Numerous studies have presented that curcumin could have a positive effect in the prevention of cancer and then in tumor therapy. Several hypotheses have highlighted that curcumin could decreases tumor growth and invasion by acting on both chronic inflammation and oxidative stress. This review focuses on the interest of use curcumin in cancer therapy by acting on the WNT/β-catenin pathway to repress chronic inflammation and oxidative stress. In the cancer process, one of the major signaling pathways involved is the WNT/β-catenin pathway, which appears to be upregulated. Curcumin administration participates to the downregulation of the WNT/β-catenin pathway and thus, through this action, in tumor growth control. Curcumin act as PPARγ agonists. The WNT/β-catenin pathway and PPARγ act in an opposed manner. Chronic inflammation, oxidative stress and circadian clock disruption are common and co-substantial pathological processes accompanying and promoting cancers. Circadian clock disruption related to the upregulation of the WNT/β-catenin pathway is involved in cancers. By stimulating PPARγ expression, curcumin can control circadian clocks through the regulation of many key circadian genes. The administration of curcumin in cancer treatment would thus appear to be an interesting therapeutic strategy, which acts through their role in regulating WNT/β-catenin pathway and PPARγ activity levels. The complex process of cancer can be defined in terms of three stages: initiation, promotion and progression –3. Many In parallel, oxidative stress promotes DNA damages in cancers . Since fCurcuma longa L. [The recent therapeutic strategies in cancer are associated with several limitations, such as high risk of relapse, drug resistance, poor outcomes and unavailability of therapy. However, plants are the site of promise sources of bioactive natural components . These nlonga L. . Curcumilonga L. . Its yellonga L. . Severallonga L. . Curcumilonga L. .The chronic inflammatory microenvironment of tumors could be targeted by curcumin. It is well known that the human body is capable of self-healing after a short-term inflammatory response, but a long-term chronic inflammation could lead to initiation of the cancer process. Many studies have shown that inflammatory factors  and the ROS production-induced inflammation infiltrate the inflammatory microenvironment leading to DNA damages and ultimately initiation of cancer . By actiCurcumin acts as peroxisome proliferator-activated receptor gamma (PPARγ) agonists and thus downregulate the aberrant WNT/β-catenin pathway observed in cancers . PPARγ aIn parallel, dysregulation of circadian rhythms (CRs) has been observed in cancers . This dyThis review focuses on the interest of use curcumin in cancer therapy by acting through the opposed interaction between the canonical WNT/β-catenin pathway and PPARγ to repress chronic inflammation and oxidative stress, and to control circadian rhythms.Curcuma longa L. and presents surprising wide range of beneficial properties, such as anticancer, chemopreventive and chemotherapeutic activities [Phytotherapy has claimed importance globally in cancer therapies Table . Curcumitivities . The heaEven if some studies have related that pharmacokinetics of curcumin have revealed poor bioavailability , strong Numerous studies have presented that chronic inflammation leads to DNA damages and tissue injury . ChronicThe immune system is also regulated by several inflammatory factors, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and tumor growth factor-β (TGF-β) . TNF-α eChronic inflammation is also responsible for an increase in cyclooxygenase 2 . Numerous cytokines  activate COX-2 . COX-2 sOxidative stress is considered as an imbalance between the production and elimination of ROS and RNS , 66. ROS+. Superoxide anion is then produced, and molecular oxygen phenomenon is reduced due to the transfer of electron through the mitochondrial membrane.The NADPH oxidase (NOX) enzyme increases ROS production through the oxidation of intracellular NADPH to NADPROS production has a key role in numerous signaling involved in changes of microenvironment . Thus, dSeveral researches have demonstrated the mechanism by which oxidative stress can lead to chronic inflammation, which in turn could cause cancers . The imbMany studies have shown that canonical the WNT/β-catenin pathway stimulates inflammation . MoreoveDrosophila melanogaster and its mouse homolog Int. The WNT pathway is involved in several signaling and regulating pathways, such as embryogenesis, cell proliferation, migration and polarity, apoptosis, and organogenesis [WNT name is derived from Wingless ogenesis . During ogenesis . Recent ogenesis .Fig. 2ThWNT ligands are lipoproteins that activate specific co-receptors. These WNT ligands activate the canonical WNT pathway through the action of β-catenin. WNT ligands activate Frizzled (FZD) receptors and low-density lipoprotein receptor-related protein 5 and 6 (LRP 5/6) . The comDuring the “off-state” of the WNT/β-catenin pathway, WNT ligands do not bind FZD and LRP 5/6. The β-catenin destruction complex, formed by AXIN, APC (adenomatous polyposis coli) and GSK-3β (glycogen synthase kinase 3β), phosphorylates β-catenin. Thus, phosphorylated β-catenin is degraded into the proteasome.Several WNT inhibitors inactivates the canonical WNT/β-catenin pathway. GSK-3β is the major inhibitor of the WNT pathway. GSK-3β is a neuron-specific intracellular serine-threonine kinase that regulates several signaling pathways such as inflammation, neuronal polarity and cell membrane signaling . GSK-3β Positive interplay