Murraya exotica (L.) on OA. The rat OA models were duplicated to prepare for separating OA chondrocytes, synovial fluid (SF), and serum containing M. exotica , M. exotica showed the activity of decreasing the contents of TNF-α and IL-1β in SF and the chondrocyte apoptosis in a dose-dependent manner. To investigate the probable mechanism, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to determine gene expression and protein profiles, respectively. The results reveal that M. exotica can downregulate mRNA and protein expressions of β-catenin and COX-2 and reporter activity significantly. Conclusively, M. exotica exhibits antiapoptotic chondroprotective activity probably through inhibiting β-catenin signaling.Osteoarthritis (OA) is a degenerative joint disease that affects millions of people. Currently, there is no effective drug treatment for it. The purpose of this study is to investigate the chondroprotective effects of  It had been elucidated that the signaling pathways directing joint formation and homeostasis were the key molecular players in OA . Wnt proβ-catenin signaling in the pathogenesis of OA [β-catenin in OA had not been reported, because tissue-specific activation of the β-catenin gene (target by Col2a1-Cre) was embryonic lethal. In Col2a1-CreERT2  β-cateninfx(Ex3)/wt mice, overexpression of β-catenin protein was detected by immunostaining in the 3th month, reduction of Safranin O and Alcian blue staining in the 5th month, and cell cloning, surface fibrillation, vertical clefting, and osteophyte formation were observed in the 8th month. In addition, expression of chondrocyte marker genes, such as aggrecan, MMP-9, MMP-13, Alp, Oc, colX, and Bmp2, was significantly increased [ColX-expressing chondrocytes were detected in Col2a1-Smurf2 transgenic mice which might represent a mechanism of Smurf2-induced OA that Smurf2 mainly induced ubiquitination of GSK-3β and its proteasomal degradation, and hence upregulation of β-catenin [Cumulating studies mainly based on experimental animal models for OA have suggested an important procatabolic role for Wnt/is of OA , 4. DireMurraya exotica (L.) (Rutaceae) is widely grown in the southern China, and it has been well documented in Pharmacopoeia of the People's Republic of China, 2010 Edition (Ch.P 2010) for treating stomachache, rheumatic arthralgia, toothache, body swelling, and pain [M. exotica show antinociceptive and anti-inflammatory activities in rat knee osteoarthritis models. It can downregulate the expressions of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the rat serum significantly [M. exotica, which might influence the apoptosis and the β-catenin signaling in OA chondrocytes.and pain . In our ficantly . In thisM. exotica leaves employed in this study were collected at Zhangzhou  in 2011. The plants were identified by Jialin Li. The voucher specimen (ID: GMU-M20081008) was deposited in the herbarium of College of Pharmacy, Gannan Medical University, Jiangxi Province, China. The leaves were harvested, air-dried, and then grounded into fine powder (150–200 mesh) with a laboratory scale mill.The Approximately 1 kg of the above-mentioned fine powder was extracted with 10 L of 70% ethanol for 48 h by maceration at room temperature. The extract was evaporated in vacuum to generate a crude ethanol extract  [The study was approved by the Institutional Animal Care and Use Committee of Gannan Medical University. Each rat was intragastrically administered with the 70% ethanol extracts at different doses. The control group animals received the same experimental handling as those of the treating groups except that the drug treatment was replaced by appropriate volumes of the dosing vehicle. Indomethacin (10 mg/kg) was used as positive reference.M. exotica was as follows. Rats were intragastrically administered with the 70% ethanol extracts at 50 mg/kg, 100 mg/kg, and 200 mg/kg doses for one week, respectively. Rats were sacrificed, 5 mL of blood was taken from the heart, and serum was separated by centrifuge and ready for cell culture.Preparation of rat serum containing M. exotica, cells were starved for 24 hours with serum-free medium. The separated rat serum containing M. exotica was added into cells and incubated for another 24 hours.Before administration of β and TNF-α. Other segments of the cartilage were taken for chondrocytes separation and culture.Rat OA model was established by using Hulth's (1999) method . The proM. exotica, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C with 5% CO2. The chondrocytes were identified by toluene blue stain and type II collagen immunohistochemistry reaction. Cells from the first passage were used.Eight-week-old OA model group rats were sacrificed. Immediately, cartilage was harvested from the knee joint under sterile conditions as digested with 0.25% pancreatic enzymes for 30 min to remove other tissues and cells and then digested with 0.2% collagenase II at 37°C for 4 h. Cells were grown to confluence in DMEM (low glucose) supplemented with 10% fetal bovine serum (FBS) or rat serum containing M. exotica, cells (1 × 106/mL) were collected by centrifugation and incubated in buffer containing FITC annexin V and PI. Apoptotic cells were measured by a flow cytometer .The changes of cell apoptosis were quantified by loading FITC annexin V/PI double—fluorescence labeling and using flow cytometry. Flow cytometry was performed according to the apoptosis detection kit  procedures. After being treated by 5/mL). After incubation for 24 h in media containing different doses of M. exotica, MTT (5 mg/mL) was added (20 μL/well). Cells were then incubated with MTT for 4 h, and DMSO (150 mL/well) was added after removing the culture medium. Absorbance was measured at 570 nm. This step was repeated for four times to get average results.OA chondrocytes were cultured in a 96-well plate  to synthesize the first-strand of cDNA following standard protocols. To detect the expression level of COX-2, β-catenin, and caspase-3 genes, EzOmics SYBR qPCR kits were purchased from Biomics in a Mastercycler (Eppendorf). Their respective primer sequences . For each sample, 2 2+ concentrations had been optimized to render efficiency for each target near one per assumption underlying the 2−ΔΔCT method [All of the PCR reactions were performed in sets of four. GAPDH was used as an internal control. Primer and template designs followed the same criteria for each target, and primers and MgT method .4 cells in 200 μL/well) and transfected with Wnt/β-catenin reporter plasmid   in the presence of Lipofectamine 2000. In all experiments, cells were cotransfected with Renilla luciferase plasmid  to control for transfection efficiency. Cultures were transfected for 4 h, prior to addition of 200 μL FBS containing media, and incubated overnight. On the next day, cells were administrated by M. exotica at different doses for 24 h. Cultures were then lysed with 1 × Passive Lysis Buffer . The luciferase activities of both Topflash and pRL-TK-luc reporters were measured using a dual luciferase assay kit  in an L-max II microplate reader .Chondrocytes were resuspended in serum-free culture medium and plated on 48-well dishes (3.4 × 10μg) were electrophoresed on 10% SDS-PAGE gel for anticleaved caspase 3, β-catenin, and COX-2. Proteins were western-blotted onto polyvinylidene difluoride (PVDF) transfer membranes, and blots were blocked with Tris-buffered saline (TBS) containing 5% nonfat milk for 1 h and incubated with anti-cleaved caspase 3, β-catenin, and COX-2 at 4°C overnight. The blots were then rinsed and incubated with HRP-conjugated IgG goat anti-rat for 1 h. The blots were then washed and developed by use of a Super Enhanced chemiluminescence detection kit , and the protein bands were visualized after the exposure of the membranes to Kodak film (USA). GAPDH was used as the internal control in all Western blot analyses.Cells were lysed in lysis buffer  and then subjected to immunoblot. Before sampling, the protein concentrations were measured using a BCA Protein Assay Kit  with bovine serum albumin as a standard. After being combined with gel loading buffer  and boiled for 5 min, samples (80 t-test. Differences were considered significant at P < 0.05.All data were expressed as mean ± standard deviation (SD). Statistical analysis of gene expression data was analyzed by a paired β and TNF-α at the time of harvest  is a nucleic acid dye that cannot normally pass through the intact cell membrane, but in the middle and late stages of apoptosis, it can stain the nucleus due to breaks in the cell membrane. Flow cytometry using the FITC annexin V/PI double-staining method was used to generate an apoptotic cell scatter plot of different doses of M. exotica groups  activity, indicating that M. exotica elicited a significant decrease in β-catenin regulated-reporter activity in chondrocyte  for 72 h, Western blot analysis was used to measure the expression of the β-catenin signaling-associated proteins β-catenin and COX-2 and the apoptotic effecter protein caspase 3. The protein expressions of β-catenin and COX-2 and the apoptotic effecter caspase 3 in condrocytes were dose-dependently downregulated by exposure to M. exotica, compared to the model group  or related to a known medical condition or event. There is now strong evidence that the structural changes globally observed in OA are due to a combination of factors, ranging from mechanical to biochemical . It is iα and IL-1 are proinflammatory cytokines, which are associated with cartilage degeneration, synovial inflammation, and bone changes. IL-1β is known as playing a pivotal role to trigger apoptosis, which leads to further cartilage degradation. Chondrocytes stimulated with IL-1β  in vitro have been used to mimic the microenvironment that occurs in OA [β stimulus enhances the expression of paracrine pro-inflammation, including TNFα and IL-1β genes expression, which provides evidence for a positive feedback loop [M. exotica has been reported to exhibit chondroprotective activity by decreasing the contents of TNFα and IL-1β in rat serum [2 can upregulate NF-κB through EP4/G protein/MAPK signaling to promote pro-inflammatory factors expressions; on the other hand, PGE2 is the product of COX-2, a target gene of Wnt/β-catenin