Wnt proteins comprise a large family of highly conserved glycoproteins known for their role in development, cell fate specification, tissue regeneration, and tissue homeostasis. Aberrant Wnt signaling is linked to developmental defects, malignant transformation, and carcinogenesis as well as to inflammation. Mounting evidence from recent research suggests that a dysregulated activation of Wnt signaling is involved in the pathogenesis of chronic inflammatory diseases, such as neuroinflammation, cancer-mediated inflammation, and metabolic inflammatory diseases. Recent findings highlight the role of Wnt in the modulation of inflammatory cytokine production, such as NF-kB signaling and in innate defense mechanisms as well as in the bridging of innate and adaptive immunity. This sparked the development of novel therapeutic treatments against inflammatory diseases based on Wnt modulation. Here, we summarize the role and function of the Wnt pathway in inflammatory diseases and focus on Wnt signaling as underlying master regulator of inflammation that can be therapeutically targeted. This could be explained by the fact that the tumor microenvironment contributes to β-catenin accumulation in the cytoplasm. However, besides tumor or inflammatory microenvironments  or by the use of small molecule inhibitors or activators or by exogenous modulation through DNA engineering (recombinant Wnt) conditioned media by adding Wnt regulators to cell culture  rendered the mice more sensitive to inflammation. This was associated with increased numbers of inflammatory cells, including activated macrophages, with a predominance of eosinophils in the lung and also a significant increase of the Th2 response as indicated by an increase of the specific markers IL-4, IL-3, and GATA3 as well as the chemoattractant CCL2 involved in Th2 polarization. It is interesting to note that genetic deletion of Wnt10b in HDM-sensitized mice enhanced the response of T cell effectors, shown by the increase of CD44+CD4+ regulatory cells Treg response and increased the Th1 and Th17 inflammatory response . This suggests a regulatory role of the canonical Wnt pathway during ongoing neuroinflammation in which activation of this pathway regulates the differentiation of naïve CD4+ T cells into Th1 and Th17 through regulation of DC-specific cytokine production and limitation of uncontrolled differentiation. Moreover, prophylactic and therapeutic treatment of EAE mice with a β-catenin agonist restores the injury by significantly delaying EAE onset and by markedly reducing its severity. This was explained by the reduction of the expression of pro-inflammatory cytokines and the increase of anti-inflammatory cytokines. It seems that β-catenin activation during EAE limits CNS pathology via the suppression of Th1 and Th17 response, the increase of Treg cells frequency, and the promotion of the regulatory response, suggesting that immunotherapy targeting Wnt/β-catenin could represent a promising therapeutic approach for clinical management of multiple sclerosis  stage, increase of their proliferation, and the blockade of their apoptosis. In addition, a very strong decrease of naïve T cell marker CD62L concomitant with an enhancement of activation memory marker CD44 and CD69 on CD4+ and CD8+ T cells in the thymus and in the splenic and peripheral lymph node was detected. β-catenin overexpression recruits activated T cells to initiate a Th1-type inflammatory cascade by increasing IFNγ, IL-2, and TNFα expression and activates involving microglia and astroglia, leading to neurologic abnormalities and motor impairment in mice  provides an experimental model of tissue injury and intestinal inflammation. Recent studies highlight that LRP5/6 are still expressed in colonic DC and macrophages in this model. Accordingly, selective deletion of LRP5/6 in CD11c+ DCs as discussed above, high levels of β-catenin in T cells can cause chronic T cell activation by the expression of several activation markers including CD69, CD122, and NK62D and causes Th17 commitment by the expression RORγT, the signature transcription factor of the Th17 lineage. The activation of the latter transcription factor is responsible for Treg dysfunction, which loses the anti-inflammatory functions and contributes to systemic inflammation, colitis, and polyposis  was able to significantly suppress the LPS effect on BEAS-2B human bronchial epithelial cells by decreasing pro-inflammatory gene expression, including IL-6, IL-8, TNFα, IL-1β, MCP-1, MMP-9, iNOS, and COX2. Although the expression of proinflammatory genes are increased, Ik-β is phosphorylated and degraded, followed by NF-kB release and its translocation from the cytoplasm to the nucleus. Even though XAV939 was able to significantly block Ik-β phosphorylation and NF-kB translocation from the cytoplasm to nucleus, this effect was rescued by β-catenin expression  as well as long-term overexpression of Wnt ligands is a promising approach that could help to reveal the long-term effect of these ligands in the remodeling process. In addition, unraveling the pathophysiological function of Wnt signaling components in an organ-specific manner by targeting tissue-specific expression using, for example, Cre-recombinase techniques would be useful. Such studies that yield another level of manipulating Wnt signaling allow the understanding of the exact inflammatory pathology and identification of therapeutic opportunities and also reveal potential risks and side effects of pharmacological interference with the Wnt signaling network.IJ wrote the first draft, which was amended, completed and corrected by FS, KP-O, and KC-B. All authors saw and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we provide clear evidence of the potentiation of canonical WNT signaling by RSPO during mouse facial development by analyzing compound Wnt9b and Rspo2 gene knockout mice and utilizing ex vivo facial explants. Wnt9b;Rspo2 double mutant mice display facial defects and dysregulated gene expression pattern that are significantly more severe than and different from those of Wnt9b or Rspo2 null mutant mice. Furthermore, we found suggestive evidence that the LGR4/5/6 family of the RSPO receptors may play less critical roles in WNT9b:RSPO2 cooperation. Our results suggest that RSPO-induced cooperation is a key mechanism for fine-tuning canonical WNT/β-catenin signaling in mouse facial development.The R-spondin (RSPO) family of proteins potentiate canonical WNT/β-catenin signaling and may provide a mechanism to fine-tune the strength of canonical WNT signaling. Although several  Ctnnb1 (β-Catenin) gene function within facial primordia results in severe defects in many facial structures  family of proteins are known for their roles in potentiating or synergistically activating canonical WNT/β-catenin signaling in the presence of the WNT ligands . RSPOs iRspo2 gene results in reduced WNT/β-catenin signaling mainly within the mandibular branchial arch 1 (MdBA1), resulting in cleft palate accompanying the deformation of MdBA1-derived bone structures  and maxillary process of branchial arch 1 (MxBA1) due to significantly diminished WNT/β-catenin signaling  mice exhibited defective facial phenotypes that were much more severe than those of Wnt9b or Rspo2 single-gene knockout mice. We found that WNT/β-catenin signaling within the facial primordia is activated by the cooperative action between ectoderm-derived WNT9b and mesenchyme-derived RSPO2 and is crucial for growth and patterning of the facial structures in mice. Intriguingly, our results suggest that the LGR4 family of RSPO receptors may play less significant roles in WNT9b:RSPO2-mediated WNT signaling regulation in the development of facial structures.In the present study, we systematically investigated how Wnt9b gene KO mice . We detected Wnt9b gene expression within the ectoderm of both the BA1 and NPs adjacent to the Rspo2 expression domain  may potentiate canonical WNT/β-catenin signaling with unknown WNT ligand(s) expressed in ectodermal epithelial cells of BA1 . Conside KO mice , 2012, wn domain . The exp the BA1 . Their eSuperTopFlash (STF) in HEK293T cells treated with recombinant RSPO2 and WNT9b proteins. While WNT9b or RSPO2 alone only induced a weak to mild activation of the STF reporter, combined treatment of RSPO2 and WNT9b produced a strong synergistic activation of the STF reporter in a dose-dependent manner  embryos at E10.5 and E11.0, respectively  is not present. Taken together, these results confirm cooperation between WNT9b and RSPO2 in the regulation of WNT/β-catenin signaling in developing facial structure.To determine whether WNT9b and RSPO2 together can potentiate WNT/β-catenin signaling ectively . As prevectively , 2012, T mutants . Furthere manner . SimilarFgf8 and Msx1, two WNT target genes expressed in the ectoderm and mesenchyme, respectively  that were either upregulated or downregulated by more than 1.5-fold were identified in Wnt9bKO, Rspo2KO, and DKO mice, respectively . In contrast, –/–Wnt9b mice in a Rspo2± background showed the phenotype identical to –/–Wnt9b in a wild-type background. In addition, DKO mice exhibit some defects that were neither evident nor present in either –/–Rspo2 or –/–Wnt9b mice. Upper jaw hypoplasia and open eyelids were never detected in Wnt9b or Rspo2 single-mutant mice  play a central role as RSPO receptors for WNT:RSPO cooperation in many cellular contexts . Howeverat E10.5 , suggestat E10.5 . This suSTF reporter assay  mutant protein failed to induce both marker genes , and Wnt9b mutant mice . The genotyping of these mice was performed as described previously  according to the manufacturer’s instructions. To evaluate cell proliferation, immunofluorescent staining with anti-phospho-histone H3  was performed on cryosections. The nuclei positive for TUNEL or phospho-histone H3 were counted, and the percentage of total nuclei was calculated.To determine apoptotic cell death, a TdT-mediated dUTP nick-end labeling (TUNEL) assay was carried out on 10 μm cryosections using an For facial process explant culture, the facial primordial tissues were dissected from E10.5 embryos in cold phosphate-buffered saline (PBS). When necessary, the ectodermal layer was removed by incubating the explants with Dispase II  for 30 min. Isolated tissues were cultured for 24–36 h on filters floating in the transwell plates  containing α-MEM  with 0.1 μg/ml ascorbic acid , 10% (v/v) fetal bovine serum , and 1% (v/v) streptomycin/penicillin .2 at 37°C. Recombinant RSPO2 and WNT9B proteins were obtained from R&D Systems  and treated at the concentrations of 200 and 20 ng/ml, respectively.Human embryonic kidney 293T (HEK 293T) cells were acquired from the American Type Culture Collection  and maintained in DMEM  containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin under 5% COTotal RNA was isolated from freshly dissected and cultured facial primordial explants of mouse embryos using TRIZOL , and cDNA was synthesized from 0.8 μg RNA using a Proscript cDNA synthesis kit . qRT-PCR was performed using cDNA  as described previously . The seqTopFlash plasmid DNA transfection was performed using TransIt-LT1 reagent  according to the manufacturer’s instructions. Briefly, DNA-TransIT mix was incubated with cells for 16 h, and the transfected cells were harvested 24 h after transfection for use in the luciferase assay.Protein lysates for Western blot analysis were prepared by using a RIPA  lysis buffer. Protein lysates (10 μg) were resolved on 8% polyacrylamide gel and transferred to PVDF membranes. The membranes were probed with various primary antibodies against phospho-LRP6 , LRP6 , and α-tubulin . Species- and isotype-matching secondary antibodies conjugated with horseradish peroxidase (HRP) were incubated, and the HRP signal was developed using the Pierce Super Signal West Dura