between WNT/β-catenin and NF-ϰB has been highlighted . The actNF-ϰB over-expression inactivates GSK-3β whereas it stimulates β-catenin signaling , 95. GSKThe over-activated PI3K/Akt pathway observed in the cancer process is stimulated by ROS production . PTEN isThe ligand-activated transcriptional factor peroxisome proliferator receptor γ (PPARγ) is a member of the nuclear hormone receptor super family. It forms a heterodimer with retinoid X receptor (RXR), leading to a PPARγ-RXR complex which binds to specific peroxisome proliferator response element (PPRE) regions in the DNA and activating several target genes involved in fatty acid transport (FABP3), cholesterol metabolism , glucose homeostasis  and lipid catabolism (SCD-1). This dimer interacts with other coactivators proteins such as PGC-1α, and induces specific genes expression . GlucoseThe action of PPARγ agonists remains unclear in cancer cells even if their role is well understood in the regulation of differentiation and stemness programs . In physNumerous biological processes in the body are regulated by the circadian “clock” (circadian locomotors output cycles kaput). The circadian clock is in the hypothalamic suprachiasmatic nucleus (SCN). CRs are endogenous and entrainable free-running periods that last approximately 24 h. Numerous transcription factors are responsible for the control of CRs. These are called circadian locomotors output cycles kaput (Clock), brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 , Period 1 (Per1), Period 2 (Per2), Period 3 (Per3), and Cryptochrome (Cry 1 and Cry 2) , 120. ThEpidemiological and fundamental evidence supports the idea of linking circadian disruption with cancer . DNA repMelatonin has been used in the treatment of chronic bowel inflammation resulting in decreasing inflammation through inhibition of COX-2 and iNOS . MoreoveRecent studies have indicated that the hypoxic response in cancer could be directly controlled by the circadian rhythm Clock/Bmal1 . In a siWNT/β-catenin pathway is the downstream target of the RORs control factors and has several putative Bmal1 clock-binding sites within its promoter . ThroughIn physiological conditions, the core circadian genes work in accurate feedback loops and keep the molecular clockworks in the SCN. They allow the regulation of peripheral clocks . Per1 anPPARγ acts directly with the core clock genes and presents diurnal variations in liver and blood vessels . In miceNumerous studies have shown that curcumin inhibits the precursors of angiogenesis in cancers . ChemicaThe complex process of metastasis involves numerous alterations and degradations of the ECM by MMP which leads to the over-expression of chemokine receptors, inflammation and then angiogenesis. Curcumin inhibits cell migration in colorectal cancer by inhibiting MMP-9 activity and NF-ϰB and in the same time by activating AP-1 . FurtherCell-cycle is arrested in G2/M step in medulloblastoma cells by using curcumin inhibiting the WNT/β-catenin pathway . CurcumiIn Burkitt’s lymphoma cells, curcumin increases radiation-induced apoptosis through the inhibition of the PI3K/Akt pathway . MoreoveFew studies have reported the PPARγ agonist role of curcumin. However, curcumin is known to induce apoptosis, and to inhibit cell proliferation and inflammation by stimulating PPARγ . ThroughSeveral studies have suggested that curcumin can alleviate oxidative stress and inflammation through the Nrf2-keap1 pathway . In variCancer process is responsible for the activation of the NF-κB pathways leading to the over-expression of pro-inflammatory factors, including COX-2, iNOS, cytokines, TNF-α . CurcumiN-acetylcysteine, a biosynthetic precursor of glutathione (GSH), the effect of curcumin was decreased, probably due to GSH-mediated scavenge and thus inactivating of curcumin-derived electrophile [Recent findings have shown that curcumin presents anti-inflammatory effects mediated by the inactivation of the NF-ϰB pathway , but rattrophile . Oxidatitrophile . The rectrophile . Indeed,trophile . Due to trophile . In additrophile . Inhibittrophile . In the trophile .Few studies have investigated the role of curcumin with circadian clock in cancers . HoweverThe numerous interactions between clock dysregulation and cancer underline the interest of circadian therapeutic actions . The temIn mouse ovaries, melatonin administration protects against ROS production and mitochondrial damage . In coloCancers are associated with chronic inflammation, oxidative stress and circadian clock disruption. The over-activation of the WNT/β-catenin pathway increases these pathological phenomena. In cancers, the WNT/β-catenin pathway is upregulated whereas PPARγ is downregulated. These two signaling pathways act in opposing manners and this could explain their unidirectional profile observed in cancers. Moreover, in cancers, the disruption of circadian clock leads to the increase of the WNT/β-catenin pathway and to decrease of PPARγ expression. The strongly link between circadian rhythms, chronic inflammation and oxidative stress appears to be a major mechanism underlying cancers. The use of curcumin, which acts as PPARγ agonists, could be interesting in the reduction of both chronic inflammation and oxidative stress, and in the control of circadian clock by inhibiting the WNT/β-catenin pathway. Due to the considerable impact of cancers on mortality and morbidity rates worldwide, it would appear of the utmost importance to better understand the action of curcumin in cancers and particularly its role in the inhibition of the major signaling system known as the WNT/β-catenin pathway.


