Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/β-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/β-catenin pathway and colon cancer cell growth in vitro.Overactivation of the Wnt/β-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/β-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/β-catenin pathway. Both chalcones are able to affect the cell distribution of β-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in  Wnt/β-catenin signaling is the main cause of many different neoplasias, and has a close association with colorectal cancer (CRC). Approximately 80% of all CRCs have mutations in Wnt components, which results in overactivation of the pathway in colon cells. The most common mutations result in APC loss of function which are negative regulators of this signaling pathway; and gain of function mutations in β-catenin, the main effector protein of this signaling ,2,3,4. TCRC is a malignant disease with high prevalence, and the third most commonly diagnosed cancer in the world . It is dFlavonoids are polyphenolic compounds found in many plants and have a wide range of biological effects. Because flavonoids are a large part of the human diet, they have been intensively studied in regard to their beneficial effects in many human diseases, such as cancer. They have anti-proliferative, anti-invasive and pro-apoptotic effects ,11,12. MLonchocarpus sericeus [in vitro, by controlling the cell cycle progression. Moreover, derricin and derricidin strongly inhibited Wnt8-induced double axis in Xenopus embryos, which strongly indicates that these flavonoids are modulators of the Wnt/β-catenin pathway.Here, we investigated the effect of two little-known flavonoids, derricin and derricidin, which were extracted from sericeus . DerriciRenilla [All cell culture reagents were purchased from Gibco-Invitrogen . Dimethyl sulfoxide (DMSO) and anti-β-catenin were purchased from Sigma . Secondary antibodies were purchased from Life Technologies . Cell lines used were HEK293t, L-cell, L-Wnt3a, HCT116, DLD-1 and IEC-18 (ATCC) and RKO-pBAR/Renilla . The chaRenilla .Renilla cells were cultured on 96-well plates, with 1.0 x 104 cells/well in DMEM-High Glucose with 10% fetal bovine serum (Gibco). After confluence, cells were treated with derricin  or derricidin  in the presence of Wnt3a conditioned medium [Renilla luciferase activities were detected according to the manufacturer’s protocol .RKO-pBAR/d medium , for an 4 cells/well in DMEM-F12 with 10% fetal bovine serum (Gibco). After 70% confluence was reached, each well was transfected with 50 ng TOP-Flash or FOP-Flash plasmids, 5 ng TK-Renilla-luciferase, with or without β-catenin [Renilla luciferase activities were detected according to the manufacturer’s protocol .HEK293t and HCT116 were cultured on 96-well plates with 1.0 x 10-catenin . The traXenopus embryos were obtained by in vitro fertilization and staged according to Nieuwkoop and Farber [Renilla to perform the embryo luciferase assays. After injection, embryos were maintained in 0.1x Barth 2.4H2O; 0.04 mM CaCl2.2H2O; pH 7.7), until stage 27, when the phenotypes were analyzed or until gastrula stage (st 10) when the luciferase activity was detected according to the manufacturer’s protocol .Frog experiments were carried out according to the guidelines granted by the Animal Care and Use Ethic Committee (Comissão de Ética no Uso de Animais—CEUA) of the Federal University of Rio de Janeiro and were approved by this committee under the permission number 152/13. Adult frogs  were stimulated with human chorionic gonadotropin . d Farber . All exp4 cells/well in 96-well tissue culture plates in DMEM F-12 medium containing 10% fetal bovine serum and cultured for 24 h before treatment with chalcones  for 0, 24, 48, or 72 h. MTT was added to each well at a final concentration of 150 mg/ml for 4 h before cell harvesting. The formazan reaction product was dissolved with DMSO and quantified spectrophotometrically at 570 nm (Modulus II microplate multimode reader).3--2,5-diphenyl tetrazolium bromide (MTT) was used to assay mitochondrial activity in viable cells. Cells were plated at a concentration of 1.0 x 10HCT116 cells were fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100. Samples were then blocked for 1 h with 5% bovine serum albumin. A rabbit anti-β-catenin (1:200) primary antibody was incubated overnight. Specific secondary antibodies conjugated with Cy3 fluorochrome (1:5000) were incubated for 2 h at room temperature. After PBS washes, DAPI staining   was performed for 5 min, and then slides were mounted with FluorSave  and observed in a Nikon TE 2000-S inverted microscope . Images were captured using a CoolSNAP-Pro  digital camera, with a zoom of 100x and 600x.4 cells were plated on the previous day and treated with 30 μM or 50 μM derricin or derricidin for 24 h. Click-iT EdU (Life Sciences) assay was performed according to manufacturer’s protocol. DMSO was used as a vehicle to solubilize the flavonoids and was added to control cultures conditions at 0,5%.For cell proliferation assay, 5,0 x106 cells were suspended in 0.5 mL ice-cold VindeLov solution [Cell cycle analysis was performed using flow cytometry. Cells were rinsed briefly with calcium and PBS and detached with trypsin at room temperature. After centrifugation, 1×10solution  containiXenopus Wnt/β-catenin reporter assay we used one-way ANOVA following a Kruskal-Wallis . Statistical significance set at *P<0.05, **P<0.01, and ***P<0.001.Each experiment was performed at least three times. The luciferase, cell cycle and MTT assays were performed in triplicate. Cell staining quantification was performed by counting the number of DAPI and the number of non-nuclear and nuclear β-catenin stained cells in randomly chosen microscope fields, and then the percentage of non-nuclear and nuclear cells of the total cells was calculated. Statistical analysis was performed using the unpaired Student’s t test. In the MTT assays, we used two-way ANOVA following a Bonferroni post-test . In the Many natural compounds, including flavonoids, are able to affect cell proliferation and apoptosis and thus are important candidates for cancer control. We performed a low throughput Wnt specific reporter assay to screen natural compounds extracted from different Brazilian plants (data not shown). Among the inhibitors, we found a detectable Wnt/β-catenin inhibition activity displayed by the chalcones derricin and derricidin . Here, wOur first analyses revealed that derricin and derricidin were able to decrease cell viability, suggesting that both flavonoids affect cancer cell growth. To test whether derricin and derricidin could inhibit proliferation of CRCs cell lines, we performed Edu-incorporation assay. We noted that both chalcones were able to reduce the number of proliferating cells, at 30 and 50 μM, in all cell lines . After qWe next asked if these effects could be related to cell cycle arrest. To do this, we treated HCT116, DLD-1 and IEC-18 cells with each flavonoid for 96 h and quantified the percentage of cells in different cell cycle phases . DMSO-trDeregulation in canonical Wnt signaling is closely related to colon cancer cells ,3. For iRenilla cells and HEK293t cells. These cells were treated with 10, 20 or 50 μM of derricin in the presence of Wnt3a conditioned medium. Derricin decreased the Wnt reporter activity in a concentration-dependent manner in both cell lines (Our experiments using HCT116 strongly suggest that derricin and derricidin are potent inhibitors of canonical Wnt signaling. To address this effect more specifically, we performed classical experiments to analyze Wnt pathway regulation, i.e., luciferase reporter-gene assays in RKO-pBAR/ll lines . Derricill lines . We obsell lines . These dXenopus is related to overactivation of Wnt/β-catenin signaling during development of Xenopus embryos, and thus, negative modulators of this pathway are able to reverse or reduce the double axis phenotype [Xenopus embryos. We co-injected embryos with xWnt8 mRNA and derricin or derricidin in the ventral blastomeres of 4-cell-stage Xenopus embryos. We observed that approximately 73% of the embryos displayed a secondary axis after xWnt8 injection (xWnt8 with derricin or derricidin reduced the induction of ectopic axis (Xenopus embryos. This siamois-reporter is able to respond to the Wnt signaling activation induced by the co-injecting of xWnt8 mRNA. We injected concomitantly 0.4 pmol/embryo of derricin and derricidin to test whether they can inhibit the reporter activation. As a result, both chalcones reduced the reporter activity significantly (in vivo in the Xenopus laevis embryo model.The Wnt-induced secondary axis formation in henotype ,27. As onjection . Howeverpic axis . Co-injepic axis . In addificantly . These din vitro (Flavonoid consumption has long been associated with prevention, control and treatment of various human cancers . In regain vitro  and alsoin vitro . We alsoDerricin and derricidin effects are related to modulation of the cell cycle progression, mainly by changing the percentage of cells in the G2/M and S phases of cell cycle in HCT116 and G0/G1 in DLD-1 . InteresRenilla and HEK293T cell lines, which are classically used to investigate this pathway [Recent studies have improved understanding of the mechanisms of action of flavonoids. For instance, many flavonoids are modulators of different signaling pathways, such as Wnt/β-catenin, which is commonly associated with colorectal tumor progression ,2,3,5,36 pathway ,26. Derr pathway . Similar pathway ,39. Morein vivo effects of these chalcones on Wnt/β-catenin modulation in Xenopus embryos, which are widely used as an in vivo model to search for new Wnt regulatory molecules [Xenopus embryo (in vivo assays again demonstrated that these chalcones have potential effects on the modulation of the Wnt/β-catenin pathway.We also analyzed the olecules ,27. Derrolecules ,41. Alsos embryo . In summTaken together, our results show for the first time that derricin and derricidin are potential modulators of the Wnt/β-catenin pathway. These results suggest questions about the mechanisms of action of these chalcones in Wnt/β-catenin pathway, specifically with which Wnt component they can interact. As derricin and derricidin are chalcones with very similar chemical structures, it is also interesting to investigate their differences in regard to structure-activity relationships. As chemical screenings of Wnt regulators based on structure-activity relationships are a recent issue in investigations of Wnt signaling, these important questions should be further addressed and will certainly open new routes toward understanding Wnt pathway interactions.In conclusion, our results show that the Wnt pathway modulation exerted by derricin and derricidin make these chalcones promising candidates for the prevention or treatment of Wnt signaling-related pathologies, such as colorectal cancer.