kit . The signals were measured using Image J software, and each experiment was performed in duplicate.Rspo2 open-reading frame (ORF) tagged with Myc-His at the C-terminus. Within the FU-CRD1 domain of RSPO2, we specifically introduced two alanine mutations into R65 and Q70 residues, which have been reported in human patients diagnosed with inherited anonychia. This domain is required for the interaction with ZNRF3/RNF43 receptors  WNT reporter assay in HEK 293T cells. Concentrations of mutant forms of RSPO2 were normalized with wild-type RSPO2 by Western blot analysis using anti-Myc antibody.Various RSPO2 mutant constructs carrying specific amino acid mutations were generated using the QuikChange Mutagenesis Kit  from the pCDNA3.1A-mRspo2MH plasmid template encoding the wild-type mouse eceptors . Within eceptors . Wild tyMus Musculus GRCm38 reference genome using STAR. Expression levels for each gene were quantified using HTSeq-Count. DEGs showing more than 1.5-fold change with p < 0.05 in Wnt9bKO, Rspo2KO, and DKO mice were identified using DESeq2. Analysis of gene ontology for the biological processes was performed with the database for annotation, visualization, and integration discovery  online tool.Facial process explants, including mandibular and maxillary branchial arch and nasal processes, from E10.5 embryos were dissected in cold PBS, and total RNA was extracted using TRIZOL reagent (Invitrogen). RNA sequencing was performed on three samples per genotype at the Genome Technology Access Center at Washington University , and data analysis was performed by our colleagues at the Israel National Center for Personalized Medicine at Weizmann Institute of Science and Macrogen Inc. . More than 25 million reads per sample were acquired from the single-ended 50 bp sequencing on the HiSeq 2500. Poly-A/T stretches and Illumina adapters were trimmed from the reads using Cutadapt, and resulting reads shorter than 30 bp were discarded. Reads were mapped to the t-test using GraphPad Prism software . Statistical significance was set at p < 0.05.Three to four embryos and fetuses for each genotype were used in most experiments unless otherwise stated. The experimental data were analyzed by non-paired Student’s GSE147474.The datasets generated for this study can be found in NCBI GEO accession The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Soonchunhyang University and Maine Medical Center.Y-RJ: conceptualization, investigation, and writing-original draft. XH: investigation. KN: resource. DB-A: data analysis. YO: investigation and formatting. JS: conceptualization, writing – reviewing and editing, and supervision. JY: conceptualization, writing – reviewing and editing, supervision, and funding.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Additionally, accumulating evidence indicates that reactive oxygen species (ROS) mediate EMT. Our results showed that the level of ROS was significantly increased after chrysophanol treatment. We further speculated that chrysophanol-mediated EMT and metastasis are involved in the Wnt-3-dependent signaling pathway. The inhibition of the EMT phenotype and metastasis and accumulation of ROS caused by chrysophanol was reversed by treatment with the Wnt-3 agonist Bml 284. Therefore, our findings indicated that chrysophanol altered EMT formation, ROS accumulation, and metastasis via the Wnt-3-dependent signaling pathway.Oral cancer belongs to the group of head and neck cancers. If not diagnosed or treated early, it can be life threatening. Epithelial-mesenchymal transition (EMT) plays an important role in tumor formation and progression. An increase in the presence of the EMT phenotype causes tumor cell proliferation, migration, invasion, and poor prognosis. Therefore, attenuating carcinogenesis via EMT inhibition is a good strategy. Herein, we will determine the pharmacological effects of chrysophanol on the EMT in FaDu cells. To analyze EMT, we detected the expression EMT markers, including  It incl-catenin , as welldhern-11 , 6. Therdhern-11 . MoreoveRheum palmatum, a well-known traditional Chinese herb used for chronic kidney disease and liver disease [κB/cyclin D1 and NF-κB/Bcl-2 signaling cascade in breast cancer cell lines [Moreover, Li et al. suggested that chemokine (C-C motif) ligand 2 (CCL2) is critical for the regulation of endogenous ROS production in OSCC SCC-9 and CAL-27 cells . Accumul disease . Chrysopll lines . Howeverll lines , 13. Thell lines ; howeverChrysophanol, glutathione, and NAC were supplied by Sigma . Bml 284 .α-SMA , β-catenin , vimentin , N-cadherin , E-cadherin , Wnt-3 , p-GSK3β , GSK3β , β-actin .Antibodies for western blot analyses and the final working dilutions were as follows: rabbit polyclonal antibodies to p65 , and fibrillin . All HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Inc. .2 atmosphere at 37°C.The human oral cell line FaDu was purchased from the ATCC . Cells were analyzed for mycoplasma contamination and tested negative. Cells were cultured in Dulbecco's Modified Eagle Medium containing 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin and incubated in a 5% CO3VO4, 1 mM phenylmethylsulphonyl fluoride, a 1% dilution of Sigma protease inhibitor cocktail, and 1% Triton X-100), and boiled in (sodium dodecyl sulfate) SDS sample buffer. Equal amounts of protein were separated on SDS-PAGE and then transferred to polyvinylidene difluoride membranes . The membrane was blocked with 1% BSA and incubated with specific primary antibodies overnight. The appropriate HRP-conjugated secondary antibodies  were incubated for 1 h at room temperature, and expression of protein was detected using an ECL kit .Cells were collected, resuspended in ice-cold relaxation buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM NaF, 2 mM Na2; 0.2 mM PMSF; 20 μg/ml aprotinin; 0.5 mM DTT; and 0.5% NP-40) on ice for 15 minutes. After centrifuging at 6,000 × g for 15 minutes at 4°C, the pellet was collected and then washed with basal buffer (hypotonic buffer without 0.5% NP-40). After centrifuging again at 6,000 × g for 15 minutes at 4°C, the pellet was collected and resuspended in a hypertonic buffer  at room temperature for 30 minutes. After centrifuging at 10,000 × g for 30 minutes at 4°C, the nuclear fraction contained in the supernatant was collected.The nuclear fraction was extracted from FaDu cells. The cells were collected and resuspended in a hypotonic buffer  were seed in the 48-well plate with serum-free medium . After treatment with different conditions, the ability of adhesion was measured according to the manufacturer's instructions . Finally, the fluorescence was read with a fluorescence plate reader at 485/530 nm .The adhesion analysis was used according to previous study  with somμm pore size, Corning, NY, USA) was used to measure the metastasis ability of the FaDu cells in three conditions. First, FaDu cells were seeded (1 × 105) in the upper well. After incubation for 48 hours, cells were treated with chrysophanol or Bml 284 for 24 hours in serum-free medium supplemented with 5% bovine serum albumin . Then, a noncoated membrane in serum-free medium supplemented with 5% bovine serum albumin  was added to the lower well. After 24 hours, the migratory cells on the underside of the membrane were stained with 0.1% crystal violet for 5 minutes and washed with H2O; the membrane was analyzed using a Nikon E400 phase-contrast microscope . The crystal violet was destained with methanol for 15 minutes, and the absorbance values were measured at OD570. All measured values were detected by Synergy HT .A 24-well transwell plate  after treatment with chrysophanol. The results showed that the decrease in α-SMA, β-catenin, N-cadherin, and vimentin and increase in E-cadherin and phospho-GSK-3β were reversed in the presence of Bml 284   . As suchμM)   . These rα-SMA, β-catenin, vimentin, N-cadherin, E-cadherin, Wnt-3, and phospho-GSK-3β in the presence of Bml 284 (0.7 μM) (Supplemental β-catenin nuclear translocation (Supplemental β-catenin and TCF-dependent transcriptional activity [The poor prognosis of OSCC is mainly caused by its high recurrence and metastasis rates and remains a significant medical challenge, regardless of advances in diagnostic and therapeutic procedures , 22. Accactivity . Since tβ were significantly upregulated in OSCC in comparison with control tissues [β-catenin was detected in oral leukoplakia with dysplasia but not in epithelial cells without dysplasia [β-catenin nuclear import pathway may provide an opportunity to identify specific drug targets via inhibition of the β-catenin nuclear function or translocation in cancer progression [β-catenin in FaDu cells (Wnt-3 and GSK-3 tissues . Uraguch tissues . On the ysplasia . Jamiesogression . Our resDu cells , which iDu cells , which iIn conclusion, chrysophanol significantly inhibited EMT formation and metastasis via a Wnt-3-dependent signaling pathway. On the other hand, chrysophanol caused ROS accumulation via a Wnt-3-dependent signaling pathway . Our pha


Dehydroeffusol (DHE) is a phenanthrene compound that possesses anti-tumor activity. However, the effect of DHE on non-small cell lung cancer (NSCLC) has not been investigated previously. Therefore, the objective of our study was to explore the role of DHE in NSCLC and the underlying mechanism. Our results showed that DHE significantly inhibited the cell viability of A549 cells in a dose- and time-dependent manner under normoxic condition. Moreover, A549 cells were more sensitive to DHE under hypoxic condition compared with the A549 cells cultured in normoxic condition. Hypoxia-induced increased migration and invasion abilities were mitigated by DHE in A549 cells. Treatment of DHE caused increased E-cadherin expression and decreased N-cadherin expression in hypoxia-induced A549 cells. DHE also suppressed hypoxia-induced increase in both protein and mRNA levels of hypoxia inducible factor-1α (HIF-1α) expression in A549 cells. Furthermore, DHE inhibited hypoxia-induced activation of Wnt/β-catenin pathway in A549 cells. The inhibitory effect of DHE on hypoxia-induced EMT was reversed by LiCl, which is an activator of Wnt/β-catenin signaling pathway. In conclusion, these findings demonstrated that DHE prevented hypoxia-induced EMT in NSCLC cells by inhibiting the activation of Wnt/β-catenin pathway, suggesting that DHE might serve as a therapeutic target for the NSCLC metastasis. Lung cancer is one of the most deadly cancers in the world . Lung caHypoxia in the tumor microenvironment has been extensively studied for decades and found to be critical for driving the tumorigenesis of NSCLC . HypoxiaIn vitro and in vivo biological evaluation reveal that DHE is a bioactive phytochemical with broad activities, including antimicrobial [Dehydroeffusol (DHE) is a phenanthrene compound isolated from the Traditional Chinese Medicine (TCM) Juncus effusus. icrobial ,11, anxiicrobial  and antiicrobial . In receicrobial . DHE efficrobial . DHE wasicrobial ,15. HoweHuman NSCLC cell line (A549 cells) obtained from the  were cultured in RPMI-1640 medium  with 10% fetal bovine serum  and 1% penicillin/streptomycin .2) for 7 days as previously described [Cells in control group were maintained in a normoxic condition. Cells in the hypoxia-induced group were exposed to hypoxia condition (1% Oescribed . Cells i3 cells/well) were seeded into 96-well culture plates and then treated with different concentration of DHE  under a normoxic or hypoxic condition. After indicated incubation time points, 20 μl of MTT  was added to each well for 4 h. Then the supernatant was discarded, and the formazan crystals were solubilized with 150 μl of dimethyl sulfoxide (DMSO). Subsequently, the absorbance at 490 nm was measured using a microplate reader  and expressed as percentages relative to untreated controls.A549 cells .Transwell assays