APC inactivation or β-catenin mutations. Both processes promote β-catenin nuclear accumulation, which up-regulates epithelial-to-mesenchymal transition (EMT). We investigated β-catenin localization, transcriptome, and phenotypic differences of HCT116 cells containing a wild-type (HCT116-WT) or mutant β-catenin allele (HCT116-MT), or parental cells with both WT and mutant alleles (HCT116-P). We then analyzed β-catenin expression and associated phenotypes in CRC tissues. Wild-type β-catenin showed membranous localization, whereas mutant showed nuclear localization; both nuclear and non-nuclear localization were observed in HCT116-P. Microarray analysis revealed down-regulation of Claudin-7 and E-cadherin in HCT116-MT vs. HCT116-WT. Claudin-7 was also down-regulated in HCT116-P vs. HCT116-WT without E-cadherin dysregulation. We found that ZEB1 is a critical EMT factor for mutant β-catenin-mediated loss of E-cadherin and Claudin-7 in HCT116-P and HCT116-MT cells. We also demonstrated that E-cadherin binds to both WT and mutant β-catenin, and loss of E-cadherin releases β-catenin from the cell membrane and leads to its degradation. Alteration of Claudin-7, as well as both Claudin-7 and E-cadherin respectively caused tight junction (TJ) impairment in HCT116-P, and dual loss of TJs and adherens junctions (AJs) in HCT116-MT. TJ loss increased cell motility, and subsequent AJ loss further up-regulated that. Immunohistochemistry analysis of 101 CRCs revealed high (14.9%), low (52.5%), and undetectable (32.6%) β-catenin nuclear expression, and high β-catenin nuclear expression was significantly correlated with overall survival of CRC patients (P = 0.009). Our findings suggest that β-catenin activation induces EMT progression by modifying cell-cell junctions, and thereby contributes to CRC aggressiveness.WNT signaling activation in colorectal cancers (CRCs) occurs through  Up to 80% of sporadic CRCs show inactivating mutations or allelic loss of adenomatous polyposis coli (APC), is a key WNT pathway regulator, resulting in persistent WNT pathway activation2. Additionally, approximately 5% of sporadic CRCs show activating mutations in CTNNB1 (β-catenin-encoding gene), which are extremely rare in CRCs with APC mutations. Although β-catenin mutations are infrequent in sporadic CRCs, these have been reported in about 18% of hereditary non-polyposis colorectal cancers (HNPCCs)4. Most β-catenin mutations occur in exon 3, which encodes an E3-ligase-binding region that helps β-catenin escape degradation and, consequently, results in WNT pathway activation5. Although it is unclear how cytosolic accumulation of β-catenin induces its translocation into the nucleus, both APC and β-catenin mutations commonly lead to nuclear overexpression of β-catenin6. Notably, since β-catenin is the acting downstream effector of WNT pathway, an enhanced understanding of its targeting and function could provide direct insight into how WNT activation promotes CRC tumorigenesis.WNT signaling pathway alterations play key roles in colorectal cancer (CRC) tumorigenesis. Specifically, WNT pathway activation promotes transcriptional activation of target genes involved in various tumorigenic processes, such as proliferation, stemness, and cell motility7. During EMT, loss of cell junction molecules leads to perturbation of cell-cell interactions; this is considered the most critical step for cancer cells to dissociate from the primary tumor, invade surrounding tissues, and metastasize to secondary sites8. In normal cells, β-catenin promotes adherens junction (AJ) formation by binding to E-cadherin, but it can also function to induce EMT when released from the E-cadherin-β-catenin complex9. Notably, although the clinical significance of abnormal E-cadherin expression in prognosis, invasive potential, and metastasis of CRC is known, the expressional and functional relationship between E-cadherin and β-catenin remains poorly understood10.Nuclear β-catenin acts as a coactivator of T-cell and lymphoid enhancer factors (TCF-LEF), and thereby stimulates expression of target