migrating cells and depletion of β2-adaptin by RNAi increases cell spreading and inhibits directional cell migration via a loss of cellular polarity. Knockdown of β2-adaptin in both U2OS osteosarcoma cells and MCF10A normal breast epithelial cells promotes the formation of matrix degrading invadopodia, adhesion structures linked to invasive migration in cancer cells. These data therefore suggest that actopaxin-dependent recruitment of the AP-2 complex, via an interaction with β2-adaptin, to focal adhesions mediates cell polarity and migration and that β2-adaptin may control the balance between the formation of normal cell adhesions and invasive adhesion structures. Directed cell migration is a highly coordinated and dynamic process that involves polarization of the cell in response to an external stimulus, followed by the extension of membrane protrusions in the direction of migration Endocytic trafficking of activated receptors serves to compartmentalize, amplify or terminate their downstream signaling pathways. The importance of endocytic trafficking in regulating growth factor signal transduction is well established The adaptor protein-2 complex (AP-2) is a heterotetrameric complex consisting of two large (α and β 2), one medium (μ2) and one small (σ2) subunit that is involved in clathrin-mediated endocytosis of select receptors by directly linking clathrin with cargo proteins Herein we demonstrate a novel interaction between the focal adhesion protein actopaxin (α-parvin) 2.U2OS cells (ATCC) were cultured in DMEM, 10% FBS, 1 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin, 1 mM sodium pyruvate and kanamycin. MCF10A cells (ATCC) were cultured in DMEM/F-12 (50∶50), 15 mM HEPES pH 7.5, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 5% horse serum, 0.02 µg/ml EGF, 0.5 µg/ml hydrocortisone and 10 µg/ml insulin. Cells were maintained at 37°C and 5% COThe following antibodies were used for immunofluorescence and Western blotting: β-adaptin, α-adaptin, γ-adaptin, clathrin heavy chain, GM130, ILK, paxillin clone 349 (BD Transduction), paxillin clone H114, Src, GFP (Santa Cruz Biotechnology), α-actinin, actopaxin (Sigma), Src pY418, FAK pY397 (Invitrogen). The Src inhibitor, PP2 , was used at 2 µM.5 cells per well of a 6-well dish the day before transfection. Transfections were performed using Oligfectamine (Invitrogen) according to manufactures instructions. Sequences for siRNAs (Ambion) used are as follows: human β2-adaptin 5′-CAACUUAAUUGUCCAGAAA-3′ and 5′-UUUCUGGACAAUUAAGUUG-3′; control 5′-ACUCUAUCUGCACGCUGACUU-3′ and 5′-GUCAGCGUGCAGAUAGAGUUU-3′. Cells were used 72 hrs post transfection.For siRNA transfections, cells were plated at 1.2×10GST binding assays were carried out as previously described 2, 10 mg/ml leupeptin, 1 mM NaF, 1 mM PMSF). Clarified lysates were incubated end over end for 2 hours at 4°C with primary antibody then an additional hour with protein A/G beads (Santa Cruz). Immunoprecipitates were solubilized in sample buffer and analyzed by Western blot.U2OS cells were lysed in co-immunoprecipitation buffer  at room temperature. Western blots were visualized by chemiluminescence using ECL (GE).Coverslips were fixed in 3.7% formaldehyde and then permeabilized in 1% Triton-X-100 in PBS . Primary antibodies were used at 1∶250 in 3% BSA in PBS for 90 minutes at 37°C. Rhodamine phalloidin (1∶1000) (Invitrogen) was used to visualize F-actin. Secondary antibodies (Jackson Immunoresearch Laboratories) were used at 1∶250 for 1 hour at 37°C. Images were acquired on a Nikon Eclipse TE2000-U inverted microscope with a Nikon Apo oil 60x/1.40NA objective using a Spot RT Slider camera and Spot Advance software. Image analysis was performed using NIH ImageJ.Cells were seeded at 10,000 cells per well of a 10 µg/ml collagen coated 24 well tissue-culture dish in complete media. Time-lapse live cell images were captured every 2 minutes over a 4 hour time period on a Nikon Eclipse Ti microscope using a 10X/0.30 PL FLUOR Nikon objective and equipped with a Hamamatsu Orca R2 camera  and Nikon NS-Elements