were performed using standard protocol with transwell chambers . A549 cells with 200 μl serum-free medium at the density of 2.5 × 10Total RNA was isolated from A549 cells using Trizol reagent (Invitrogen). Reverse transcription was performed to synthesized cDNA using the total RNA and a First Strand cDNA Synthesis Kit . Quantitative determination of HIF-1α mRNA level was conducted by real-time RT-PCR with SYBR Green Master Mix . HIF-1α, forward primer: 5′-CAGAGCAGGAAAGAGAGTCATAGAAC-3′, reverse primer: 5′-TTTCGCTTCCTCTGAGCATTC-3′; vimentin, forward primer: 5′- TGAAGTGGATGCCCTTAAAGGAA-3′, reverse primer: 5′- GCAGGCGGCCAATAGTGTCT-3′; snail, forward primer: 5′-CACCTCCAGACCCACTCAGATGT-3′, reverse primer: 5′-GCAGGGACATTCGGGAGAAGGT-3′; slug, forward primer: 5′-GCGAACTGGACACACATACAGTG-3′, reverse primer: 5′-GCTGAGGATCTCTGGTTGTGGT-3′; β-actin, forward primer: 5′-CTCTTCCAGCCTTCCTTCCT-3′, reverse primer: 5′-AGCACTGTGTTGGCGTACAG-3′.Total protein was extracted from A549 cells using RIPA lysis buffer  and then the concentration was measured by BCA Protein Assay Kit (Beyotime). Approximately 50 μg protein samples were processed for Western blotting analysis as described in detail elsewhere . Primaryt-test or one-way analysis of variance (ANOVA). P<0.05 was considered as statistically significant difference.Data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS statistical software 18.0  with Student’s Initially, we evaluated the influence of hypoxia on A549 cells. A549 cells were maintained under normoxic or hypoxic condition for 12, 24, or 48 h. We found that cell viability was markedly decreased after incubation for 48 h under hypoxic condition A. Thus, Next, we investigated the effect of DHE on A549 cells viability under normoxic or hypoxic condition. A549 cells were treated with different concentrations of DHE  for 24 h and exposed to normoxic or hypoxic condition. As shown in Next, we performed transwell assays to explore the effects of DHE on A549 cells migration and invasion. Untreated A549 cells under hypoxia condition and A549 cells treated with 10 and 20 µM of DHE for 24 h under hypoxic condition. The results revealed that migration and invasion abilities were dramatically increased in hypoxia-induced A549 cells as compared with the A549 cells under normoxic condition A,B. TreaThen, we determined the effects of DHE on hypoxia-induced EMT in A549 cells. The cellular morphology of A549 cells was shown in Supplementary Figure S1A. The expression levels of EMT markers including E-cadherin and N-cadherin were detected. Western blotting analysis indicated that exposure to hypoxia caused a significant decrease in E-cadherin expression and an increase in N-cadherin expression. However, these effects were reversed by DHE . In addiSubsequently, we aimed to explore whether HIF signaling was involved in the effects of DHE. The protein and mRNA levels of HIF-1α were respectively determined using Western blot and qRT-PCR. The hypoxia-induced mRNA HIF-1α expression level was significantly suppressed by DHE A. As it Available literature has demonstrated that Wnt/β-catenin pathway plays a crucial role in HIF signaling ,20. HencThen we utilized a pharmacological activator of Wnt/β-catenin pathway, LiCl, to further verify the role of Wnt/β-catenin pathway. A549 cells were pretreated with LiCl (10 μM) for 2 h, followed by incubation with 20 μM of DHE for 24 h under hypoxic condition. Results in in vitro.Hypoxia is a common condition presented in tumor environment due to the fast growth of tumor cells and inadequate blood supply . The hypHIF signaling is crucial for the regulation of genes involved in angiogenesis, glycolytic metabolism, and other biological mechanisms in tumorigenesis . There iHIF is sufficient to induce EMT and invasion through both direct and indirect mechanisms . For exaIn conclusion, our results demonstrated that DHE prevented hypoxia-induced EMT in NSCLC cells by inhibiting the activation of Wnt/β-catenin pathway. These findings suggested that DHE might be a therapeutic target for the metastasis of NSCLC.Click here for additional data file.


Foxg1 is essential for the proliferation of certain precursors and inhibits premature neurogenesis during brain development. To date, very little attention has been paid to the role of Foxg1 in the proliferation and differentiation of OPCs in demyelinating diseases of the CNS. Here, for the first time, we examined the effects of Foxg1 on demyelination and remyelination in the brain using a cuprizone (CPZ)-induced mouse model. In this work, 7-week-old Foxg1 conditional knockout and wild-type (WT) mice were fed a diet containing 0.2% CPZ w/w for 5 weeks, after which CPZ was withdrawn to enable remyelination. Our results demonstrated that, compared with WT mice, Foxg1-knockout mice exhibited not only alleviated demyelination but also accelerated remyelination of the demyelinated corpus callosum. Furthermore, we found that Foxg1 knockout decreased the proliferation of OPCs and accelerated their differentiation into mature oligodendrocytes both in vivo and in vitro. Wnt signaling plays a critical role in development and in a variety of diseases. GSK-3β, a key regulatory kinase in the Wnt pathway, regulates the ability of β-catenin to enter nuclei, where it activates the expression of Wnt target genes. We then used SB216763, a selective inhibitor of GSK-3β activity, to further demonstrate the regulatory mechanism by which Foxg1 affects OPCs in vitro. The results showed that SB216763 clearly inhibited the expression of GSK-3β, which abolished the effect of the proliferation and differentiation of OPCs caused by the knockdown of Foxg1. These results suggest that Foxg1 is involved in the proliferation and differentiation of OPCs through the Wnt signaling pathway. The present experimental results are some of the first to suggest that Foxg1 is a new therapeutic target for the treatment of demyelinating diseases of the CNS.The massive loss of oligodendrocytes caused by various pathological factors is a basic feature of many demyelinating diseases of the central nervous system (CNS). Based on a variety of studies, it is now well established that impairment of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is a vital event in the failed treatment of demyelinating diseases. Recent evidence suggests that  In the central nervous system (CNS), oligodendrocytes (OLs) are myelin-forming glial cells that play vital roles in rapid impulse conduction and normal axonal functions. Current studies have demonstrated that inflammatory stimuli or immune attacks can damage the myelin sheath, leading to OL death and myelin sheath loss, such as that observed in multiple sclerosis (MS), the most common demyelinating disease . It has Foxg1, also referred to as brain factor-1, is an important member of the Forkhead box transcription factor family, which plays dual roles in transcriptional activation and transcriptional repression [Foxg1 is widely expressed in the cerebral cortex and is critical to the development of the CNS [Foxg1 gene translocations, deletions, or mutations are closely related to various diseases associated with the occurrence and development of the CNS [Foxg1 reduces the proliferation of neural precursor cells, accompanied by early differentiation into neurons [et al. found that Foxg1 deletion increases the numbers of neurons and astrocytes at an early stage after birth in conditional Foxg1 gene-knockout mice, and they speculated that Foxg1 might inhibit neurogenesis and glial cell formation in the early stage after birth [Foxg1 in the development and differentiation of OPCs is still uncertain.pression . Foxg1 i the CNS . One or  the CNS , 8. Prev neurons , 10. In er birth . HoweverFoxg1 on the development and maturation of OPCs is associated with the Wnt signaling pathway has not been reported.The Wnt signaling pathway plays an important role in the development and myelination of OPCs , 13. GSKFoxg1 in the proliferation and differentiation of oligodendrocyte lineage cells and myelin regeneration, we conditionally knocked out Foxg1 in vivo to assess the loss and repair of myelin during demyelinating injury in mice, and we further explored its regulatory mechanism in vitro. Our findings revealed a previously-unknown function of Foxg1 in oligodendrocyte lineage cells, suggesting that downregulating the expression of Foxg1 in the treatment of demyelinating CNS diseases may alleviate demyelination and promote remyelination, thus providing a new gene therapy target for demyelinating CNS diseases such as MS.To investigate the role of Foxg1 cKO mice  were donated by Professor Chunjie Zhao from the Medical School of Southeast University. Wild-type ICR mice were purchased from the Experimental Animal Center of Xuzhou Medical University. All mice were housed with free access to food and water under a 12/12-h dark/light cycle and specific pathogen-free conditions. For Foxg1 conditional disruption in neural stem/progenitor cells (NSPCs), Nestin-CreERTM mice were crossed with Foxg1fl/fl mice and induced with tamoxifen . The genotypes of all mice were determined by PCR analysis of tail genomic DNA with appropriate primers. Male and female mice were used for all experiments without bias. All experiments were performed according to the guidelines approved by Xuzhou Medical University Experimental Animal Ethics Committee.TM-mediated recombination, TM was intraperitoneally injected into mice at 1 mg/20 g body weight [The TM was dissolved in corn oil to a concentration of 10 mg/mL by shaking the solution for 3 h at 37°C. For CreERy weight , 17 3 tiExperimental demyelination was induced by feeding 7-week-old male mice 0.2% (w/w) cuprizone  mixed into ground standard rodent chow , 19. TheFoxg1, 8 wild-type mice (WT group) and 8 Foxg1 cKO mice (cKO group) were sacrificed on day 7 after TM injection; (2) at the end of 5 weeks of CPZ administration, 32 mice in 4 groups  were sacrificed to explore the effects of Foxg1 on CPZ-induced demyelination and OPC differentiation; (3) at the end of 4 weeks of CPZ administration, 24 mice in 3 groups  were sacrificed to explore the effects of Foxg1 on OPC proliferation; and (4) another 24 mice from the same groups (8 mice per group) were sacrificed to investigate the effects of Foxg1 on remyelination after 5 weeks of CPZ administration and another 2 weeks of recovery with normal chow. All mice received the same amount of chow each day.Depending on the experimental purpose, mice were sacrificed at different times as follows: (1) to assess the knockout efficiency of In the OPC proliferation study, the mice were intraperitoneally injected with 5-bromo-2’-deoxyuridine   twice a day for 7 consecutive days from week 3 to week 4, and then sacrificed  was used to evaluate spatial location learning and memory as described in previous studies , 21. AllLuxol fast blue staining and degrees of demyelination in three non-serial sections from each mouse were assessed semi-quantitatively in a blinded manner, as described previously . The demFor immunofluorescence, the brain sections were blocked with 5% bovine serum albumin (BSA) and 0.3% Triton X-100  in 0.01 mol/L phosphate-buffered saline (PBS) for 30 min at 37°C, followed by incubation with primary antibodies overnight at 4°C. The primary antibodies used were anti-CNPase , anti-MAG , anti-Nestin , anti-MBP , and anti-BrdU , anti-O4 , anti-Foxg1 , and anti-NG2 . The sections were treated with the appropriate FITC- or TRITC-conjugated secondary antibodies  overnight at 4°C and then counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Finally, the sections were mounted with anti-fade mounting medium. The slides for BrdU staining were pretreated with solution containing 50% formamide, 280 mM NaCl, and 30 mM sodium citrate at 65°C for 2 h, incubated with 2 M HCl at 37°C for 30 min, and rinsed with 0.1 