In vitro expansion of adult human islet β cells is an attractive solution for the shortage of tissue for cell replacement therapy of type 1 diabetes. Using a lineage tracing approach we have demonstrated that β-cell-derived (BCD) cells rapidly dedifferentiate in culture and can proliferate for up to 16 population doublings. Dedifferentiation is associated with changes resembling epithelial-mesenchymal transition (EMT). The WNT pathway has been shown to induce EMT and plays key roles in regulating replication and differentiation in many cell types. Here we show that BCD cell dedifferentiation is associated with β-catenin translocation into the nucleus and activation of the WNT pathway. Inhibition of β-catenin expression in expanded BCD cells using short hairpin RNA resulted in growth arrest, mesenchymal-epithelial transition, and redifferentiation, as judged by activation of β-cell gene expression. Furthermore, inhibition of β-catenin expression synergized with redifferentiation induced by a combination of soluble factors, as judged by an increase in the number of C-peptide-positive cells. Simultaneous inhibition of the WNT and NOTCH pathways also resulted in a synergistic effect on redifferentiation. These findings, which were reproducible in cells derived from multiple human donors, suggest that inhibition of the WNT pathway may contribute to a therapeutically applicable way for generation of functional insulin-producing cells following ex-vivo expansion. Beta-cell replacement represents a promising approach of treatment of type 1 diabetes. However, tissue donor shortage remains a major obstacle. In-vitro expansion of functional β-cells from adult human islets is an attractive approach for generating an abundant source of cells for β-cell replacement, however cell proliferation is associated with a rapid loss of β-cell phenotype CDH1 transcription In the canonical WNT pathway, WNT ligands regulate gene transcription by controlling protein levels and nuclear localization of β-catenin. In the absence of WNT signaling, glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli protein (APC), AXIN, and β-catenin form a destruction complex, enabling GSK3β to phosphorylate β-catenin and target it to degradation Ccnd1 and Myc, both of which are WNT target genes WNT pathway activity is necessary for normal pancreas development HES1 shRNA treatments, resulting in enhanced cell redifferentiation.In this work we analyzed the role of WNT pathway signaling in BCD cell proliferation and dedifferentiation. Our findings demonstrate that the WNT pathway is activated in BCD cells, while inhibition of β-catenin expression by shRNA leads to BCD cell growth arrest and redifferentiation. β-catenin inhibition synergizes with RC and This study was conducted according to the principles expressed in the Declaration of Helsinki. The Institutional Review Boards of the following medical centers, which provided human islets, each provided approval for the collection of samples and subsequent analysis: University of Geneva School of Medicine; San Raffaele Hospital, Milan; Faculty of Medicine, Lille 2 University; Massachusetts General Hospital; Washington University; University of Pennsylvania; Scharp/Lacy Institute; University of Illinois; University of Wisconsin; University of Miami; Southern California Islet Consortium. All donors provided written informed consent for the collection of all samples and subsequent analysis.Human Islets were received 2–6 days following isolation. Islets from individual donors  were disβ-catenin shRNAs  and a non-target shRNA cloned in plko.1-PURO lentiviral vector were obtained from the RNAi Consortium (Sigma-Aldrich). Virus was produced in 293T cells as described Expanded human islet cells or sorted BCD cells in passages 5–7 were infected with β-catenin shRNA or nontarget shRNA viruses. Five to 6 days following infection cells were trypsinized, pelleted, and seeded at 3.8×104 cells/cm2 in ultra-low attachment plates (Corning) in CMRL 1066 medium containing 5.6 mM glucose and supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml gentamycin sulphate , 1% BSA fraction V (Sigma), 1× insulin/transferrin/selenium , D-Glucose , 8 nM exendin-4 (Acris), 8 nM activin A (Cytolab/PreproTech), 1× B27 supplement (Stem Cell Technologies), and 10 mM nicotinamide (Sigma)  for the indicated periods. Half-volume medium changes were performed every other day.Total RNA was extracted using the High Pure RNA isolation kit (Roche), or with ZR RNA MiniPrep RNA Isolation Kit (Zymo), and treated with RNase-free DNase1 (Thermo), or with TRI©Reagent (Sigma) followed by treatment with DNA-Free (Ambion). cDNA was produced using SuperScript III First-Strand Synthesis System (Invitrogen) or High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was carried out in triplicates using the Universal Probe Library System (Roche) in 7300 Real-time PCR system (Applied Biosystems). Results were normalized to the TATA-box-binding protein (TBP) or Ribosomal protein large P0 (RPLP0) transcripts. These genes were selected as normalization controls since their detection threshold occurred at the same cycle in all the samples studied. Data analysis was performed with qBase software. Cells were spotted on slides using Shandon Cytospin4 centrifuge (Thermo Scientific), and fixed for 15 minutes at room temperature in 4% paraformaldehyde. For nuclear antigens, cells were incubated in 0.25% NP40 for 10 minutes at room temperature prior to blocking. Samples were blocked for 15 min at room temperature in blocking buffer  and incubated overnight at 4°C with primary antibodies diluted in blocking buffer as follows: mouse anti-β-catenin ; mouse anti-active β-catenin ; rat anti-human C-peptide ; rabbit anti-eGFP ; rabbit anti-Ki67 ; mouse anti-Ki67 ; and mouse anti- human PDX1 . Slides were washed in blocking buffer 5 times, and incubated with a secondary antibody conjugated to Alexa fluorophores . DNA was stained with DAPI. The slides were mounted with Fluorescent Mounting Medium (GBI Labs). Images were visualized under a fluorescent BX61 microscope or TCS SP5 confocal fluorescent microscope. To demonstrate antibody specificity, a minus-primary antibody control was employed.Cellular protein was extracted for 10 min in 50 mM Tris-HCl buffer, pH 7.4, containing 0.5% NP-40, 0.7% NaCl, 0.2% EDTA, and protease inhibitors. Samples of 20–40 µg protein were resolved by SDS-PAGE and transferred to Immobilon-P Membrane (Millipore). Non-specific sites were blocked for one hour at room temperature in blocking buffer containing 5% skim milk in TTBS buffer. The membrane was then incubated with rabbit anti-β-catenin , mouse anti-SNAI2 , rabbit anti-FOXO1 , or rabbit anti-phospho FOXO1 . The bound antibody was visualized with appropriate horseradish peroxidase-conjugated anti-IgG (Jackson) and SuperSignal West Chemiluminescent Substrate kit (Pierce). Signal intensity was quantitated using TINA software.TUNEL assay was performed using In Situ Cell Death Detection Kit (Roche), according to the manufacturer's protocol. The fluorescence was visualized under a fluorescent BX61 microscope.Total RNA was isolated as above. Hybridization to Affymetrix GeneChip Human Gene 1.0 ST Arrays, washing, and scanning were performed according to the manufacturer. Microarray analysis was performed on CEL files using Partek Genomics Suite TM (Partek). Data were normalized with the multi-average method. Batch effect removal was applied for the different samples, followed by one-way ANOVA. Clustering analysis was performed by Partek Genomics Suite software with Pearson's dissimilarity correlation by average linkage methods. The raw data has been deposited in the GEO database (accession number GSE60803).Significance was determined using Student's t test. To approach a normal distribution of the qPCR data, a logarithmic transformation was performed. To account for multiple testing, the Bonferroni correction was applied.CCND1 and MYC , which stimulate cell proliferation; PITX2 (130-fold), which activates CCND1 and MYC; and SNAI2 (49-fold), which participates in inhibition of CDH1 expression. Analysis of sorted eGFP+ cells, labeled by an insulin promoter-driven lineage tracing system +-labeled BCD cells  shRNA . Analysi2–5-fold . Thus, i2–5-fold .SNAI2 transcript levels  e+ cells . Analysiet cells . Expandeet cells . A 3.6–7et cells . FinallyRC alone , suggestRCN-3845 , indicatOur previous work has shown that inhibition of the NOTCH pathway mediator HES1 induces BCD cell redifferentiation CDH1 inhibition. Activation of WNT receptors by WNT ligands present in the serum may further stimulate nuclear β-catenin levels by inhibiting its degradation.Our findings demonstrate that proliferation and dedifferentiation of BCD cells in culture are associated with activation of the WNT pathway, and that partial inhibition of this pathway is sufficient for induction of BCD cell growth arrest, MET, and redifferentiation. These results are reproducible in cells derived from multiple human donors. WNT pathway activation may be initiated by islet dissociation into single cells, which would release membrane-associated β-catenin into the cytoplasm, from which it can enter the nucleus and activate target gene transcription CCND1 and MYC expression, which resulted in upregulation of the cell cycle blockers p21 and p27 CDH1 transcription, were significantly inhibited.Inhibition of β-catenin expression reduced its cellular levels to <40% of control cells. This reduction was sufficient for a significant decrease in cell proliferation rates, likely mediated by downregulation of NEUROD1 and MAFA expression, and promote growth arrest by activating expression of CDKN1B, encoding the cell cycle blocker p27 Our findings suggest that the mechanism underlying the activation of insulin transcripts in a striking quantitative inverse proportion to the decrease in β-catenin transcripts may involve AKT-FOXO1 interaction. Treatment with β-catenin shRNA resulted in a decrease in AKT transcript levels, which could be responsible for the observed reduction in inactive phospho-FOXO1, likely leading to upregulation of FOXO1 activity. FOXO1 activity has been demonstrated to play important roles in maintenance of mouse β-cell identity β-catenin shRNA potentiated the redifferentiation effects of RC treatment, which itself downregulates expression of WNT pathway components. This synergistic effect further supports a quantitative inverse correlation between WNT pathway activity and insulin expression in BCD cells. The finding that simultaneous inhibition of both WNT and NOTCH pathways results in a synergistic effect on β-cell gene expression reflect activity of the two pathways through different mediators, and/or potentiation of effects on the same mediators. Alternatively, this synergy may reflect the mutual stimulatory effects these two pathways exert on each other Taken together, these results suggest that inhibition of the WNT pathway induces significant BCD cell redifferentiation following expansion in vitro, and thus may contribute to therapeutic approaches based on expanding the functional β-cell mass obtained from scarce donor islet tissue for transplantation into multiple recipients. This prospect will require replacement of the β-catenin shRNA viral vector with small-molecule inhibitors of the WNT pathway, functional assessment of the redifferentiated cells in vitro and in vivo, and development of immunoprotective approaches. In addition, inhibition of the WNT pathway may be applicable for reversing β-cell dedifferentiation in vivo, which has been implicated in early stages of both type 1 and type 2 diabetes Figure S1β-catenin shRNA does not induce cell apoptosis. TUNEL assay of expanded human islet cells at passages 5–6 infected with β-catenin shRNA. Cells treated with DNase I served as positive control. Apoptotic cells were labeled with FITC. Bar = 50 µm.(TIF)Click here for additional data file.Figure S2β-catenin shRNA does not induce expression of non-β islet cell transcripts in BCD cells. qPCR analysis of RNA extracted from eGFP+ BCD cells at passages 4–5, 7 days following infection with β-catenin or NT shRNA viruses. Data are mean±SE (n = 3 donors). P values are relative to NT shRNA.(JPG)Click here for additional data file.


