Preoperative chemoradiotherapy (CRT) is the cornerstone of treatment for locally advanced rectal cancer (LARC). Although high local control is achieved, overall rates of distant control remain suboptimal. Colorectal carcinogenesis is associated with critical alterations of the Wnt/β-catenin pathway involved in proliferation and survival. The aim of this study was to assess whether CRT induces changes in the expression of β-catenin/E-cadherin, and to determine whether these changes are associated with survival.2) or capecitabine (825 mg/m2) during RT treatment, followed by total mesorectal excision (TME). Disease-free survival (DFS) was analysed using the Kaplan-Meier method and a multivariate Cox regression model was employed for the Multivariate analysis.The Immunohistochemical expression of nuclear β-catenin and membranous E-cadherin was prospectively analysed in tumour blocks from 98 stage II/III rectal cancer patients treated with preoperative CRT. Tumour samples were collected before and after CRT treatment. All patients were treated with pelvic RT (46–50 Gy in 2 Gy fractions) and 5-fluorouracil (5FU) intravenous infusion . After a median follow-up of 25 months, patients that overexpressed nuclear β-catenin after CRT showed poor survival compared with patients that experienced a decrease in nuclear β-catenin expression . In the multivariate analysis for DFS, increased nuclear β-catenin expression after CRT almost reached the cut-off for significance (p = 0.06).In our study, preoperative CRT for LARC induced significant changes in nuclear β-catenin expression, which had a major impact on survival. Finding a way to decrease CRT resistance would significantly improve LARC patient survival. Preoperative chemoradiotherapy (CRT) is the standard treatment for locally advanced rectal cancer (LARC). However, although high local control is achieved with multi-modality treatment, overall rates of distant control remain suboptimal in 30% of patients, and it is considered the leading cause of treatment failure.Nowadays, molecular pathways of tumour resistance in rectal cancer are not fully understood and research focused on these mechanisms is urgently needed to improve patient survival. Colorectal carcinogenesis is associated with critical alterations of the Wnt/β-catenin signalling pathway. β-catenMutations occur in APC as an early event in the carcinogenesis of colorectal cancer, which results in an accumulation of β-catenin in the cytoplasm and translocation of β-catenin to the nucleus. Nuclear β-catenin binds to transcription factors of the high-mobility-group (HMG) box TCF/LEF family and results in enhanced proliferation and survival. β-catenin forms an adherens complex with E-cadherin, which is regulated by tyrosine phosphorylation and whicThe association between the expression of nuclear β-catenin and patient survival has been previously described; however, the conclusions vary dramatically. Lugli et al. studied more than 1000 colorectal tumours initially treated with surgery, showing that an increase in nuclear β-catenin and a loss of membranous E-cadherin expression were independent prognostic factors for poor survival. HoweverRadiation has been shown to induce different molecular changes in both cellular RNA and proteins, resulting in increased proliferation, migration and cell cancer invasiveness. These effects counteract cell death, rendering the tumour more aggressive and decreasing the efficacy of radiation. Some stIn the present study, we aimed to prospectively evaluate changes in the expression profile of β-catenin and E-cadherin after CRT and the impact on survival in LARC patients treated with combined RT and 5-fluorouracil based CT.Between January 2008 and December 2010, 98 patients with stage II-III (T2-T4 and/or N1-N2) rectal adenocarcinoma who were candidates for preoperative RT combined with CT were prospectively recruited in two centres.2) or capecitabine (825 mg/m2) during RT treatment, followed by total mesorectal excision (TME) 6 weeks after CRT treatment. Local response to CRT was pathologically staged using criteria described by Mandard et al.[2 once a day for 5 days repeated every 21 days, or capecitabine: 4 cycles of 1250 mg/m2 every 12 h for 14 days).Pretreatment evaluation included a complete history and physical examination with a digital rectal examination, colonoscopy with biopsy, abdomen and pelvic scan, chest X-ray, and magnetic resonance image (MRI) of the pelvis. Additionally, in 40% of patients, an endorectal ultrasound was performed. All patients were treated according to the routine protocol with pelvic RT (46–50 Gy in 2 Gy fractions) and 5-fluorouracil (5FU) intravenous infusion  and during surgery (post-CRT). Samples were embedded in paraffin for immunohistochemistry (IHC) and serial cross-sections of each tumour sample were cut and stained with hematoxylin and eosin (H&E). β-catenin and E-cadherin IHC was performed on formalin-fixed, paraffin-embedded (FFPE) tissue. For the qualitative detection of β-catenin  and E-cadherin  a Dako Autostainer  was used. E-cadherin and nuclear β-catenin were examined by staining consecutive sections of each sample.To study the expression of these proteins before CRT, a number of endoscopic biopsies ranging between 5 and 10 per tumour were fixed in formalin and embedded in paraffin. To investigate the expression after CRT, different paraffin blocks were obtained. After H&E staining, the block with the most representative part of the tumour was selected. Thus, in every section, the central and peripheral parts of the tumour were considered in order to measure the protein expressions. Two colon cancer sections known to be β-catenin and E-cadherin positive were used as positive controls, and omission of primary antibody was used as the negative control. The expression of β-catenin and E-cadherin were semi-quantitatively evaluated independently by two different pathologists without knowledge of the clinical and pathological parameters of the patients.β-catenin expression in the nucleus was evaluated, and the percentage of tumour cells that expressed β-catenin was determined. We calculated the ratio between the number of tumour cells that expressed β-catenin and the whole number of tumour cells in the tissue section, before and after CRT Figure . The expE-cadherin expression in the membrane was evaluated based on the percentage of tumour cells that expressed E-cadherin Figure . The expTo ascertain the tumours that presented changes in the expression of β-catenin, we compared the expression levels of β-catenin before and after CRT. Changes in expression were categorised as follows: increase ; decrease ; or equal (no change in category). To assess the differential expression of E-cadherin between pre-CRT and post-CRT samples, changes were categorised as follows: increase ; decrease  or equal (no change of category).Patients, type of treatment and disease characteristics were tabulated by means of frequency tables. Qualitative variables are expressed as a percentage with a 95 confidence interval of the percentage, and quantitative variables are expressed as the median and range. The association between qualitative dichotomised data of protein expression and clinico-pathological prognostic factors were compared using the chi-square test and Fisher’s exact test when appropriate. The Wilcoxon paired test was used for paired samples to compare pre-CRT and post-CRT protein expression levels. The end points of interest were tumour relapse and disease-free survival (DFS). DFS was defined as the time from first treatment to first documented relapse, secondary tumour or death by any cause. To investigate the pattern of occurrence over time of any of the aforementioned end points, descriptive analyses were carried out by estimating Kaplan-Meier survival curves, whereas inferential analyses relied on cumulative hazards. The threshold for significance for two-sided analysis was set to p > 0.05. Multivariate survival analysis was conducted using a multivariate Cox regression model. P values below the conventional 5% threshold were regarded as significant. All of the analyses were conducted using R and SPSS  version 15.0 software.http://www.wma.net/en/30publications/10policies/b3/index.html). All subjects provided informed consent for study inclusion, and the study was approved by our hospitals’ Ethics Committees .This study was carried out in compliance with the Declaration of Helsinki . After CRT, 44 patients (45%) presented a TRG 1–2, and 54 patients (55%) a TRG 3–5. After preoperative treatment, 28 patients (29%) presented lymph node metastases compared with 55 patients (56%) of lymph node metastases detected by imaging tests before CRT (P < 0.05). Table Of the 98 patients initially included in the study, a total of 69 were fully assessable in terms of availability of the tumoral specimen pre- and post-CRT. Twenty-nine patients were excluded from the analysis for several reasons: 17 patients presented a complete response (TRG 1), and 12 patients harboured only a few residual tumour cells that could not be assessed for IHC (5 patients with TRG 2 and 7 patients with unknown TRG). Clinico-pathological data of the 69 patients and distribution of the scores are described in Table Preoperative CRT significantly increased nuclear β-catenin expression . No significant changes in the expression of E-cadherin were observed after preoperative treatment , we observed 22 recurrences (23%): 6 (6%) locoregional failures and 16 (16%) distant failures . No significant association was found between disease recurrence and nuclear β-catenin or E-cadherin expression at diagnosis (p = 0.4).We analysed the pattern of recurrence in association with the increase or decrease of nuclear β-catenin expression after CRT. Of the 69 patients included, we observed 20 recurrences (29%): 5 (7%) locoregional failures and 15 (22%) distant failures . Interestingly, of the 20 patients with recurrent disease and available tumoral sample, 19 (95%) presented an increased nuclear expression after CRT. 100% of patients with metastatic disease presented an increase in nuclear β-catenin expression after CRT. On the other hand, considering all the patients that presented a decrease in the expression of nuclear β-catenin after CRT, 92% were free of disease at 3 years (p = 0.03).Of 98 patients initially included, with a median follow up of 25 months (range 5 to 58 months), 13 patients had died: 6 (46%) because of primary cancer and 7 (54%) for other causes. The 3-year OS and DFS rates were 90% and 78%, respectively.No differences in survival were observed in patients with high nuclear β-catenin compared with those with low nuclear β-catenin    and a trend in poor survival compared with those who experienced decreased nuclear β-catenin expression after CRT (p =0.06).Although RT is a major modality in the treatment of cancer, little is known about the molecular changes induced by RT with or without CT. Radiation has been shown to induce different molecular mechanisms to counteract cell death, and several preclinical studies have shown that radiation may promote proliferation, migration and tumour cell invasiveness, which could offset the