mol/L boric acid (pH 8.5) at room temperature for 10 min [in vitro, each dish was scanned with a 20 × objective lens in 600 μm × 450 μm format in the x–y direction. The number of NG2/GFP, O4/GFP or CNPase/GFP double-positive cells among GFP-positive cells in each field was counted by a blinded observer [Fluorescent images were captured using a confocal laser scanning microscope  with appropriate excitation wavelengths. The digital images were measured using Image-Pro Plus 6.0 software . In the statistical analysis, at least 6 representative fields from randomly-selected images were acquired from each sample by a blinded observer, and then the average cell count/integrated optical density values were calculated . To meas2 on poly-L-lysine-coated flasks and cultured in DMEM/F12 medium supplemented with 2% B27, 10 ng/mL platelet-derived growth factor AA , and 10 g/mL basic fibroblast growth factor  for 3 days; then, the medium was replaced with DMEM/F12 medium without PDGF-AA and bFGF for 1 day to generate preOLs. For oligodendrocyte differentiation, 10% fetal bovine serum (FBS) was added to the preOL medium and cultured for 7 days. The medium was changed every 2 days.The rat OPC proliferation culture was maintained as previously described , 25. IsoFoxg1 mRNA expression was calculated according to the standard 2−ΔΔCt method by normalization to the 18S rRNA mRNA level.Genomic RNA was extracted with TRIzol reagent  according to the manufacturer’s instructions and as previously described . The senWestern blot analysis was performed as previously described , 28. TotFoxg1 locus on chromosome 2 and its 200-bp flanking sequences were amplified by PCR from genomic rat DNA and inserted into the GV248 vector . The shRNA sequence for Foxg1 was 5′-TCGGGCCAAGCTAGCCTTTAA-3′. To generate the construct, the scrambled sequence 5′-TTCTCCGAACGTGTCACGT-3′ was inserted. In the scrambled sequence, the nucleotides were randomly added with no target sequence tracks. Preparations of the recombinant lentivirus were made by transient co-transfection of HEK 293T cells accompanied by proper transfer vectors and lentiviral helper plasmids  using Lipofectamine 2000  according to the manufacturer’s instructions. OPCs were transfected with lentiviral Foxg1 shRNA 72 h before experiments.A lentivirus encoding small hairpin RNA (shRNA) for Foxg1 was designed and synthesized by GeneChem Co., Ltd . The 4 cells were seeded into each well of a 48-well plate in DMEM/F-12 medium (1:1) supplemented with 10% FBS. Twenty-four hours later, EdU  was added to the medium at 10 μmol/L. Another 24 h later, the cells were fixed with 4% paraformaldehyde for 30 min. For EdU staining, the cells were then incubated with freshly-made 1× Apollo®567 reaction cocktail for 30 min at room temperature in the dark. Then, the cells were incubated in 0.5% Triton X-100 in PBS for 10 min at room temperature. After washing twice with PBS, the cells were fixed in methanol and then stained with Hoechst 33342. The percentage of EdU-positive cells was calculated as the number of Apollo®567-stained cells/the number of Hoechst 33342-stained cells.For EdU labeling, 3.5×10T-tests or one-way analysis of variance (ANOVA) followed by the Newman-Keuls or Tukey’s HSD post tests were used for comparisons between two or multiple groups, respectively. Statistical significance was set at P < 0.05.Data were analyzed with SPSS software version 19.0  and are expressed as the mean ± SEM. Foxg1 gene knockout  of CPZ treatment, the mice were intraperitoneally injected with TM to induce  in mice , 30. TheFoxg1 cKO+CPZ group than in the WT+CPZ group (Fig. Foxg1.LFB staining showed that myelin was intact in the corpus callosum of WT mice, but it had almost entirely disappeared in the WT+CPZ mice Fig. A. The deoup Fig. D. The stoup Fig. B, E. TheFoxg1 cKO+CPZ group than the WT+CPZ group (Fig. + cells (Fig. + cells (Fig. Foxg1-cKO treatment. The number of proliferating OPCs, indicated by NG2+/BrdU+ (Fig. +/O4+ (Fig. +/MBP+ (Fig. +/CNPase+ (Fig. +/MAG+ (Fig. Foxg1 cKO+CPZ group than in the WT+CPZ group. These data suggested that Foxg1 cKO decreases the production or proliferation of OPCs but promotes their differentiation.BrdU labeling was used to assess cell proliferation Fig. A and difoup Fig. C, D. Furlls Fig. E, F and lls Fig. J cells idU+ Fig. G, H or BO4+ Fig. I, K, wasFoxg1 in the regeneration of OLs after CPZ-induced myelin sheath injury, TM was administered on day 1 of CPZ withdrawal (Fig. Foxg1 deletion in CPZ-treated mice clearly decreased the latency (Fig. Foxg1 cKO+CPZ than the WT+CPZ group (Fig. Foxg1 cKO significantly promotes the recovery of learning and memory after CPZ withdrawal in mice.To study the role of wal Fig. A. Behavincy Fig. B, C. In oup Fig. D. These Foxg1-cKO mice. These results indicated that Foxg1 deficiency favors myelin repair after CPZ-induced demyelinating injury.After 2 weeks of feeding without CPZ, the demyelination score was decreased Fig. F, G and In vitro, GFP-tagged shRNA knockdown of Foxg1 using lentiviral transfection was required to confirm its function in the proliferation and differentiation of cultured OPCs. The results of immunofluorescence staining (Fig. Foxg1 knockdown strongly decreased the expression of Foxg1 in primary cultured rat OPCs. The lentiviral transfection efficiency was defined by GFP fluorescence (Fig. + cells was significantly smaller in the Foxg1 shRNA than the control group (Fig. Foxg1 decreases the proliferation of OPCs in vitro, suggesting that Foxg1 participates in the regulation of OPC proliferation.ing Fig. A, B and ing Fig. D demonstnce Fig. C. The reoup Fig. E, F. TheFoxg1 regulates OPC differentiation, we downregulated the level of Foxg1 in OPCs and analyzed the number of immature and mature OLs. In contrast to the control group, most of the cells in the shRNA group were labeled with CNPase (Fig. Foxg1 shRNA than in the Ctrl group (Fig. Foxg1 plays a negative regulatory role during OPC differentiation into OLs.To confirm whether ase Fig. I, and onase Fig. G or O4 (ase Fig. H. The nuoup Fig. J, suggesFoxg1 influences Wnt signal activation, we assessed the expression of GSK-3β and β-catenin. The results showed that the level of GSK-3β mRNA (Fig. Foxg1 shRNA than in the control group, while the expression of β-catenin was significantly decreased in Foxg1-knockdown OPCs (Fig. Foxg1 knockdown (Fig. + cells (Fig. + (Fig. + cells (Fig. Foxg1 shRNA group. These results indicated that Foxg1 might be involved in the proliferation and differentiation of OPCs through the Wnt signaling pathway.To explore whether RNA Fig. A and proRNA Fig. B was sigPCs Fig. C. The GSown Fig. C. Moreovlls Fig. D, E and . + Fig. F, I and lls Fig. G, I and lls Fig. H, I whenFoxg1 is abundantly expressed in the CNS [Foxg1 in the oligodendroglial lineage and the underlying mechanisms remain unclear. Therefore, the present study was designed to determine the effect of Foxg1 and the mechanisms underlying the regulation of the Wnt signaling pathway following CPZ-induced demyelination.As described in previous reports,  the CNS  and play the CNS . HoweverFoxg1 in NSPCs reversed the demyelination and spatial learning and memory impairments; moreover, a more evident remyelination was found after CPZ withdrawal. It has been reported that different subcellular localizations of Foxg1 control the machinery that causes cell differentiation, replication, and bioenergetics. A fraction of Foxg1 is recruited to mitochondria and plays a major role in neuronal survival, differentiation, and plasticity [Foxg1+/− mice [Foxg1 might participate in the repair of myelin sheath damage in mice with CPZ-induced demyelination.CPZ is a copper chelator that impacts cell metabolism and leads to oligodendrocyte death and demyelination accompanied by weight loss and behavioral disorders . The CPZasticity . In addiasticity , as well+/− mice . Thus, iFoxg1 on the regulation of demyelination and remyelination remain to be clarified. Here, we found that CPZ administration increased the number of OPCs, which indicated that CPZ injury initiates the endogenous repair process and promotes the production of OPCs from neural stem cells of the subventricular zone region and migration to the injured area [Foxg1 cKO. Moreover, double labeling using BrdU and cell-specific markers showed that Foxg1 cKO promoted OPC differentiation into mature OLs. Previous research has demonstrated that Foxg1 may be necessary for maintenance of the NSC pool in the CNS and that genetic inactivity of Foxg1 promotes both gliogenesis and neurogenesis [Foxg1 cKO in nestin-positive NSCs promotes neurogenesis and gliogenesis [Foxg1 cKO clearly promoted OPC differentiation. In vitro, purified primary OPCs were used to assess the effects of Foxg1 inhibition on OPC proliferation and differentiation. It has been demonstrated that the loss of Foxg1 leads to cycle exit and promotes the differentiation of premature cells [Foxg1 knockdown inhibited the proliferation and promoted the differentiation of OPCs under normal culture conditions. As a result, the number of OPCs in the CPZ-treated corpus callosum area was reduced due to accelerated differentiation. The differentiated mature OLs encapsulated the axons of the neurons to form new myelin sheaths, thereby alleviating the myelin damage caused by CPZ or accelerating remyelination.Oligodendrocytes are derived from OPCs , which pred area . In the ogenesis . NSCs arogenesis . Althougogenesis , it did re cells , 49. Conre cells , Foxg1 kFoxg1 knockdown. We used 10 μmol/L SB216763 to inhibit the function of GSK-3β [Foxg1 on OPC proliferation and differentiation and the nuclear level of β-catenin. These results are in agreement with reports in the literature [in vivo experiments performed here, it can be concluded that conditional knockout or knockdown of Foxg1 effectively facilitates the differentiation of OPCs into OLs and promote the recovery of damaged myelin, and the Wnt/β-catenin signaling pathway is involved in this process. Given the increasing importance of glia-glia crosstalk and the inflammatory microenvironment in demyelination or remyelination [The Wnt/β-catenin signaling pathway plays an important role in regulating cell proliferation and differentiation –52. Studf GSK-3β , 56, andterature . Togethelination –59, futuFoxg1 knockout inhibits the proliferation but promotes the differentiation of OPCs into mature OLs by regulating the activity of the Wnt/β-catenin signaling pathway, which ultimately reduces the severity of demyelination and promotes remyelination. Therefore, Foxg1 might be a promising novel target to enhance endogenous remyelination in patients with demyelinating diseases such as MS. Further experiments are required to investigate the precise targets of Foxg1 in the Wnt/β-catenin signaling pathway and its interactions with other types of glial cells involved in demyelinating diseases in the CNS.In summary, the present study provides evidence that, in CPZ-induced demyelinating mice,