However, the exact underlying mechanisms remain unknown. Here, we reported that SENP2 inhibited nuclear translocation of β-catenin, which targeted the promotor of MMP13 to activate MMP13 to enhance BC cell metastasis. WNT ligands induced TBL1/TBLR1 SUMOylation to form complexes with β-catenin to facilitate β-catenin nuclear translocation, which could be efficiently inhibited through suppression of SUMOylation of TBL1/TBLR1. Together, our data suggest that SENP2 inhibits MMP13 expression in BC cells through de-SUMOylation of TBL1/TBLR1, which inhibits nuclear translocation of β-catenin. Thus, SENP2 may be a promising therapeutic target for BC.Bladder cancer (BC) is the most popular malignant urinary cancer in China. BC has the highest incidence and mortality among all genitourinary system tumors. Although the early-stage BC could be treated with advanced electron flexible systourethroscope, early metastasis of the BC occur frequently, and often results in poor prognosis. Recently, we reported that small ubiquitin related modifier (SUMO)-specific protease 2 (SENP2) was downregulated in BC specimen. SENP2 appeared to inhibit migration and invasion of bladder cancer cells  The WNT-β-catenin signaling pathway is essential for cell differentiation and proliferation during development and homeostasis, and during tumorigenesis1235678123123123TBL1 and its related gene TBLR1 both contain a LisH/WD-40 motif1011121314151610111213141516101112131415161011121314151610111213141516SUMO is a small ubiquitin-like protein which is covalently attached to proteins through forming isopeptide bonds with specific lysine residues of target proteins18192021181920211819202118192021181920211819202123242526272324252627in vitro, through suppressing MMP13 in BC cellsRecently, we reported that SENP2 was downregulated in BC specimen. SENP2 appeared to inhibit bladder cancer cells migration and invasion Here, we reported that SENP2 inhibited nuclear translocation of β-catenin, which targeted the promotor of MMP13 to activate MMP13, which enhanced BC cell metastasis. WNT ligands induced TBL1/TBLR1 SUMOylation to form complexes with β-catenin to facilitate β-catenin nuclear translocation, which was efficiently inhibited through suppression of SUMOylation of TBL1/TBLR1. These data suggest that SENP2 inhibits MMP13 expression in BC cells through de-SUMOylation of TBL1/TBLR1, which inhibits nuclear translocation of β-catenin. SENP2 may be a promising therapeutic target for BC.All the experimental methods in the current study has been approved by the research committee at Medical College of Shanghai Jiao Tong University. All the experiments have been carried out in accordance with the guidelines from the research committee at Medical College of Shanghai Jiao Tong University.2 at 37 °C. Recombinant human WNT5a (used at 400 ng/ml) was purchased from R&D Systems .Human BC cell line T24 cells were purchase from American Type Culture Collection  and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) in a humidified atmosphere of 5% COCytoplasmic and nuclear fractions were prepared by using the NE-PER nuclear and cytoplasmic extraction kit  according to the manufacturer’s protocol. In brief, the cell pellet was re-suspended in ice-cold reagent and incubated on ice for 10 min. After incubation, the contents of the tube were mixed and centrifuged at 16,000 × g for 5 min, after which the supernatant was collected (cytoplasmic fraction). The insoluble fraction, which contains nuclei, was re-suspended in another reagent, incubated on ice for 40 min, centrifuged at 16,000 × g for 10 min, after which the supernatant (nuclear fraction) was collected. Each of these fractions was then analyzed for purity by immunoblotting for specific protein markers . Samples containing 10 μg of total protein were separated by SDS-PAGE, transferred to nitrocellulose, and probed for specific proteins. Immunoreactive bands were detected using the ECL system. All immunoblotting experiments were repeated at least 4 times with similar results.SENP2-overexpressing T24 cells have been prepared as has been described beforeIn vitro SUMOylation assays were performed using the SUMOylation kit  according to the manufacturer’s protocol. In brief, 200 nmol/l of purified recombinant human TBL1 or TBLR1 protein was incubated with reaction mixture containing 50 mmol/ Tris-HCl (pH 7.4), 2 mmol/l DTT, 5 mmol/l ATP, 10 mmol/l MgCl2, Aos-1 (150 ng), His-Uba2 (400 ng), GST-Ubc9 (500 ng), and GST-SUMO-1 for 1 hour at 30 °C. The control reaction was carried out in the absence of ATP. After the incubation, protein SUMOylation was identified by immunoblotting using the anti-SUMO antibody provided with the kit. For in vivo assays, T24 cells were treated with WNT5a for 30 minutes, after which the nuclear extracts were prepared as described above. Extracts (200 μg of protein) were immunoprecipitated with anti-SUMO-1 antibody  followed by immunoblotting with anti-TBL1 or anti-TBLR1.in vivo DNA-protein interactions at the MMP13 promoter, using a ChIP assay kit  according to the manufacturer’s instructions. Briefly, 5 × 106 cells were harvested and fixed in 1% (v/v) formaldehyde for 10 min at room temperature. After washing with cold phosphate-buffered saline, cells were lysed in 1% SDS for 30 min on ice. The lysates were sonicated to shear DNA using a Branson 250 sonicator . These shearing conditions generate DNA fragments ranging in size from 500 to 1,000 bp. Chromatin solution was precleared with protein G-coated magnetic beads for 2 hours at 4 °C. Ten microliters of the chromatin solution was reserved as the “input” sample. The remaining chromatin was immunoprecipitated overnight at 4 °C with 3 μg of antibody specific to β-catenin, rabbit immunoglobulin G (IgG) as a negative control (provided with the kit). The chromatin-antibody complexes captured on the beads were washed several times and then eluted in 50 μl of elution buffer. The immunoprecipitated and input sample cross-links were reversed by incubation for 2.5 hours at 65 °C. After treatment with proteinase K at for 1 hour at 37 °C, the reaction was stopped and the resulting DNA stored at −20 °C until analyzed by standard and real-time quantitative PCR (RT-qPCR) as described below.A ChIP assay was performed to assess 2, 0.2 μmol/l dNTP, and 0.04 units/μl of AmpliTaq Gold DNA polymerase. The PCR conditions were as follows: 1 cycle at 94 °C for 3 min, 35 cycles at 94, 60, and 72 °C for 30 seconds each, and a final cycle at 72 °C for 10 min. PCR products were analyzed by agarose gel electrophoresis. The primers used in the PCR were designed to amplify the promoter region of the human MMP13 gene .PCR analysis was performed in a 50-μl volume containing 5 μl of ChIP DNA, 1 μmol/l of each primer, 2 mmol/l MgClTotal RNA was isolated using an RNeasy Mini kit . Two micrograms of total RNA was reverse transcribed for 1 hour at 42 °C using an avian myeloblastosis virus reverse transcriptase and oligo(dT) primer. Then 1 μl of reverse transcriptase solution was combined in a reaction mixture with 1 μl of the specific primer pair, 7.5 μl of 2X SYBR Green PCR Master Mix, and water to a final reaction volume of 15 μl. Primers were all purchased from Qiagen. RT-qPCR were then run in duplicate, with 40 cycles of amplification, on an ABI Prism 7000 real time PCR instrument . A negative control containing primers, water, and Master Mix, but no complementary DNA, was included. The relative gene transcripts were first normalized to the internal control TATA box-binding protein, calculated with the comparative cycle threshold (ΔΔCt) method, and then compared to experimental controls. Relative fold changes were presented.Data were expressed as means ± standard deviation (SD). Statistical significance was evaluated by Student’s t test or one-way analysis of variance (ANOVA) followed by the Fisher’s Exact Test. Differences were considered statistically significant at p < 0.05.In order to molecular mechanisms that regulate the effects of SENP2 on MMP13 and BC cell metastasis, we have prepared SENP2-overexpressing T24 cells. T24 cells that were transduced with a null construct were used as a control (null). The SENP2 levels in these cells were analyzed, which confirmed the modification of SENP2 in T24-SENP2 cells, by RT-qPCR and semi-quantitative PCR  and by WT24-SENP2 and control T24-null cells were then treated with WNT5a for 30 min and nuclear translocation of β-catenin was analyzed by immunoblotting. We detected a band with increased density with β-catenin antibody in the nuclear fraction and a band with decreased density with β-catenin antibody in the cytosolic fraction after WNT5a treatment in T24-null cells, suggesting that WNT5a induces nuclear translocation of β-catenin . HoweverWe next examined whether nuclear β-catenin binds to chromatin that contains the MMP13 promoter region. Thus, we immunoprecipitated endogenous nuclear β-catenin from T-24 cells treated with or without WNT5a and amplified the co-purified DNA using PCR. The ChIP isolates contained the promoter region of MMP13, as detected by primers used in semi-quantitative RT-PCR . Taken tThen we studied the molecular mechanisms underlying the WNT5a-induced nuclear translocation of β-catenin that is inhibited by SENP2-mediated SUMOylation suppression. Based on literature, we hypothesize that the WNT signaling-associated proteins TBL1/TBLR1 may be the targets of SUMOylation, since their SUMOylated complex has been shown to enhance nuclear translocation of β-catenin. Thus, T24-SENP2 and control T24-null cells were treated with WNT5a for 30 min and total cell extracts (200 μg of protein) were immunoprecipitated with anti-SUMO-1 antibody followed by immunoblotting with anti-TBL1 or anti-TBLR1. Significant increases in immunoreactive bands for both TBL1 and TBLR1 were seen in the extracts after a 30-min stimulation with WNT5a in T24-null cells, suggesting that both TBL1 and TBLR1 undergo SUMOylation by WNT5a . Howeverin vitro SUMOylation assay. Slow moving immunoreactive bands of both TBL1 and TBLR1 were recognized by the anti-SUMO antibody  that can accept SUMO moieties. Mutating these residues or overexpression of SENP2, an effective SUMOylation inhibitor, not only inhibited WNT5a-mediated SUMOylation of corresponding protein (TBL1 or TBLR1), but also blocked the nuclear translocation of β-catenin.We previously reported that overexpression of SENP2 in BC cells inhibited production of MMP13. Here we further show that that disruption of SUMOylation of TBL1/TBLR1 inhibited nuclear translocation of β-catenin, which is necessary for WNT5a-stimulated MMP13 activation. Our ChIP data show that nuclear β-catenin is associated with the region of the MMP13 promoter. Although our studies indicate that β-catenin may interact with the MMP13 promoter, we speculate that this interaction may be direct or indirect. β-catenin does have a DNA binding motif in their primary structure. However, they may also play a role in transcriptional regulation of genes by interacting with certain transcription factors, which contain the basic helix-loop-helix domain to direct bind MMP13 promoter. Further studies are needed to clarify the details.Together, our data suggest that SUMOylation of both lysine residues in TBL1 and TBLR1 is required for translocation of β-catenin into the nucleus upon WNT5a stimulation. Hence, SUMOylation of TBL1 and TBLR1 is required for WNT5a-induced β-catenin nuclear translocation in BC cells.How to cite this article: Tan, M. et al. SENP2 regulates MMP13 expression in a bladder cancer cell line through SUMOylation of TBL1/TBLR1. Sci. Rep.5, 13996; doi: 10.1038/srep13996 (2015).