therapeutic effects of radiation. β-catenOur results have shown that preoperative CRT significantly increases nuclear β-catenin expression in tumour cells, which confers significantly poorer survival compared with those who experienced a decrease in nuclear β-catenin expression (p = 0.02). This finding almost reached the cut-off for significance in multivariate analysis (p = 0.06). Moreover, 93% of the patients who presented with recurrent disease also showed an increase in the expression of nuclear β-catenin (p = 0.03). On the other hand, patients who experimented a decrease of nuclear β-catenin expression after CRT showed an excellent prognosis, with 3 year DFS of 92% vs. 29% . To the best of our knowledge, this study provides the first clinical evidence to support the hypothesis that preoperative CRT in LARC increases nuclear β-catenin expression in tumour cells, which confers a significantly higher risk of recurrence and poor survival.In accordance with other series, our results show that metastatic recurrence is the main pattern of recurrence for our patients and isolated locoregional recurrence occurs rarely after TME plus RT. NowadayPrevious prognostic data on nuclear β-catenin expression at diagnosis in colorectal cancer have shown conflicting results,8. Our rOur study implies some difficulties that should be mentioned. Characterising a tumour that has been treated with CRT is a challenge for several reasons: patients with TRG1 do not show residual tumour cells after CRT, and no tumoral tissue is available for the analysis. Furthermore, in some cases, preoperative CRT leads to histological changes with no gross tumour visible in the mucosa or a scarce number of cells that may make analysis difficult. MoreoveThese factors render evaluation of the number of cells that harbour nuclear β-catenin difficult, hindering a comparison of the pre- and post-treatment expression of this protein in the same tumour. For all these reasons, our results should be taken with caution and should be confirmed with further studies.However, some strengths of our study include the homogeneity of our treatment approach, the prospective design, and the assessment by two independent pathologists. Despite the limited sample size, the poor prognostic value of nuclear β-catenin after CRT reached statistical significance.In summary, our study provides the first evidence that preoperative CRT in LARC patients induces increased nuclear β-catenin expression in tumour cells and confers poor survival compared with patients who experience decreased nuclear β-catenin expression. Overexpression of nuclear β-catenin after CRT may help identify a subgroup of patients in whom adjuvant therapies may be tested for a better control of systemic disease and an improvement in survival.LARC: Locally advanced rectal cancer; CRT: Chemoradiotherapy; RT: Radiotherapy; DFS: Disease-free survival; OS: Overall survival; IHC: Immunohistochemistry; TRG: Tumoral regression grade.The authors declare that they have no competing interest.JG contributed with the concept, design and draft of the manuscript. IA, LP, MD, SRG, VD, SP contributed with acquisition and analysis of data. AR, ET, JM, EG contributed with the draft of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/192/prepub


We have reported that p120-catenin could regulate β-catenin transcription in lung cancer cells, but the specific mechanism is unclear.In this study, bisulfite sequencing PCR showed that the β-catenin promoter region in SPC-A-1 and LTEP-a-2 lung cancer cell lines has Kaiso binding sites sequences and CpG islands which may combine with Kaiso. The demethylating reagent 5-Aza-2′-deoxycytidine significantly upregulated β-catenin mRNA expression in lung cancer cell lines, whereas expression was significantly reduced following transfection with Kaiso. However, the upregulation of β-catenin mRNA expression after treatment with 5-Aza-2′-deoxycytidine was not reduced by subsequent transfection with Kaiso cDNA. Chromatin immunoprecipitation showed that, in lung cancer cell lines, methylated CpG-dinucleotides sequences combined with Kaiso and the Kaiso binding sites sequence did not. The capacity of Kaiso to combine with p120-catenin isoforms was confirmed by immunoprecipitation.Based on these results, we concluded that Kaiso participates in the regulation by p120ctn of β-catenin mRNA expression in the lung cancer cell lines. P120-catenin (p120ctn) and β-catenin are important members of the catenin family, which bind directly with E-cadherin to form the E-cadherin/catenin complex which regulates cell adhesion Human p120ctn isoforms 1 to 4 differ according to the start codon from which translation is initiated. Additional isoforms are derived from the variable inclusion of exons A, B, and C In our previous study of p120ctn in lung cancer, we not only found expression of p120ctn and its isoforms in lung cancer cells but also, unexpectedly, found that p120ctn regulated β-catenin mRNA expression Kaiso is a member of the broad complex, Tramtrak, Bric à brac/Pox virus and zinc finger (BTB/POZ) subfamily of zinc finger proteins, that was originally identified in a yeast-two-hybrid screen for binding partners of p120ctn Human lung cancer cell lines A549, NCI-H460 (H460), SPC-A-1(SPC) and LTEP-a-2(LTE) were cultured in either DMEM or RPMI 1640 medium  supplemented with 10% fetal calf serum , 100 IU/ml penicillin , and 100 µg/ml streptomycin (Sigma). Cells were grown on sterile culture dishes and passaged every 2 days, using 0.25% trypsin (Invitrogen).The A549 and H460 cell lines were obtained from the American Type Culture Collection . SPC and LTE cell lines were obtained from Shanghai Cell Bank .Cultivated lung cancer cells were treated either with 5-Aza-2′-deoxycytidine (5-Aza-CdR) (Sigma) at different concentrations dissolved in culture medium. methylation specific PCR (MSP) and MTT assays were used to select the 5-Aza-CdR concentration which enables β-catenin promoter CpG island demethylation without significant effects on cell growth (7 µmol/L) which was then used for subsequent demethylation processing and cells were collected after continued culture for 48 h.p120ctn-1A and p120ctn-3A cDNA plasmids and Kaiso cDNA plasmid , and p120ctn-1A and p120ctn-3A cDNA plasmids with C-terminal MYC and DDK Tagged in pCMV6-Entry  were transfected using Lipofectamine 2000 (Invitrogen) into cells, following the manufacturer’s instructions. The empty plasmid was used as a negative control. Protein expression by the transfected cells was confirmed by Western blot analysis.5′-CATCCGGAAGAAACTGGT-3′; 5′-TCCCACA AAGCCAACTC-3′ GAPDH: 5′-TACTAGCGGTTTTACGGGCG-3′; 5′-TCG AACAGGAGGAGCAGAGAGCGA-3′ After electrophoresis on a 1.5% agarose gel, the PCR product bands were visualized with BioImaging Systems and quantitated with Labworks Image Acquisition and Analysis Software . The mRNA levels were normalized to the amount of GAPDH mRNA.Total RNA was extracted from cells with TRIzol Reagent (Invitrogen). RT-PCR was performed with the RNA PCR Kit (AMV) Version 3.0 , according to the manufacturer’s instructions. The primer sequences were as follows: β-catenin: Cells were lysed in Radio Immune Precipitation Assay (RIPA)  with 1 mM phenylmethylsulfonyl fluoride . The protein concentration was determined using Coomassie brilliant blue (Sigma), with bovine serum albumin (BSA) (Invitrogen) as the standard. Each sample (50 µg) was separated by 8% or 12% SDS-PAGE for 60 min and transferred  to a polyvinylidene fluoride (PVDF) membrane . After blocking with 1% BSA in Tris-buffered saline-Tween , the membrane was incubated overnight at 4°C with a mouse monoclonal antibody against either p120ctn , Kaiso (H-154) , anti-Kaiso antibody [6F/6F8]  or myc . After incubation with peroxidase-coupled anti-mouse-IgG  at 37°C for 2 h, the protein bands were visualized using ECL  and detected using the BioImaging Systems (UVP Inc.). The relative protein levels were calculated by comparison to the amount of GAPDH protein.△△Ct method. Experiments were repeated in triplicate. GAPDH was used as the reference gene, the sequences of the primer pairs were as described in Section 2.4.Total RNA was extracted from cultivated lung cancer cells using a Total RNA Isolation Classic Kit . For quantitative RT-PCR (qRT-PCR), first-strand cDNA was synthesized from total RNA using the TIANScript RT Kit  according to the manufacturer’s instructions. The resulting cDNA was used as a template for qRT-PCR using an ABI 7900 sequence detector (Applied Biosystems). The relative levels of gene expression were calculated by the 2-Methyl Primer Express (v1.0) was used to analyze the CTNNB1 gene promoter region .DNA from lung cancer cells was extracted using a cell/tissue genomic DNA extraction kit  and then treated with sodium bisulfite using the EZ DNA Methylation-Gold Kit  according to the manufacturer’s instructions. The β-catenin promoter-specific primers for bisulfite sequencing were constructed using promoter sequence data and primer design software. Primer sequences are shown in 7 cells were cross-linked by adding formaldehyde to the culture medium at a final concentration of 1% (v/v). After fixation at room temperature for 10 minutes, L-glycine  was added to terminate the cross-linking reaction. Then cells were washed twice with cold PBS containing Protease Inhibitor Cocktail II, and collected by centrifugation at 720 ×g for 10 minutes at 4°C. Chromatin of fixed cells was solubilized by EZ-Zyme™ Chromatin Prep Kit (Millipore), and the lysates were subjected to electrophoresis on a 2% agarose gel to verify the DNA fragments with an average size of 200 bp to 500 bp in length. The chromatin solution was subjected to EZ-ChIP™ Chromatin Immunoprecipitation (Millipore). The chromatin solution was precleaned by incubation with Protein G agarose for 1 h at 4°C and then incubated with anti-Kaiso antibody - ChIP Grade (Abcam), normal mouse IgG (Santa Cruz Biotechnology), or anti-RNA Polymerase II (Santa Cruz Biotechnology). Antibody complexes were precipitated with Protein G agarose, washed with ChIP wash buffer, and eluted twice with 200 µl elution buffer. Cross-linking was reversed by adding 5 M NaCl and incubating at 65°C overnight. DNA was purified by incubation with 0.5 M EDTA, 1 M Tris-HCl, and proteinase K at 45°C for 2 h and then subjected to PCR amplification using a primer set designed to amplify β-catenin promoter sequences containing the CpG sites and the KBS element.Chromatin immunoprecipitation (ChIP) was performed using a ChIP kit  according to the manufacturer’s instructions with some modifications. Briefly, 4×105′-GAAATTAAATCTCCTGCAATAGACTATA-3′) were amplified from genomic DNA by PCR, inserted between the restriction enzyme sites XhoI and KpnI of the Firefly luciferase reporter vector pGL3-Basic , and validated by sequencing. The construct with a mutation of the KBS sequence (ATGCAGCTGCTCTCC-3′5′-GCGCGCCGAGTC) was generated using mutagenic oligonucleotide primers, according to the manual of the GeneTailor Site-Directed Mutagenesis System (Invitrogen). All the reporter plasmids were purchased from Biotime Biotechnology .The human CTNNB1 gene promoter  and the KBS sequence  into the cell lines with