pathway, which is the classical target of NSAIDs available for OA treatment. To study on M. exotica modulating Wnt/β-catenin pathway in OA chondrocytes, indomethacin was identified as the positive control. In this study, pro-inflammatory cytokines in OA SF were further investigated, and M. exotica was demonstrated to exhibit downregulation of TNFα and IL-1β expression, although not statistical significant difference. In vitro, the separated OA chondrocytes were cultured with rat serum containing different doses of M. exotica, and then the chondrocytes apoptosis with FITC annexin V/PI double staining were evaluated by flow cytometry. It demonstrated that M. exotica could significantly protect chondrocytes from initiating apoptotic processes in a dose-dependent manner. To support this result, MTT assay was employed. The viability of chondrocytes, incubated with different concentrations of M. exotica, was showed in dose-dependent manner, which was consistent with that by flow cytometry.TNFrs in OA . Measurirs in OA . IL-1β sack loop . M. exotat serum . On one β-catenin is a key molecule in the canonical Wnt signaling pathway and plays a critical role in multiple steps during chondrocyte formation and maturation. Several drugs and synthetic or natural compounds have been reported to inhibit and/or modulate β-catenin signaling [β-catenin stability [β-catenin-TCF interaction [β-catenin-CREB binding protein interaction [β-catenin and T-cell factor (ICAT) is an 82-amino-acid small molecule [β-catenin. This binding disrupts the complex formation of β-catenin with TCF/LEF [In vitro transfection assays demonstrated that sFRP-3 could inhibit β-catenin nuclear translocation and TCF/LEF-dependent transcriptional activation [COX-2 gene expression was transcriptionally modulated by the β-catenin-TCF/LEF pathway, and β-catenin was bound to AU-rich elements (ARE) in the 3′-UTR of COX-2 mRNA and stabilized the mRNA [β-catenin with TCF/LEF-1 [M. exotica are flavones and coumarins, and most of flavones are quercetin analogues. We found that M. exotica significantly downregulated mRNA and protein expressions of β-catenin and COX-2 and reporter activity. However, the detail mechanism is yet to be investigated.It is shown that both constitutive up- or downregulation of the canonical Wnt pathway negatively influence cartilage development and maintenance resulting in OA-like features . This suignaling . Howevertability , blockineraction  or β-cateraction , stabilieraction , preventeraction , or otheeraction . For insmolecule  whose cr TCF/LEF , 27 and tivation . Rodriguthe mRNA . QuercetCF/LEF-1 . The mai2), which is then further processed to PGE2, PGI2, PGD2, or thromboxane A2 by specific synthases. In general, increased COX-2 levels are associated with augmented PGE2 production. Goessling reported that PGE2 modified the wnt signaling cascades at the level of β-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events [M. exotica exhibits significant chondroprotective activity by decreasing the expressions of iNOS, IL-1β, and TNF-α  in vivo, and antinociceptive activity in animal models of acetic acid induced writhing response, hot-plate latent pain response test, carrageenan-induced hind paw edema, and xylene-induced ear edema [2 positive feedback to β-catenin signaling by M. exotica in chondrocytes.COXs catalyze the conversion of arachidonic acid to prostaglandin H2 (PGHn events . Previouar edema . HoweverM. exotica decreased the contents of TNFα and IL-1β in rat OA SF and the chondrocytes apoptosis in vitro, probably due to inhibiting β-catenin signaling.Conclusively,


Although the role of glycogen synthase kinase 3β (GSK3β) in osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) is well-characterized as a negative regulator of β-catenin, its effect on osteogenesis of adipose-derived stromal cells (ADSCs) is poorly understood. Here, we show that GSK3β positively regulates osteogenic differentiation of murine ADSCs. Gain-of-function studies showed that GSK3β promotes in vitro osteogenesis of ADSCs. Regulation of GSK3β activity in ADSCs, either by small interfering RNA (siRNA)-mediated GSK3β silencing or by pharmacological inhibitors, blunted osteogenesis and the expression of osteogenic markers. Importantly, we demonstrated that transgenic mice, engineered to overexpress the constitutively active GSK3β (GSK3β-S9A) mutant, exhibited a marked increase in osteogenesis, whereas expression of the catalytically inactive GSK3β (GSK3β-K85A) in mice inhibits osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by GSK3β was mediated by downregulation of β-catenin. Remarkably, β-catenin silencing enhances osteogenesis and osteoblast marker gene expression such as alkaline phosphatase (ALP) and osterix. Taken together, these findings demonstrate a novel role for GSK3β in the regulation of osteogenic differentiation in ADSCs. Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells Osteogenesis is defined by a series of events, which starts with a commitment to an osteogenic lineage by mesenchymal cells. Subsequently, these cells proliferate and demonstrate an upregulation of osteoblast-specific genes and mineralization In this study, we found that GSK3β in ADSCs has a positive effect on osteogenic differentiation. We uncovered that GSK3β-mediated β-catenin protein levels determine the osteogenic capacity of ADSCs.All animal experiments were done with the approval of the ethical committees at the Ewha Womans University.g for 10 minutes and the cell pellet, suspended with phosphate-buffered saline (PBS), was filtered through a 100 μm cell strainer  to remove tissue debris. Collagenase was removed by dilution with PBS and centrifuged twice at 300×g for 10 minutes. The cell pellet was suspended in 0.83% NH4Cl and incubated to remove contaminating red blood cells. The residual cells were washed and centrifuged twice with PBS under same conditions, and cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 μg/ml streptomycin (HyClone) in tissue culture dishes at 37°C in a 5% CO2 humidified incubator. After 4 hours, non-adherent cells were removed by two to three washes with PBS, and adherent cells further cultured in complete medium until the cells reached 80 to 90% confluence. ADSCs from passage 3 to passage 5 were used in all experiments described. HEK293T cells were maintained in Dulbecco's modified Eagle's medium  with 10% FBS and antibiotics. All other chemicals including SB216763 and LiCl were purchased from Sigma .ADSCs were isolated from 6 to 8-week-old C57BL/6 mice (The Jackson Laboratory) as described previously −7 M dexamethasone (Sigma) and 25 ng/ml human recombinant bone morphogenetic protein 2 . The culture medium was changed three times per week for 1 to 2 weeks, respectively. Alkaline phosphatase (ALP) activity was detected by BCIP/NBT color development substrate  according to the manufacturer's instructions.The potential of the isolated cells to differentiate into osteogenic lineages was examined. For osteogenesis, cells were allowed to grow to 70% to 90% confluence and were then cultured in osteogenic medium containing 10 mM β-glycerol phosphate (Sigma), 50 μg/ml ascorbate-2-phosphate (Sigma), 10The degree of extracellular matrix calcification was estimated using ARS staining and VK staining. Briefly, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and stained with 2% ARS, pH 4  for 5 minutes at room temperature. For VK staining, cells were fixed with methanol for 10 minutes at room temperature and overlaid with 1% silver nitrate solution (Sigma) under UV light for 1 hour.ACACGAAAGTGATTGGAAA; si-β-catenin, GTTGCTTTGCTCAACAAAA; scrambled nontargeting siRNA, ACGTGACACGTTCGGAGAA, as a negative control. ADSCs were transfected with the gene-specific siRNA at a concentration of 10 nM using LipofectamineTM RNAiMAX (Invitrogen) according to the manufacturer's protocol.Double-stranded, siRNAs (21-mer) targeting mouse GSK3β and β-catenin were synthesized from Genolution Pharmaceuticals Inc. . The corresponding target mRNA sequences for the siRNAs were as follows: si-GSK3β, HA-tagged cDNAs of wild-type GSK3β (GSK3β-WT) and two mutants, catalytically inactive GSK3β (GSK3β-K85A) and constitutively active GSK3β (GSK3β-S9A) cloned into the retroviral vector, pMX-puro were previously described 9-GSK3β, hemagglutinin (HA) , β-catenin (BD Biosciences), tubulin (Santa Cruz Biotechnology), TATA-binding protein , and glyceraldehyde 3 phosphate dehydrogenase  in Tris-buffered saline containing 0.05% tween 20 (TBST) with 1% bovine serum albumin (BSA). Following wash with TBST, the membrane was incubated with either horseradish peroxidase-conjugated anti-rabbit antibody or anti-mouse antibody (Thermo Scientific) in TBST. Proteins were detected using an ECL detection kit .Cells were lysed in cell lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml of leupeptin, aprotinin, and pepstatin A, 1 mM sodium orthovanadate and 1 mM sodium fluoride (NaF) for 20 minutes on ice. The lysates were centrifuged at 14,000×g for 20 minutes at 4°C. The supernatants were boiled in SDS sample buffer containing 0.5 M β-mercaptoethanol. Cytosol and nuclear protein fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce) according to the manufacturer's instructions. Protein concentration was determined by the Bradford assay (Bio-Rad). Equal amounts of proteins were separated by 10% SDS-PAGE and electrotransferred to a PVDF membrane . The membrane was blocked in PBS containing 5% nonfat dry milk for 1 hour and then immunoblotted overnight at 4°C on a shaker with antibodies against GSK3α/β (Invitrogen), phospho-SerT method (ΔΔCT) and values were normalized to actin level as an internal control gene. Melting curve analysis was included to assure that only one PCR product was formed.Total RNA was isolated from ADSCs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. After denaturation of total RNA at 70°C for 10 minutes, first-strand cDNA were synthesized with oligo (dT) primers and MMLV-reverse transcriptase . PCR amplifications were performed using the specific primers. Primer sequences are shown in HEK293T cells were cotransfected with the experimental TCF/LEF reporter constructs (TOPFLASH or FOPFLASH) and other expression plasmids, as specified in the figure legands. Luciferase