software.For wound healing assays, cells were plated to confluence in a 35 mm dish for migration rate analysis or on a glass coverslip for Golgi polarization. The monolayer was wounded using a pipette tip. For migration rate analysis, images were acquired every 2 hours to determine the area of wound closure. For Golgi polarization, coverslips were fixed 4 hrs post wounding and stained for GM130, paxillin, actin and DAPI.Gelatin matrix degradation assays were performed as previously described Cells were treated with 50 µm monodansylcadaverine (MDC) (Sigma) or vehicle for 1 h at 37°C. TRITC-transferrin 25 µg/ml (Invitrogen) was added to the media and cells were placed at 37°C for 20 min to allow for internalization. Cells were placed at 4°C and the surface bound transferrin was stripped using an acid wash . Cells were than fixed and mounted for visualization of internalized TRITC-transferrin.For gelatin degradation, cells were plated on 488-gelatin coverslip as described above in the presence of vehicle or 50 µm MDC.Pearson’s correlation coefficient was performed using the Image J software. Pearson’s correlation coefficients were calculated from the TRITC and Cy5 channels comparing paxillin and β-adaptin staining. The Pearson’s correlation reflects the linear relationship between the localized intensities of the fluorophore labeled proteins.Values were calculated from at least 3 independent experiments and were compared by student t-test and P<0.05 was considered statistically significant. Error bars represent the standard error of the mean (SEM).Clathrin-coated pits have been shown to be enriched around focal adhesions in vitro binding experiments (To identify which protein(s) facilitate the recruitment of β-adaptin to focal adhesions, a biochemical screen was conducted using GST-tagged focal adhesion scaffold proteins to pull down β-adaptin. This approach revealed that β-adaptin interacts with the focal adhesion protein actopaxin  and the eriments . Actopaxeriments . Endogeneriments , while teriments . TogetheThe AP-2 adaptor complex is responsible for cargo selection from the plasma membrane, while AP-1 is involved in cargo selection from the trans-Golgi network and endosomes To determine if β2-adaptin localizes to focal adhesions via its interaction with actopaxin, we generated a GFP-actopaxin 1–95 fragment that is able to bind β2-adaptin , but canTo examine the role for β2-adaptin in adhesion-mediated responses, siRNA knockdown of β2-adaptin in U2OS cells was utilized . ConsistDuring directional cell migration, cells maintain a polarized morphology and typically extend a single lamellipodia at the leading edge. Given that siRNA knockdown of β2-adaptin resulted in the formation of multiple lamellipodia, this suggested that depletion of β2-adaptin may suppress the ability of cells to migrate in a polarized and directional manner. Immunolocalization of β2-adaptin in U2OS cells migrating directionally into a scrape wound revealed that β2-adaptin is enriched in paxillin-containing adhesions at the wound edge/leading lamellipodia  and is lin vivoWhile performing the initial characterization of cell spreading in control and β2-adaptin siRNA treated cells, macromolecular structures consisting of actin puncta surrounded by a ring of paxillin were observed, that were reminiscent of invadopodia . Invadopin vitroThe acquisition of an invasive phenotype is a key step in cancer progression. Furthermore, the ability of cancer cells to invade surrounding tissue and to metastasize correlates with the formation of invadopodia The requirement for Src-tyrosine kinase activity in the formation of invadopodia has been well documented Global clathrin-mediated endocytosis can be inhibited with monodansylcadaverine (MDC) as shown using a TRITC-transferrin uptake assay . InteresIn this study, the endocytic adapter protein β2-adaptin was shown to bind directly to the focal adhesion protein actopaxin. Furthermore, GST-actopaxin pull downs also contained α-, but not γ-adaptin, confirming a selective interaction with the