Kaiso-pcDNA3 vector or control vector (empty-pcDNA3 vector) using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, the relative luciferase activity, expressed as the ratio of Firefly to Renilla, was measured by Dual-Luciferase Reporter Assay System , according to the manufacturer’s instructions.Cells were lysed in precooled IP cell lysis buffer  with 1 mM PMSF (Sigma) on ice. The lysate was centrifuged at 12,000 rpm for 10 min at 4°C and the supernatant was quantified by BSA, and equal amounts of total protein were used for immunoprecipitation with the anti-Kaiso (Abcam), mouse IgG , and PBS. Following the addition of ProteinA+G agarose , and slowly shaking for 3 h at 4°C, the immunocomplexes were centrifuged at 2,500 rpm for 5 min, washed with PBS and subjected to SDS-PAGE.P values <0.05 were considered statistically significant.All statistical analyses were performed using SPSS 11.5 for Windows software. Data from cells in different experimental groups were compared using the independent samples t-test or ANOVA. Analysis of the CTNNB1 gene promoter region  revealed the presence of two CpG islands , and treEach lung cancer cell line was transfected with the Kaiso cDNA plasmid and Kaiso protein expression confirmed by Western blot analysis at different time points  for LTE.Based on our experimental results, we found that the regulatory effects of Kaiso on β-catenin transcription are related to methylation, and we observed that the β-catenin promoter region contains methylated CpG dinucleotide sequences. Therefore, we investigated the presence of methylated CpG dinucleotide sequences within the β-catenin promoter region and the effects on Kaiso binding in lung cancer cell lines. ChIP studies showed that Kaiso binds with the β-catenin promoter region through methylated CpG dinucleotide sequences. We also identified the KBS sequence within the β-catenin promoter region, although specific binding of this sequence with Kaiso was not detected by ChIP. Therefore, we speculate that Kaiso binds to the β-catenin promoter region via methylated CpG dinucleotide sequences, rather than the KBS sequence  for LTE.Kaiso, as a binding partner of p120ctn, exhibit dual-specificity DNA binding: Kaiso recognizes a sequence-specific DNA consensus, TCCTGCnA, as well as methylated CpG-dinucleotides, in addition to its ability to combine with p120ctn. In the present study, we introduced p120ctn isoforms 1A and 3A into lung cancer cell lines by transfection with cDNA plasmids containing a DKK-MYC tag and the effects of transfection were confirmed by Western blot analysis  for LTE.We have previously reported that decreased expression of p120ctn down-regulates β-catenin mRNA expression in the lung cancer cell lines LTEP-a-2(LTE) and SPC-A-1(SPC) Cancers often exhibit aberrant methylation of gene promoter regions, which is linked to abnormal gene transcription However, the proportion and location of CpG islands and specifically, the presence of CpG dinucleotide sequences in the β-catenin promoter region remain to be established. Therefore, we used Methyl Primer Express v1.0 to analyze the CTNNB1 gene promoter region . We identified two CpG islands in the promoter region, containing 189 single CG sites, including 19 CG-dinucleotides. Among the 19 CG-dinucleotides, only one CG-dinucleotide was shown to be methylated by BSP sequencing. We also unexpectedly identified the KBS sequence in the CTNNB1 gene promoter region.Kaiso, which is an important member of the BTB-POZ protein family, has been confirmed with transcriptional repressor function, such as its ability to directly repress canonical Wnt gene targets  Based on these results, we investigated whether the transcriptional repressor function of Kaiso depends on its binding with the KBS sequence or the methylated CpG dinucleotide sequence within the β-catenin promoter region in lung cancer cells. ChIP and Luciferase analysis confirmed that Kaiso combines with the β-catenin promoter region via the CpG dinucleotide sequence rather than the KBS sequence.It is known that Kaiso combines with p120ctn in addition to recognizing and binding specific DNA sequences within the promoter region. Our previous study demonstrated that Kaiso binds to p120ctn in lung cancer cells, and that p120ctn 3 is likely to be the major isoform We then speculated that the mechanism by which decreased expression of p120ctn down-regulated β-catenin mRNA expression in the lung cancer cell lines SPC and LTE is based on the decreased binding of p120ctn with Kaiso. This results in increased combination of Kaiso with the methylated CpG dinucleotide sequence within the β-catenin promoter region. This would result in increased binding of the β-catenin promoter region with Kaiso and thus increased transcriptional inhibition of β-catenin.However, it is interesting that we also found that p120ctn isoform 1A noticeably increased the expression of β-catenin mRNA in the lung cancer cell lines A549 and NCI-H460 (H460)


Objective. To demonstrate the role of Wnt/β-catenin canonical pathway in postmenopausal osteoporosis by evaluating serum β-catenin levels in patients with postmenopausal osteoporosis and analyzing their possible relationship with serum OPG, RANKL, the ratio of RANKL/OPG, sclerostin, and bone turnover markers. Methods. 480 patients with postmenopausal osteoporosis and 170 healthy postmenopausal women were enrolled in the study. Serum β-catenin, OPG, RANKL, and sclerostin levels were measured by enzyme-linked immunosorbent assay. Bone status was assessed by measuring bone mineral density and bone turnover markers. Estradiol levels were also detected. Results. Serum β-catenin levels were lower in postmenopausal osteoporotic women compared to nonosteoporotic postmenopausal women . Serum β-catenin was positively correlated with osteoprotegerin  and negatively correlated with the ratio of RANKL/OPG, body mass index, and sclerostin  in patients with postmenopausal osteoporosis. Conclusion. The results indicate that lower serum β-catenin and concomitantly higher ratio of RANKL/OPG may be involved in the pathogenesis of postmenopausal osteoporosis. Functional communication between RANKL/RANK/OPG system and Wnt pathways plays an important role in postmenopausal osteoporosis. The Wnt/β-catenin pathway is essential for normal osteogenesis [β-catenin canonical pathway is modulated by a number of factors that include Dickkopf (Dkk-1) and sclerostin, which compete with the Wnt/β-catenin for binding to LRP5/6, disrupting (Dkk-1) or antagonizing (sclerostin) LRP5/6 mediated Wnt signaling [κB ligand (RANKL) is highly expressed on the surface of bone marrow stromal cells (BMSCs) and preosteoblasts [β-catenin in colon cancer cells [β-catenin antagonist, stimulates expression of RANKL [β-catenin signaling and RANKL/RANK/OPG system in PMOP.Postmenopausal osteoporosis (PMOP) is a highly prevalent disease, characterized by reduced bone mass, leading to increased bone fragility and fracture risk, caused by estrogen deficiency. A lot of recent reports provide evidence that the Wnt/ogenesis –3. The Wignaling . Receptoeoblasts , 8. de Ter cells . Recent of RANKL , 11. Basβ-catenin canonical pathway in postmenopausal osteoporosis, we evaluated the levels of serum β-catenin, OPG, RANKL, sclerostin, and bone turnover markers in postmenopausal osteoporotic patients and compared them to those in postmenopausal nonosteoporotic women. In addition, we analyzed the relationships of β-catenin with OPG, RANKL, the ratio of RANKL/OPG, sclerostin, and bone turnover markers.To demonstrate the role of Wnt/Our cross-sectional study included 480 postmenopausal women with osteoporosis and 170 healthy postmenopausal women as a control group. According to the principle of statistics, the PMOP patients were enrolled from 4 hospitals  by advertisement recruiting during the period of December 2009 and March 2012 in China. Eligible PMOP participants were required to have a natural menopausal history of 2–10 years and a BMD T-score of <−2.5 at the lumbar spine using dual-energy X-ray absorptiometry (DXA). Exclusion criteria were as follows: (1) treatment with calcitonin, bisphosphonates, raloxifene, estrogen, or estrogen/progestogens within 12 months, (2) coexistence of any other metabolic bone disease except for osteoporosis, (3) severe chronic disease, including malignancy, (4) medication that could affect bone metabolism, (5) previous radiation therapy, and (6) abnormal liver and kidney function tests. Healthy postmenopausal women were enrolled from physical examination center in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. After a medical examination, they were excluded from osteoporosis and other diseases affecting bone metabolism. The protocol was approved by the responsible Clinical Trial Ethics Committee. Written informed consent was obtained from each participant.In all subjects, we measured height and weight and calculated body mass index (BMI) using the Quetelet formula (weight in kilograms divided by the square of height in meters). Bone mineral density (BMD) was measured for the anteroposterior lumbar spine (L1–L4) by dual-energy X-ray absorptiometry (DXA) . A control phantom was scanned every day, and all DXA measurements were performed by experienced operators in every hospital. Osteoporosis was defined as a T-score of <−2.5 at the lumbar spine.Venous blood samples were taken in the morning between 8:00 AM and 9:00 AM after an overnight fast. The samples were centrifuged for 10 minutes at approximately 3000 r/min within 30 minutes, and the serum was separated and stored at −80°C prior to analysis. β-catenin, RANKL, OPG, and sclerostin levels were measured by enzyme-linked immunosorbent assay . According to the manufacturer's instructions, the minimum detectable amount of human β-catenin and OPG kit was less than 1.0 pg/mL. The minimum detectable amount of human sclerostin and RANKL kit was less than 1.0 pmol/L. Intra- and interassay coefficients of variation were less than 15%. No significant cross-reactivity or interference was observed. Serum β-isomerized C-terminal crosslinking of type I collagen (CTX), intact N-terminal propeptide of type I collagen (PINP), N-mid fragment of osteocalcin (N-MID-OT), and 25-hydroxyvitamin D (25(OH)D) levels were measured using automated Roche electrochemiluminescence system. Serum estradiol levels were measured by electrochemiluminescence system in the Department of Nuclear Medicine. Intra- and interassay variations were <6% in our laboratory.Serum β-catenin and other parameters between patients and controls were compared by independent-samples t-test. Spearman's coefficient of correlation was used for correlation between serum levels of β-catenin and other parameters in both groups. Multiple regression analysis was used to determine the influence of one independent variable after correcting for others. All statistics were analyzed using SPSS 16.0 software. A P value of less than 0.05 was considered statistically significant in all tests.All data for continuous variables were described as mean ± SD. Serum levels of β-catenin, sclerostin, and RANKL levels between two groups were significant, with P < 0.001.The main characteristics and