activity was measured by the luciferase assay system (Promega) and normalized to the activity of the control (pRenilla). The data were obtained from three independent transfections and presented as the -fold induction in luciferase activity (mean ± S.D.) relative to the control.5′-ATGGTAATCGTGCGAGAGGG; AG-R, 5′-CACTGTTGTCACCTTGCTGC; GP-F, 5′-GAGACCGTGGACAGACCAATA; GP-R, 5′-AGCCAGAAGTCAGATGCTCAAG) to detect the transgene.A cDNA encoding HA-tagged human GSK3β mutants was cloned into the expression vector pCAGGS, which carries the cytomegalovirus (CMV) enhancer and chicken β-actin promoter (CAG) and the polyadenylate (polyA) DNA fragments. For generating transgenic mice, we used the standard pronuclear injection method with C57BL/6 mice (The Jackson Laboratory). Genomic DNA isolated from the tail was analyzed by polymerase chain reaction (PCR) using the specific primers  and Sca-1, as well as CD105 (endoglin) but not the hematopoietic markers CD45 and CD11b, and the endothelial markers CD31 . FurtherTo establish the role of GSK3ß in osteogenic differentiation of ADSCs, we analyzed the effect of GSK3β on osteogenesis using a small interfering RNA (siRNA)-mediated knocked down experiment. Silencing of GSK3β expression decreased GSK3β mRNA by 60–70% and GSK3β protein level by 70–80%, whereas a control siRNA had no effect . In contTo further confirm the role of GSK3β in the differentiation of ADSCs into osteoblasts, we investigated the effects of pharmacological inhibition of GSK3 on osteoblast formation under osteoinductive media. Consistently, incubation of GSK3 inhibitors SB216763 or lithium chloride (LiCl), with ADSCs blocked osteogenic capacity as indicated by both ARS and VK staining . As showTo clarify the role of GSK3β in vivo, we generated transgenic (Tg) mice expressing GSK3β-S9A or GSK3β-K85A under control of the ß-actin gene promoter. Human GSK3β-S9A or GSK3β-K85A cDNA tagged hemagglutinin (HA) was fused with a 1.3-kb sequence located upstream of the ß-actin gene . GSK3β TInhibition of GSK3β decreases phosphorylation of β-catenin, preventing its degradation by the proteasome To investigate the role of β-catenin in osteogenic differentiation of ADSCs, we determined whether siRNA-mediated silencing of β-catenin regulates expression of osteoblast marker genes in ADSCs. Silencing of β-catenin decreased β-catenin mRNA by 60% and β-catenin protein level by 90%, whereas a control siRNA had no effect . In contThe characterization of regulatory mechanisms that direct osteogenic differentiation of ADSCs is of prime interest for developing therapeutic strategies to enhance bone formation and regeneration The effect of Wnt pathway on osteogenic differentiation remains more controversial. Early studies have yielded a wealth of information describing that activation of the Wnt/β-catenin signaling pathway promotes osteogenic differentiation In light of a series of experiments carried out through this study, our results suggest that the regulation of GSK3β-mediated β-catenin level is critical for osteogenic differentiation of ADSCs. It is possible that high levels of β-catenin negatively regulate the differentiation of ADSCs into an osteoblast precursor. Therefore, an early phase of differentiation process of ADSCs may maintain lower levels of β-catenin. However, in order that the precursors further differentiate into osteoblasts, they may elevate β-catenin levels. In agreement with this hypothesis, we observed differential expression patterns of β-catenin over a period of time during osteogenesis . At the In conclusion, here we provide evidence supporting the finding that GSK3β positively regulates osteogenic differentiation of ADSCs. The identified effects of GSK3β were due to regulation of β-catenin levels. Enhanced GSK3β expression had a significant positive effect on osteogenic capacity through downregulation of β-catenin. It might be clear that maintenance of an appropriate physiological level of GSK3β activity is crucial since either too little or too much GSK3β activity can modulate cell fate changes. Our data now suggest another level of regulation in osteogenic differentiation of ADSCs and may help to explain the complexity of cellular responses to GSK3β/β-catenin signaling.Figure S1Immunophenotypic characterization and differentiation assays of mouse adipose-derived stromal cells (ADSCs). (A) ADSCs were stained with antibodies against surface markers or control antibodies and subjected to flow cytometry analysis. Cells were homogenously positive for mesenchymal markers CD44 and CD105; progenitor cells markers Sca-1 but negative for hematopoietic markers CD11b and CD45 and endothelial markers CD31. The respective isotype control is shown as a thick black-line histogram.  ADSCs were cultured with or without differentiation medium for 1 to 2 weeks to induce cell differentiation. Osteogenesis was detected by Von Kossa (VK) staining and adipogenesis was detected by Oil red O (ORO) staining . Expression of osteoblast and adipocyte marker encoding genes was analysed by RT-PCR. The osteoblast markers, alkaline phosphatase (ALP), osteopontin, and osterix were expressed in differentiated cultures, whereas control undifferentiated cultures were negative for these genes (B). The adipocytic markers, adipsin and PPARγ2 were expressed in differentiated cells (C). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) is shown as a loading control.(PDF)Click here for additional data file.Figure S2Generation of transgenic mice expressing GSK3β mutants. (A) Schematic representation of the transgenic construct. The HA-tagged human GSK3β  cDNA was cloned into the expression vector pCAGGS, which carries the cytomegalovirus (CMV) enhancer, the chicken β-actin promoter (CAG) and the polyadenylate (polyA) DNA fragments. (B) Schematic diagram of the GSK3β constructs shown in (A) and arrows indicate a set of primers used for confirming integration of the transgene. Genomic DNA isolated from the tail of transgenic mice (TG) and wild-type littermates (WT) was analyzed by PCR using specific primers for the transgene.(PDF)Click here for additional data file.Figure S3Three-dimensional microstructural analysis of the femurs of wild- type (WT) and Tg mice (Tg) by microcomputed tomography (μCT). (A) S9A #17 Tg and WT littermates. (B) K85A #8 Tg and WT littermates. (C) K85A #34 Tg and WT littermates. Histograms represent the three-dimensional trabecular structural parameters in femurs: bone volume fraction (BV/TV), trabecular number (Tb. N), and bone mineral densities (BMD). Data represent means ± SD. n = 6. n.s., not significant.(PDF)Click here for additional data file.Figure S4Effect of GSK3β on the proliferation of ADSCs in transgenic mice expressing GSK3β mutants. ADSCs were isolated from either GSK3β-K85A (#8 and #34 line) or GSK3β-S9A (#17 line) Tg mice and WT littermates. ADSCs were maintained in medium supplemented with 10% FCS. Cell proliferation was determined by the MTT assay during 7 days of culture.(PDF)Click here for additional data file.Figure S5GSK3 inhibitors induce β-catenin accumulation in ADSCs. ADSCs were treated with 5 μΜ SB216763 (SB21) or 20 mM LiCl in osteogenic differentiation medium for the indicated periods of time. Whole cell extracts were immunoblotted with total β-catenin, phospho-Ser9-GSK3β antibody and GSK3β antibody. Membranes were reblotted with GAPDH antibody to ensure equal protein loading. These results are representative of at least three independent experiments.(PDF)Click here for additional data file.Figure S6Effects of GSK3β on osteoblast differentiation in BMSCs. (A) BMSCs were infected with either the retrovirus expressing GFP, catalytically inactive GSK3β (GSK3β-K85A) or constitutively active GSK3β (GSK3β-S9A). After 48 hours, cells were harvested for immunoblot analysis for GSK3β expression using antibodies specific for GSK3β or HA. GAPDH served as a loading control. (B) Retroviral infected cells were cultured in osteogenic differentiation medium for 2 weeks. Alkaline phosphatase activity and matrix mineralization were visualized by ALP (ALP) and Alizarin red S (ARS) staining. (C) Total RNA was isolated and analyzed for ALP, osterix, and osteopontin expression by real time PCR. Data represent mean ± S.D. *p<0.05, **p<0.001. Significant differences from the GFP infected group are seen. (D) BMSCs were transiently transfected with 10 nM GSK3β siRNA or a control siRNA (scramble). After 48 hours, GSK3β silencing was examined by immunoblot analysis. GAPDH is used as a loading control. Silencing GSK3β enhanced ALP activity and matrix mineralization as determined by ARS staining. Further, GSK3β silencing increased ALP, osterix and osteopontin mRNA expression as determined by real time PCR analysis. Data represent mean ± S.D. *p<0.001 and show significant difference from scramble siRNA transfected group.(PDF)Click here for additional data file.Figure S7Immunophenotypic characterization and differentiation assays of BMSCs. (A) BMSCs were stained with antibodies against surface markers or control antibodies and subjected to flow cytometry analysis. Cells were homogeneously positive for mesenchymal markers CD44 and CD105 as well as for progenitor cells markers Sca-1 but negative for hematopoietic markers CD11b and CD45 and endothelial markers CD31. The respective isotype control is shown as a thick black-line in the histogram.  BMSCs were cultured with or without differentiation medium for 2 weeks to induce cell differentiation. Osteoblastogenesis was detected by Alizarin red S (ARS) staining, and adipogenesis was detected by Oil red O (ORO) staining. Original magnification, X 10.(PDF)Click here for additional data file.Figure S8Change of β-catenin levels during osteoblast differentiation. ADSCs were cultured in osteogenic differentiation medium for the indicated periods of time. Whole cell extracts were immunoblotted with total β-catenin. Membranes were reblotted with GAPDH antibody to ensure equal protein loading. These results are representative of at least three independent experiments.(PDF)Click here for additional data file.Methods S1More information on the Flow cytometric analysis, BMSC isolation, Adipogenic differentiation and oil red O (ORO) staining, and MicroCT analysis.(DOC)Click here for additional data file.


BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of β-catenin immunohistochemical expression in pretreatment  BRAF, with V600EBRAF and V600KBRAF representing approximately 70–90% and 10–30% of mutations, respectively V600E/K-mutant metastatic melanoma, with objective response rates of approximately 50%, and in phase III trials, a significant improvement in progression-free survival (PFS) and overall survival (OS) compared with dacarbazine chemotherapy BRAF mutations in these trials exhibit some degree of tumor reduction, even if they do not meet the criteria for an objective clinical response.The incidence and mortality associated with melanoma has risen steadily since the 1970s in the USA, Europe and Australia BRAF mutations and how can these determinants be utilized to predict clinical responses and tailor therapies? Such determinants may be utilized to develop molecular assays that facilitate the identification or selection of optimized drug combinations for patients.Despite the promise of these targeted BRAFi, most patients develop recurrence and relapse at a median of 6–7 months. Studies utilizing patient tumor samples and preclinical models have identified several pathways to the development of BRAFi resistance, and the majority of resistance mechanisms identified to date appear to result in reactivation of the MAP kinase (MAPK) pathway as demonstrated by high levels of phosphorylated ERK1/2 AXIN2, which encodes a core pathway protein that promotes the degradation of β-catenin V600EBRAF-mutant cell lines, the activation of Wnt/β-catenin signaling in combination with BRAFi synergistically enhanced apoptosis in vitro and increased inhibition of tumor growth in vivoThe Wnt/β-catenin signaling pathway has been implicated as an important regulator of melanoma despite the fact that activating mutations in core pathway members appear to be rare in this disease. This signaling pathway is activated by secreted ligands including WNT3A, which is the WNT isoform most often used for activating Wnt/β-catenin signaling in laboratory studies. Frequently, the activation of Wnt/β-catenin signaling has been detected through the measurement of endogenous downstream target genes such as Given that elevated Wnt/β-catenin signaling has been associated with improved melanoma survival outcomes in molecularly unselected patients along with enhancement of apoptosis with BRAFi in laboratory melanoma models, we hypothesized that higher levels of Wnt/β-catenin signaling in pre-treatment melanoma tumors (as measured by increased nuclear β-catenin) might predict a better clinical response to BRAFi. To address this hypothesis, we performed a retrospective analysis of Wnt/β-catenin signaling of pretreatment melanoma specimens from patients treated clinically with BRAFi for metastatic melanoma. In parallel, we studied the effects of long-term BRAFi treatment in cultured melanoma cells. Our results extend the previous model for how Wnt/β-catenin and BRAF/MAPK signaling interact in melanoma.BRAF mutation at the V600 position (confirmed by DNA sequencing) who received treatment with BRAFi (n = 32) were included in this study. Cohort characteristics are summarized in r = 0.83; Patients with metastatic melanoma carrying a Given the limited sample size, we performed post-hoc exploratory analysis comparing levels of nuclear β-catenin with the survival endpoints using different methods of patient stratification that subdivided the cohort into two or three groups. Comparison of patients in the lowest quartile of nuclear β-catenin (lowest 25%) to the remaining 75% of patients within the cohort revealed a significant difference in OS  and in the presence or absence of WNT3A . Two of AXIN2 transcript) are uncoupled from the enhancement of apoptosis by Wnt/β-catenin signaling at or above the level of GSK3B.Interestingly, long-term treatment of cells with BRAFi alone did not affect activation of Wnt/β-catenin signaling by the GSK3 inhibitor CHIR99021 . A similV600EBRAF mutation  were transduced with lentiviral constructs encoding either GFP or an epitope-tagged BRAFV600E (V600EBRAF (but not GFP) led to a dose-dependent increase in phosphorylation of ERK1/2, confirming increased activation of the MAPK signaling cascade  or inhibition of MEK using U0126  contributes to the decreased survival observed in patients with tumors harboring Patient specimens were formalin-fixed, paraffin-embedded tumors. Informed written consent was obtained for each patient under approved protocols (Protocol No X10-0305 &HREC/10/RPAH/539 and Protocol No X10-0300 HREC/10/RPAH/530) governed by the Human Research Ethics Committee of the Royal Prince Albert Hospital . All clinical investigation was conducted according to principles outlined in the Declaration of Helsinki.V600E was purchased from Biomyx . The coding sequence for HA-BRAFV600E was inserted using standard cloning techniques into third-generation replication-deficient lentivirus  and cultured using the vendor's suggested media and culture conditions. Melanoma cell lines and their culture conditions have been previously described Patients were selected on the basis of availability of baseline melanoma tumor samples. All patients received a BRAFi via enrolment in a clinical trial; either the GlaxoSmithKline (GSK) Phase 1/2 trial of dabrafenib (12 patients) Objective response to BRAFi treatment was assessed with CT scanning 6–9 weekly, using RECIST 1.0 Immunohistochemical cytoplasmic and nuclear β-catenin stains were conducted on tumor samples (either primary melanomas or melanoma metastases) from this cohort collected prior to the initiation of BRAFi. Five micron-thick tumor sections were labeled immunohistochemically with a mouse monoclonal antibody to β-catenin  at a dilution of 1∶1000, which was experimentally determined to be the optimal concentration using standardized specimens. Fluorescent visualization was performed using an anti-mouse Cy5-conjugated antibody . Nuclei were visualized using Prolong Gold DAPI . Initial tumor specimens were examined, dissected, processed and interpreted at the Melanoma Institute of Australia prior to antigen retrieval, immunostaining, quantification and imaging of these samples by HistoRX (Branford CT), who were blinded to all outcomes data. S100 staining was used to identify a tumor mask, defined as the cellular area of the tumor , and thea priori by the team that conducted the stains, blinded to the clinical outcome data. Scores for nuclear β-catenin were averaged for each tumor based on an average signal from each tumor grid Click here for additional data file.


Finally, we examined the effects of this training on imagery vividness. Thirty-nine participants were randomly allocated to imagery CBM-I, a non-imagery control program, or a no treatment control group. Imagery CBM-I led to greater improvements in depressive symptoms, interpretive bias, and imagery vividness than either control condition at post-treatment (n = 13 per group), and improvements were maintained at 2-week follow-up (n = 8 per group). This pilot study provides first preliminary evidence that imagery CBM-I could provide positive clinical outcomes in an Iranian psychiatric setting, and further that the imagery component of the training may play a crucial role.Cognitive bias modification paradigms training positive mental imagery and interpretation (imagery CBM-I) hold promise for treatment innovation in depression. However, depression is a global health problem and interventions need to translate across settings and cultures. The current pilot study investigated the impact of 1 week of daily imagery CBM-I in treatment-seeking individuals with major depression in outpatient psychiatry clinics in Iran. Further, it tested the importance of instructions to  Depression is a global health problem . A second aim was to investigate whether the requirement to We addressed the primary aim by investigating the imagery CBM-I in a sample of treatment-seeking patients with major depression in outpatient psychiatry clinics in Iran. The initial theoretical and experimental work underpinning this imagery CBM-I approach was largely carried out in European settings, and although there are no specific theoretical reasons to expect that the approach would be ineffective in a non-European population or culture, a straightforward equivalence of effects cannot be assumed. With some exceptions  in which participants were presented with an identical set of training stimuli  over an identical schedule of sessions, but were given no training in imagery or instruction to imagine the scenarios. Instead they were told to listen to the scenarios and not instructed to use any particular form of processing. We further included an additional no treatment control condition in order to provide a comparison for the active control condition  in everyday life in order to more fully characterise any effects of the CBM-I on imagery and verbal processing. For example, it may be that the repeated requirement to use imagery and not verbal processing within the task would generalize to a greater tendency to use mental imagery and reduced tendency to use a verbal (ruminative) processing style in everyday life. Although these are not hypothesised to be key mechanisms by which the CBM-I paradigm improves clinical outcomes, understanding the broader potential impact of the program is helpful in describing its effects more fully.The imagery CBM-I would be successfully translated to and applied in the new population, such that participants in the imagery condition would show improvements in symptoms of depression and anxiety, and reduction in negative interpretive bias over the 1-week training and at 2-week follow-up.Participants in the imagery condition would show greater improvement in outcome measures than those in the non-imagery active control condition.Participants in the imagery condition would show increased vividness of mental imagery following the imagery CBM-I compared to those in the control conditions.We hypothesised that:A mixed design was used, in which participants were randomly allocated to one of three groups: imagery-focussed positive CBM-I (imagery condition), the identical program but with no instruction to use imagery (non-imagery condition), or a no treatment control condition (no treatment condition). Following the baseline assessment, participants in the imagery condition received practice in imagery and then completed a session of imagery-focussed positive CBM-I every day from home on their home computer. Participants in the non-imagery condition completed an identical program, but without the prior instruction to use imagery or imagery practice. Participants in the no treatment condition simply returned 1 week later. Measures of depressive symptoms, anxiety, interpretive bias, use and vividness of imagery, and rumination were completed pre and post the intervention week, and in the imagery and non-imagery conditions 2 weeks later at follow-up.Treatment-seeking patients with major depressive disorder (MDD) were recruited from five outpatient psychiatry clinics in Isfahan, Iran. Diagnoses were determined by experienced psychiatrists based on Structured Clinical Interview for DSM-IV axis I disorders .There