APC, whereas other cancer types  have activating mutations in β-catenin (CTNNB1). We have compared the dynamics and the potency of β-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of β-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated β-catenin.Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in  GT Chen & ML Waterman (September 2015)See also: APC (adenomatous polyposis coli) gene is the most common event in colorectal cancer (CRC). A recent study where a large number of CRCs (200) were sequenced showed mutation in over 70% of CRC  for degradation. In the absence of a Wnt ligand, APC in complex with AXIN, casein kinase 1 (CK1) and GSK3beta is required for the phosphorylation of β-catenin by GSK3, which targets β-catenin for degradation. Following a Wnt signal or APC mutation, this complex does not form and β-catenin is no longer targeted for degradation and accumulates in the nucleus. Within the nucleus, β-catenin interacts with T-cell factor-1/lymphoid-enhancing factor-1 (TCF/LEF1) transcription factors to drive transcription of TCF/LEF1 target genes , ∼75% compared to ∼5%, respectively  but also to reduced adhesion alongside cell loss from the villus . We have previously shown that genetic deletion of both copies of the Apc tumour suppressor (Apcfl/fl) rapidly generates a crypt-progenitor cell-like phenotype (CPC) within the intestinal epithelium, with cells failing to differentiate, retaining proliferative capacity and failing to migrate up the crypt-villus axis  phenotype with high nuclear β-catenin, increased proliferation and upregulation of Wnt target genes in both the small intestine and colon  and GSK3beta (GSK3B), and data from ES cells suggested that in the absence of one isoform, the other can compensate  and found following Cre deletion a significant fraction of these mice spontaneously developed intestinal tumours, in contrast to the single isoform mice /lox(ex3))showed the CPC phenotype with nuclear localisation of β-catenin and increased proliferation along the crypt-villus axis , activation of a single copy of β-catenin did not yield a robust CPC phenotype at day 5 or day 10. Immunohistochemical staining of β-catenin showed very little nuclear localisation and no increase in proliferation /+ mice and a number of crypts became enlarged. At time points past day 20, a single copy of Catnblox(ex3)/+ was able to induce a robust CPC phenotype in the small intestine /+ and AhCreERCatnblox(ex3)/lox(ex3) crypts  which were induced in vivo to form organoids/spheres in culture and their R-spondin1 dependence. In contrast to organoids where Apc was deleted, AhCreERCatnblox(ex3)/+ crypts did not show R-spondin1 independence and died in culture when plated 5 days following Cre recombination in vivo /lox(ex3) formed spheres in an equivalent manner to homozygous Apc deletion, independent of R-spondin1. Crypts sampled at later time points (day 10) from AhCreERCatnblox(ex3)/+ mice were able to survive in culture without R-spondin. At this time point, we observed a more organoid-like structure phenotype (∼85%) than the expected typical round spheres (∼15%) /lox(ex3) almost completely formed spheres, and only few organoid-like structures were observed.To probe the kinetics of β-catenin activation, we used intestinal “organoid” crypt culture /+ mice. In contrast to AhCreERApcfl/fl mice, which rapidly develop a CPC phenotype with nuclear β-catenin accumulation and Wnt target gene activation, this did not occur in the AhCreERCatnblox(ex3)/+ mice. Colons from AhCreERCatnblox(ex3)/+ mice even 25 days post-induction did not show nuclear β-catenin or Wnt target gene activation despite a clear CPC phenotype in the small intestine  of VilCreERCatnblox(ex3)/lox(ex3) and VilCreERApcfl/fl mice with increased levels of nuclear β-catenin and SOX9. However, no phenotype or accumulation of β-catenin or Sox9 was observed in the colons  of VilCreERCatnblox(ex3)/+ mice at this time point /lox(ex3) and VilCreERApcfl/fl needed to be harvested due to signs of ill health. In contrast, VilCreERCatnblox(ex3)/+ developed signs of ill health after about 25 days which was associated with a small intestinal CPC phenotype. In the colon, there was not an obvious CPC phenotype at this stage /+ to Lgr5CreER mice to allow inducible Cre-mediated recombination within the LGR5-positive intestinal stem cells. The Lgr5CreER delivers highly penetrant recombination in the small intestinal Lgr5-positive stem cell population and lower penetrant recombination in colon Lgr5-positive stem cell population. We have previously shown that targeting Apc loss to the Lgr5 compartment led to rapid small intestinal adenoma formation /+ and Lgr5CreERCatnblox(ex3)/lox(ex3) mice were induced with a single intraperitoneal injection (IP) injection of tamoxifen and rapidly developed small intesti-nal tumours /+, Lgr5CreERCatnblox(ex3)/lox(ex3) and Lgr5CreERApcfl/fl mice when they were harvested due to small intestinal disease burden. We found that the majority of Lgr5CreERCatnblox(ex3)/lox(ex3) mice (88%) and Lgr5CreERApcfl/fl mice (91%) had lesions in the colon, whereas none of the Lgr5CreERCatnblox(ex3)/+ mice (0%) had any lesions /+ mice, we first examined whether E-cadherin levels might be higher in the colon than the small intestine. This would suggest that E-cadherin might be playing a role in limiting the free β-catenin. Investigating expression by both qRT–PCR and immunoblotting showed markedly higher levels of E-cadherin in the colon compared to the small intestine in wild-type mice . Using antibodies for β-catenin and E-cadherin allowed us to quantify the number of complexes in the small intestine and the colon of wild-type mice. Consistent with the increase in E-cadherin expression, there was a 2- to 3-fold increase in the number of E-cadherin:β-catenin complexes in the colon compared to the small intestine /lox(ex3) mouse, all newly produced β-catenin in intestinal epithelial cells will be smaller (Δex3). Using co-immunoprecipitation for E-cadherin, the relative amounts of mutant β-catenin complexed with E-cadherin can be assessed. We induced VilCreERCatnblox(ex3)/lox(ex3) mice and sampled the small intestine and the colon after 24 and 48 h. After just 24 h  produces a smaller protein which is easily detected by immunoblotting. Thus, in a VilCreERCatnblox(ex3)/+ mice. Here, only half of the newly produced β-catenin will be of the mutant form, so we hypothesised that saturation would take longer, especially given the protracted time taken to generate the CPC phenotype. However, we would expect the mutant form to accumulate as this would not be turned over by the destruction complex. First, we examined E-cadherin:β-catenin complexes 4 days post-Cre induction. We showed that 75% of the β-catenin bound to E-cadherin is of the mutated form in the small intestine (75:25 Δex3:wt), whereas the colon has the mutant and the wild-type β-catenin in a roughly similar ratio (53:47 Δex3:wt). At day 8 after induction, approximately 85% of β-catenin bound to E-cadherin in the SI is mutated (85:15 Δex3:wt) compared to 55:45 Δex3:wt in the colon. Therefore, this relative increase in the mutant form of β-catenin compared to the wild-type protein showed that mutant β-catenin was specifically accumulating with E-cadherin at the adherens junction. This suggested that this could be acting as a sink for β-catenin, stopping the mutant β-catenin being translocated into the nucleus where it would bind TCF/LEF transcription factors and drive Wnt targets and the CPC phenotype.We next analysed the ratio in If E-cadherin was acting as a sink and limiting mutant β-catenin from entering the nucleus, this would predict the following: (i) when β-catenin can be targeted for destruction, reduction in E-cadherin levels should have no phenotype, (ii) when there is mutant β-catenin (Δex), reduction in E-cadherin should cause this sink to be saturated quicker and hence lead to a translocation of β-catenin to the nucleus and a CPC phenotype.AhCreERCatnblox(ex3)/+ mice to mice carrying a conditional knockout E-cadherin allele to generate AhCreERCatnblox(ex3)/+Cdh1fl/+ mice and controls  showed a reduction in E-cadherin expression to ∼60% and importantly a reduction in E-cadherin:β-catenin complexes assessed by PLA /+Cdh1fl/+ mice. In contrast to the AhCreERCatnblox(ex3)/+ 10 days post-induction, AhCreERCatnblox(ex3)/+Cdh1fl/+ showed a CPC phenotype in both the small and large intestine with nuclear β-catenin and increased expression of the Wnt target Sox9 /+Cdh1fl/+ mice which had a CPC phenotype 4 days post-induction with increased expression of several Wnt target genes /+Cdh1fl/+ mice formed spheroids in an R-spondin1-independent manner 5 days post-induction /+ mice. Moreover, targeting a copy of E-cadherin loss in combination with a β-catenin mutation to Lgr5 ISC (Lgr5CreERCatnblox(ex3)/+Cdh1fl/+) significantly accelerated tumourigenesis when compared to Lgr5CreERCatnblox(ex3)/+ mice. Most importantly, when these mice with reduced E-cadherin were analysed for colonic lesions, 6 of 7 mice had lesions with high nuclear β-catenin /+ mice  in colorectal cancer are very rare, approximately 5% (11/212). Closer analysis showed that many of these were not in exon 3 and therefore unlikely to affect β-catenin degradation. In total, only 2 of 11 mutations were in the most common exon 3 hotspot  or solid pseudopapillary tumours of the pancreas show very different patterns of mutations  have a β-catenin mutation in exon 3, this suggests deregulation of Wnt signalling as an essential step for tumourigenesis in this cancer. From our model, we would predict a change in E-cadherin levels for a β-catenin activating mutation to have the greatest transforming properties. Importantly in these SPT tumours, in addition to the nuclear accumulation of β-catenin, an aberrant localisation of E-cadherin has been reported  where approximately 20% of tumours have a mutation in exon 3 of β-catenin. Analysing expression data from 269 patients , we saw a significant negative correlation of E-cadherin with several Wnt target genes  that activation of Wnt signalling by loss of the destruction complex or bi-allelic β-catenin mutation is sufficient to drive rapid intestinal transformation. In contrast, upon monoallelic deletion of exon 3, the mutant form of β-catenin takes much longer to accumulate in the small intestine and lead to a CPC phenotype. In the colon, monoallelic mutation of β-catenin did not drive a CPC phenotype (even 25 days post-induction) and did not cause lesions when targeted to the Lgr5 compartment. This suggests that the level of Wnt deregulation required to transform the colorectal epithelium is higher than in the small intestine. This idea often referred to the “just right” model has previously been associated with different levels of Wnt signalling induced by different APC mutations  have an increased tumour burden when crossed to the Apc1638N/+ mouse model, a long latency model of Apc-loss induced tumourigenesis , then on many occasions, cells carrying Apc mutations would be lost. From our work, we would suggest that as it takes a protracted time for the β-catenin exon 3 mutation to have a phenotype in the small intestine, this mutation would probably be neutral (0.5) for some time. Thus, given neutral drift within the intestine, one might predict that these mutant cells would be lost.Another potential reason for the lack of β-catenin mutations in both small intestinal and colon cancer is suggested from recent work investigating the selective advantage of different tumour-promoting mutations in murine small and large intestinal epithelium. The Winton group showed that a single inactivating ApcMin mice (a model of FAP) mainly develop small intestinal tumours rather than FAP where patients preferentially develop colon tumours, though they will eventually develop duodenal tumours. Our data show acute Apc loss manifests phenotypes in both the small intestine and colon which does not explain the tropism for small intestine in the mouse. A recent study by Tomasetti and Vogelstein  , pGSKB-Ser9 , β-catenin , cMyc , Sox9 , lysozyme  and E-cadherin . Staining for nuclear β-catenin was performed on tissue samples fixed at 4°C for less than 24 h in 10% formalin prior to processing.g, 3 min). The following antibodies were used: E-cadherin ; β-actin .Standard Western blot techniques were used. Crypts from the small and large intestine were purified by incubation with 2 and 25 mM EDTA/PBS (respectively) for 30 min at 4°C. Crypts were further purified by centrifugation with low speed (50 × g for 10 min at 4°C. Magnetic beads conjugated to anti-mouse IgG (Dynabeads) were incubated with 1 mg of protein lysates, 1 μg of either monoclonal anti-E-cadherin (BD Biosciences #610182) or IgG isotype control (Sigma) for 1 h at 4°C with constant rotation. After several washes with lysis buffer, bound proteins were eluted from the beads by boiling at 100°C for 5 min in SDS reducing buffer. Bound proteins and 5 μg of total lysates (Input) were run on 4–12% Bis–Tris Gradient SDS gel and probed for β-catenin .Small intestinal and colonic tissue (∼4 cm) was lysed in lysis buffer . Lysates were clarified by centrifugation at 16,000 × Z-stacks with 40× objectives were taken. PLA dots in crypts were analysed with ImageJ and either calculated as area fraction or count/nuclei.PLA was performed on tissue samples fixed at 4°C for < 24 h in 10% formalin prior to processing using the Duolink Detection kit (Sigma) according to the manufacturer's instructions. Briefly, after citrate buffer-mediated antigen retrieval, the slides were incubated with goat E-cadherin  and mouse β-catenin  overnight. Detection was performed with PLA probes (anti-goat and anti-mouse) conjugated to oligonucleotides. After ligation, amplification detection with a fluorescent probe, slides were imaged on a Zeiss LSM confocal microscope. et al, Crypts were purified from mice as previously described (Sato et al, Tissue microarray of 18 SPT patients was used as described before, and normal tissue was used as control (Serra For detailed description and primer sequences, see


The cadherin–catenin complex is vital cell adhesion molecules and it plays a fundamental role in maintaining the cells differentiation and the normal architecture of epithelial tissues . ConsequTo achieve this, paraffin blocks of 103 CRC patients were retrieved. The expression of E-cadherin, N-cadherin, α-catenin and β-catenin were analyzed by immunohistochemmistry (IHC) and promoter hypermethylation of E-cadherin gene CDH1 was analyzed using Methy-Light Assay. Statistical analyses were performed to determine the association of these markers with clinico-pathological variables.The results showed that the expressions of both α-catenin and N-cadherin were significant sign of poor outcome and recurrence as evaluated by univariate Kaplen-Meier for disease-free survival (DFS) . In multivariate survival (Cox) analysis, α-catenin was significantly independent predictor of DFS (P=0.008). On the other hand, β- catenin and E-cadherin exhibits no prognostic power for recurrence and poor survival. Promoter methylation of CDH1 was observed in 45% of our samples and correlation with E-cadherin expression revealed a 60% concordance.These results implicate the usefulness of α-catenin and N-cadherin in predicting outcome of patients with CRC.This work was financially supported by King Abdulaziz City for Science and Technology (KACST) under research no (ARP -30-262).