laboratory data of the study population were listed in β-catenin levels and various parameters in patients and controls. As shown in β-catenin levels were positively correlated with OPG and negatively correlated with sclerostin, the ratio of RANKL/OPG, BMI, and BMD. No correlation between serum β-catenin and age, estradiol, N-MID-OT, 25(OH)D, CTX, PINP, and RANKL was found. No correlation between serum β-catenin and other parameters was observed in control group. A multiple regression analysis was performed to check correlations among the potential determinant variables. β-catenin was designated as the dependent variable, whereas age, BMI, BMD, estradiol, N-MID-OT, 25(OH)D, CTX, PINP, sclerostin, OPG, and RANKL were included as independent variables. In this analysis, BMD, PINP, sclerostin, and OPG were found to be independent predictors of serum β-catenin levels in PMOP, after adjusting for age, BMI, estradiol, N-MID-OT, 25(OH)D, CTX, and RANKL  [β-catenin serum levels lower than controls. β-catenin, a pivotal signaling molecule of the Wnt pathway, has been shown to be important in osteoblast differentiation, proliferation, and apoptosis [β-catenin increased Wnt signaling activity [β-catenin levels may reflect the lower Wnt signaling activity in our PMOP cohort. Recent evidence has indicated that the Wnt/β-catenin pathway plays an important role in skeletal development and growth [β, and promote the accumulation of β-catenin in osteoblasts [β-catenin translocates into the nucleus and together with TCF/LEF induces the expression of OPG to inhibit RANK-RANKL-mediated osteoclastogenesis [β-catenin and the ratio of RANKL/OPG. It suggested that some cross-links were present between Wnt/β-catenin signaling pathway and RANKL/RANK/OPG system and provided evidence that increased RANKL/OPG expression was related to reduction of Wnt/β-catenin signaling activity in PMOP.The cross-sectional study confirms that s (T2DM) , and indpoptosis . Overexpactivity . Lower βd growth , 22, pard growth , 24. Thed growth . Loss-ofd growth , 26. Caneoblasts . The accogenesis . In our β-catenin and sclerostin, which in fact agrees with a major contribution of sclerostin to the impairment of the Wnt/β-catenin signaling pathway in this setting. The pattern was unchanged in a multiple regression model. Sclerostin, an osteocyte-derived and -secreted glycoprotein, has been shown to influence the activity of Wnt signaling pathways [We also found a significant negative correlation between pathways . The mecpathways , 30–33. pathways –37.β-catenin and CTX and the PINP was observed in both the PMOP group and the control group. Furthermore, we observed no correlation of serum β-catenin levels with age. Different from our findings, the recent report by Gaudio et al. [β-catenin levels with age and serum sclerostin levels in T2DM patients and with age in controls. These discrepancies may be related to differences in the population characteristics. Our cohort in the study was limited to postmenopausal women with the age of 50.5–65.5 years old.Both CTX and PINP significantly increased in PMOP cohort, confirming that higher bone turnover took place in this population compared to controls. However, no association between the o et al.  showed tβ-catenin levels with lumbar spine BMD. For the contradictory phenomenon, the rational explanation would be that β-catenin is increased in a compensatory manner. Another interesting thing was that we observed a negative correlation between serum β-catenin and BMI. The result may be related to the role of Wnt/β-catenin signaling on inhibiting adipogenesis of mesenchymal precursors [In our PMOP cohort, it is difficult to explain the negative correlation of serum ecursors , 39.β-catenin with OPG and the ratio of RANKL/OPG. We hypothesized that the function communication between Wnt/β-catenin signaling and RANKL/RANK/OPG system was not established in postmenopausal non-osteoporosis women.In our control group, we found no associations of β-catenin, RANKL/OPG, and other variables cannot be established. We analyzed serum β-catenin, RANKL/OPG levels which may not be sensitive enough to reflect their expression in bone cells. The study has some limitations. It was a cross-sectional design, and the causative nature of the associations between β-catenin signaling on bone metabolism has stimulated a number of studies on the effects of this signaling on treatment of PMOP. However, the effects of Wnt signaling on bone metabolism and the involved molecular mechanisms remain unclear. According to our findings, functional communication between RANKL/RANK/OPG system and Wnt/β-catenin signaling pathway plays an important role in postmenopausal osteoporosis. Targeting the Wnt/β-catenin signaling to change the ratio of RANKL/OPG to alter bone turnover can potentially provide an approach for postmenopausal osteoporosis therapy.Given the existence of multiple pathogeneses of PMOP, the recognition of the role of Wnt/


Computational biology educators are leading and raising the next generation of scientists and, in doing so, are in need of new tools, methods, and approaches. The need for education in science, and in computational biology in particular, is greater than ever. Large datasets, -omics technologies, and overlapping domains permeate many of the big research questions of our day. PLOS Computational Biology originally created the Education section to highlight the importance of education in the field PLOS Computational Biology Education section. In our minds, educational initiatives in computational biology and bioinformatics serve two important goals: to communicate digital biology with each other, and to educate others on how best to do this. These are themes we practice as educators in our university teaching, in our involvement with the bioinfomatics.ca workshops series, and in our outreach efforts. We are very excited to continue Fran's great vision as we continue her work with the PLOS Computational Biology staff.The etymology of the word “education” in Wikipedia is enlightening: “a rearing” and “I lead forth” .These initiatives help to extend computational biology beyond the domain of specialized laboratories. Researchers, at all levels, need to keep themselves up-to-date with the quickly changing world of computational biology, and trainees need programs where bioinformatics skills are embedded so they can have comprehensive training. New bioinformatics workflows can be adopted more widely if education efforts keep pace. As previously pointed out We would like to involve the community in this endeavor. With this editorial, we are calling out to educators and researchers who have experience in teaching, specifically, those keen to raise the expectations and the inquisitiveness of the next generation of biologists. The Education collection will continue to publish leading edge education materials in the form of tutorials that can be used in a “classroom” setting . We will continue to encourage articles set in the context of addressing a particular biological question and, as mentioned above, we welcome new “primers” and “quick guides.” We will also be inviting tutorials from the various computational meetings. A new category of papers that is in the pipeline for the Education collection is the “Quick Tips” format, the first of which was just published We also hope, and plan, to incorporate new thinking and perspectives in the greater field of education of computational biology and bioinformatics. For example, articles that highlight the use of new tools such as those used in cloud computing or methods for using third and fourth generation sequencing technologies are encouraged. We would also like to see articles that incorporate best practices in teaching, including the use of new media, flexible online teaching tools, and the use and re-use of large well-defined data sets that are computed on in classes, courses, and programs. We encourage articles that highlight new types of training initiatives, the use of workflows to help students in the path to reproducibility in science, and open course materials (open lecture notes and open course notes and datasets for exercises) that reach more learners.ploscompbiol@plos.org with your ideas for the kind of articles you would like to see in the PLOS Computational Biology Education section. We hope to see you in the classroom soon, where we learn together.In the end, the Education section belongs to the community and thus comes with responsibilities. We need to identify the gaps and the material with which we want to educate ourselves; we need to recognize and encourage great teachers and writers to communicate openly about what works best with the specific methods. We invite you to contact us via Joanne A. Fox  has a PhD in Genetics from the University of British Columbia (UBC). As a faculty member at the Michael Smith Laboratories and in the Department of Microbiology and Immunology at UBC, she is involved in a range of education and outreach initiatives at the undergraduate and secondary school levels, and teaches a variety of courses. She is a former instructor and current review committee member of the Canadian Bioinformatics.ca Workshops.B.F. Francis Ouellette (@bffo on Twitter) did his graduate studies in Developmental Biology and is now an Associate Professor in Cell and Systems Biology at the University of Toronto, as well as a senior scientist and Associate Director of Informatics and Biocomputing at the Ontario Institute for Cancer Research. He was one of the founders and is still the scientific director and an instructor for the Canadian Bioinformatics.ca Workshops.The authors have worked together in the past, and have known each other for more than 15 years.