AP-2 complex. Beta2-adaptin also localized to focal adhesions during cell spreading and migration in an actopaxin-dependent fashion. In contrast, localization of actopaxin to coated pits was not observed, suggesting that actopaxin may be involved in mediating AP-2 specific endocytosis at sites of adhesion via its interaction with β2-adaptin.The interaction of β2-adaptin with actopaxin occurred within the N-terminal 1–95 region, that lacks the paxillin or ILK-binding site Alternatively, actopaxin may be binding β2-adaptin, and therefore the AP-2 complex, in order to ensure that selective endocytosis of integrins and associated proteins occurs specifically at focal adhesions. A number of integrin subunits contain the internalization signal NPXY motif that is associated with clathrin-mediated endocytosis Knockdown of β2-adaptin resulted in enhanced spreading and the formation of multiple lamellipodia in U2OS cells spread on collagen. Cells failed to polarize their Golgi when migrating into a scrape wound, demonstrating that β2-adaptin plays a key role in the establishment of cell polarity and consequently a single lamellipodium during migration. In migrating U2OS cells, β2-adaptin colocalized with paxillin in adhesions at the leading edge, but not at the cell rear, consistent with previous observations made with the μ2 subunit of AP-2 Interestingly, post-endocytic trafficking of internalized integrin receptors has been shown to mediate persistent cell migration and lamellipodia formation via the re-delivery of integrins to the leading edge of migrating fibroblasts In addition to the profound effect of β2-adaptin knockdown on polarized cell migration, depletion of β2-adaptin resulted in the formation of matrix-degrading invadopodia. Invadopodia are adhesion structures that occur in invasive tumor cells and other transformed cells. They consist of an actin core surrounded by a ring of adhesion-associated proteins, including integrins, Src, FAK, paxillin, vinculin, talin Knockdown of β2-adaptin resulted in activation of Src tyrosine kinase and inhibition of Src activity suppressed the ability of β2-adaptin knockdown cells to degrade matrix. Elevated Src expression and activity promotes the formation of invadopodia An alternative mechanism by which β2-adaptin depletion may result in the formation of invadopodia is via the regulation of Arf6-dependent trafficking. Arf6 is a small GTPase that binds the α-adaptin subunit of AP-2 In summary, this paper demonstrates an interaction between β2-adaptin and the focal adhesion protein actopaxin and a role for β2-adaptin in regulating adhesion-mediated events including spreading and polarized cell migration. Depletion of β2-adaptin also results in the formation of invadopodia, suggesting that β2-adaptin controlled endocytic trafficking of specific cargo acts to regulate focal adhesion turnover and suppress the formation of invasive structures. Elucidating the molecular mechanisms by which actopaxin and β2-adaptin regulate trafficking and signaling of focal adhesion components will therefore provide critical insights into cellular events that contribute to a number of pathological situations.Figure S1U2OS cells transfected with GFP-actopaxin 1–95 and dsRed clathrin heavy chain demonstrate that although β2-adaptin is lost from focal adhesions, it remained localized with non-adhesion associated clathrin-coated pits.(TIF)Click here for additional data file.Movie S1Control siRNA treated U2OS cells were seeded onto collagen in complete media and spread for 4 hours. Time-lapse live cell images were taken every 2 minutes on a Nikon Eclipse Ti microscope using a 10X/0.30 PL FLUOR Nikon objective and Nikon NS-Elements software.(AVI)Click here for additional data file.Movie S2Beta 2-adaptin siRNA treated U2OS cells were seeded onto collagen in complete media and spread for 4 hours. Time-lapse live cell images were taken every 2 minutes on a Nikon Eclipse Ti microscope using a 10X/0.30 PL FLUOR Nikon objective and Nikon NS-Elements software.(AVI)Click here for additional data file.