were 448 different positive training paragraphs, which had previously been used by Blackwell and Holmes . These wEach day, participants were presented with 64 different auditory training descriptions in eight randomized blocks of eight paragraphs. All descriptions were followed by a 2 s pause. Task instruction reminders were given between blocks, with short breaks allowed between these blocks. The same training paragraphs were used in both the imagery and non-imagery condition. In order to focus participants on their assigned task (see below), after each training paragraph (and 2 s gap), participants in the imagery condition were asked to rate the vividness of their imagery (“How vividly could you imagine the situation that was described?”) as in previous studies , use of imagery  as you were listening to the sentences?”, where 1 = not at all and 9 = all the time), use of verbal analysis , and difficulty maintaining focus . They were then interviewed in more detail about their use of imagery or verbal processing  and returned to the psychiatry clinics 1 week later to repeat them (post-treatment). They were then referred on for treatment. Following the initial assessment session, participants in the imagery and non-imagery conditions completed the outcome measures (pre-treatment), followed by a first session of the relevant CBM-I program. In the imagery condition, this session included the imagery practice described above. Over the subsequent week participants completed a session of the CBM-I program every day from home. At the end of this intervention week they returned to the psychiatry clinics to repeat the outcome measures (post-treatment) and manipulation check questionnaire, and were interviewed about their experience of completing the corresponding CBM-I program. They returned to the psychiatry clinics 2 weeks later to repeat the outcome measures (follow-up). All of the assessments and the first session of CBM-I were completed individually at one of the psychiatry clinics.ps > .2; see Tables n = 26) < 0.01, p = 1.000. Reasons for drop-out from post-treatment to follow-up were, in the imagery condition: Moving out of the area (n = 2), not able to attend due to exams starting (n = 2), starting treatment for depression (n = 1), and in the non-imagery condition: Starting treatment for depression (n = 2), family crisis (n = 1), did not wish to attend the assessment (n = 2).The three groups did not statistically differ with regard to demographic or clinical characteristics, or any of the baseline measures  and non-imagery condition  on the number of CBM-I sessions completed, t(24) < 1. Participants were included in the analyses regardless of how many sessions they had completed.Of participants assigned to the imagery or non-imagery conditions, all completed at least six of the possible seven sessions. There was no significant difference between imagery condition  and non-imagery  conditions, t(24) < 1. Participants in the imagery condition reported more use of imagery than those in the non-imagery condition  = 2.93, p = .007). Participants in the imagery condition reported less use of verbal analysis than those in the non-imagery condition  = 4.25, p < .001). Participants rated maintaining focus as equally difficult in both the imagery  and non-imagery  conditions, t(24) < 1. Information gathered during the debriefing interview further suggested that participants in the imagery condition had predominantly used field perspective, rather than observer perspective, imagery; conversely within the non-imagery condition all but one participant reported that when they did use imagery it had been predominantly observer rather than field perspective. Thus it appeared that the condition manipulation had been successful in leading to different styles of processing in the two CBM-I conditions. Participants in the imagery condition, and hence engaging in more imagery and less verbal processing, were more likely to describe the sessions as enjoyable. This is consistent with the feedback from a previous study that participants who struggled to engage in imagery and used a more verbal processing style tended to find the sessions more tedious  = 22.14, p < .001, η2 = .38, but not of condition, F < 1. There was a significant interaction of time with condition, F = 5.26, p = .010, η2 = .23. There was a significant decrease from pre to post-treatment within both the imagery condition, M = 12.46, SD = 9.23, t(12) = 4.87, p < .001, d = 1.58, and within the non-imagery condition, M = 3.92, SD = 5.78, t(12) = 2.45, p = .031, d = 0.42, but no decrease within the no treatment condition, M = 2.62, SD = 9.66, t(12) < 1. The reduction in depressive symptoms in the imagery condition was significantly greater than that in both the non-imagery, t(24) = 2.82, p = .009, d = 1.11, and the no treatment condition, t(24) = 2.66, p = .014, d = 1.04. The reduction in depressive symptoms in the non-imagery condition was not significantly different from that in the no treatment condition, t(24) < 1.For the BDI-II there was a significant main effect of time, F = 8.17, p = .007, η2 = .19, but not of condition, F < 1, and there was no significant interaction of time with condition, F = 1.59, p = .219.For the STAI-T there was a significant main effect of time, F < 1, and no significant effect of condition, F < 1, but there was a significant interaction of time with condition, F = 6.82, p = .003, η2 = .28. There was a significant decrease from pre to post-treatment within the imagery condition, M = 21.23, SD = 20.62, t(12) = 3.71, p .003, d = 0.89, but not within the non-imagery condition, M = 7.20 (increase), SD = 15.39, t(12) = 1.69, p = .117, nor within the no treatment condition, M = 3.75 (increase), SD = 26.76, t(12) < 1. The reduction in negative interpretive bias in the imagery condition was significantly greater than that in both the non-imagery, t(24) = 3.99, p = .001, d = 0.77, and the no treatment condition, t(24) = 2.67, p = .013, d = 0.73. There was no significant difference in change between the non-imagery and no treatment condition, t(24) < 1.For the SST, there was no main effect of time, F = 2.66, p = .112 or condition, F < 1. However, there was a significant interaction of time with condition, F = 4.32, p = .021, η2 = .19. There was a significant increase from pre to post-treatment within the imagery condition, M = 9.00, SD = 13.18, t(12) = 2.46, p = .030, d = 0.52, but not within the non-imagery condition, M = 0.77, SD = 8.72, t(12) < 1, nor within the no treatment condition, M = 2.00 (decrease), SD = 6.77, t(12) = 1.07, p = .308. The increase in vividness of visual imagery in the imagery condition was at trend level compared to the non-imagery condition, t(24) = 1.88, p = .073, d = 0.73, and significantly greater than that in the no treatment condition, t(24) = 2.68, p = .013, d = 0.67. There was no significant difference in change between the non-imagery and no treatment condition, t(24) < 1.For the VVIQ there were no significant main effects of time, F < 1 or condition, F < 1, and no significant interaction of time with condition, F = 2.28, p = .117.For the SUIS there were no significant main effects of time, F = 11.89, p = .001, but not of condition, F = 1.42, p = .254. There was a significant interaction of time with condition, F = 5.36, p = .009, η2 = .23. There was a significant decrease in ruminative responses from pre to post-treatment within the imagery condition, M = 19.08, SD = 17.12, t(12) = 4.02, p = .002, d = 1.68, but not within the non-imagery condition, M = 3.31, SD = 14.22, t(12) < 1, or within the no treatment condition, M = 2.08, SD = 12.60, t(12) < 1. The decrease in ruminative responses in the imagery condition was significantly greater than that in both the non-imagery condition, t(24) = 2.56, p = .017, d = 1.00, and that in the no treatment condition, t(24) = 2.88, p = .008, d = 1.13. There was no significant difference in change between the non-imagery and no treatment condition, t(24) < 1.For the RRS there was a significant main effect of time, n = 8 in the imagery condition, and n = 8 in the non-imagery condition) are included in these analyses.The follow-up analyses compare only the imagery and non-imagery conditions, as participants in the no treatment condition were not requested to return for a follow-up. Only those participants who provided follow-up data  = 26.14, p < .001, η2 = .65, but not of condition, F < 1. There was a significant interaction of time with condition, F = 12.26, p = .004, η2 = .47. There was a significant decrease from pre-treatment to follow-up within the imagery condition, M = 17.38, SD = 7.82, t(7) = 6.29, p < .001, d = 2.17, but not within the non-imagery condition, M = 3.25, SD = 8.31, t(7) = 1.11, p = .305. The reduction in depressive symptoms in the imagery condition was significantly greater than that in the non-imagery condition, t(14) = 3.50, p = .004, d = 1.75.For the BDI-II there was a significant main effect of time, F = 7.41 p = .017, η2 = .35, but not of condition, F < 1. There was a significant interaction of time with condition, F = 5.06, p = .041, η2 = .27. There was a significant decrease from pre-treatment to follow-up within the imagery condition, M = 7.88, SD = 5.17, t(7) = 4.31, p = .004, d = 0.87, but not within the non-imagery condition, M = 0.75, SD = 7.32, t(7) < 1. The reduction in trait anxiety in the imagery condition was significantly greater than that in the non-imagery condition, t(14) = 2.25, p = .041, d = 1.13.For the STAI there was a significant main effect of time, F = 1.06, p = .321, or condition, F = 2.40, p = .143. However, there was a significant interaction of time with condition, F = 21.57, p < .001, η2 = .61. There was a significant decrease from pre-treatment to follow-up within the imagery condition, M = 25.76, SD = 24.46, t(7) = 2.98, p = .021, d = 1.34, but a significant increase within the non-imagery condition, M = 16.41, SD = 7.82, t(7) = 5.93, p = .001, d = 0.59. The reduction in negative interpretive bias in the imagery condition was significantly greater than that in the non-imagery condition, t(14) = 4.65, p < .001, d = 2.32.For the SST there was no significant main effect of time, F = 3.51, p = .082, d = 0.20, but no significant effect of condition, F < 1. There was a significant interaction of time with condition, F = 7.58, p = .016, η2 = .35. There was an increase from pre-treatment to follow-up within the imagery condition, M = 10.75, SD = 9.77, t(7) = 2.53, p = .039, d = 0.63, but not within the non-imagery condition, M = 2.50, SD = 6.52, t(7) = 1.08, p = .314. The increase in vividness of visual imagery in the imagery condition was significantly greater than that in the non-imagery condition, t(14) = 2.75, p = .016, d = 0.99.For the VVIQ there was a trend level effect of time, F < 1 or condition, F < 1, and no significant interaction of time with condition, F = 1.76, p = .205.For the SUIS there were no significant main effects of time, F = 7.20, p = .018, η2 = .34 but not of condition, F = 1.83, p = .198. The interaction of time with condition was not significant, F = 2.57, p = .131.For the RRS there was a significant main effect of time, F = 1.05, p = .324, and no significant interaction between