Colorectal cancer is the fourth most common cancer and the second leading cause of cancer death. Transformation begins with activation of Wnt signaling through mutations in APC (80%) or its degradation target β-catenin (15%), producing a gain-of-function in TCF-dependent nuclear transcription underlying epithelial dysfunction and tumorigenesis. While a role for APC and β-catenin in colorectal cancer is well-established, steps leading from gene mutation to tumorigenesis, and their reversibility, remain incompletely defined. Guanylin is the paracrine hormone in colorectum for the receptor GUCY2C. This hormone is the most commonly lost gene product in colorectal cancer, and its universal suppression and associated silencing of GUCY2C at the earliest step in neoplasia contributes to tumorigenesis in a mechanism that is conserved across species. GUCY2C regulates homeostatic mechanisms organizing the intestinal crypt-surface axis, and its silencing through guanylin suppression drives hyperproliferation, DNA damage, metabolic reprogramming and desmoplasia contributing to tumorigenesis. Beyond this dysregulation of homeostatic mechanisms contributing to transformation, we recently discovered that guanylin suppression is required for tumorigenesis induced by mutant APC-β-catenin, reflecting a role for GUCY2C in β-catenin degradation that blocks tumorigenesis. Thus, inactivation of APC, or activation of β-catenin, induces TCF-dependent elimination of guanylin transcription and translation in human intestinal cells in vitro and in conditional genetic mouse models in vivo. Conversely, activation of GUCY2C induces cGMP protein kinase-dependent elimination of wild type or mutant β-catenin by amplifying proteosomal degradation, even in the context of mutations which inactivate the APC degradation complex. Importantly, enforced genetic expression of guanylin in intestinal epithelial cells completely eliminated tumorigenesis in all mouse models of colorectal cancer examined. These observations reveal a pathophysiologic model in which mutant APC-β-catenin signaling eliminates guanylin expression as an obligatory step in tumorigenesis. In turn, silencing GUCY2C reversibly disrupts epithelial homeostatic processes corrupted in tumorigenesis, and removes an essential block to transformation, creating a circuit which amplifies mutant APC-β-catenin signaling. These studies shift the prevailing paradigm for colorectal tumorigenesis from an irreversible oncogenomic mechanism to a reversible functional mechanism whose reconstitution abrogates those mutational defects. Indeed, they define a novel molecular pathway that is obligatory for tumorigenesis, to which the transformation process is addicted. In that context, they highlight how this addiction creates a unique disease-specific vulnerability that can be leveraged to eliminate tumorigenesis by GUCY2C hormone replacement. We have translated these observations to an NCI-funded clinical program exploring the utility of oral GUCY2C ligands to prevent colorectal cancer.


The toxicity of dusts from mechanical abrasion of multi-walled carbon nanotube (CNT) epoxy nanocomposites is unknown. We compared the toxic effects of dusts generated by sanding of epoxy composites with and without CNT. The used CNT type was included for comparison.interleukin 6 and heme oxygenase 1 was measured in the lungs and serum amyloid A1 in the liver. Printex 90 carbon black was included as a reference particle.Mice received a single intratracheal instillation of 18, 54 and 162 μg of CNT or 54, 162 and 486 μg of the sanding dust from epoxy composite with and without CNT. DNA damage in lung and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Furthermore, the mRNA expression of Pulmonary exposure to CNT and all dusts obtained by sanding epoxy composite boards resulted in recruitment of inflammatory cells into lung lumen: On day 1 after instillation these cells were primarily neutrophils but on day 3, eosinophils contributed significantly to the cell population. There were still increased numbers of neutrophils 28 days after intratracheal instillation of the highest dose of the epoxy dusts. Both CNT and epoxy dusts induced DNA damage in lung tissue up to 3 days after intratracheal instillation but not in liver tissue. There was no additive effect of adding CNT to epoxy resins for any of the pulmonary endpoints. In livers of mice instilled with CNT and epoxy dust with CNTs inflammatory and necrotic histological changes were observed, however, not in mice instilled with epoxy dust without CNT.Pulmonary deposition of epoxy dusts with and without CNT induced inflammation and DNA damage in lung tissue. There was no additive effect of adding CNT to epoxies for any of the pulmonary endpoints. However, hepatic inflammatory and necrotic histopathological changes were seen in mice instilled with sanding dust from CNT-containing epoxy but not in mice instilled with reference epoxy.The online version of this article (doi:10.1186/s12989-016-0148-2) contains supplementary material, which is available to authorized users. Carbon nanotubes (CNTs) are very promising nanomaterials due to their many technically applicable properties. When CNTs are added to epoxy resins to form epoxy/CNT nanocomposites, these nanocomposites exhibit improved properties such as increased strength combined with reduced weight of the product , 2. DuriSeveral studies on rodents have shown that pulmonary exposure to different types of CNTs induces an asbestos-like toxicological response characterized by persistent inflammation, granulomas and fibrosis with low no-effect levels –9. It haKnowledge is currently developing on the process-specific particle emissions and release of fibrous nanomaterials during the life-cycle processes  of carbon-based nanocomposites. Recently, it was shown that significant fractions of carbon fibers of μm-size diameters were clearly separated from matrix during industrial-scale grinding and sanding of layered silica-carbon epoxy composite . ConversThe purpose of the present study was to assess the toxicity, by inflammatory and DNA damaging effect, of sanding dusts from epoxy composites with and without CNT for dose-responses following pulmonary exposure at different time points in mice. In order to be able to assess if the toxicological changes induced by dust from epoxy/CNT nanocomposites were similar to changes induced by the pristine CNT, data on the same CNT (Nanocyl NC7000) as used in the epoxy/CNT nanocomposite were included for comparison. Some of the data on the pristine CNT have been published previously , 22 and We tested sanding dusts from three different types of epoxy composite boards with and without CNT: The Danish Technological Institute provided two epoxy/CNT nanocomposite boards and one epoxy matrix board for the study: 1) One epoxy nanocomposite contained 0.2 % w/w CNT Nanocyl NC7000 (EPOXY-CNT), 2) One corresponding epoxy board contained no CNT (EPOXY-REF) and was considered a reference for the epoxy nanocomposite matrix; and 3) EPOCYL™NC R128-04 (EPOCYL) containing less than 20 wt.% Nanocyl NC7000 . The prChemical characterisation by WDXRF was performed on disks cut out of the original boards. It was confirmed by SEM that these pieces (EPOXY-CNT and EPOCYL) contained CNT  by wave-length dispersive X-ray fluorescence analysis (WDXRF). The results are shown in Additional file Table Scanning Electron Microscopy (SEM) of the test materials showed that the sanding dusts from epoxy/CNT nanocomposites were dominated by angular and sub-angular particles with upper sizes around 10 μm Figs.  and b. DThe test materials were dispersed by probe-sonication in Nanopure-filtered water with 2 % v/v C57BL/6 mice serum and further diluted by serum-water into the concentrations used for instillation. All suspensions used for intratracheal instillation were analyzed by DLS (Dynamic Light Scattering). The DLS correlation plots suggested that the test materials dispersed well in the batch dispersions and instillation mediums, but the dispersions were often unstable as indicated by variable hydrodynamic sizes as well as trending hydrodynamic zeta-sizes and intensity counts  as previously described [The endotoxin content in supernatants from particle suspensions used for intratracheal instillation was measured using the escribed . The amointerleukin 6 (Il6) and heme oxygenase 1 (Hmox-1) was measured in lung tissue and Serum amyloid A1 (Saa1) was measured in liver tissue.Mice received a single intratracheal instillation of 18, 54 and 162 μg of CNT or 54,162 and 486 μg of EPOXY-REF, EPOXY-CNT and EPOCYL. DNA damage in lung and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Furthermore, the mRNA expression of To assess the recruitment of inflammatory cells into the lung lumen, we determined the total number of BAL cells and the number of macrophages, neutrophils, eosinophils, lymphocytes, and epithelial cells in the BAL Table . The neuSanding dust from all epoxies resulted in increased numbers of total BAL cells in mice 1 day after intratracheal instillation of all doses . Significantly higher numbers of total BAL cells were also observed at all doses in mice 3 days after intratracheal instillation of the two CNT containing epoxies, while only 162 and 486 μg of the reference epoxy resulted in increased number of total BAL cells. Only the highest dose (486 μg) resulted in an increase in the total number of BAL cells 28 days after instillation of dust from the three epoxies.Sanding dust from all epoxies resulted in increased neutrophil cell numbers in mice 1 day after intratracheal instillation of all doses . Significantly higher numbers of neutrophils were also observed at all doses in mice 3 days after intratracheal instillation of the two CNT containing epoxies, while only 162 and 486 μg of the reference epoxy resulted in increased number of neutrophils. Higher numbers of neutrophils were observed in mice 28 days after instillation of 486 μg of dust from EPOXY-REF, 162 and 486 μg of EPOXY-CNT and 486 μg of EPOCYL.In mice instilled with sanding dust from epoxies, significantly higher number of macrophages were only seen 3 days after intratracheal instillation of EPOXY-CNT (486 μg) and EPOCYL (162 μg).Sanding dust from all epoxies resulted in increased eosinophil counts in mice 1 and 3 days after intratracheal instillation of all doses . None of the tested materials resulted in increase in eosinophil counts 28 days after instillation.One day after instillation, increased numbers of lymphocytes were only seen in mice instilled with the highest dose of EPOXY-REF and EPOXY-CNT (486 μg). Sanding dust from all epoxies resulted in increased lymphocyte counts 3 days after intratracheal instillation of the two highest doses (162 and 486 μg). In addition, instillation of 54 μg of dust from the two CNT containing epoxies resulted in increased number of lymphocytes. Only, instillation of EPOCYL resulted in increased lymphocyte numbers 28 days after instillation (54 and 486 μg).Increased numbers of epithelial cells were only seen in mice 1 day after instillation of CNT and 3 days after instillation of 486 μg of two of the epoxy dusts (EPOXY-REF and EPOCYL).Instillation of the reference particle (162 μg CB) resulted in a similar response as observed in our previous study , 29–31: Thus, pulmonary inflammation