The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells. Therefore, understanding the events that promote tumor invasion and metastasis will help us prevent the spread of malignant tumors especially in case of early stage and perhaps oligometastatic disease. As a member of adherens junction, p120-catenin (p120ctn) plays an important role in cell-cell adhesions and loss of p120-catenin expression results in destabilization of the cadherin-catenin complex thereby promoting tumor invasion and metastasis p120-catenin (CTNND1) transcription in NSCLC cells through interaction with transcription factor SP1 p120-catenin (CTNND1) promoter, thereby promoting tumor progression, invasion and metastasis.c-Crk (CRK) belongs to a family of widely expressed adaptor proteins that are involved in signal transduction from a variety of receptors and oncoproteins  itself was also highly phosphorylated mainly on serine and about five percent on tyrosine residues. Several intracellular signaling pathways contribute to CRK phosphorylation. For example, c-Abl phosphorylates Crk on tyrosine 221 causing disassociation of Crk from the Crk-associated substrate (CAS) thereby inhibiting cell migration and promoting apoptosis in normal and malignant cells In addition to CRK overexpression as a proto-oncogene and its role in cell transformation, CRK phosphorylation is also apt to contribute to its biochemical activity. As an adaptor protein, CRK does not contain a catalytic domain however both tyrosine and serine kinase activities have been associated with CRK With regards to CRK serine phosphorylation, two serine/threonine kinases, , members of PAK serine/threonine kinase super family, are known to mediate phosphorylation of CRK family members and possibly participate in CRK mediated JNK activation p120-catenin (CTNND1) in NSCLC cells. CRK can be phosphorylated in both tyrosine and serine residues, which then influences its interaction with downstream targets p120-catenin (CTNND1) transcriptional repression. A close correlation of either serine or tyrosine CRK phosphorylation with p120-catenin expression would indicate the presence of an upstream serine/threonine or a tyrosine kinase that would mediate CRK phosphorylation thereby p120-catenin downregulation. For this purpose, we examined the phosphorylated CRK-II level both on tyrosine 221 and serine 41 and correlated that with p120-catenin levels in a panel of NSCLC and BEAS-2B cells (p120-catenin (CTNND1) transcriptional downregulation through serine phosphorylation of the CRK adaptor protein.We recently reported that CRK mediates transcriptional repression of 2B cells . In casep120-catenin (CTNND1), we decided to examine whether CRK-II serine 41 manipulations might have any effect on the p120-catenin expression level. Therefore, we proceeded to prepare CRK-II serine 41 phosphodeficient and phosphomimetic mutants. A site directed mutagenesis reaction was utilized to replace CRK-II serine 41 with either Glycine [Ser41Gly (phosphodeficient)] or Aspartic acid [Ser41Asp (phosphomimetic)]. A CRK-II construct fused with a Myc-tag was used as the backbone of the site directed mutagenesis reactions therefore, all resulting mutants contained a Myc tag as well. All constructs were checked by sequencing. Subsequently, A549, Rh2 and H157 cells were transiently transfected with the resulting CRK-II mutants and we measured the p120-catenin (CTNND1) promoter activity as well as p120-catenin protein level by western blotting in the transfected cells. The expression levels of CRK-II mutants and the endogenous CRK-II are presented in (p120-catenin (CTNND1) promoter activity was observed in cells expressing phosphodeficient CRK-II. On the other hand, a decrease in p120-catenin (CTNND1) promoter activity was observed in cells expressing phosphomimetic CRK-II serine 41 mutant .In order to further assess the role of CRK serine 41 phosphorylation in the transcriptional regulation of ented in . Compare1 mutant . p120-cap120-catenin (CTNND1) transcriptional regulation, it is not clear whether phosphorylation of CRK-II on serine 41 has any other significant biological relevance. Since expression of CRK has been associated with more aggressive NSCLC tumors Even though we observed CRK-II serine 41 phosphorylation is engaged in In order to examine whether changes in cell proliferation are contributing to the observed biological behavior of the cell lines with CRK-II mutants, cell proliferation rate of the resulting cell lines were measured . Followip120-catenin (CTNND1) transcriptional repression and also promotion of motility and invasiveness in NSCLC cells, we sought to identify the upstream kinase(s) that might mediate CRK-II serine 41 phosphorylation. CRK serine phosphorylation has been reported by several members of the PAK serine/threonine family of kinases Considering the role of CRK-II serine phosphorylation on the p120-catenin (CTNND1) transcriptional repression, we asked whether PAK kinases are in fact involved in p120-catenin (CTNND1) repression. In order to verify this notion, we silenced PAK1 and PAK2 by siRNA and measured the p120-catenin (CTNND1) promoter activity in a dual luciferase assay and also measured p120-catenin protein level by western blotting (p120-catenin (CTNND1) promoter reporter compared to cells transfected with non-silencing siRNA (p120-catenin (CTNND1) promoter activity following siRNA mediated PAK2 silencing. These data further institute a role for PAK1 in transcriptional regulation of the p120-catenin (CTNND1).Considering the involvement of PAK1 kinase in mediating CRK-II phosphorylation on serine 41 and also the role of CRK-II serine 41 phosphorylation in blotting . Following siRNA . Moreoveng siRNA . We did p120-catenin (CTNND1) transcriptional regulation. Accordingly, we silenced PAK1 in A549 cells that stably express CRK-II serine 41 phosphomimetic and phosphodeficient mutants (p120-catenin (CTNND1) transcriptional activity following PAK1 silencing. This effect of PAK1 on p120-catenin (CTNND1) transcriptional activity was abrogated in cells expressing either phosphomimetic or phosphodeficient CRK-II serine 41 mutants suggesting that the inability of CRK-II to alter its phosphorylation status on serine 41 (as a result of inserted mutations) abolishes the effect of PAK1 on p120-catenin (CTNND1) transcription. These findings further supports the role of CRK-II serine 41 phosphorylation on PAK1 mediated p120-catenin (CTNND1) transcriptional regulation. The minor changes in the p120-catenin (CTNND1) promoter activity that are observed in CRK-II serine 41 phosphomimetic mutant expressing cells  could be as a result of the endogenous CRK-II phosphorylation alteration following PAK1 silencing. It is worth mentioning that RNAi mediated silencing of the endogenous CRK does not result is a complete knock down of the endogenous CRK-II  CRK-II serine 41 phosphorylation promotes cell motility and invasiveness and (ii) PAK1 mediates CRK-II serine 41 phosphorylation, we were facing he question whether PAK1 is indeed promotes cell motility and invasiveness through CRK-II seine phosphorylation. To answer this question we examined the motility and invasiveness of A549 cells following simultaneous manipulations of PAK1 and CRK-II serine 41 phosphorylation . As expep120-catenin (CTNND1). Through detailed analysis of the p120-catenin (CTNND1) promoter, the role of transcription factors FOXC2 and SP1 in the p120-catenin (CTNND1) downregulation were recognized. Further analysis of the binding partners of SP1 lead us identify the role of adaptor protein CRK in this pathway p120-catenin (CTNND1) promoter activity, expression level and also the invasive property of NSCLC cells.Understanding the signaling events that promote metastasis in solid tumors will help us pin point therapeutic targets in order to intervene in the metastasis process. Loss of cell-cell adhesions in epithelial cells is one of the earliest steps of tumor spread. Therefore, losing the integrity of adherens junction and its members  plays a pivotal role in the promotion of metastasis. One of the frequent observed events in NSCLC tumors is the p120-catenin downregulation. Since p120-catenin repression in NSCLC is transcriptionally mediated Regarding CRK-I, it seems that higher expression of CRK-I oncoprotein by itself is correlated with tumorigenesis. This observation is in contrast with CRK-II where phospho-CRK-II establishes a strong association with tumorigenesis. A significant increase in CRK-I oncoprotein level and phosphorylated isoform (CRK-II) were observed in NSCLC p120-catenin (CTNND1) and could only identify PAK1 as a regulator of p120-catenin (CTNND1). PAK1 silencing reduced CRK-II serine 41 phosphorylation in A549 and H157 cells and enhanced p120-catenin (CTNND1) promoter activity and protein level in NSCLC cells. Additionally, PAK1 silencing reduced cell motility and invasiveness via CRK-II serine 41 phosphorylation.CRK serine phosphorylation is not well studied although there are anecdotal reports regarding the role of PAK family of serine/threonine kinases in CRK serine phosphorylation PAK1 has a number of downstream targets that regulate actin organization and polymerization, cell proliferation and apoptosis. For instance, Pak1 interacts with filamin A and LIM kinase In summary, these data describe one of the metastatic promoting pathways in NSCLC cells involving PAK1 kinase, CRK-II, SP1 and p120-catenin. In other words, CRK-II phosphorylation seems to play a part in conveying signals downstream of PAK1. In conjunction with other report that highlights PAK1 overexpression in squamous cell NSCLC A549, H157, Rh2 and H358 cells were routinely cultured in RPMI supplemented with antibiotics and 10% heat-inactivated FBS . Immortalized normal human epithelial cells (BEAS-2B) were cultured in BEBM medium supplemented with all the additives .p120ctn promoter construct was transfected into NSCLC cell lines by Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection cells were washed with PBS and lysed using a Branson Sonifier in 1x passive lysis buffer (Promega) at room temperature (RT). Reporter gene expression was assessed by using the Dual-Luciferase Reporter Assay System kit (Promega) according to manufacturer’s instructions in a TD-20/20 Luminometer . We normalized for transient transfection efficiency (i.e. firefly luciferase activity) by co-transfection of a Renilla luciferase expressing control vector (pRL-SV40). All experiments were performed in triplicate and were reported as means ± standard deviation (SD), and each experiment was performed at least twice.A549 and H157 cells were transfected with siRNA against CRK, PAK1 and PAK2. Transfections were performed by using Lipofectamine 2000 (Invitrogen). The siRNA duplex sequences that we used to silencing the above mentioned genes are as follows:CRK: 5′-CUGCUUACCCUGAUUUAUUdtdt-3′5′-AAUAAAUCAGGGUAAGCAUdtdt-3′.PAK1: 5′-GAAAGAGCGGCCAGAGAUUdtdt-3′5′-AAUCUCUGGCCGCUCUUUCdtdt-3′.PAK2: 5′-GGAUUUCUUAAAUCGAUGUdtdt-3′5′-ACAUCGAUUUAAGAAAUCCdtdt-3′.As a control, scrambled non-silencing siRNA was used (Sigma-Aldrich Cat. number SIC002-10NMOL). Concentrations of siRNA were kept at 100 nM among groups. In case of silencing the endogenous CRK, the above siRNA is directed against CRK mRNA in a region outside the open reading frame (approximate siRNA start site 1210 based on reference NM_005206). Therefore using this CRK siRNA has no effect on the CRK expressing plasmids.We used TaqMan probes and SYBR Green methods for measurement of mRNA level following gene silencing. In case of p120ctn, mRNA level in NSCLC cell lines were measured by TaqMan probe; assay IDs: Hs00931670_m1 . For CRK, PAK1 and PAK2 we used SYBR Green method with the following primers sets.PAK1-forward: 5′-CGTGGCTACATCTCCCATTT-3′PAK1-reverse: 5′-TCCCTCATGACCAGGATCTC-3′PAK2-forward: 5′-GAAGAATCCTCAGGCTGTGC-3′PAK2-reverse: 5′-GGGTCACCTATGCTCACGAT-3′CRK-forward: 5′-GCAAGAGAGGGATGATTCCA-3′CRK-reverse: 5′-ATGGGAAGTGACCTCGTTTG-3′Actin-forward: 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′Actin-reverse: 5′-CTAGAAGCATTTGCGGTGGACGATGGA-3′In each cell type, total RNA was extracted by using Trizol method and cDNA was prepared by an RT-PCR reaction using SuperScript-II RT Kit (Invitrogen) and Oligo (dT)s according to the manufacturer’s instructions. For each cDNA sample an internal control (beta actin) was also measured by TaqMan probe. In order to compare the expression level of mRNA between samples, we used the Comparative Ct Method (ΔΔCt). Relative expression of mRNA compared to beta actin in each sample was calculated (ΔCt) and relative expression of mRNA among samples was determined by calculating