time and condition, F < 1, suggesting that the improvement in symptoms of depression occurred during the week of training and was maintained at follow-up. An ANOVA examining change from post-treatment to follow-up for the STAI showed no significant effects of time, condition, or their interaction . For the RRS, such an ANOVA showed no significant effect of time or time by condition interaction (Fs < 1), but there was a significant main effect of condition, F = 6.66, p = .022, η2 = .32.In response to helpful reviewer suggestions, to further understand where the clinical change occurred, we carried out a similar ANOVA examining the change from post-treatment to follow-up for the BDI-II. There was no significant main effect of time, ps > .1).There was no difference between participants who did or not drop out at follow-up in terms of age, gender, or baseline depression severity (BDI-II score at pre-treatment), within either the imagery or within the non-imagery condition  = 5.57, p = .062. Further exploration demonstrated that significantly more participants showed clinically significant change over the 1-week intervention in the imagery condition compared to the non-imagery condition, p = .047, Fisher’s exact test, but not compared to the no treatment condition, p = .428, Fisher’s exact test. There was no significant difference in rates of clinically significant change between the non-imagery and no-treatment conditions, p = .41, Fisher’s exact test. From pre-treatment to follow-up, significantly more participants in the imagery condition demonstrated clinically significant change compared to those in the non-imagery condition, 87.5 versus 12.5 %, p = .010, Fisher’s exact test.Clinically significant change was defined as a shift to a lower category of depressive symptom severity accompanied by a reduction greater than the reliable change index of 7.16, calculated according to the guidance provided by Jacobson and Truax  and as ar(37) = .33, p = .042), and increase in score on the VVIQ (r(37) = .39, p = .014) over this week.To investigate whether changes in symptoms of depression from pre to post-treatment were related to the potential mechanisms of change targeted by the CBM-I  we carried out correlations between the relevant change scores in the whole sample. There was a significant correlation between reduction in BDI-II over the 1 week from pre to post-treatment and both reduction in negativity score on the SST  but 3 of the positive imagery group were clustered at the top end of the “responders” in terms of change in BDI–II from pre to post-intervention, compared to only 15 % (n = 2) of the active control group or 30 % (n = 4) of the no treatment group in this range (top 16).One potential concern with small samples is that analyses of means can sometimes be unreliable, as the means may be disproportionately influenced by outliers. We therefore examined our data for the main outcome (change in BDI-II from pre to post-treatment) on an individual-level basis. The pattern of individual level change on the BDI-II from pre to post-treatment showed a high level of consistency, demonstrative of systematic differences between the groups in their response rather than sampling bias, increasing our confidence in the reliability of our main statistical analyses. As a graphic example of this, all  mental imagery ability, as measured by self-reported imagery vividness. This study therefore provides preliminary evidence for the potential cross-cultural applicability of a positive imagery training paradigm in depression, and furthers our knowledge of the parameters and effects of this form of training. The implications of the current study and how it builds on previous work will be discussed first in relation to the clinical outcomes, then the role of imagery in the training, and finally the impact of the training on imagery.n = 13 per group) and subsequent 2 week follow-up (n = 8 per group), corresponding to large effect sizes. Rates of clinically significant change in symptoms of depression were generally high in this study in comparison to previous studies. For example, in the imagery condition, the percentage of participants showing clinically significant change were 69.2 and 87.5 % from pre to post-treatment and pre-treatment to follow-up respectively, whereas these figures were 46.2 and 53.8 % in the study by Lang et al.  in mind when interpreting the results from this study. As a first attempt (to our knowledge) to implement imagery CBM-I in this novel population, a small pilot study was appropriate in order to provide the initial evidence that could justify the time, resources, and participant burden of larger clinical trial in this new setting. However, this small sample size means that the results can only be interpreted as providing encouraging preliminary evidence. The results from pre-treatment to follow-up in particular must be interpreted with caution, due to the lack of outcome measurement in the no treatment group at follow-up, and the attrition in the imagery and non-imagery groups at this time point. The high rate of attrition at follow-up was mostly due to practical difficulties in attending another face-to-face assessment session, and thus future studies could enhance collection of follow-up data by having this data completed remotely, e.g. via online questionnaires or phone interviews. One concern when such promising clinical results are obtained with a small study relates to whether they can be replicated, as a small sample limits the generalizability of a study’s findings. We note that the results of this study are consistent with the two previous clinical studies  the participants engaged in modified their interpretive bias or imagery vividness, the more they experienced a reduction in symptoms of depression. The relationship between the putative active mechanisms of change in this CBM-I, imagery and interpretation, and clinical outcomes will be important to investigate more fully in larger samples and over longer time periods. It will also be important to investigate the potential interaction between the cognitive biases targeted (e.g. Everaert et al. Mental imagery represents a number of complex cognitive processes and plays numerous roles in daily functioning (Holmes and Mathews The development and dissemination of novel treatment approaches to help tackle the global health problem presented by depression requires that novel interventions translate from one country to another. This pilot study provides some preliminary evidence that the benefits of training positive imagination could possibly transcend national and cultural boundaries and encourages further investigation of the application of imagery CBM-I in novel populations. Further, it indicates the potential importance of mental imagery in translating the positive resolutions of the training scenarios into positive clinical outcomes in depression.


Preconditioning via heat acclimation (34°C 30 d) results in neuroprotection from traumatic brain injury due to constitutive as well as dynamic changes triggered by the trauma. Among these changes is Akt phosphorylation, which decreases apoptosis and induces HIF1α. In the present study we investigated the Akt downstream GSK3β/β -catenin pathway and focused on post injury alternations of β catenin and its impact on the cellular response in preconditioned heat acclimated mice. We found that the reduction in motor disability is accompanied with attenuation of depressive like behavior in heat acclimated mice that correlates with the GSK3β phosphorylation state. Concomitantly, a robust β catenin phosphorylation is not followed by its degradation, or by reduced nuclear accumulation. Enhanced tyrosine phosphorylation of β catenin in the injured area weakens the β catenin-N cadherin complex. Membrane β catenin is transiently reduced in heat acclimated mice and its recovery 7 days post TBI is accompanied by induction of the synaptic marker synaptophysin. We suggest a set of cellular events following traumatic brain injury in heat acclimated mice that causes β catenin to participate in cell-cell adhesion alternations rather than in Wnt signaling. These events may contribute to synaptogenesis and the improved motor and cognitive abilities seen heat acclimated mice after traumatic brain injury. After the acclimation period, HA animals cope better with insults due to reprogrammed gene expression and altered post transcriptional mechanisms Since the HA model induces Akt phosphorylation, reduced apoptosis and transient hypothermia-all are involved in the determination of the fate of β catenin, this study was designed to investigate whether HA affects β catenin along one or both of its main pathways: (i) Wnt signaling (ii) cell-cell adhesion.ad libitum. The mices were divided into two groups: control normothermic (NT) maintained at an ambient temperature of 24°C±1°C and heat acclimated (HA) held in a climatic chamber at 34°C±1°C for 30 days, a period that ensures acclimatory homeostasis has been achieved The study was approved by the Institutional Animal Ethics Committee of the Hebrew University and complied with the guidelines of the National Research Council Guide for the Care and Use of Laboratory Animals . Male Sabra mice weighing 38 to 53g were used. Animals were kept under controlled light conditions with a 12 h/12 h light/dark cycle. Food and water were provided Experimental closed head injury (CHI), is a subtype of TBI without skull penetration. CHI was induced under isoflurane anesthesia using a modified weight drop device developed in our laboratory The functional status of the mice was evaluated according to the Neurological Severity Score (NSS) by an observer unaware of the treatment. This score is a 10 point scale assessing functional neurological status based on the presence of some reflexes and the ability to perform motor and behavioral tasks such as beam walking, beam balance and spontaneous locomotion In a calm quite room with an ambient room temperature of 25°C, mice were gently placed for 6 minutes in a cylindrical glass container (diameter 17 cm) filled with 24°C water at a depth of 22 cm. Mice were gently dried using towel and the water was changed after every test. Tests were recorded using a video camera and analyzed offline. Behavioral analysis was done from 2 to 6 minutes after placing the mouse in the water. Immobility was defined as minimal tail and limb movements to keep head above water. Mobility was defined as any movement other than those necessary to keep head above water. In order to avoid false results due to physical disability, only mice with moderate injury (NSS≤7) were subjected to the test (n = 7–9 mice/group).2 1.5 mM, glycerol 10%, EDTA 1 mM, samples were centrifuged at 5000 rcf for 10 min. the supernatants were then homogenized for 30 min at 30,000 rcf and pellets were re-suspended in 40 µL homogenization, and used for membrane protein enriched subcellular fraction. Validation of membrane enrichment was conducted using anti synaptophysin  that was almost absent in cytosolic extraction. After cellular fractionation, homogenates were stored at −80°C until analysis. Protein concentration was determined using the Bradford method . Equal protein