was observed for all tested materials. There were no differences between mice intratracheally instilled with dust from the CNT containing epoxy (EPOXY-CNT) and the reference epoxy (EPOXY-REF) at any time point for any of the measured cell types, or between the two CNT containing epoxies (EPOXY-CNT and EPOCYL).Il-6 mRNA expression level in lung tissue 1 day after intratracheal instillation of the two highest doses (162 and 486 μg)  and the reference epoxy (EPOXY-REF) at any time point, or between the two CNT containing epoxies (EPOXY-CNT and EPOCYL).Sanding dust from all epoxy dusts resulted in increased  162 and 86 μg  and the reference epoxy (EPOXY-REF) at any time point, or between the two CNT containing epoxies (EPOXY-CNT and EPOCYL).Sanding dust from EPOXY-REF (the highest dose) and EPOXY-CNT  resulted in increased re Table . There wSaa1 mRNA expression in livers at the same time point from mice instilled with 486 μg of epoxy sanding dusts or 162 μg CB or CNT. Pulmonary exposure to CNT, EPOXY-REF, EPOXY-CNT and EPOCYL induced significant increases in the hepatic Saa1 mRNA expression levels compared to controls, while there were no effects in mice exposed to CB , while there were no effects 3 and 28 days after instillation. EPOXY-CNT did not induce any significant increases in DNA strand break levels. In contrast, dust from EPOCYL induced significantly increase in DNA strand break levels 1 day after instillation of 54 and 162 μg, and 3 days after intratracheal instillation of 162 and 486 μg. There were no statistically significant differences in response between the three epoxy dusts. Previously published data on the CNT used in the epoxy showed that CNT induced pulmonary DNA damage on day 1 (18 and 54 μg) and day 3 (54 and 162 μg) .Compared to the vehicle controls, none of the test materials induced significantly changes in DNA strand break levels in liver tissue. There were no statistically significant differences between the three sanding dusts.Several histological changes were observed in the liver Fig. , Table 6The histological changes in the livers from mice exposed to EPOXY-REF were similar to the controls with regard to the lack of the inflammatory and necrotic changes. For the other types of lesions recorded for the groups exposed to either CNT, EPOXY-CNT or EPOCYL low incidences were also noted in the EPOXY-REF group. Mice instilled with CB displayed hepatic changes of the type that we have reported before .In the present study, we investigated the dose–response relationships of inflammation and DNA damage of respirable dust generated and sampled directly during sanding of epoxy boards with (EPOXY-CNT) and without CNT (EPOXY-REF) 1, 3 and 28 days after a single intratracheal instillation in mice. In addition, an industrial epoxy product with CNTs was included (EPOCYL). Our results show that pulmonary deposition of epoxy dust results in inflammation and pulmonary DNA damage up to 28 days and 3 days after exposure, respectively. There was no additive effect of adding CNTs to the epoxy compared to the reference epoxy for any of the measured pulmonary toxicological endpoints. In contrast, instillation with dusts from epoxy boards with CNT (EPOXY-CNT and EPOCYL) was associated with histological inflammatory and necrotic changes in the liver. These changes were also observed for CNT-instilled mice but not for mice instilled with dust from EPOXY-REF.We chose to study composite materials reinforced with the CNT Nanocyl NC 7000 because this CNT is widely used as reinforcement for many different applications including industrial components, such as rollers, medical knives and windmill blades, and for applications in the following markets; automotive, sports, marine and aerospace , 23. The2) [2 which made it possible to compare the toxicity of the NanoTiO2-containing paint dust to the toxicity of the same amount of both dust from paint without NanoTiO2 and to the toxicity of the same dose of pristine NanoTiO2 (eg. 18 μg of pristine NanoTiO2 corresponds approximately to the amount of NanoTiO2 in 162 μg of paint). A set-up enabling this comparison was not possible in the present study because of the low CNT content in EPOXY-CNT (0.2 %). Thus the 486 μg dose of EPOXY-CNT should be compared to the toxicity of less than1 μg of CNT and this dose was expected to be too low to generate a response. Higher doses than 486 μg of sanding dust was expected to result in overload. On the basis of these considerations and for comparison, we therefore chose to use the same doses of epoxy dusts as used in the previous study on paint dusts.The chosen doses  and time points  are similar to our previous study on sanding dusts from paints with and without nano titaniumdioxide (NanoTiO2) . In that3 for respirable inert mineral dust, respectively . For comparison, the doses of CB  equal pulmonary deposition in mice after 1, 3 and 9 working days of 8 h at the Danish occupational exposure limit of 3.5 mg/m3 for CB, respectively, (with same assumptions as above except for a higher pulmonary deposition of CB in the pulmonary region (33 %)) [3 [We have not been able to identify any studies on exposure levels to sanding dust in epoxy resin workers in the scientific literature. However, the dust doses  equal pulmonary deposition in mice after 8, 23 and 68 working days of 8 h at the Danish occupational exposure limit of 5 mg/m (33 %)) . When coMice intratracheally instilled with epoxy dust responded with a massive influx of polymorph nuclear cells into the lung lumen and the same response was observed for mice instilled with dust from EPOXY-CNT and EPOXY-REF. Two studies have been published on the pulmonary toxicity of dusts derived by machining of composite epoxy materials (graphite fiber-epoxy and fiberglass-epoxy) , 33. BotMice intratracheally instilled with CNT and epoxy dust responded with a massive influx of eosinophils (previously discussed in ). EosinoWe observed a number of histological lesions (including granulomas) in the liver from mice exposed to EPOXY-CNT and EPOCYL dust compared to dust from EPOXY-REF. Compared to controls, none of the dusts caused increased levels of DNA strand breaks in liver tissue. Several histological liver changes, although of low incidence, were recorded in groups exposed to the dusts from either epoxy resin without CNT (EPOXY-REF) or from CNT containing epoxy resins (EPOXY-CNT and EPOCYL), as well as to CNT. The noteworthy finding was that inflammatory and necrotic changes were solely recorded in the CNT group and the groups exposed to the CNT-containing epoxy dusts. This indicates that the pulmonary deposition of these resins is associated with stronger hepatic effects compared to the pulmonary deposited epoxy without CNT (EPOXY-REF).These hepatic changes could hypothetically be caused by systemic inflammation, other types of signalling and/or translocation of CNTs. The two latter possibilities are considered most likely, since the pulmonary inflammatory response was similar for EPOXY-CNT and EPOXY-REF and slow translocation of CNTs from the lungs to the liver and other distant organs has been reported , 39. ThiSaa1 as the most differentially regulated acute phase gene [Saa1 expression as biomarker of a hepatic acute phase response. Our results show that the hepatic mRNA expression levels of the acute phase gene Saa1 were increased for all three sanding dusts and the CNT, while no increased Saa1 mRNA expression levels were detected in the CB exposed mice  induced a strong pulmonary acute phase response in a dose-dependent manner . The pulase gene . In addiase gene  and was ice Fig. . During egulated . In accois study . FurtherNo significant differences in the particle size distribution between EPOXY-REF and EPOXY-CNT was observed during sanding . This isSanding epoxy boards generated two different size ranges below and above 100 nm. Similar size distributions have been observed in previous studies on particle generation during sanding of different types of materials by sanding machine engines , 28, 42.Sanding dusts from the epoxy/CNT composites were characterized by having single CNTs protruding from the surface of the nanocomposite particles. Protruding CNTs were also observed in the few other studies characterizing sanding dusts from epoxy/CNT composites –20. The We do not have information on the specific content of CNT in the industrial product, EPOCYL (stated to be <20 %) compared to the experimental CNT epoxy product. However, based on the very similar amounts of Fe and Al (the major trace elements in the used CNT) detected in the two types of CNT epoxy boards do not indicate that the content of CNT should be very different. Since we do not know the content of CNT in EPOCYL and we do not have a control sample without CNT for this epoxy, we cannot make any conclusions regarding the cause of the histological changes following exposure to EPOCYL dust.It has been shown previously that the inflammatory response induced by low-toxicity low-solubility particles correlates well with the instilled surface area of the particles , 45–47. Saa1 mRNA expression level and some of the inflammatory lesions recorded for CNT were not observed following exposure to CB. Furthermore, our results also show that all three sanding dusts induced increased hepatic mRNA expression levels of the acute-phase gene Saa1 while no increased Saa1 mRNA expression levels were detected in the CB exposed mice. This suggests that the shape of nanomaterials with similar chemical composition is of importance for the toxicity. Shape has previously been shown to be a determinant of pulmonary response e.g., [We always include CB as an internal reference particle in our studies to be able to compare our results across studies. Moreover, the inclusion of CB in the study makes it also possible to compare the toxicity of spherical CB with fiber-shaped CNT. These two carbon based nanomaterials resulted in very different hepatic effects. Compared to CB, CNT induced much stronger hepatic se e.g.,  but we dData on the toxicity of epoxy dust are scarce and to the best of our knowledge, no in vivo studies on the toxicity of particles generated during mechanical processing of epoxy/CNT nanocomposites have been published. However, in addition to the present study on epoxy/CNT composites, two other studies testing the same CNT (Nanocyl NC7000) as part of other matrices have been published by Wohlleben and co-workers: 1) testing of sanding dust from CNT/cement and CNT/polyoxymethylene matrices by pulmonary deposition in rats  and 2) iThe level of pulmonary inflammation and DNA damage in mice pulmonary exposed to sanding dust from epoxy boards was not increased by the addition of CNT to the epoxy matrix. However, sanding dust from CNT-containing epoxy induced inflammatory and necrotic lesions in the liver that were not induced by EPOXY-REF but similar to the histological changes observed following pulmonary exposure to the same CNT.Female C57BL/6 mice 5–7 weeks old  were acclimatized for 1–3 weeks before the experiment. Mice were given food  and water ad libitum during the whole experiment. The mice were randomly divided into housing groups of 10in polypropylene cages (425 mm x 266 mm x 150 mm) with pinewood sawdust bedding and enrichment in form of sticks of aspen wood and rodent tunnels. The cages were stored