the difference in (ΔCt) between samples (ΔΔCt). All relative quantitative PCRs were performed, recorded, and analyzed by using the ABI 7300Prism Sequence Detection System . All samples were carried out in triplicate  and repeated at least twice. Controls without template or reverse transcriptase were run in each experiment.5 cells per dish, which resulted in 30–40% confluency 24 hours after plating. Cells were harvested at 24 hours by adding trypsin, pelleted and lysed in 100 µl of lysis buffer  using a Branson Sonifier. Cell debris was collected by centrifugation at 4°C, and protein concentration was measured by the BCA method. Protein was resolved by SDS-PAGE and was transferred to a nitrocellulose membrane. The membrane was blocked with TBS with 5% nonfat powdered milk.NSCLC cell lines were seeded in 10 cm Petri dishes at 5×10Membranes were immunoblotted with the following primary antibodies: p120ctn (BD biosciences Cat. number 610133); E-cadherin (BD biosciences Cat. number 61081); CRK II (Santa Cruz Biotechnology Cat. number sc-289); p-Ser41 CRK-II (Santa Cruz Biotechnology Cat. number sc-130186); PAK1 (Sigma-Aldrich Cat. number SAB4300427) and c-Myc (Santa Cruz Biotechnology Cat. number sc-40).Horse radish peroxidase conjugated secondary antibodies were used for detection of bands by chemiluminescence .We obtained a CRK-II expression plasmid (OriGene Technologies Cat. number RC201701). A sight directed mutagenesis kit (Strategene Cat. number200523) was used according to the manufacturer instructions in order to replace serine 41 with either glycine or aspartic acid by replacing the corresponding nucleotides. The following primer sets were used for site directed mutagenesis.G GCA CCA GCC CCG GGG ACT ATG TGC-3′Ser41Gly Forward: 5′-TCC TGG TGC GGG ACT CGSer41Gly Reverse: 5′-GCA CAT AGT CCC CGG GGC TGG TGC CCG AGT CCC GCA CCA GGA-3′G ACA CCA GCC CCG GGG ACT ATG TGC-3′Ser41Asp Forward: 5′-TCC TGG TGC GGG ACT CGSer41Asp Reverse: 5′-GCA CAT AGT CCC CGG GGC TGG TGT CCG AGT CCC GCA CCA GGA-3′All constructs were checked by sequencing for accuracy of mutagenesis. Subsequently, A549 cells were transfected with the above mentioned plasmids by using Lipofectamine 2000 (Invitrogen). 24 hours after transfection, cells were treated with G418 at 1500 µg/ml in order to generate stable cell lines. The pooled transfected and selected cells were used for the subsequent experiments.5 cells per well and were grown to confluent stage. By using a sterile P1000 pipette tip, a straight scratch was made along the largest diameter of each well and a baseline photomicrograph was taken from this scratch with two different magnifications. A follow up photomicrograph was taken at 24 hours. Photomicrographs of the cells were obtained by a Nikon Eclipse TS100 inverted microscope equipped with a Cannon A510 digital camera. The digital camera was connected with a Max View Plus adaptor (ScopeTronix) to the inverted microscope. An equal area of the photomicrographs from each condition was imported into the Adobe Photoshop software and the resolution of the pictures were set at 25 pixels/inch. The resulting low-resolution pictures were made of countable squares pixels. The surface area of the wound in each condition was measured by counting the number of pixels in each wound area. Experiments were repeated three times and the average wound surface area was compared among the groups.A549 and H157 cells were plated in a 6 well plate dish at 1×105 cells were incubated in the upper chambers in RPMI in the absence of serum in triplicates. As the chemoattractant, RPMI with 10% FBS was used in the lower chambers. Cells were incubated for 24–36 hours in the 37°C incubator. Cells were harvested and stained with Diff-Quik™ kit according to the manufacturer’s instructions. Photomicrographs were taken by an Olympus BH2 microscope equipped with a Cannon A510 digital camera. The invaded cells in five corresponding regions of each Matrigel membrane were counted and the mean of invaded cells were compared among the groups.Matrigel cell invasion assays were performed by using Matrigel Invasion Chamber (BD Biosciences Cat. number354480) according to the manufacturer instructions. 1×10Following plating equal number of A549 cells transfected with CRK-II and CRK-II mutants in triplicates, cells were harvested at 24 hour intervals. Cells were stained by Trypan blue and counted by using a hemocytometer.A photograph of the western blots were imported into Adobe Photoshop software. The photographs were inverted in order to present the protein bands as the brighter signal compared to the background. An equal area of each band was selected and the mean and standard deviation of signal intensity were recorded by the histogram tool of the software. In case the signal intensity of any selected band was reaching the maximum range of the histogram thereby causing signal saturation, the contrast of the image was adjusted to avoid signal saturation.Figure S1Western blots showing the endogenous CRK-II and CRK-II-Myc mutants following transient transfection of CRK-II-Myc mutant constructs in A549, Rh2, and H157 cells. Measurement at 48 hours post transfection.(TIF)Click here for additional data file.Figure S2Western blots showing the endogenous CRK-II and CRK-II-Myc mutants in A549 cells stably transfected with CRK-II mutants. The endogenous CRK-II is diminished by a CRK-II siRNA directed against a sequence outside the open reading frame of CRK. The expression level of CRK-II-Myc mutants as well as the endogenous CRK-II levels are quantified and the ratio of CRK-II mutant/endogenous CRK-II are demonstrated.(TIF)Click here for additional data file.Figure S3Wider views of wound healing assays presented in .(TIF)Click here for additional data file.Figure S4Wider views of the invasion assays presented in .(TIF)Click here for additional data file.Figure S5Line chart representing the growth rate of A549 cells stably expressing pCMV (empty vector), wild type CRK-II (WT), CRK-II (Ser41Gly) or CRK-II (Ser41Asp) mutants.(TIF)Click here for additional data file.


Fuz regulates ciliogenesis and Fuz loss of function studies reveal an array of embryonic phenotypes. However, cilia defects can affect many signaling pathways and, in humans, cilia defects underlie several craniofacial anomalies. To address this, we analyzed the craniofacial phenotype and signaling responses of the −/−Fuz mice. We demonstrate a unique role for Fuz in regulating both Hedgehog (Hh) and Wnt/β-catenin signaling during craniofacial development. Fuz expression first appears in the dorsal tissues and later in ventral tissues and craniofacial regions during embryonic development coincident with cilia development. The −/−Fuz mice exhibit severe craniofacial deformities including anophthalmia, agenesis of the tongue and incisors, a hypoplastic mandible, cleft palate, ossification/skeletal defects and hyperplastic malformed Meckel's cartilage. Hh signaling is down-regulated in the Fuz null mice, while canonical Wnt signaling is up-regulated revealing the antagonistic relationship of these two pathways. Meckel's cartilage is expanded in the −/−Fuz mice due to increased cell proliferation associated with the up-regulation of Wnt canonical target genes and decreased non-canonical pathway genes. Interestingly, cilia development was decreased in the mandible mesenchyme of Fuz null mice, suggesting that cilia may antagonize Wnt signaling in this tissue. Furthermore, expression of Fuz decreased expression of Wnt pathway genes as well as a Wnt-dependent reporter. Finally, chromatin IP experiments demonstrate that β-catenin/TCF-binding directly regulates Fuz expression. These data demonstrate a new model for coordination of Hh and Wnt signaling and reveal a Fuz-dependent negative feedback loop controlling Wnt/β-catenin signaling.The planar cell polarity effector gene  Fuzzy (Fuz) as an important regulator of Hedgehog (Hh) signaling suggests that there may be substantial crosstalk between the different molecular cues. Furthermore, Fuz can coordinate ciliogenesis and secretion, two processes that affect a variety of signaling pathways Fuz plays a pivotal role in the Wnt and Hedgehog pathways Fuz leads to dramatic defects in craniofacial development.Vertebrate craniofacial development is a complicated process requiring the coordination of multiple signaling pathways and tissue interactions among the three germ layers and neural crest cells in three dimensions. A number of signaling pathways have been implicated in craniofacial development including Hedgehog (Hh), Wnt/β-catenin, TGF-β, Fibroblast Growth Factor (Fgf), Notch, and Planar Cell Polarity (PCP) signaling Drosophila, controls a diverse range of polarized cellular behaviors PCP signaling, initially discovered in Drosophila have shown that fuzzy, along with inturned, plays a role in maintaining cytoskeletal integrity in the wing hairs; this role is genetically downstream of dishevelled and frizzledThe PCP pathway makes use of the Wnt signaling components Frizzled and Dishevelled, but is β-catenin-independent Recent studies suggest that mutations affecting ciliogenesis lead to congenital human anomalies. Cilia appear to act as mechanical and chemical sensors that interpret extracellular cues via signaling pathways such as Hh, PDGF and Wnt Several proteins involved in PCP signaling, including Dishevelled, localize to the base of cilia Fuz during vertebrate craniofacial development. We found that −/−Fuz mice display massive craniofacial defects including anophthalmia, agenesis of the tongue and incisors and a hypoplastic mandible leading to cleft palate. In contrast, we found that Meckel's cartilage, an embryonic structure that develops into the mandible and portions of the inner ear, was hyperplastic and malformed. Some aspects of the craniofacial phenotype, such as missing teeth and cleft palate, could be attributed to decreased Hh signaling. However, the increased growth of Meckel's cartilage was associated with increased Wnt signaling.In this study, we analyzed the expression, regulation and functional requirements of Fuz loss of function, we analyzed expression of Wnt target genes in −/−Fuz mice. We found that a number of β-catenin/TCF target genes were up-regulated in our mutants, suggesting that Fuz plays a dual role in Wnt signaling, in both the canonical and non-canonical pathways. Furthermore, we found that the Fuz protein could repress expression of Wnt pathway genes as well as a Wnt-dependent reporter, suggesting a direct role within the canonical pathway. Finally, most interesting, we identified β-catenin/TCF-binding sites in the Fuz gene, which we confirmed by chromatin immunoprecipitation. Together, these data imply that Fuz may be a critical factor linking the Hh, PCP and Wnt/β-catenin signaling pathways and may function as a switch to balance the activities of these pathways during craniofacial development.To further examine the molecular consequences of LacZ/+Fuz mouse embryos revealed LacZFuz expression was restricted to the dorsal tissue and brain , and tongue (TE) has increased from E12.5 , C3H10T1/2 cells (mouse embryonic fibroblast), HEK 293 FT cells (human embryonic kidney fibroblast) and