samples (40 µg for cytosolic and 20 µg for nuclear extracts) were separated on 10% SDS-polyacrylamide gels with 4.5% SDS stacking gels and electrotransferred onto nitrocellulose membranes . Blots were probed with a rabbit polyclonal anti–GSK3β , anti-p-GSK3βser9 , anti-β catenin , anti-p-β cateninser33/37/Thr41,, anti JNK  or anti p-JNK Thr183/Tyr185 antibody . Rabbit polyclonal antibody against β -actin antibody  was also used to confirm equal protein loading (Xilouri and Papazafiri 2006). Appropriate peroxidase-coupled immunoglobulin G  was used for secondary incubations. Reactive bands were visualized using the enhanced chemiluminescence system . Optical density of reactive bands was quantified using Tina software  and protein levels were expressed as the optical density of the examined factor relative to β -actin in the same lane.After 30 days of heat-acclimation or normothermic conditions, mice were subjected to CHI or sham surgery and sacrificed 6, 12, 24, 72 h or 7 days later (n = 5–6/group). After decapitation, brains were rapidly removed and frontal segments (40–60 mg) from left, injured hemispheres were separated and frozen at −80°C until analysis. Sample preparation was performed as previously described tyr654 antibody ,N-cadherin , or anti β catenin antibody .The co-immunoprecipitation (co-ip) method was used to determine β catenin-N cadherin interactions and y654 phosphorylation of β catenin. Mice were subjected to CHI, and sacrificed 6, 12 or 24 h later. Brains were rapidly removed and frontal segments from the injured left hemisphere were homogenated in buffer containing 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol, pH = 7.4. Protein concentrations were determined using the Bradford method  and homogenates were kept at −80°C until analysis. For co-ip, 200 µg protein in 200 µl was incubated overnight at 4°C with rotation with anti β catenin antibody . 30 µl of cleaned Protein Immobilized rProtein  was added and incubated with rotation for 30 minutes at 4°C. Following 3 minutes centrifugation at 14,000 rpm the pellet was washed five times with homogenization buffer. Finally sample buffer was added, samples were centrifuged at 12,000 rpm for 5 minutes and supernatants were collected. Western blot analysis was performed using either β cateninAt 24 h post injury, brains were perfused and fixed and 10 µm frozen sections were prepared. Slices were cut in the injured area (bregma +0.26). Brain slices were double stained for immunohistochemical evaluation using specific antibodies for β catenin tyr654  and DAPI . Alexa 488 was used as the secondary antibody . Pictures were taken using an epifluorescent Olympus microscope.P-values<0.05 were considered significant for all comparisons. Data are expressed as mean ± s.e.m.For statistical analyses, we used commercially available computer software (SigmaStat 2.03). Treatments were the independent variables and the outcomes of the TBI parameters were the dependent variables. Significance was tested using two-way ANOVA for repeated measures in NSS, followed by Tukey post-hoc tests. For protein levels and forced swimming test, significance was calculated using two-way ANOVA between HA and NT, or one-way ANOVA within the same group (NT or HA) followed by Tukey post-hoc tests. HA mice recovered significantly better than NT controls after TBI , signifiSince we observed HA induced behavioral changes and Akt phosphorylation, our next step was to evaluate the Akt downstream target and depression mediator GSK3β using Western blot analysis. We found that non injured sham HA mice have lower levels of non-activated p-GSK3β (higher ratio p-GSK3β/t-GSK3β) than the NT control mice.There was a significant increase in p-GSK3β 24 h after injury in HA mice, hence HA induces GSK3β inactivation after TBI, . No signAfter examination of the phosphorylation state of GSK3β and JNK, we examined their phosphorylation target, β catenin. TBI led to robust phosphorylation of β catenin at residues ser33/37thr44 6 h after injury. β catenin phosphorylation lasted up to 24 h post injury . TBI didA band at 70–80 kD, under the predicted 95 kD band of β catenin was only observed after TBI . This baSince tyrosine phosphorylation of β catenin regulates the integrity of the cadherin-catenin complex, we evaluated total N cadherin levels and cadherin-catenin complex integrity. N cadherin levels were up-regulated following HA as shown in Next we examined the levels of membrane bound β catenin, i.e. its role in cell-cell adhesion and synaptic establishment. Membrane bound β catenin levels slowly decreased in injured NT mice, reaching significance at 7 d post injury . In contThe present study is focused on the role and the dynamics of β catenin after traumatic brain injury. β catenin is a member of the canonical Wnt signaling, regulated by GSK3β. Post traumatic alternations in this cascade can lead to apoptosis and depression in TBI victims. β catenin is also crucial for cell-cell adhesion as a part of the catenin-cadherein complex. Following TBI, interference in the complex can lead to motor and cognitive deficits. Here we show that following TBI, heat acclimation mediated cross-tolerance is reflected by attenuated motor disability and depressive like behavior in the injured mice. We provide evidence that in the acclimated - preconditioned mice, β catenin returns to form the catenin-cadherein complex, induces synaptophysin levels and, in turn, post injury synaptogenesis. These issues are discussed below.Motor disability and depression are among the long lasting consequences of TBI. Our present findings corroborate our earlier reports on the beneficial effects of HA pre-conditioning on recovery of neurobehavioral function (measured using ΔNSS) Akt is a “master regulator” of GSK3β and β catenin, independent of Wnt-mediated GSK3β inactivation. The PI3K/Akt pathway inactivates GSK3β via direct Akt-mediated phosphorylation of Ser 9, hence it inhibits β-catenin phosphorylation. This phosphorylation designates β-catenin for degradation and also prevents direct detrimental cellular activity of GSK3β such as apoptosis. Here we show induced GSK3β phosphorylation in HA mice, corresponding to the known enhanced Akt phosphorylation. Also, attenuated GSK3β activity partly explain the reduced apoptosis and attenuated pro-inflammatory cytokine levels seen in injured HA mice ser33/37/Thr41 degradation. A similar phenomenon was documented under hypothermic conditions of focal brain ischemia GSK3β is also partially regulated by the canonical Wnt signaling. The Wnt signaling includes GSK3β translocation from the active cellular complex that includes APC (adenomatous polyposis coli), Axin and CKIα (casein kinase Iα), or by phosphorylated LRP, keeping GSK3β away from its substrate. This allows β catenin stabilization Recent studies suggest the presence of other kinases that phosphorylate β catenin at the same positions as GSK3β. Lee et al., ser33/37/Thr41 degradation in HA mice is counteracted by the increased β catenin cleavage in NT mice. However this explanation is doubtful due to the relatively low amounts of the fragmented β catenin as measured by optical density, when compared with higher optical density of total or β catenin ser33/37/Thr41. Increased β catenin ser33/37/Thr41, as seen in HA mice, with no further alternation in cytosolic or nuclear protein levels may imply that HA blocks β catenin ser33/37/Thr41 degradation. In that case, it is unlikely that the Wnt signaling plays an important part in the brain response after TBI or in the facilitation of HA induced neuroprotection. Another explanation may include the role of HIF1α. Studies suggest that under hypoxic conditions HIF1α can directly bind β catenin and regulate Wnt signaling HA mice displayed robust phosphorylation of β catenin after TBI, however its cytosolic levels remained unchanged and there was no further degradation of β catenin. In order to reconcile this conflict we looked for evidence of β catenin cleavage. It is known that β catenin can be cleaved by cellular proteases including caspase 3, and the cleaved fragments of β catenin are seen at the molecular weight 70–80 kD in WB +2dependent proteolysis +2 levels after TBI and subsequent protease activation β catenin is crucial not only in the canonical Wnt signaling, but also in cadherin mediated cell-cell adhesion. The cadherin-catenin complex is localized in synaptic junctions and is involved in synapse formation, stabilization and plasticity In conclusion, to our knowledge this is the first study to examine β catenin dynamics after TBI and to associate these dynamics with post injury synaptogenesis and cognitive function. This is also the first study to imply that dynamic changes in β catenin after TBI paly a role in synaptic alternations following TBI, in the same way, but on a different time scale to memory formation process


Aberrant accumulation of intracellular β-catenin is a well recognized characteristic of several cancers, including prostate, colon, and liver cancers, and is a potential target for development of anticancer therapeutics. Here, we used cell-based small molecule screening to identify CGK062 as an inhibitor of Wnt/β-catenin signaling. CGK062 promoted protein kinase Cα (PKCα)-mediated phosphorylation of β-catenin at Ser33/Ser37, marking it for proteasomal degradation. This reduced intracellular β-catenin levels and consequently antagonized β-catenin response transcription (CRT). Pharmacological inhibition or depletion of PKCα abrogated CGK062-mediated phosphorylation and degradation of β-catenin. In addition, CGK062 repressed the expression of the genes encoding cyclin D1, c-myc, and axin-2, β-catenin target genes, and thus inhibited the growth of CRT-positive cancer cells. Furthermore, treatment of nude mice bearing PC3 xenograft tumors with CGK062 at doses of 50 mg/kg and 100 mg/kg (i.p.) significantly suppressed tumor growth. Our findings suggest that CGK062 exerts its anticancer activity by promoting PKCα-mediated β-catenin phosphorylation/degradation. Therefore, CGK062 has significant therapeutic potential for the treatment of CRT-positive cancers. The Wnt/β-catenin pathway, which is activated by the interaction of Wnt1, Wnt3a, and Wnt8 with Frizzled (Fz) receptors and low-density lipoprotein receptor-related protein5/6 (LRP5/6) co-receptors, plays important roles in cell proliferation, differentiation, and oncogenesis c-myc, cyclin D1 and metalloproteinase-7 (MMP-7), which play important roles in tumorigenesis and metastasis Abnormal activation of the Wnt/β-catenin pathway and subsequent up-regulation of β-catenin response transcription (CRT) is thought to contribute to the development and progression of certain cancers In the present study, we identified CGK062, which markedly inhibits the Wnt/β-catenin pathway and cell proliferation of CRT-positive cancer cells. CGK062 promoted the degradation of intracellular β-catenin through PKCα-mediated β-catenin phosphorylation.50 = 12.3 µM). CGK062 did not affect FOPFlash activity in HEK293 control cells and cell viability in HEK293 reporter cells  plasmids. After the incubation of these reporter cells with Wnt3a-CM and each compound, we measured firefly luciferase activity using a microplate