at controlled temperature 21 ± 1 °C and humidity 50 ± 10 % with a 12-h light:12-h dark cycle.. Female mice were studied at 8 weeks of age. The average weight at the day of instillation was 18 ± 1.2 g. All animal procedures followed the guidelines for the care and handling of laboratory animals according to the EC Directive 86/609/EEC and the Danish law. The experiments were approved by the Danish “Animal Experiment Inspectorate” under the Danish Ministry of Justice (2012-15-2934-00223).The tested pristine nanomaterials comprised of a multiwalled CNT material , and carbon black, Printex 90 (CB), which was included as an internal reference particle. Printex 90 was a gift from Degüssa (Germany). In addition, we tested sanding dusts from three different types of epoxy boards with and without CNT: The Danish Technological Institute provided three different types of epoxy boards: 1) an epoxy product with 0.2 % content of CNT (referred as EPOXY-CNT), 2) corresponding product without CNT (referred to as EPOXY-REF) and 3) EPOCYL  with a Tiger S8 4 kV instrument and SpectraPlus Vs.3 software . The three epoxy materials were measured as solid disks .Disks of 4 cm in diameter were cut out of the original material and polished using a polishing machine LaboPol . Samples were imaged in a Helios EBS3 dual-beam SEM  at 2 kV, 86pA in in-lens mode.3 dust reservoir made of plastic, and a commercial electrostatic precipitator (ESP), previously characterised by Sharma et al. [Figure a et al. , for parThe size distribution of the generated particles was measured from the chamber using an ELPI+ , covering the range from 6 nm to 10 μm. The total particle concentration was measured using a CPC . Sampling for analysis with electron microscopy was done with TEM-grids mounted in a 25 mm filter holder with a flow-through of 1 lpm.Sanding was performed using a commercial hand-held orbital sander (Metabo Model FSR 200 Intec) with an internal fan for dust removal. Grit size 120 sanding paper was used as recommended by the paint and lacquer manufactures. For our purpose the sander outlet was modified to connect a 60 cm long and 32 mm ID flexible plastic tube after the sander to lead the dust to the dust reservoir chamber from which particle measurement and sampling was done.The ESP was attached at the side of the chamber sampling air through a 21 cm deep, 37 cm wide and 15 cm high tunnel made in aluminium . The particles (3.25 mg/ml) and dust suspensions (9.75 mg/ml) were sonicated using a Branson Sonifier S-450D  equipped with a disruptor horn (Model number: 101-147-037) as described previously . In brieNitrogen sorption isotherms were measured at liquid nitrogen temperature (77 K) using a Micromeritics ASAP2020 volumetric adsorption analyzer. Before the measurements, the samples were degassed under vacuum for 10 h at 80 °C. The BET (Brunauer- Emmett-Teller) equation  was usedThermogravimetric analyses were performed on Mettler TGA instrument by heating the samples from 25 to 900 °C at a heating rate of 5 °C min-1 on an alumina holder under the flow of air at 20 ml min-1.The SEM analyses were performed by using a Zeiss NVision 40 Cross-Beam Focused Ion Beam machine, equipped with a high resolution Gemini Field Emission Gun (FEG) scanning electron microscope column. The instrument was also equipped with an Oxford INCA 350 Energy Dispersive X-Ray Spectrometer (EDS) incorporating an X-act silicon drift detector with an energy resolution of 129 eV at the Mn kα line.A sample of the as-received carbon nanotubes was prepared by dispersing the powders directly on to a SEM stub covered with conductive carbon tape. SEM images were acquired at accelerating voltages of either 3 kV or 8 kV. EDS spectra were obtained from the samples at an accelerating voltage of 15 keV and a beam current 0.33 nA.Samples of the sanding dusts were prepared on carbon tape on which a TEM grid was also mounted. The conductive tape was intended to facilitate SEM investigations but the dusts still exhibited highly non-conductive behaviour, which required careful adjustment of the working conditions. Accelerating voltages of 1–10 keV were used to minimize charging effects.The average sizes of the materials in instillation vehicle were determined by Malvern Nano ZS Dynamic Light Scattering (DLS) equipment mounted with a 633 nm red laser. The optical and dielectrical parameters of water were used for the medium while an optical refraction index of 2.5 and an optical absorption of 0.3 was used for the sanding dusts. The optical refraction index for CNT was set to 2.02 and the absorption was set to 2. Samples were thermally equilibrated to 25 °C in the DLS equipment before analysis. Each data point is the average of six consecutive analyses for each dispersion to measure the hydrodynamic size-distribution and the evolution of the derived count rate to assess the stability of the dispersions. The final data set was calculated considering the measured viscosity of the dispersion mediums using a SV-10 Vibro Viscometer The dispersion state of the test materials in the instillation vehicle were characterized by scanning electron microscopy (SEM). Samples of CNTs in suspension (doses 18 μg and 162 μg) and dusts (doses 54 μg and 486 μg) used in the instillation experiments were prepared for SEM by depositing a small amount of the liquid on SEM-stubs covered with an Aluminum foil and allowing it to dry before examination. The SEM procedure was performed as described above.An amount of 3.24 mg/ml of each type of nanoparticle and 9.72 mg/ml of each type of dust was suspended in pyrogen free water with 0.05 % Tween 20 and suspended by sonication as described above. The endotoxin contents were analysed using the kinetic Limulus Amebocyte Lysate test  as described previously in .n = 5–7 per group). Because the CNT and sanding dust instilled mice were exposed in two overlapping experiments the number of control mice differs between the different time points: day 1 (12 mice), day 2 (22 mice) and on day 3 (24 mice). Before the intratracheal instillation, the mice were anesthetized using isoflurane. The instillation procedure has been described previously [The mice were treated with a single intratracheal instillation with 18, 54 and 162 μg for the nanoparticles and 54, 162 and 486 for the epoxy dusts  using the MagNA Pure Compact RNA Isolation kit (Roche) according to the manufacturer’s protocol. In brief, the RNA isolation procedure is based on the MagNA Pure Magnetic Glass Particle (MGP) Technology (Roche): nucleic acids are bound on the surfaces of MGPs whereas unbound molecules are removed by several washing steps. Genomic DNA molecules are degraded by incubation with DNase. Total RNA was stored at − 80 °C until analysis.Total RNA was isolated from lung tissue of 144 mice in total (cDNA synthesis was performed using the Enhanced Avian HS RT-PCR kit (Sigma-Aldrich), with total RNA as template, as described in the manufacturer’s protocol. A total of 500 ng was used for each cDNA synthesis. The heating cycle was 25 °C (15 min)/50 °C (50 min)/85 °C (5 min) and the obtained cDNA solutions were further diluted to a final concentration of 10 ng/μl.The expression of the target genes, compared to a reference (GAPDH), was determined with real time-PCR using a LightCycler® 480 Instrument (Roche) according to the manufacturer’s protocol. The relative expression was calculated using the Livak–Schmittgen method . The staSaa1) and 18S was measured using a modified TaqMan Fast 2x Universal PCR Master Mix protocol . The primer/probe mix for Saa1 was Mm00656927_g1  and Saa1 mRNA levels were normalised to 18S. The samples were run in triplicates on 384-well reaction plates . A negative (minus reverse transcriptase), a positive and a blank control were added on each plate. The plate was run in the ViiA 7 Real-time PCR system . The relative expression was calculated using the Livak–Schmittgen method [RNA was prepared from liver tissue using using the Maxwell 16 LEV simplyRNA tissue kit as as described by the manufacturer .cDNA synthesis was performed using the TaqMan® Reverse Transcription Reagents kit , with total RNA as template, as described in the manufacturer’s protocol. The expression of hepatic serum amyloid A 1  using NIS-Elements BR 2.30 programme .P < 0.05 level. The statistical analyses were performed in SAS version 9.2 .For each particle, the data were assessed by non-parametric two-way ANOVA with post-hoc Tukey-type multiple comparison test for effects showing statistical significance in the overall ANOVA test. A comparison of the three dusts was assessed by non-parametric three-way ANOVA. Statistical significances were tested at 2, nano titaniumdioxide; Saa1, serum amyloid a1; SEM, scanning electron microscopy; Vtot, total pore volume; WDXRF, wave-length dispersive x-ray fluorescenceBAL, bronchoalveolar lavage; BET, Brunauer-Emmett-Teller; BJH, Barrett-Jouner-Halenda; CB, carbon black (Printex 90); CNT, carbon nanotubes; CPC, condensation particle counter; DLS, dynamic light scattering; EDS, energy dispersive x-ray spectrometer; ELPI, ELECTRICAL LOW-PRESSURE IMPACTOR; EPOCYL, industrial epoxy composite with CNT; EPOXY-CNT, epoxy composite with CNT; EPOXY-REF, epoxy composite without CNT; ESP, ElectroStatic Precipitator; FEG, field emission gun; Hmox-1, heme oxygenase; IL-6, interleukin 6; LAL, limulus amebocyte lysate; NanoTiOAdditional file 1: Figure S1.SEM images of A) EPOXY-CNT and B) EPOCYL polished surfaces with CNT (torn-off ends) sticking out. The materials are very similar in appearance. (PPTX 713 kb)Additional file 2: Figure S2.Inorganic chemical composition given as elemental weight% measured by standardless WDXRF. The three epoxy materials were measured as solid disks . For comparison, the results for CNT powder, previously published in , were adAdditional file 3: Figure S3.Correlation between neutrophil influx and mass (A) or surface area (B) of the instilled particles and sanding dusts. (PPTX 122 kb)