Progressive fibrosis of the dermal tissues is a challenging complication of radiotherapy whose underlying mechanism is not fully understood, and there are few available treatments. The canonical Wnt/β-catenin signaling pathway plays an important role in fibrosis as well as in the epithelial-to-mesenchymal transition (EMT). We investigated whether inhibition of Wnt/β-catenin signaling with sLRP6E1E2, a molecule that binds to extracellular Wnt ligands, ameliorated radiation-induced fibrosis both in vitro and in vivo. Radiation with a single dose of 2 Gy not only facilitated fibrosis in cultured human dermal fibroblasts via activation of the Wnt/β-catenin pathway but also initiated EMT in cultured keratinocytes, developing collagen-producing mesenchymal cells. sLRP6E1E2-expressing adenovirus treatment exerted anti-fibrotic activity in irradiated cultured dermal fibroblasts and keratinocytes. In a mouse model, a single fraction of 15 Gy was delivered to the dorsal skins of 36 mice randomized into three groups: those receiving PBS, those receiving control adenovirus, and those receiving decoy Wnt receptor-expressing adenovirus (dE1-k35/sLRP6E1E2). The mice were observed for 16 weeks, and excessive deposition of type I collagen was suppressed by sLRP6E1E2-expressing adenovirus treatment. These results demonstrate that the modulation of the Wnt/β-catenin pathway has the potential to decrease the severity of radiation-induced dermal fibrosis. Since the skin is usually the first site of entry of radiation, various skin reactions can occur. In the acute phase of radiation injury, erythema may be evident within hours of irradiation, and desquamation and ulceration appear at higher doses. These acute events can be addressed symptomatically and generally resolve without exacerbation2. However, progressive fibrosis of the dermal tissues is a late adverse effect of radiation that is not always resolved. Pain, limited range of motion, and poor cosmesis are well-described adverse events attributed to radiation-induced fibrosis of the skin3. Unfortunately, there are few anti-fibrotic therapies available.Radiotherapy is an effective treatment for controlling solid tumors such as head and neck cancer, breast cancer, and soft tissue sarcoma. However, collateral injury to the surrounding healthy tissues may result in toxicity and frequently limits the therapeutic dose of radiation that can be delivered4. Several mediators are involved and act on fibroblasts to cause tissue fibrosis. In addition to the TGF-β signaling pathway, which is a key mediator of fibroblast activation, the Wnt/β-catenin signaling pathway plays an important role in fibrotic reactions5. In the setting of radiation, β-catenin mediates the effects of radiation in fibroblasts, and its modulation has the potential to decrease the severity of radiation-induced complications4.Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components following a breakdown in the normal balance of extracellular matrix synthesis and degradation6. Low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt ligand, is required for activation of the canonical Wnt/β-catenin pathway7. A novel soluble Wnt receptor, sLRP6E1E2, which is composed of the LRP6 E1 and E2 regions, was used to inhibit the pathway8. Therefore, we explored the biological effects of the binding of sLRP6E1E2 to extracellular Wnt ligands after irradiation.In this study, we tested the hypothesis that inhibition of the Wnt/β-catenin signaling pathway would ameliorate radiation-induced fibrosis. Inhibition of the pathway was accomplished using adenovirus gene therapy expressing a Wnt antagonist that binds Wnt ligands to block interactions with receptorsDermal fibroblasts and keratinocytes, representative cells that compose the skin, were selected to determine the effect of irradiation on skin tissues. We began with the premise that the Wnt/β-catenin signaling pathway would be activated in the setting of radiation. To verify that radiation contributed to the activation of the Wnt/β-catenin pathway, we examined Wnt-3a and β-catenin expression level in human dermal fibroblasts (HDF) and HaCaT keratinocytes at 48 h after a single dose of 2-Gy irradiation. Both Wnt3a and β-catenin are upregulated at the protein level Fig. a,b. This6. Therefore, we examined the extent of cytosolic and nuclear localization of β-catenin by immunofluorescence staining of irradiated HDFs and HaCaT keratinocytes after transduction with dE1-k35/sLRP6E1E2 (sLRP6E1E2-expressing virus) or dE1-k35 (control virus). In absence of radiation, β-catenin was rarely observed in the cytosol and nucleus. Upon radiation, control groups (radiation + PBS and radiation + sLRP6E1E2 (−) virus) showed increased β-catenin localization on the cytosol and nucleus, while dE1-k35/sLRP6E1E2-transduced cells showed lower levels of nuclear localization of β-catenin than control groups  virus) Fig. b, indicaWe next evaluated changes in the Wnt/β-catenin pathway in the setting of radiation in a mouse model treated with PBS, control adenovirus (dE1-k35), or decoy Wnt receptor-expressing adenovirus (dE1-k35/sLRP6E1E2) using western blot analysis of Wnt-3a and β-catenin. In the PBS control, levels of Wnt-3a began increasing after irradiation and peaked at 12 weeks, while β-catenin increased rapidly at 4 weeks, peaked at 8 weeks, and decreased 12–16 weeks after irradiation . As we sought to focus on radiation-induced fibrosis in this study, fibrosis caused by wound healing should be excluded. After 15 Gy radiation was delivered to the dorsal skin, tiny dry desquamations appeared in approximately 1/3 of the mice in each group within 4 weeks, and fields receiving radiation healed without ulceration in all mouse. Upon examination by planimetric analysis and laser Doppler flowmetry, there were no significant differences between groups in almost values , and sLRP6E1E2 (+) groups) compared to those in normal dermal tissue. Thus, radiation seems to be responsible for increasing collagenous fibrosis. Among the irradiated groups, a relatively thin dermis and many miniaturized follicles were observed in the sLRP6E1E2-treated group, while mice in the control groups ) had thicker dermal tissues and fewer follicles. In addition, adnexa were replaced by collagenous fibers in both control groups Fig. a.Figure To test the potential effect of sLRP6E1E2 on in vivo fibrosis, mRNA expression of collagen type I α1, collagen type III α1 and TGF-β1 was examined at 12 weeks post-radiation, and immunohistochemistry for type I collagen was performed at each time point. The mRNA expression of collagen type I α1, collagen type III α1 and TGF-β1 was significantly reduced in dE1-k35/sLRP6E1E2-transfected mouse compared to controls In conclusion, radiation facilitates the fibrotic reaction in cultured keratinocytes as well as cultured human dermal fibroblasts, while sLRP6E1E2 exerts anti-fibrotic activity in irradiated cultured cells (in vitro study). In a mouse model, excessive collagen deposition was controlled in the sLRP6E1E2-treated group (in vivo study). Taken together, these results support the idea that modulation of the Wnt/β-catenin pathway has the potential to decrease the severity of radiation-induced dermal fibrosis.2 until forming a confluent monolayer. An X-RAD320 irradiator  was used to irradiate cell cultures with a single dose of 2-Gy radiation.HDF and HaCaT keratinocytes  were seeded at passage 4 in 6-well plates and grown in a complete cell culture medium consisting of Dulbecco’s modified Eagle medium (DMEM) with 100 U/ml penicillin, 100 g/ml streptomycin sulfate, and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO6. The replication-incompetent dE1-k35 was used as a negative control8. Titers of generated adenoviruses were determined using the optical density of the viral genome. For both dE1-k35/sLRP6E1E2 (sLRP6E1E2-expressing virus) and dE1-k35 (control virus), a multiplicity of infection (MOI) of 100% was transfected into cultured dermal fibroblasts and HaCaT keratinocytes. Irradiation of cell cultures was followed immediately by viral transfection.The virus dE1-k35/sLRP6E1E2 was obtained as previously describedCOL1A1), collagen type 3 α1 (COL3A1), TGF-β1 (TGFB1) in cultured HDF cells and those of E-cadherin (CDH1), vimentin (VIM), and twist (TWIST1) in cultured HaCaT keratinocytes, qRT-PCR was performed 48 h after irradiation. Total RNA was prepared with the RNeasy Mini Kit , and complementary DNA was prepared from 0.5 µg of total RNA by random priming using a first-strand cDNA synthesis kit  under the following conditions: 95 °C for 5 min, 37 °C for 2 h, and 75 °C for 15 min. Applied Biosystems TaqMan primer/probe kits were used to analyze mRNA expression levels with an ABI Prism 7,500 HT Sequence Detection System . For cDNA amplification, AmpliTaq Gold DNA polymerase was activated by 10-min incubation at 95 °C; this was followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C for each cycle. The mRNA expression levels were normalized to the levels of the GAPDH housekeeping gene, and then relative quantities were expressed as fold-inductions compared with the control gene after determining the threshold cycle and drawing standard curves. The following primers were used: Hs00164004_m1 (COL1A1), Hs00164103_m1 (COL3A1), Hs00998133_m1 (TGFB1), Hs 01023894_m1 (CDH1), Hs 00185584_m1 (VIM), Hs 00361186_m1 (TWIST1), and Hs99999905_m1 .To evaluate mRNA levels of collagen type 1 α1 , 1% Nonidet P-40, 150 mM sodium chloride, and 0.1 mM zinc acetate in the presence of protease inhibitors. Protein concentration was determined by the Lowry method , and 20 µg of each sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gels were then transferred electrophoretically onto a polyvinylidene difluoride membrane . The membrane was blocked with blocking buffer for 1 h and then incubated overnight at 4 °C with primary antibodies against β-catenin , Wnt , and actin . After a 2-h incubation with the secondary antibodies horseradish peroxidase-conjugated rabbit antibody (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated mouse antibody (Santa Cruz Biotechnology), protein bands were visualized using an ECL detection kit  according to the manufacturer’s instructions. Protein expression was analyzed using ImageJ software .After irradiation, HaCaT keratinocytes were cultured for 48 h, and phenotypic changes were examined using phase-contrast microscopy .After 48 h post-radiation, cultured cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 15 min at room temperature, and then permeabilized by incubation for 15 min with 0.01% Tween 20 in PBS. Samples were blocked with 5% bovine serum albumin followed by incubation with anti-collagen I, anti-phospho-Smad 2/3 and anti-β-catenin  overnight at 4 °C. The next day, cells were washed with PBS and incubated with bovine anti-rabbit IgG-FITC  secondary antibody for 2 h at room temperature. Cells were mounted on slides with mounting solution containing DAPI , and cells were viewed under a confocal microscope system .Animals were handled according to national and international guidelines in an animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The number of animals used was minimized, and all necessary precautions were fulfilled to mitigate pain and suffering. Protocols were approved by the Institutional Animal Care and Use Committee in Yonsei Biomedical Research Institute.10 viral particles (VP)/ml was administrated. The mice were housed and observed for 16 weeks to allow toxic effects of radiation to develop. At 2, 4, 6, 8, 10, and 12 weeks after irradiation, digital photographs and laser Doppler flowmetry  measurements were taken. At 4, 8, 12, and 16 weeks, mice were euthanized (n = 3 each week), and tissues were harvested for histological analysis.A total of 36 four- to five-week-old mice were randomized into three groups according to the injection materials: PBS, control adenovirus (dE1-k35), and decoy Wnt receptor-expressing adenovirus (dE1-k35/sLRP6E1E2). A single fraction of 15 Gy was delivered to the dorsal skin (dimension: 1.5 × 2 cm) of each mouse using an X-RAD320 irradiator, ensuring that > 90% of the prescribed dose would be limited to skin depth. Post-irradiation, PBS or virus was directly injected into each irradiated dorsal skin at 1, 3, 5, 7, 9, and 11 weeks. For each mouse, 5 × 10Gross changes of the skin after a single fraction irradiation of 15 Gy were observed, and complications related to irradiation such as erythema, dry desquamation, moist desquamation, ulceration, hair loss, and necrosis were recorded. Irradiated areas were marked on the dorsum of each mouse before radiation, and the marked areas were later assessed by digital photographs. The Scion  image program