reader and then identified that CGK062 -acrylic acid 2,2-dimethyl-8-oxo-3,4-dihydro-2H,8H-pyranochromen-3-yl ester) as an inhibitor of the Wnt/β-catenin pathway  and S1. er cells  and S2A.er cells . These rIn the Wnt/β-catenin pathway, CRT is largely dependent on the level of intracellular β-catenin, which is controlled by ubiquitin-dependent proteolysis Next, to explore whether down-regulation of β-catenin by CGK062 is mediated by the proteasome, we used MG-132 to inhibit proteasome-mediated protein degradation in HEK293 reporter cells. As shown in Given that the phosphorylation of β-catenin by GSK-3β and its subsequent association with β-TrCP leads to β-catenin degradation β-TrCP ubiquitin ligase complex We then determined whether β-TrCP is necessary for CGK062-induced degradation ofβ-catenin. As shown in in vitro, when we used synthetic peptide containing PKCα phosphorylation site as a substrate , SNU475 (hepatoma), and SW480 (colon cancer). Consistent with results from HEK293 cells, CGK062 promoted the translocation of PKCα to the plasma membrane in these cancer cells . To detecyclin D1, which is a known β-catenin-dependent gene, was evaluated. A reporter construct containing the cyclin D1 promoter, which contains a β-catenin/TCF-4 responsive region, was transfected into PC3, SNU475, and SW480 cells followed by treatment with different concentrations of CGK062. As shown in cyclin D1 promoter activity was repressed by CGK062 in these CRT-positive cancer cells. We also evaluated the protein level of cyclin D1 in CGK062-treated CRT-positive cancer cells. Consistent with our result for the cyclin D1 promoter, a dose-dependent decrease in cyclin D1 protein expression was observed in response to CGK062 , treatment of mice with CGK062 significantly inhibited PC3 tumor growth  and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 120 µg/ml penicillin, and 200 µg/ml streptomycin. Wnt3a-conditioned medium (Wnt3a-CM) was prepared as previously described The HEK293 reporter cells were inoculated into 96-well plates at 15,000 cells per well in duplicate and grown for 24 h. Wnt3a-CM was added, and then compounds (800 compounds) including coumarins, flavonoids, naphthoquinones, and terpenoids were added to the wells at a final concentration of 30 µM. After 15 h, the plates were assayed for firefly luciferase activity and cell viability.g for 2 min at 4°C. The supernatant was centrifuged at 100,000× g for 30 min at 4°C in a 100Ti rotor . The pellet was suspended in extraction buffer containing 0.5% (w/v) Triton X-100.Cells grown in 100-mm culture dishes were washed with ice-cold PBS. The cells were then suspended in 1 ml of ice-cold extraction buffer (20 mM Tris (pH 7.5), 0.5 mM EDTA, and 0.5 mM EGTA); homogenized using a syringe (26G); and incubated on ice for 30 min. The homogenate was centrifuged at 13,400× The cytosolic fraction was prepared as previously described Total RNA was isolated with Trizol reagent  in accordance with the manufacturer's instructions. cDNA synthesis, reverse transcription, and PCR (Polymerase chain reaction) were performed as previously described HEK293cells were cultured on glass chamber slides and then treated with DMSO or CGK062 for 15 h. After treatment, the cells were washed with PBS, fixed with 4% formaldehyde, permeabilized in 0.3% Triton X-100, and blocked in 4% bovine serum albumin for 1 h. The cells were stained with anti-PKCα or anti-E-cadherin (BD Transduction Laboratories) antibodies and then analyzed by confocal microscopy using a Zeiss LSM510 Meta microscope.Kinase assays were performed with purified PKCα (Sigma) using GST-β-catenin (100 ng) as a substrate as previously described Cells were inoculated into 96-well plates and treated with CGK062 for 48 h. The cell viability from each treated sample was measured in triplicate using CellTiter-Glo assay kit  according to the manufacturer's instructions.7) cells were injected s.c. into 6-week-old female athymic nude mice. One week after cell implantation, mice were randomly assigned to three experimental groups (n = 5 each) and then treated with CGK062 (50 mg/kg/day or 100 mg/kg/day) by i.p. for 4 weeks. Tumors were measured with a caliper and their volumes were calculated using formula, width2×length×0.52.Animal experiment was performed with approval of the Animal Study Committee of Inje University (Permit Number: 2008-051). PC3 prostate cancer cells -Decursinol was prepared form the roots of A. Gigas and was added to a solution of caffeic acid  in pyridine  was added acetic anhydride  at room temperature. The reaction mixture was stirred for 1 day and extracted with ethylacetate/H2O (1∶1) solution. The organic layer was dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude mixture was solidified with ethylacetate/n-hexane (1∶1) solution to give 3--acrylic acid in 74.8% yield (5.48 g). Thionyl chloride  was added to a solution of 3--acrylic acid  in anhydrous benzene (18 ml) and catalytic amount of DMF (1 drop). The reaction mixture was refluxed for 6 h and then allowed to cooling to room temperature. After removal of solvent by rotary evaporation, the residue (intermediate A) was used next reaction without further purification. To a solution of decursinol  and pyridine  in 30 ml of anhydrous methylene chloride was added a solution of intermediate A in 20 ml of anhydrous methylene chloride. After stirring for 5 h at room temperature, the solvent was removed in vacuo to give intermediate B, which was purified by a silica gel column chromatography in 84.5% yield (2.08 g). White Solid, mp: 92°C, Rf = 0.27 (1∶1 n-hexane-ethyl acetate); [α]25 D+28.0 ; 1H NMR: δH 7.60, 7.59, 7.38-7.33, 7.25-7.17, 6.82, 6.35, 6.23, 5.19, 3.24, 2.93, 2.29, 2.29, 1.42, 1.38; 13C NMR  δC 168.5(OC = O-6'), 168.4(OC = O-7'), 166.2(C-1'), 160.8(C-2), 157.1(C-9a), 155.0(C-10a), 145.0(C-3'), 144.3(C-6'), 144.3(C-7'), 143.7(C-4), 133.7(C-4'), 130.2(C-5), 127.4(C-9'), 124.9(C-8'), 123.9(C-5'), 119.4(C-2'), 116.7(C-5a), 113.8(C-3), 113.7(C-4a), 104.6(C-10), 77.5(C-8), 70.9(C-7), 28.2(C-6), 25.0(CH3-8), 23.6(CH3-8), 20.4(OCOCH3-6'), 20.4(OCOCH3-7'); ESI-MS: m/z = 493 [M+H]+. Anal. Calc. for C27H24O9: C, 65.95; H, 4.91. Found: C, 65.88; H,4.90. To a solution of intermediate B  in 60 ml of acetone was added 3N HCl (3 ml) and the reaction mixture was refluxed for 18 h. After cooling to room temperature, the reaction mixture was evaporated, extracted with ethylacetate/H2O (1∶1) and washed with brine. The washed solution was dried over anhydrous sodium sulfate, and concentrated in vacuo. The desired compound (CGK062) was obtained by short silica gel column chromatography in 93.2% yield (1.31 g). White Solid, mp: 115°C, Rf = 0.36 (1∶2 n-hexane-ethyl acetate); [α]25 D+19.3 ; 1H NMR: δH 9.63, 9.10, 7.90, 7.46, 7.45, 7.00, 6.99, 6.81, 6.71, 6.25, 6.22, 5.14, 3.24, 2.88, 1.35, 1.31; 13C NMR : δC 166.9(C-1'), 162.3(C-2), 156.6(C-9a), 154.0(C-10a), 147.0(C-7'), 146.2(C-6'), 144.2(C-3'), 143.9(C-4), 128.8(C-5), 126.8(C-4'), 122.5(C-9'), 116.0(C-5a), 115.3(C-8'), 114.3(C-2'), 114.1(C-3), 112.8(C-4a), 112.7(C-5'), 104.7(C-10), 76.8(C-8), 70.0(C-7), 27.8(C-6), 24.8(CH3-8), 23.3(CH3-8); ESI-MS: m/z = 409 [M+H]+. Anal. Calc. for C23H20O7: C, 67.64; H, 4.94. Found: C, 67.58; H, 4.95.(TIF)Click here for additional data file.Figure S2CGK062 is not cytotoxic to HEK293 reporter cells and does not affect other pathways. (A) HEK293 reporter cells were incubated with CGK062  for 15 h in the absence or presence of Wnt3a-CM, and cell viability was measured by Cell titer-Glo (Promega). (B) HCT116 cells were co-transfected with p53-FL and pCMV-RL plasmids and incubated with CGK062 in the presence or absence of doxorubicin, an activator of p53 pathway, for 15 h. Luciferase activities were measured 39 h after transfection and reported as relative light unit (RLU) normalized to Renilla luciferase activities. (C) HEK293 cells were co-transfected with NF-κB-FL and pCMV-RL plasmids and incubated with CGK062 in the presence or absence of aspirin, an activator of NF-κB pathway, for 15 h. Luciferase activities were measured 39 h after transfection and reported as relative light unit (RLU) normalized to Renilla luciferase activities.(TIF)Click here for additional data file.Figure S3CGK062 induces β-catenin degradation through a mechanism independent of the lysosomal degradation pathway. Cytosolic proteins prepared from HEK293 reporter cells, which were incubated with vehicle (DMSO) or CGK062 (25 µM) in the presence or absence of Wnt3a CM, exposed to NH4C1 (10 mM), were subjected to Western blotting with anti-β-catenin antibody.(TIF)Click here for additional data file.Figure S4The N-terminus of β-catenin is required for CGK062-mediated β-catenin degradation. HEK293 reporter cells were transfected with wild-type β-catenin (A), β-catenin S45A (B) or β-catenin S37A (C) plasmids, incubated with CGK062 for 15 h, and then luciferase activities were measured.(TIF)Click here for additional data file.Figure S5CGK062 induces the translocation of PKCα to the membrane. The cellular location of PKCα in HEK293 cells was determined by immunofluorescence analysis. HEK293 reporter cells were incubated with vehicle (DMSO) or CGK062 (25 µM) for 15 h. After fixation, the cells were stained with anti-E-cadherin and anti-PKCα antibody and observed at 400× magnification.(TIF)Click here for additional data file.Figure S6CGK062 phosphorylates Ser45 residues of β-catenin. HEK293 reporter cells were incubated with CGK062 for 15 h in the absence or presence of Wnt3a-CM. Cytosolic fractions were prepared and subjected to western blot analysis with anti-phospho-p45-β-catenin or anti-β-catenin antibody. The same amount of β-catenin was loaded in each lane.(TIF)Click here for additional data file.Figure S7CGK062 promotes β-catenin phosphorylation and degradation. Cytosolic proteins prepared from HEK293 reporter cells, which were incubated with vehicle (DMSO) or CGK062 in the presence of Wnt3a-CM for indicated periods of time, were subjected to Western blotting with anti-β-catenin antibody (A) or anti-phospho-p33/37/41-β-catenin antibody (B). In (B), the same amount of β-catenin was loaded in each lane.(TIF)Click here for additional data file.Figure S8CGK062 promotes PKCα-mediated β-catenin degradation in SW480 cells. SW480 cells were incubated with CGK062 (25 µM) and BIM (10 µM) for 15 h. Cytosolic fractions were prepared and subjected to Western blot analysis with anti-phospho-p33/37-β-catenin or anti-β-catenin antibodies. The same amount of β-catenin was loaded in each lane.(TIF)Click here for additional data file.Figure S9CGK062 does not affect the expression of PKCα. PC3 (A), SNU475 (B), and SW480 (C) cells were incubated with the vehicle (DMSO) or CGK062 for 15 h and then cell extracts were prepared for Western blotting with anti-PKCα antibody. To confirm equal loading, the blots were reprobed with anti-actin antibody.(TIF)Click here for additional data file.Figure S10CGK062 inhibits the proliferation of CRT-positive cells. Cells were incubated, in the indicated concentrations of CGK062, for 48 hrs in 96-well plates. Cell viability was examined using the CellTiter-Glo assay (Promega). To calculate the inhibition of growth, the value at time 0 was subtracted.(TIF)Click here for additional data file.