SW1353 cells (human chondrocyte). It had relatively weak expression in MDPC-23 cells , and no expression in CHO cells (hamster ovary)  .Fuz function during craniofacial development, we used the Fuz null mouse created with a gene trap cassette inserted in the second exon of the Fuz gene Fuz gene in homozygotes (−/−Fuz) was lethal for mice immediately after birth. At E18.5, all null embryos have a hypoplastic mandible and maxilla, and anophthalmia  seen in the heterozygous mice  suggesting a delay of elevation, a lack of eyes, however the upper and lower molar (ML) tooth buds have formed  and an expansion of other structures (Meckel's cartilage).To examine the developmental progress of craniofacial formation, embryos were harvested at different developmental time points and analyzed. At E12.5, the mandible of the  embryos . The norus mouse . These sabnormal . In conttermates . The tritermates . Meckel'termates . The upptermates . The malous mice . Coronale formed . Meckel'e formed . There wFuz null mice, E16.0 embryos were stained with Alcian Blue/Alizarin Red and compared to their heterozygous littermates , and Gli1 expression levels while Shh levels were unchanged  and Cyclin D2 (Ccnd2) expression Immunohistochemistry experiments revealed that Ptch1 was highly expressed in the tongue, oral and dental epithelium and mesenchyme, but weakly expressed in Meckel's cartilage in E14.5 heterozygous embryos . In the artilage . Real-tiartilage . Real-ti embryos . At E14. embryos , though  embryos . Shh traFuz null embryos, which could affect cell proliferation. Immunofluorescence with a β-catenin antibody was performed using sagittal sections of E14.5 embryos. These experiments revealed that β-catenin was increased in the −/−Fuz oral epithelium, mesenchyme  expression is also regulated by Wnt/β-catenin signaling Fuz null Meckel's cartilage at E14.5, and further confirmed by Real-time PCR  −/−Fuz Meckel's cartilage  within the murine Fuz 2.4 kb promoter  to the cell culture medium stimulates β-catenin nuclear localization and caused significant increase of Fuz promoter activity in both cell lines  is a downstream target gene of Sox9 Col2a1 expression was also increased in Fuz null Meckel's cartilage measured by Real-time PCR  and other craniofacial structures.In the Fuz null mice, the Wnt/β-catenin pathway was up regulated whereas the non-canonical Wnt5a/Ror2 pathway was down regulated. These data suggest that Fuz is a potent repressor of Wnt/β-catenin signaling. The data also indicate that Fuz is critical to maintain the non-canonical Wnt signaling pathway, which is overlapped with PCP signaling. It provides a possible explanation of the neural tube defect  previously reported in Fuz null mice −/−Fuz mice can be attributed to increased Wnt/β-catenin signaling. Thus, PCP, Wnt/β-catenin and Hh signaling pathways may converge to provide cues for and instruct specific cell differentiation, tissue patterning and morphogenesis.In the Fuz regulation of Wnt/β-catenin signaling may be mediated by primary cilia and interaction of multiple signaling pathways. Previous studies have reported that the basal component of primary cilium could repress Wnt/β-catenin signaling through interaction of Dishevelled protein Fuz could cause the secondary up-regulation of Wnt/β-catenin signaling. Previous studies have shown that Hh signaling antagonizes Wnt/β-catenin during development of the tongue and cartilage Fuz null mice. Non-canonical Wnt signaling is known to antagonize the canonical Wnt/β-catenin activity Wnt5a was shown to repress Wnt/β-catenin signaling in the limb buds Ror2 receptor was necessary for Wnt5a repression of Wnt/β-catenin activity Fuz null mice, down-regulation of Wnt5a and Ror2 could attribute to activation of Wnt/β-catenin signaling as well. Whether it is one of those mechanisms or a combination requires further investigation.Fuz, mice mutants for cilia intraflagellar transport (IFT) proteins; IFT88/polaris and Kif3a have increased Hh activity Kif3a mice mutants display a range of similar developmental defects and a conditional knockout of Kif3a in the neural crest results in an increase in Hh activity associated with truncated cilia Fuz expression is predominantly expressed in the oral and dental epithelium at early stages and in epithelial cell lines. The −/−Fuz mice do not present with a wide facial prominence, which is indicative of increased Hh signaling −/− miceFuz have reduced and truncated cilia, but not a complete loss of primary cilia. The presence of truncated cilia in the Fuz null mice may play a limited role in the signaling activities. Fuz interacts with a Rab-similar GTPase (RSG1) to affect trafficking from the cytoplasm to basal bodies and to cilia tips Fuz null mice more than defective cilia.In contrast to loss of Runx2 null mice have a complete loss of bone tissue and regulation of Runx2 dosage during development can result in the cleidocranial dysplasia phenotype Runx2 mutant mice have reduced ossification of the calvarial bones, reduced basisphenoid bone and non-osseous tissue between the parietal bones, similar to the Fuz null mice Runx2 expression is increased in the Fuz null mice and Runx2 over-expression newborn mice do not reveal defects in calvarial bone development Fuz null mice demonstrated a delayed calvarial bone development, which may appear normal at later stages of newborn mice . We speculate that increased Runx2 expression may compensate for reduced Hh expression, which is also required for bone development.Runx2 plays a major role in bone development and specifically intramembranous bone formation during craniofacial development. Sox9 are associated with campomelic dysplasia, which is characterized by skeletal defects and cranial dismorphology Sox9 null mice die midway through gestation, however the Sox9 conditional knockout with Wnt1-Cre reveal craniofacial defects including a short mandible and a large cleft palate Sox9 leads to reduced Meckel's cartilage growth. Sox9 and Col2a1 expression are increased in the Fuz null mice, which correlates with increased Meckel's cartilage growth. The expanded Meckel's cartilage would cause increased endochondral ossification as well giving rise to the boney mandible structure observed in the Fuz null mouse mandible. Sox9 expression also occurs in the mouse head skeleton and some of the bone defects could be due to the activity of Sox9 in these tissues Meckel's cartilage is a transient structure responsible for mandible formation and undergoes endochondral-like ossification to produce several bones during craniofacial development FUZ mutations have not been reported or associated with syndromes with known ciliary pathology. However, Oral-Facial-Digital syndrome, type1  appears to represent similar phenotypic features to the craniofacial defects associated with the Fuz null mouse Cxorf5 transcript termed OFD1OFD1 is also mutated in X-linked Joubert syndrome OFD1 mutants reproduce the main features of the human syndromes and have some features not observed in the Fuz null mouse FUZ in human disorders and the molecular mechanisms of FUZ are currently being investigated.There are multiple ciliary genes and proteins associated with ciliopathies,  Repository and injected into blastocysts by the Texas A&M Institute for Genomic Medicine. Four chimeras were obtained and mated to wild-type C57BL/6 mice. Two chimeras yielded germ-line transmission. Offspring from both chimeras were used for X-gal staining and exhibited the same expression pattern. Embryos were collected at various time points, considering the day of observation of a vaginal plug to be embryonic day (E) 0.5. Genotyping PCR primers for +/−Fuz and −/−Fuz mice and embryos were described previously LacZFuz are as below: forward, 5′- CTCTCCTGGCAGCATGTCCT -3′; reverse, 5′- TCCTCCTACATAGTTGGCAGTG -3′. The resulting PCR product represents the LacZ knockin allele, whereas the wild-type allele does not generate any products. All PCR products were sequenced to confirm their identity.The 2, 5 mM EDTA pH 8.0 and 100 mM NaH2PO4 pH 7.3) and washed three times in rinse solution . Embryos were stained for 72 hour at 37°C in staining solution , rinsed in PBS and postfixed in 4% paraformadehyde. Heads of both E12.5 and E14.5 embryos were dehydrated through alcohol, embedded in paraffin and sectioned at 16 µm thickness.Whole embryos of different stages were fixed for 20–40 minutes at room temperature in the fix solution  and stained with Hematoxylin and Eosin. Some embryos were fixed in Bouin's solution and subjected to Wilson section. Immunofluorescence was done on 7 µm paraffin sections with standard procedure. Antigen retrieval was done by boiling samples in 10 mM Sodium Citrate, pH 6.0. Antibodies were obtained and diluted as follows: Ki67  1∶500; Ptch1  1∶300; Gli2  1∶300; β-catenin  1∶500; Lef1  1∶500; Tcf4  1∶500; Sox9  1∶500. Secondary antibodies Alexa Fluor 488 goat anti-rabbit HCA were from Invitrogen (A11034) and used at 1∶500 dilution. The Arl13b antibody  for cilia staining was used at 1∶500 and visualized using a Zeiss Axiovert 200 confocal microscope. Skeletal defects are shown by using Alcian Blue/Alizarin Red staining of cartilage and bone in mouse .p-values were calculated. No-template control was run in each experiment. Melting curve analyses were performed to confirm amplification specificity of the PCR products. All PCR products were sequenced to confirm their identity.Meckel's cartilage and surrounding mesenchyme were dissected from E14.5 mouse embryos. The Real-time PCR was performed with different probes listed in the Fuz full length cDNA reverse-transcribed from total RNA of NIH-3T3 cells was constructed into pcDNA3.1 vector (Invitrogen). The 7xTopFlash reporter plasmid was constructed into luciferase vector by inserting seven Lef/Tcf binding sites upstream of the minimal TK promoter Fuz 2.4 kb promoter was constructed into the TK-luciferase vector by replacing the minimal TK promoter The expression plasmid containing the cytomegalovirus (CMV) promoter linked to the mouse CHO, HEK 293 FT, LS-8 and SW1393 cells  and confirmed by sequencing. As controls the primers were used without chromatin, normal mouse IgG was used replacing the β-catenin antibody to reveal non-specific immunoprecipitation of the chromatin.The ChIP analyses were performed as described Chotteau-Lelièvre, 2006]. Expression analysis of murine genes using in situ hybridization with radioactive and non-radioactively labeled probes. In: I.A. Darby and T.D. Hewitson, Editors, Methods Mol. Biol. (third ed.), In Situ Hybridization Protocols, Humana Press, Totowa, NJ (2006), pp. 61–87.Chotteau-Lelièvre et al., 2006, using an Intavis InSitu Pro robot. The detailed robotic procedure can be found at http://empress.har.mrc.ac.uk/browser/ (gene expression section), . Expression analysis of murine genes using in situ hybridization with radioactive and non radioactively labeled probes. , In Situ Hybridization Protocols, Humana Press, Totowa, NJ (2006), pp. 61–87).Whole-mount in situ hybridizations with digoxigenin-labelled riboprobes were performed as described [Statistics were performed by two-sample t-test. P-values less than 0.05 were considered to be significant.