was used by two blinded observers to digitally measure each affected area. Using the program, the length of each image was converted to the actual length, and the surface area was calculated. Sequential changes in the irradiated area were analyzed.To assess changes in blood flow within the irradiated area, the Periflux system 5,000 was used. Skin vascularity was assessed from the center of the irradiated area using laser Doppler flowmetry. Data were measured three times at 1-min intervals, and the mean values were obtained. Data are presented as perfusion units (PU).To analyze the histology of collagen fibers, Masson’s trichrome staining was performed. The staining solution was prepared with a standard protocol.Immunohistochemistry was performed to measure the amount of type I collagen. Slides were incubated overnight at 4 °C with rabbit anti-collagen I antibody (Abcam) and rabbit anti-fibronectin (Santa Cruz Biotechnology) diluted in blocking serum. After several stages of washing with PBS, the tissue sections were incubated with peroxidase-conjugated goat anti-rabbit polyclonal antibody  at a dilution of 1:200 in PBS for 30 min at 37 °C. After several washes in PBS, the signals on the tissues were revealed by incubating the tissues with diaminobenzidine in PBS for 10 min.Semi-quantitative analysis of the synthesis of type I collagen was executed using MetaMorph image analysis software . Results are expressed as the average optical density (OD) of five different digital images.t-test for unpaired observations or Bonferroni’s correction for multiple comparisons. P < 0.05 was considered significant.Each experiment was performed in triplicate. Results are expressed as mean ± standard error of the mean (SEM). Group results were compared by one-way analysis of variance, followed by post hoc Student’s Supplementary file1 (PDF 648 kb)


Studies conflict concerning the use of enzyme immunoassays (EIA) for plasma free metanephrines (P-MNs) vs. other methods for pheochromocytoma/paraganglioma (PPGL) diagnosis. We compared commercially available EIAs for P-MNs with high-pressure liquid chromatography (HPLC) for 24 h-urinary MNs (U-MNs) and -catecholamines (U-CATs). 943  patients  were studied. Simultaneous measurements of all parameters analyzed at the central lab of our university hospital was mandatory for inclusion. Sensitivity of P-MNs (94.4%) was similar to that of U-MNs (100%), and both were higher than of U-CATs (77.8%), specificity of P-MNs (100%) higher than of U-MNs (73.6%), and similar to U-CATs (99.8%). With the recently proposed downward adjusted ULN of P-MNs to correct for the reported negative bias of the EIAs sensitivity (98.1%) raised non-significantly, but specificity decreased significantly (94.8%). Areas under receiver-operating characteristic curves indicated comparable diagnostic performance of P-MNs (0.989) vs. U-MNs (0.995), both better than U-CATs (0.956). In summary, the EIAs to measure P-MNs performed similarly to U-MNs by HPLC, and both better than U-CATs by HPLC. The post-test probability of PPGL given a positive test result was best for P-MNs, and higher than for the other pairs of analytes. Downward corrections of ULN of P-MNs did not improve test performances. Pheochromocytomas (PHEO) and paragangliomas  are rare catecholamine-producing tumors estimated to occur in about 2–8 out of 1 million people per year , in an uThe 2014 Endocrine Society Clinical Practice Guideline with a level A quality of evidence by the GRADE system . Nonethehttps://en.wikipedia.org/wiki/Austria), has been offering chromatographic/MS methods for quantification of plasma-free metanephrines for routine patient care, neither in private institutions nor in university hospitals. Capital costs of instrumentation, the level of operator expertise, and the need for in-house validation are higher than with EIA methods  Guideline QMS24. Plasma MNs were measured by the commercially available 2-MET Plasma ELISA Fast Track Assay kit of LDN ,17. The Concentrations of test parameters are given as percent (%) of ULN to allow for easy comparisons between the methods. The ULN used for analyses are given in the Sensitivity (defined as the fraction of patients with the disease identified as positive by the test), and specificity (fraction of those without the disease correctly identified as negative by the test) of the biochemical parameters were calculated for each analyte alone and as pairs (combined model). For the combined model, a positive finding was defined as a concentration above the ULN of at least one analyte, MN or NMN and A or NA, respectively. For a negative result, the concentrations of both analytes had to be below the respective ULN. Ninety-five percent confidence intervals (CI) of sensitivities and specificities were computed by the method of Wilson  and compTo evaluate the diagnostic performance independent of ULN, ROC curves based on logistic regression analysis were created for each analyte as well as for the pairs, and 95% CI calculated by the method of Newcombe . The ROCwww.graphpad.com. Means and medians were compared by the t-test and the Mann-Whitney U test (both two-sided), respectively, as appropriate. For the comparison of three or more means, one-way ANOVA and Tukey´s multiple comparisons test was used, for non-parametric comparisons of three or more medians the Kruskal–Wallis and Dunn´s multiple comparisons test. P-values < 0.05 were considered significant.To determine the probability of a patient suffering from PPGL given a positive and a negative test result, Fagan´s nomogram for Bayes theorem based on the pre-test probability  and the diagnostic sensitivity and specificity was used ,25. PostThe study has been approved by the ethics committee of the Medical University of Vienna (No. 1089/2016).p = n.s.). A genetic disease was identified in 14 of the 54 patients with PPGL . These patients were younger (p = 0.001) than those with sporadic disease (37 ± 12 vs. 53 ± 14 years), but concentrations of biochemical parameters were comparable (p = n.s.) to patients with sporadic tumors. There were 5 patients with PGL (2 of them with metastases), 2 patients with both PGL and PHEO, and 49 PHEO among the 54 patients with confirmed PPGL  U-NMN and U-NA, but similar concentrations of the other parameters (n = 5) of PGL precludes further evaluation as to the accuracy of the diagnostic tests in PGL vs. PHEO.Age and sex were comparable between PPGL+ and PPGL− patients (med PPGL . As to trameters . The smap < 0.05) sensitivity than all other single parameters, except for P-NMN (51 of 54 PPGL positive) with the corrected ULN as proposed by others [p < 0.05) than all other single metabolites except for U-A . U-NMN  performed worst regarding specificity. U-NMN detected all 54 PPGL and provided higher  than that of the pair of U-A and U-NA (42 of 54 PPGL detected), but not (p = n.s.) vs. the combined U-MN and U-NMN . Specificity (p < 0.001) than that of the pair of 24 h-urinary metanephrines  and of plasma-free metanephrines , when using the corrected ULN as proposed by others [Clinical decisions are seldom based on a single parameter, so sensitivities and specificities were computed of combined models (pairs) as well. The sensitivity (y others .As depicted in p < 0.05) up to almost 5-fold the ULN when compared to the range of false positive concentrations (up to 3.3-fold the ULN) of P-MN and P-NMN with the corrected ULN [False positive results of U-MN and U-NMN showed a broader range (cted ULN . Comparip (area = 0.5) was equal to 0.0001 for all analytes alone and in pairs, indicating that all metabolites, determined by either method, were able to correctly classify patients with and without PPGL.The significance level p = 0.02, 0.004, and <0.0001, respectively) to U-MN , to U-A , and to U-NA , respectively, but not better (p = n.s.) than U-NMN . The AUC of P-MN  was smaller (p = 0.02) than that of U-NMN, but not different (p = n.s.) to the other 24 h-urinary analytes  and was superior  than that of the pair of U-A and U-NA , but not different (p = n.s.) when compared to each other , and of U-MN and U-NMN , were larger , the post-TP of a patient suffering from PPGL given a positive and negative test result of the pairs of analytes were 100% and 0% using P-MN and P-NMN together, 53% and 0% with the combined P-MN and P-NMN using the corrected ULN , 18% andWhen analyzed with the higher ULN stipulated by the manufacturer of the 24 h-urinary metanephrines after February 2019, post-TP of PPGL given a positive test result increased to 76%, while that of a negative result remained the same. More details including summaries of sensitivities and specificities as well as calculations of post-TP of positive and negative test results based on the data of other studies are given in the To the best of our knowledge, this is the largest study comparing the diagnostic efficacy of an immunometric method for the measurement of plasma-free metanephrines (P-MNs) to that of other methods in the biochemical diagnosis of PPGL, and the only series that reports superiority of P-MNs over another method . LC-MS/MS for the measurement of P-MNs or 24-h urinary metanephrines (U-MNs) have been recommended as one of the first-line tests in the biochemical diagnosis of PPGL . Their aOur study provides evidence of equal diagnostic performance as quantified by the AUC of ROC curves of a commercially available EIA kit for the measurement of P-MNs, when compared to a HPLC for U-MNs, and that both methods perform better than the determination of 24 h-urinary catecholamines (U-CATs) by HPLC. The use of downward corrected ULN of the former, as proposed by others , resulteThe post-test probability (post-TP) that an individual is affected by a PPGL, given a positive or negative test result on the basis of the probability of the individual having the condition before the test was run (5.7%), was almost perfect and higher for the P-MNs (100% and 0%) than for U-CATs (96% and 1%), and for U-MNs (18% and 0%), respectively. Obviously, the ULN of the U-MNs (138 μg/24 h) and of U-NMN (311 μg/24 h) was too low, and has been changed by the manufacturer to 320 μg/24 h (U-MN) and to 390 μg/24 h (U-NMN) in February 2019, long after the study has been completed. Applying these higher ULN would have increased post-TP of PPGL given a positive test result from 18 to 76%, without changing the nearly perfect exclusion of PPGL. Given the potentially different results obtained with the new assay version after February 2019, this remains to be determined, however. Increasing ULN would increase specificity of the tests but be followed by an unacceptable decrease of sensitivity. Subjecting only patients with a high pre-test probability of PPGL to biochemical testing could lead to a better specificity. However, studies have not shown consistently better specificity  with sucInitial proficiency studies have suggested that P-NMN measured by the EIA of LDN was systematically lower than determined by HPLC, even after correction for the D-enantiomer of spiked samples and was ascribed to matrix effects . FurtherThe upright position leads to an increase of sympathetic output. As compared to the supine position, both plasma-free MNs (P-NMN more than P-MN) as measured by LC-MS/MS obtained from the patient in the sitting position have been suggested to result in an up to three-fold increase of false positives ,32, so tIn comparing P-MNs by LC-ED vs. the combination of U-MNs by spectrophotometry and of U-CATs by LC-ED, is has been suggested that P-MNs may be the test of choice in high-risk patients . However, in the more common clinical situation to screen for sporadic PPGL, especially in older hypertensive patients, measurement of U-MNs in combination with U-CATs may provide comparable sensitivity with a lower rate of false-positive tests. The authors acknowledge that