To evaluate and characterize potential cellular and molecular changes of human osteoblasts, cells were exposed to sera of four groups of patients: (1) affected by obesity with normal BMD (O), (2) affected by obesity with low BMD (OO), (3) affected by obesity and sarcopenia (OS), and (4) affected by obesity, sarcopenia, and low BMD (OOS) as compared to subjects with normal body weight and normal BMD (CTL). Patients were previously investigated and characterized for body composition, biochemical and bone turnover markers. Then, sera of different groups of patients were used to incubate human osteoblasts and evaluate potential alterations in cell homeostasis. Exposure to OO, OS, and OOS sera significantly reduced alkaline phosphatase, osteopontin, and BMP4 expression compared to cells exposed to O and CTL, indicating a detrimental effect on osteoblast differentiation. Interestingly, sera of all groups of patients induced intracellular alteration in Wnt/β-catenin molecular pathway, as demonstrated by the significant alteration of specific target genes expression and by altered β-catenin cellular compartmentalization and GSK3β phosphorylation. In conclusion our results show for the first time that sera of obese subjects with low bone mineral density and sarcopenia significantly alter osteoblasts homeostasis in vitro, indicating potential detrimental effects of trunk fat on bone formation and skeletal homeostasis.Obesity and sarcopenia have been associated with mineral metabolism derangement and low bone mineral density (BMD). We investigated whether imbalance of serum factors in obese or obese sarcopenic patients could affect bone cell activity  Obesity and osteoporosis are important global health problems with high prevalence and impact on both mortality and morbidity , 2. Duriα, might play by interfering with bone cells homeostasis [Interestingly obesity has been considered a protection factor against the development of bone loss and osteoporosis, likely for increased androgen aromatization to estrogens in postmenopausal obese women , 4 and, eostasis –15. Moreeostasis , 17. Thueostasis –20, by mIn addition, obesity is associated with a chronic low-grade inflammation as depicted by increased plasma levels of C-reactive protein (CRP), proinflammatory cytokines, and osteopontin , 22 as w in vitro and, likely altering skeletal health.Since our group has recently demonstrated skeletal alterations in adult obese subjects and also an association of obesity with sarcopenia , aim of T score (CTL). Subjects' characteristics are reported in Female subjects were selected in the Department of Experimental Medicine, Section of Medical Pathophysiology, Endocrinology, and Nutrition, of Policlinico Umberto I, Sapienza University of Rome. Chronic medical conditions, use of medications affecting bone metabolism, hormonal and nutritional status, vitamin D supplementation, recent weight loss, and prior bariatric surgery interventions were assessed as exclusion criteria. Patients were investigated for metabolic, inflammatory, and bone turnover markers and their sera used for culturing osteoblasts. Subjects were characterized by Dual Energy X-ray Absorptiometry (DEXA) and divided into different groups upon bone mineral density (BMD) and muscle mass. They were divided into different groups: the first group included obese women  with normal bone mineral density and normal muscle mass (O); second group was formed by obese women with low bone mineral density for age (OO); another group included obese women affected by sarcopenia (OS); the last group included women with both low BMD and sarcopenia (OOS). A control group was also evaluated, which included healthy women with normal BMI and normal The research protocol was in accordance with the ethical requirements of the Helsinki Declaration and was approved by Ethical Committee of Policlinico Umberto I, Sapienza University of Rome. All subjects enrolled gave written informed consent.2.The anthropometric measurements were taken following the procedures described in the anthropometric standardization reference manual . Body we2).Body composition (fat mass (FM) and fat-free mass (FFM)) was estimated by bioelectrical impedance analysis (BIA). The BIA measurement was performed on the right side of the body using 800-A and 50 kHz alternating sinusoidal current and a standard tetrapolar technique . Fat-free mass index (FFMI) was calculated as FFM in kg/, considering the reduction of muscle mass, the impairment of muscle strength, and physical performance . FFM wascle mass . Abdomincle mass , 28.2 in a humidified incubator. All buffers, media, and reagents were purchased from Euroclone. For patients sera treatments, cells were grown up to 70% confluence, starved overnight in a medium serum-free, and then incubated 24 hrs with medium supplemented with 20% patient serum. For RNA extraction and quantitative real-time evaluation, cells were treated with serum of single patients whereas for protein extraction and Western blot analysis and for immunofluorescence experiments, growing media were supplemented with a pull of sera of the same group.Human SAOS-2 osteoblastic cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, 10% fetal bovine serum (FBS), at 37°C and 5% CO−ΔΔCt method [For RNA extraction, cells were washed twice with 1X phosphate buffer saline  and total RNA was immediately isolated with Ambion RNA mini KIT  according the manufacturer's instructions. The purity and integrity of total RNA were monitored by electrophoretic analysis on denaturating agarose gel; ultraviolet spectrophotometry (Biorad) was used for RNA yield evaluation. Total RNA was treated as previously described . Quantitt method  and repoβ-catenin, anti-GSK3β Ser9-phosphorilated, anti-totalGSK3β, and β-actin antibodies, diluted as indicated by manufacturers, were added overnight at 4°C. The membrane was washed three times with T-TBS and incubated for 60′ at room temperature with HRP-labeled anti-rabbit or anti-mouse in 5% nonfat dry milk. The membrane was then washed three times and antibodies were visualized using ECL Prime Western Blotting Detection Reagent . Quantitative analysis was performed using Imagequant TL Image analysis software ; sample value was normalized for housekeeping gene β-actin and was reported as arbitrary units. All antibodies were purchased by Cell Signaling.Total proteins were extracted as previously described . Twenty β-catenin  1 : 200 in PBS overnight at 4°C. After three washes, cells were incubated with 1 : 500 anti-mouse Alexa Fluor (Molecular Probes) in the dark 45′ at room temperature. Cells were rinsed three times with PBS and slides were mounted with 70% glycerol in PBS. Images were acquired by Nikon Eclipse 600 microscope and Nikon CCD camera at 20x magnification.For immunofluorescence (IF) analysis, cells were rinsed twice with cold PBS, fixed with 4% paraformaldehyde 15′ at room temperature, rinsed three times with PBS, permeabilized by incubation in 0.2% Triton in 3% bovine serum albumin (BSA), and rinsed accurately in PBS. Cells were incubated in blocking solution (6 ng/mL IgG from goat serum in PBS) for 30′ at room temperature and further incubated with anti-t-test. A P < 0.05 was conventionally considered statistically significant.Data are expressed as means ± SE. Statistical significance between data points was determined using the two-tailed Student's Patients were characterized for anthropometric measurements, BMD, muscle mass as described in  in vitro.No differences were found in hormones levels , beside To determine potential effects of the sera from obese patients on osteoblast homeostasis, cells were incubated with the sera of obese subjects to assess effect on cell differentiation and activity.P < 0.05, P < 0.05, P < 0.05, P < 0.05, P < 0.05).Osteoblasts were cultured 24 hrs in presence of the sera obtained from individuals of the different experimental groups after which total RNA was extracted and quantitative RT-PCR performed to evaluate specific genes. Expression of Runx2, an essential transcription factor expressed during bone formation and osteoblast differentiation, was decreased in all experimental group, except that in the OS  of action of the observed alteration of osteoblast homeostasis. The canonical Wnt/eoblasts . When theoblasts , 38.β-catenin pathway was involved in the osteoblastic gene expression modulation exerted by obese sera, expressions of some specific target genes were evaluated by quantitative RT-PCR and Western blot analysis. Osteoblasts were grown and exposed to the human sera for 24 hrs as described above. Expression of cMyc and axin2 decreased in osteoblasts incubated in the presence of sera of O patients compared to CTL (P < 0.05) as shown in Thus, to evaluate whether canonical Wnt/In particular, Wnt pathway inhibition was most significantly induced by exposure of cells to the sera of OS patients  as compared to the other groups, strongly indicating complex mechanism(s) underlying skeleton homeostasis in obese and sarcopenic subjects.The expression of LEF1 protein was not significantly affected in cells exposed to the patients sera, whereas LEF1-ΔN was strongly inhibited in cells exposed to all obese groups Figures .β-catenin pathway, GSK3β, a component of the β-catenin destruction complex and negative regulator of the Wnt pathway, was analyzed. In particular we checked both, by Western blotting analysis, phosphorylation at Ser-9 which inactivates GSK3β [β-catenin nuclear translocation. A strong decrease of a ratio GSK3β-Ser9/total GSK3β was observed in osteoblasts upon exposure to the sera of all obese subjects  GSK3β, and normal regulation within cells disrupted by obese subjects sera. Accordingly total β-catenin amount is decreased showing that protein is tagged for proteasomal degradation. Since nuclear localization of β-catenin is used as a marker of enhanced Wnt signaling, as expected, immunofluorescence analysis demonstrated a cytoplasmic localization of β-catenin in osteoblasts exposed to the sera of all obese subjects /lymphoid enhancer-binding factor (Lef) to regulate expression of target genes [β-catenin pathway is one of the molecular intracellular pathways involved in the gene expression modulation disrupted in the osteoblasts exposed to obese sera. Indeed, GSK3β, a key modulator of β-catenin activity, is repressed by phosphorylation at ser-9 and consequently β-catenin is slightly detectable and only in the cytoplasm. As a consequence, the β-catenin target genes are downmodulated and osteoblast differentiation and activity disrupted.Wnt signaling pathway has emerged as a critical regulatory component of the control of bone formation and bone resorption . When thet genes , 38. Our in vitro [β-catenin binding domain, retains the ability to weakly interact with β-catenin via second domain and to promote osteogenesis [In regards of the Wnt molecular pathway, an interesting result is linked to the evaluation and characterization of Lef1 modulation. Lef1 contains two promoters that drive expression of a full-length protein (Lef1) and an N-terminally truncated isoform (Lef1-ΔN). Lef1 overexpression inhibits osteoblast maturation and Runx2 activity on the osteocalcin promoterin vitro , 48, butin vitro . Lef1-ΔNogenesis . Interesβ-catenin, triggering the molecular mechanism(s) leading to an increase of osteoblast differentiation and activity. Obesity is associated with decreased GH secretion and IGF-1 deficiency is associated with increased abdominal adipose tissue accumulation, decreased BMD [Moreover, Wnt interacts with other signaling pathways in the bone cellular environment. One of these signals, IGF-1, enhances stabilization of ased BMD , 42, andased BMD . in vitro model system. However, it is well known that molecular or cellular mechanism(s) described in cellular model system is usually confirmed in primary cell lines, which we are evaluating as well.We understand that a limitation of our work is due to the fact that we have chosen a clonal osteoblastic cell line as our in vitro results further point out for a central role of IGF-1 and osteoblasts disruption in the mechanisms linking obesity to decreased bone quality.Thus, ourβ-catenin signaling in the osteoblasts leading to a disruption of normal differentiation pathway. Further investigations are needed to clarify and characterize the complex link between adipose-bone and muscle tissues and identify other potentially involved molecules, beside IGF-1, linked to the observed altered Wnt/β-catenin pathway.Indeed, these results lead to the suggestive hypothesis that lower levels of IGF-1 in subjects affected by an increased abdominal adipose tissue accumulation could contribute to the blunted levels of