Isodonrubescens var. lushanensis, a plant long been used in folk medicine to prevent inflammation and gastrointestinal disease. In the present study, we report that henryin selectively inhibits the proliferation of human colorectal cancer cells with a GI50 value in the nano-molar range. Microarray analysis and reporter assays showed that henryin worked as a novel antagonist of Wnt signaling pathway. Henryin reduced the expression of Cyclin D1 and C-myc, and induced G1/S phase arrest in HCT116 cells. Concurrently, henryin did not affect the cytosol-nuclear distribution of soluble β-catenin, but impaired the association of β-catenin/TCF4 transcriptional complex likely through directly blocking the binding of β-catenin to TCF4. We also then analyzed the structure-activity relationship among the ent-kaurane type diterpenoids. Our data suggests that henryin, as a novel inhibitor of Wnt signaling, could be a potential candidate for further preclinical evaluation for colon cancer treatment, and as such warrants further exploration.Aberrant Wnt/β-catenin signaling has been strongly associated with the tumorigenesis of human colorectal cancer. Inhibitors of this pathway may then offer therapeutic strategies as well as chemoprevention for this malignant disease. Henryin is an ent-kaurane diterpenoid isolated from  Colorectal cancer (CRC) is a common malignancy that, despite advancements in drug treatment and improved diagnosis, remains a major challenge to the medical establishment world-wide. Though the exact pathogenesis of the disease is not clearly understood, chronic inflammation bowel disease is deemed to a risk factor that contributes to the development and metastasis for colorectal cancer . NumerouIn colon cancer cells, the Wnt/β-catenin pathway is frequently aberrantly activated –6. AccorIsodon species, widely distributed plants, are used as tea drink to prevent inflammation, gastrointestinal bacterial infections and even tumors. We hypothesized that chemical substances present in some Isodon species likely possessed chemopreventive properties as well as chemotherapeutic effects against cancer. In our previous work, many chemical constituents were isolated and characterized from Isodon species, but the study of their bioactive profile is very limited and mechanisms are lacking [To date, advances in the treatment and diagnosis of colorectal cancer are still limited in their effectiveness, prompting us to consider alternative options in moving forward. In folk medicine, some  lacking –16.Isodonrubescens var. lushanensis, exhibited selective growth inhibitory effects on human colorectal cancer cells by inhibiting Wnt signaling. Our data showed that henryin interferes with the β-catenin/TCF complex interaction and induces the G1/S phase arrest in colorectal cancer cells. Accordingly, henryin, as novel inhibitor of Wnt/β-catenin pathway, could make a promising drug candidate for both the prevention of colorectal cancer as well as a novel therapeutic.Our present study focuses on screening the compounds that confer anti-tumor effects, especially on human colorectal cancer cells. We found that henryin, an ent-kaurane diterpenoid, isolated from RPMI 1640 medium, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), antibiotics solution, and Trypsin-EDTA were purchased from HyClone . The complete protease inhibitor cocktail was purchased from Roche Applied Science . Mouse monoclonal anti-CyclinD1 and anti-β-catenin antibodies were purchased from BD Biosciences . Rabbit monoclonal anti-TCF4 and polyclonal anti-phospho-β-catenin antibody were purchased from Cell Signaling Technology . Rabbit monoclonal anti-p21 was purchased from Epitomics, Inc . Mouse monoclonal anti-lamin A/C, anti-β-actin, anti-c-Myc antibodies and other reagents, unless otherwise indicated, were purchased from Sigma-Aldrich . Purified human recombinant proteins β-catenin was purchased from Sino biological inc. Purified human recombinant proteins TCF4 was purchased from OriGene. Dual-luciferase reporter gene assay system was purchased from Promega . Lipofectamine 2000 was purchased from Invitrogen . The QuantiTect SYBR, Green PCR Kit was obtained from Qiagen (Germany).Isodonrubescens var. lushanensis, as previously described [Henryin and its analogues were extracted from escribed –16. The Colon cancer cell lines , human lung cancer cell line A549, human normal lung epithelial cell line (Beas-2B) and HEK293T were purchased from ATCC. The normal colonic epithelial cell line (CCD-841-CoN)  was kind3cells/well and incubated for 12h in a CO2 incubator. Cells were treated with the dose ranging from 0.008 to 4µM of henryin and its analogues, with cisplatin as a positive control for 48h. Then, 20 µl of CellTiter 96® AQueous One Solution Reagent  was added and the cells were further incubated at 37 °C for 1-2h. Cell viability was then measured by reading the absorbance at a wavelength of 490 nm. The following formula was used to determine cell growth ratio values: 100*  – A490 ) /  – A490 ). The GI50 values—the concentrations which cause 50% cell growth inhibition—were determined by non-linear regression analysis using TableCurve software.Cells were seeded in 96-well plates at a density of 5×10SW480 cells were incubated with 4µM of henryin or dimethyl sulfoxide (DMSO) as a control for 12h. Cells were lysed with TRIzol  and total RNAs were isolated with the QIAGEN RNeasy Mini Kit  before reverse transcription, labeling and hybridization to Agilent Whole Human Genome Oligo Microarray (4×44K) . The microarray assay was conducted by Shanghai Bio Corporation. After GO annotation and pathway analysis, genes with more than 2 fold alteration in the expression level were selected for further analysis.2 incubator for 12h. SW480 and HCT116 cells were co-transfected with 100ng of plasmids in total, including 80ng of a well-characterized Wnt/β-catenin pathway responsive firefly luciferase reporter plasmid SuperTOPflash (ST-Luc) and 20ng of pRL-SV40 (Promega) control reporter plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Each well of HEK293T cells was transfected with 200ng of plasmids in total, including 80ng of ST-Luc and 20ng of pRL-SV40 and 10ng of β-catenin or 64ng of wnt1 and empty vector plasmids as indicated. Some 3h after transfection, cells were incubated with various concentrations of compounds for 24h. Cells were lysed with 50µl of Promega lysis buffer in each well and luciferase activity was measured and normalized by pRL-SV40 reporter activity. All experiments were done independently in triplicate. Results are reported as means and standard deviations (SD).For reporter gene assays, cells were seeded in 96-well plates and incubated at 37°C in 5% COTotal RNA was extracted from cultured cells with TRIzol and reverse transcription was performed using the SuperScript III Reverse Transcription Kit (Invitrogen) with oligo(dT) priming according to the manufacturer’s instructions. Gene transcripts were quantified by real-time PCR using SYBR Green I and β-actin was used as a control. The primes used were:5'-AAGTGCGAGGAGGAGGTCTT-3' and the reverse primer: 5'-GGATGGAGTTGTCGGTGTAGA-3'.Human CyclinD1 forward primer: 5'-CTTCTCTCCGTCCTCGGATTCT-3' and the reverse primer: 5'-GAAGGTGATCCAGACTCTGACCTT-3'.Human C-myc forward primer: 5'-CGCGAGAAGATGACCCAGAT-3' and the reverse primer: 5'-GATAGCACAGCCTGGATAGCAAC-3'.Human β-actin forward primer: RT-PCR experiments were performed in triplicates and PCR efficiency with given primers was between 95 and 100%. Melting curves were also performed for monitoring the primer-specific amplifications.SW480 cells were treated with the henryin in 6-well plates. Fractionated nuclear and cytosolic lysates were obtained using nuclear extraction buffer and hypotonic lysis buffer, respectively. All protein extraction buffers were supplemented with Complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). The protein concentration of each crude lysate was determined using the Enhanced BCA Protein Assay Kit . The normalized amounts of the lysate samples were loaded on SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membranes . Immunoblotting was performed using antibodies detecting phospho-β-catenin (Ser33/37/Thr41), β-catenin, Axin2, CyclinD1, C-myc, Survivin, P21, TCF4, with Lamin A/C and β-actin used as loading controls. HRP conjugated anti-mouse IgG and anti-rabbit IgG were used as secondary antibodies. SuperSignal West Pico Chemiluminescent Substrate (Thermo scientific) was used to detect chemiluminescence, and blots were imaged using the Luminescent Image Analyzer LAS-4000mini System .SW480 cells were cultured in 6-well plates, treated with henryin and its analogs enmenol and minheryin C for 12h, lysed in protein lysis buffer  and centrifuged at 14000×g for 15 min at 4°C. The supernatant was incubated with a specific primary antibody overnight at 4°C and then incubated with A/G PLUS agarose  for at least 2h. Beads were then washed five times and re-suspended in 50 µl SDS loading buffer. Western blot analysis was performed on the samples.To assay the effect of compounds in inhibiting β-catenin/TCF4 binding, purified recombinant proteins β-catenin (0.8µg) and TCF4 (0.5µg) were incubated with henryin and its analogs enmenol and minheryin C at indicated concentrations at 4°C for 2h, respectively. β-catenin/TCF4 complexes were pulled down using protein A/G PLUS agarose beads saturated with β-catenin antibody. Detection was then performed using TCF4 antibody in western blotting analysis.5cells/well in 12-well plates) were incubated with henryin for 0h, 12h and 24h, respectively. All adherent cells were collected and washed twice with PBS. Cells were fixed with 70% ethanol overnight. Fixed cells were washed with PBS, and then stained with a 50µg/ml propidium iodide (PI) solution containing 50µg/ml RNase A for 30 min at room temperature. Fluorescence intensity was analyzed by FACSCalibur1 flow cytometer . The percentages of the cells distributed in different phases of the cell cycle were determined using ModFIT LT 2.0.HCT116 cells  or <0.5 (down-regulated) as differentially expressed genes. Both GO annotation and pathway analysis showed that henryin strongly affects the Wnt signaling pathway as well as other altered pathways that are associated with colorectal cancer biological processes in both the KEGG and BioCarta databases (Following the microarray analysis, we checked the effect of henryin on Wnt/β-catenin signaling using the TOPflash reporter assay. In wnt1 transfected HEK293T cells, henryin inhibited the Wnt signaling reporter expression in a dose-dependent manner after 24h of treatment (To characterize the structure-activity relationship of henryin, we investigated the activities of additional henryin analogues (We checked the expression of endogenous Wnt target genes in our microarray data and found that the expression of Wnt target genes, including Axin2, Cyclin D1, SP5, DKK1, NOS1, PPARδ and FGF20, were significantly down-regulated after treatment with henryin (One hallmark of activation of Wnt signaling is the stabilization and nuclear accumulation of β-catenin in colorectal cancer cells. We checked the effect of henryin on the level and distribution of β-catenin in SW480 cells. SW480 cells were treated for 24hours with henryin at various concentrations and total β-catenin and phosphorylated form of β-catenin were examined (In the nucleus, β-catenin needs to bind transcription factors of the TCF family to regulate target gene expression. We checked whether henryin impairs the β-catenin/TCF interaction. SW480 cells treated with henryin were immune-precipitated by β-catenin antibody or mouse IgG as a control, and TCF4 was detected by western blotting and the blots were analyzed with ImageJ for intensities of the bands. The results showed that henryin strongly inhibited the binding of TCF4 to β-catenin in a dose-dependent manner in SW480 cells but not its analogs enmenol and minheryin C (In order to determine whether henryin can directly disrupt the interaction of β-catenin with TCF4, in vitro binding assays were performed with puriﬁed recombinant β-catenin and TCF4 proteins. The result showed that henryin strongly inhibited the binding of TCF4 to β-catenin in the in vitro binding assays, whereas its analogs enmenol and minheryin C exhibited no any effect as expected (Targeting Wnt signaling may represent a promising strategy to eradicate malignant disease with abnormal activation of this pathway. Accordingly, identifying new inhibitors of the Wnt/β-catenin signaling pathway are of great importance and significance. This inhibition may be achieved by disrupting the interaction of β-catenin/TCF  or CREB In recent years, there has been significant interest in the search for Wnt/β-catenin signaling antagonists from natural products . Indeed,Isodonrubescens var. lushanensis preferentially induced colorectal cancer cells death while leaving normal colonic CCD-CoN-841 and normal lung epithelial Beas-2B cells less affected (Henryin, an ent-kaurane diterpenoid, isolated from affected , 5F. TakAs a key target gene of Wnt signaling, the oncogene C-myc contributes to increased cell proliferation in a variety of human cancers. Recent insights into its expression and function have led to therapeutic opportunities . Cyclin Isodon species, preferentially induced colorectal cancer cell death. In the present study, we proposed and illustrated the molecular mechanisms modulated by henryin responsible for anti-tumor proliferation effects. Isodon species have long been used in folk medicine. Our findings that henryin is a novel inhibitor of Wnt/β-catenin signaling will provoke its use as a potential anticancer agent. Obviously further studies on the in vivo efficacy as well as the pharmacodynamic effects of henryin will provide potentially novel therapeutic strategies for colorectal cancer with minimum adverse effects on normal tissues.In conclusion, henryin, an ent-kaurane diterpenoid isolated from