defining the absence of PPGL by the clinicians´ evaluation or by an alternative diagnosis only was one of the limitations . In our PPGL may relapse at a rate of 1 in 100 patient years, or with a probability as high as 25% over 5 years in patients <20 years harboring PGL >15 cm at first diagnosis , and mayThere are several limitations to our study. We have performed a retrospective analysis with a limited number of patients with PPGL, which may have compromised power to detect sensitivity difference. However, the overall prevalence of PPGL is very low, and a prospective study would need a very large sample population and is not available as yet. In our study, all patients were well characterized, and the sample size was among the largest of the studies that assessed the performance of an EIA for P-MNs. Also, our study was conducted in one center, potentially limiting generalizability, although it is of note that we presented data of a commercially available EIA kit widely used. We did not measure 3-methoxytyramine, the Catechol-O-Methyltransferase metabolite of dopamine, the “sibling” of MN and NMN, that recently has become available and included in the new kits of the EIA. Thus, purely dopamine-secreting PPGLs could not be detected. Moreover, drug history was not available, so specificity may have been overestimated. Interference by medications has been studied for measurement of P-MNs by LC-MS/MS, but there are no data with EIA. Incomplete collection of 24 h-urine could have resulted in falsely low U-MNs and U-CATs measurements. The use of an inappropriately low ULN of 24 h-urinary fractionated metanephrines may have compromised routine patient care, unless other diagnostic criteria have been considered in the treatment decisions. With use of the increased ULN issued by the manufacturer after February 2019, post-TP of PPGL given a positive test of the combined U-MNs improved but was still significantly below that of the pair of P-MNs. Also, we did not study P-MNs by HPLC-MS/MS, so no inference can be drawn as to that method.One of the strengths of our study is that samples were provided by the central lab of the largest hospital in our country serving a population of more than 2 million. Additionally, all three pairs of metabolites  were available in every patient and at the same time within a broad spectrum of almost 1000 well-characterized patients suffering from refractory hypertension, anxiety disorders, or adrenal incidentaloma, which may be confused with PPGL, rendering the study sample representative of a typical population treated by endocrinologists including hereditary syndromes. Finally, for the first time, data were compared with use of a recently proposed ULN to adjust for the assumed systematic negative bias of the EIAs for P-MNs in addition to that of ULN stipulated by the manufacturer .In conclusion, we have shown that the determination of P-MNs with a commercially available EIA kit is as sensitive and more specific when compared to U-MNs by HPLC, and that both methods perform better than U-CATs, the latter resulting in a low sensitivity despite excellent specificity. In the absence of HPLC-MS/MS available, state-of-the-art care of patients suspected to harbor PPGL may thus be possible with the EIAs studied. Post-test probabilities of PPGL given a positive and negative test result of P-MNs were almost 100% and 0%, respectively, with P-MNs, but only 18% and 0% with U-MNs. The systematic negative bias of P-MNs resulting in a low sensitivity by EIAs as reported previously  could no


Oral carcinogenesis is a complex and multifactorial process that involves cumulative genetic and molecular alterations, leading to uncontrolled cell proliferation, impaired DNA repair and defective cell death. At the early stages, the onset of potentially malignant lesions in the oral mucosa, or oral dysplasia, is associated with higher rates of malignant progression towards carcinoma in situ and invasive carcinoma. Efforts have been made to get insights about signaling pathways that are deregulated in oral dysplasia, as these could be translated into novel markers and might represent promising therapeutic targets. In this context, recent evidence underscored the relevance of the Wnt/β-catenin signaling pathway in oral dysplasia, as this pathway is progressively “switched on” through the different grades of dysplasia , with the consequent nuclear translocation of β-catenin and expression of target genes associated with the maintenance of representative traits of oral dysplasia, namely cell proliferation and viability. Intriguingly, recent studies provide an unanticipated connection between active β-catenin signaling and deregulated endosome trafficking in oral dysplasia, highlighting the relevance of endocytic components in oral carcinogenesis. This review summarizes evidence about the role of the Wnt/β-catenin signaling pathway and the underlying mechanisms that account for its aberrant activation in oral carcinogenesis. Oral cancer is a subtype of head and neck cancer, representing the sixth most common malignancy in the world ,2. FolloWnt signaling pathways encompass both canonical and the different noncanonical pathways and are involved in a variety of biological functions, such as cell differentiation, migration and proliferation ,18. Unlic-myc, cyclin D1 and survivin, among others , whose main risk factors include the consumption of tobacco and alcohol . Oral caAs mentioned, OSCC is preceded by lesions that are potentially malignant and histopathologically diagnosed as oral epithelial dysplasia, a stage that encompasses cellular and tissue alterations in the oral epithelium. Following the World Health Organization’s recommendations, two ways for classifying oral epithelial dysplasia have been proposed: one is the grading system for dysplasia, which has three categories: mild, moderate and severe dysplasia ,90,91, wEarly studies by Sato et al. detected β-catenin at the nucleus in samples of oral dysplasia obtained from a murine model of carcinogenesis . In accosurvivin and cyclin D1, which raises the possibility of new therapeutic approaches for patients with oral dysplasia [Unlike other malignancies, no mutations in components of the Wnt/β-catenin pathway, such as APC, axin or β-catenin itself, have been documented at the different stages of oral carcinogenesis ,29,30,31ysplasia . Altogetysplasia ,100,101.ysplasia ,98 and sysplasia ,103; howUnlike oral dysplasia, literature about the localization and function of β-catenin in oral cancer remains a matter of debate, although most studies suggest that β-catenin is mainly cytoplasmic and correlated with poor histological differentiation ,105. FirThe membranous expression of β-catenin exerts a restrictive effect on cell proliferation and migration, and, hence, the loss of β-catenin at this location is associated with increased cell motility and proliferation, correlating with the tumor grade and histological differentiation in OSSC ,105. In Despite that several studies have explored the possibility of mutations in the components of the Wnt/β-catenin pathway in OSCC, no such mutations have been described so far ,29,30,31In relation to Wnt/β-catenin target genes, reports have explored their expressions in oral cancer, with data showing abnormal expressions in a subset of those genes ,114,115.Despite that most evidence has shown a low detection of membranous β-catenin in OSCC, and that augmented cytoplasmic β-catenin in invasive cancers is observed at the expense of decreased detection at the cell-cell junctions, this protein appears only sporadically located in the nucleus of OSCC. Hence, the assessment of functionality in this pathway becomes relevant, namely by exploring downstream transcriptions and their consequences in cell fates. This is due, in part, to the limited use of methodologies that are necessary to assess the status of Wnt/β-catenin-dependent events, such as gene expression. In this context, recent studies have explored the mechanistic aspects of β-catenin-dependent events in early lesions of the oral mucosa based on both in vitro models and in clinical samples (see the following sections).c-myc and cyclin D1 [The Wnt/β-catenin signaling pathway is subject to regulation by Wnt antagonists, which include members of the Dickkopf family (Dkk), Wnt inhibitory factor 1 (WIF-1) and secreted frizzled-related proteins (SFRPs). Thus, a balanced function between the Wnt ligands and their inhibitors contributes to cell proliferation in normal tissues ,121. In yclin D1 ,108,126.yclin D1 ,108,126,In OSCC, members of the SFRP gene family, which prevent the binding of Wnt to frizzled receptors, are epigenetically downregulated via hypermethylation in their promoters . AdditioFor years, the endocytic trafficking was considered as a mechanism of downregulation in signaling pathways, based on the fact that the internalization of the cell surface receptors and their subsequent transport from early endosomes to late endosomes and lysosomes leads to their degradation . HoweverSpecifically, the binding of Wnt ligands causes phosphorylation of the cytoplasmic tail of LRP5/6 by GSK3β and CK1α. This phosphorylation enables the binding of the remaining proteins of the destruction complex at the plasma membrane, forming a structure known as “LRP6 signalosome”, which contains aggregates of Frizzled, phosphorylated LRP5/6, Dvl, axin, APC and GSK3β and whose complex is internalized in endosomal structures ,132,133.A body of evidence accumulated in the past two decades supported the notion that uncontrolled endocytic trafficking is a recurrent phenomenon in malignancy, which is due, in part, to the fact that endosome trafficking controls a plethora of cellular processes ,136. In In oral cancer, alterations in endosomal proteins and regulators of intracellular trafficking have been reported, and these include the overexpression of Rab proteins ,151, cavBased on these observations, it is relevant to explore possible consequences derived from Rab upregulation in OSCC, especially traits associated with malignancy. Intriguingly, whether these GTPases are altered at the early stages of oral mucosal carcinogenesis remains largely unknown. In this context, our recent studies indicate that a subset of Rab-GTPases are upregulated in oral dysplasia . SpecifiDespite that the nuclear accumulation of β-catenin is recognized as a recurrent event in oral dysplasia, mechanisms accounting for such phenomenon have remained elusive ,15,16,27Although further research is still required, and that more mechanistic insights should be drawn by in vitro approaches, it must be emphasized that current evidence showing the endosomal sequestration of components of the β-catenin destruction complex in patient biopsies represents a landmark finding to understand the “deregulation” of this pathway during oral carcinogenesis ,14. IntrAs discussed, the Wnt/β-catenin pathway appears to be relevant in several malignancies, and, hence, therapies aiming to target Wnt signaling represent attractive therapeutic approaches. For instance, recombinant proteins that minimize Wnt-Frizzled interactions or Wnt inhibition have been proposed and thought to impact the outcomes of cancers with deregulated Wnt ligand secretions ,159,160.Following their translation at the endoplasmic reticulum, Wnt ligands are posttranslationally modified by palmitoylation and glycosylation ,38,161. Conversely to these descriptions, which have been focused on the cancer stage itself, no study is available about a potential use of these pharmacological inhibitors at earlier stages of oral carcinogenesis, whose targeting should be significant to preclude tumor progression. To this end, a more comprehensive understanding of the mechanisms involved in Wnt activation during oral carcinogenesis will be required to rationally propose effective therapies in oral cancer.Consequences of uncontrolled Wnt/β-catenin signaling in oral cancer are becoming elucidated, with special emphasis on the early events of oral carcinogenesis. Particularly, an increase in the secretion of Wnt ligands has a fundamental role in the progression of oral cancer through its effect on the processes of tumorigenesis and metastasis. In this context, therapeutic approaches aiming to use Wnt inhibitors appear advantageous for cancers associated with the overexpression of Wnt ligands, since the evidence supports their safety and feasibility in preclinical models, with a powerful inhibitory effect on Wnt signaling. This is expected to be impactful, not only by reducing the growth of tumors caused by the aberrant activation of this pathway, but also, by bringing promising therapies of chemoprevention in order to manage early malignant lesions and prevent their progression towards cancer.Recent studies indicate the involvement of endocytic proteins in promoting Wnt/β-catenin signaling during oral carcinogenesis, an effect namely attributed to their effects in the endosomal sequestration of the β-catenin destruction complex. Although these mechanisms are not completely understood, current models propose that augmented Rab5 activity accounts for early endosome enlargement, followed by increased sequestration of the components of the β-catenin destruction complex and the nuclear localization of β-catenin, which represents a landmark finding to understand the “deregulation” of this pathway during oral carcinogenesis . Further


Our research is designed to explore the function of brain acid soluble protein 1 (BASP1) in the progression of gastric cancer (GC) and its underlying molecular mechanisms. β-catenin in AGS and HGC-27 cells were measured by western blot. In addition, the mRNA expressions of WT1, Wnt, and β-catenin in AGS and HGC-27 cells were detected by qRT-PCR.  In this study, the expression of BASP1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) in both GC tissue and GC cells. The cell cloning, proliferation, apoptosis, migration, and invasion potential of AGS and HGC-27 cells were, respectively, determined using colony formation assay, 5-ethynyl-20-deoxyuridine (EDU) assay, flow cytometry, and Transwell assay. The protein expressions of Bax, caspase-3, Bcl-2, matrix metalloproteinases 2 (MMP-2), MMP-9, Wilms tumor 1 (WT1), Wnt, and β-catenin in AGS and HGC-27 cells.  BASP1 expression was significantly downregulated in both GC tissue and GC cells. BASP1 overexpression markedly repressed proliferation, migration, and invasion and facilitated apoptosis in AGS and HGC-27 cells. In addition, BASP1 overexpression notably promoted the protein expression of Bax and caspase-3 in AGS and HGC-27 cells and inhibited the expression of Bcl-2, MMP-2, and MMP-9. Moreover, BASP1 overexpression significantly inhibited the mRNA and protein expression of WT1, Wnt, and β-catenin pathway in GC. BASP1 could significantly suppress cell proliferation, migration, and invasion and promote apoptosis through inhibiting the activation of the Wnt/ Gastric cancer (GC) is the fifth most common cancer and the third-highest cause of death related to cancers worldwide , 2. OverBrain acid soluble protein 1 (BASP1) was initially identified as an abundant membrane-bound protein in the brain . More anβ-catenin pathway in multiple cancers [β-catenin pathway plays a key role in the process of multiple human malignancies by regulating cell proliferation, apoptosis, and metastasis [β-catenin pathway in lung cancer [β-catenin pathway is unknown.BASP1 is reported to be a component of Wilms tumor 1 (WT1) cosuppressor which can encode transcription factors . Studies cancers . Previoutastasis , 14. A sg cancer . Howeverβ-catenin pathway in GC. The findings of our study may provide a new theoretical foundation for exploring novel treatments for GC.In our study, we investigated the function and molecular mechanism of BASP1 on GC. Our results demonstrated that BASP1 could suppress cell proliferation, migration, and invasion, as well as facilitate apoptosis through inhibiting the activation of Wnt/GC tissue samples and their adjacent noncancerous tissues were collected from 30 patients who underwent surgical resection at our hospital from July 2018 to June 2019. None of the patients before this study had received chemotherapy or radiotherapy. Written informed consent was signed by all recruited patients, and the study protocol was approved by the Ethics Committee of Yucheng People's Hospital of Shandong Province.μg/ml penicillin  and 100 μg/ml streptomycin (HyClone) at 37°C in an incubator filled with 5% CO2 atmosphere.The human normal gastric epithelial cell line (GES-1) and gastric cancer cell lines  were supplied by the Type Culture Collection of the Chinese Academy of Sciences . All the cells were cultured in RPMI-1640  with 10% fetal bovine serum , 100 When AGS and HGC-27 cells were passaged at 80% confluence, the cells were seeded into 6-well plates. The pcDNA3.1-BASP1 and the empty pcDNA3.1 vectors were transfected into GC cells using Lipofectamine® 3000 Reagent  according to the manufacturer's protocol. The transfected AGS and HGC-27 cells were randomly divided into three groups: Control group (no treatment), pcDNA3.1-NC group (transfected with empty pcDNA3.1 vector), and pcDNA-BASP1 group (transfected with pcDNA3.1-BASP1). The pcDNA3.1-BASP1 and empty pcDNA3.1 vectors were obtained from Sangon .3 AGS and HGC-27 cells were seeded in 6-well plates and incubated for 2 weeks. Subsequently, the cell colonies were washed with phosphate-buffered saline (PBS) three times, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution (Sigma). The number of stained colonies was observed using a light microscope.Colony formation assay was performed using previous described methods . After tμm EDU for 2 h. And the cell nuclei were dyed with 4′,6-diamidino-2phenylindole . Finally, the EDU-positive cells were observed using a fluorescence microscope.The proliferation of AGS and HGC-27 cells was measured as described previously by using an EDU assay kit . In brief, the transfected AGS and HGC-27 cells were incubated with 50 The apoptosis of AGS and HGC-27 cells was assessed by using an Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) apoptosis detection kit , according to the manufacturer's instructions. Simply, the transfected AGS and HGC-27 cells were harvested and resuspended in binding buffer. Next, Annexin V-FITC and PI were added to the cell suspension to stain the AGS and HGC-27 cells. At the end, the apoptotic cells were examined with a flow cytometer .μm pore inserts; Corning, Corning, USA) coated with or without Matrigel . After transfection, AGS and HGC-27 cells were resuspended in serum-free medium containing 1% bovine serum albumin  and planted into the upper chamber. The lower chamber was filled with 500 μl of medium containing 10% FBS. After culturing for 24 h, the cells on the upper surface were carefully wiped away. And the migrated or invaded cells on the underside of the membrane were fixed for 15 min at room temperature with 4% paraformaldehyde and stained with 0.1% crystal violet  for 15 min at room temperature. Finally, the number of migrated and invaded cells was counted using an optical microscope.The invasion and migration abilities of AGS and HGC-27 cells were measured by using a Transwell Boyden chamber (8 β-catenin (sense): 5′-ATTTGATGGAGTTGGACATGGC-3′, (antisense): 5′-GAGGAAGAGGATGTGGATACCTCC-3′; and β-actin (sense): 5′-AGGCACCAGGGCGTGAT-3′, (antisense): 5′-GCCCACATAGGAATCCTTCTGAC-3′.Total RNA from GC tissue and GC cells was extracted using the TRIzol kit  following the manufacturer's protocols. Total RNA was then reverse-transcribed into the complementary deoxyribose nucleic acid (cDNA) using the revert aid first-strand cDNA synthesis kit . qRT-PCR was performed using the SYBR Green One-Step RT-PCR Master Mix  on a 7900 real-time PCR system . The primer sequences were designed by Sangon and listed as follows: BASP1 (sense): 5′-CTTCAGACT CAAAACCCGGC-3′, (antisense): 5′-ACGGTTTGGTCGGAATTAGC-3′; WT1 (sense): 5′-CACAGCACAGGGTACGAGAG-3′, (antisense): 5′-CAAGAGTCGGGGCTACTCCA-3′; Wnt (sense): 5′-ACGCTGACTTCGGCGTGTTAG-3′, (antisense): 5′-CGTGGCACTTGCATTTGAGG-3′; β-catenin, 1 : 1000, ab32572) overnight at 4°C. Subsequently, the membranes were incubated with the secondary antibody for 2 h. Finally, the protein bands were visualized with an ECL system .Western blotting was performed according to standard techniques as described previously . Brieflyt-test and one-way ANOVA followed by Tukey's post hoc analysis were used for the analysis of significant differences. P < 0.05 was considered to indicate a statistically significant difference.Experimental data are presented as the mean ± SD. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc.). Student's P < 0.01) (P < 0.01) (P < 0.01)  . Similar < 0.01) . Thus, w < 0.01) , suggestP < 0.01). In addition, the EDU assay results also confirmed that BASP1 overexpression significantly inhibited the proliferation of AGS and HGC-27 cells (P < 0.01)  . All datP < 0.01) (P < 0.01) but reduced the expression level of Bcl-2 (P < 0.01) (The results of flow cytometry showed that BASP1 overexpression significantly increased apoptosis in AGS and HGC-27 cells compared with the Control and pcDNA3.1-NC groups ( < 0.01) . To conf < 0.01) . These rP < 0.01) (P < 0.01) (P < 0.01) (As shown in  < 0.01) . Similar < 0.01) . To conf < 0.01) . All theβ-catenin in AGS cells were dramatically decreased in pcDNA-BASP1 group relative to the Control and pcDNA3.1-NC groups (P < 0.01). Similarly, when compared with the Control and pcDNA3.1-NC groups, the mRNA and protein expressions of WT1, Wnt, and β-catenin in HGC-27 cells were also reduced in the pcDNA-BASP1 group (P < 0.01) (β-catenin pathway in AGS and HGC-27 cells.As shown in  < 0.01) . All theβ-catenin pathway in GC.GC is a global health-threatening malignancy with more than 1,000,000 new cases diagnosed each year . In ChinBASP1 is identified as a tumor suppressor and is lowly expressed in multiple cancers. For example, the expression of BASP1 is reported to be downregulated in thyroid cancer tissues . A studyβ-catenin pathway and facilitate cell migration and invasion in tumors [β-catenin pathway is highly conserved and can regulate a series of biological processes in the progression of diseases [β-catenin pathway plays an important role in multiple cancers, including GC [β-catenin pathway has been reported to regulate cell biological behaviors including cell proliferation, apoptosis, and metastasis [β-catenin pathway in GC. Zhong et al. [β-catenin pathway. In the present study, we explored the effect of BASP1 on the expression of WT1, Wnt, and β-catenin in GC cells by qRT-PCR and western blot assays. The results confirmed that BASP1 overexpression could significantly inhibit the expression of WT1, Wnt, and β-catenin, indicating that BASP1 overexpression could inhibit the activation of Wnt/β-catenin pathway in GC. However, our study has some limitations, such as the absence of in vivo experiments. The function of BASP1 in in vivo experiments will be explored further in our future studies.In recent years, WT1 has been reported as an oncogene regulating the progression of malignancies . Carpentn tumors . The Wntdiseases , 21. In uding GC –24. In atastasis . Chen ettastasis  have repg et al.  have sugβ-catenin pathway in GC. These findings suggest that BASP1 may serve as a therapeutic target for the treatment of GC in the future.In summary, our work revealed that the expression of BASP1 was downregulated in both GC tissues and cells. We also demonstrated that BASP1 could significantly suppress cell proliferation, migration, and invasion and promote apoptosis through inhibiting the activation of the Wnt/