genes related to various oncogenic pathways, particularly the epithelial-to-mesenchymal transition (EMT). EMT results from β-catenin activation, and is directly associated with invasion and metastasis of various cancers11. Of these TJ molecules, abnormal expression of several Claudin proteins  has been associated with tumorigenesis of various cancers, including CRCs. Claudin expression has also been correlated with prognosis, invasion, and metastasis in CRCs. However, Claudin family members show heterogeneous expression patterns and even opposite roles in various types of cancers, and their expressional and functional relationships with β-catenin expression remain unclear12.In addition to AJs, tight junctions (TJs) play central roles in EMT regulation and subsequent cancer progression. In normal cells, TJs maintain cell polarity and integrity, but they are dismantled in cancers to allow dissemination. TJ proteins consist of three major groups: Claudins, Occludins, and linker molecules. Claudin and Occludin families facilitate tight sealing of cells in the epithelial sheet, whereas zonula occludins (ZO) protein-1, a linker molecule, mediates interaction between Claudins and Occludins and the actin cytoskeletonHere, we aimed to investigate the mechanism by which β-catenin activation affects cell-cell junctions during EMT progression using a panel of HCT116 cell lines with differential β-catenin mutation status.13. DLD-1, LoVo, RKO, HCT8, Hep3B, HepG2, and LS174T cell lines were purchased from the Korean Cell Line Bank . DLD-1, LoVo, RKO, HCT8, LS174T, and HCT116 cells were maintained in Roswell Park Memorial Institute (RPMI) medium, containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 °C in a 5% CO2 incubator. Hep3B and HepG2 cells were respectively maintained in Dulbecco’s Modified Eagle Medium (DMEM) and Minimum Essential Medium (MEM), both containing 10% FBS and 1% P/S. For morphological analysis, images were obtained using the 20× objective of an Olympus  IX71 camera.HCT116 cell lines were purchased from Horizon Discovery . HCT116 parental (HCT116-P) line contains one WT β-catenin allele and one mutant allele; HCT116-MT and HCT116-WT contain one mutant or one WT allele, respectively, generated by disruption of the other allele in the parent strainWhole-cell lysates were prepared in Passive Lysis Buffer , containing protease inhibitor cocktail. Proteins were electrophoresed and transferred to a membrane. Membranes were incubated overnight at 4 °C with primary antibodies against GAPDH , β-catenin, E-cadherin , FLAG , EGFP, hnRNPC, α-tubulin , Claudin-7 , SNAIL, SLUG, TWIST1, ZEB1, TCF4, Vimentin, and N-cadherin . Membranes were then washed and incubated with appropriate secondary antibodies for 1 h at room temperature. Western blot images were analyzed using a LAS 4000 mini camera , and relative band densities were quantified using ImageJ (NIH) software.Cells grown on slides were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) or 100% methanol for 15 min, and permeabilized in 0.2% Triton X-100 in PBS. Fixed slides were incubated at 4 °C overnight with primary antibodies against β-catenin, E-cadherin (both from BD Biosciences), Claudin-7 (Thermo Fisher Scientific), or FLAG (Sigma). Appropriate fluorescence-labeled secondary antibodies were then applied for 1 h at room temperature . All images were collected using a LSM700 confocal microscope .Total RNA was extracted using TRIzol Reagent (Life Technologies) according to manufacturer’s instructions. Reverse transcription was performed with 2-μg total RNA. Real-time PCR was performed using the ABI PRISM 7500 Sequence Detector (Applied Biosystems) and SYBR Premix Ex Taq II , according to manufacturer’s guidelines. All mRNA expression levels are presented as the mean ± standard deviation (SD) from two or three independent experiments. Melting curves of PCR products were assessed for quality control.14. Our microarray data are deposited in Gene Expression Omnibus (GSE126845). Differentially expressed genes (DEGs) were selected as those with P < 0.05 and fold change > 1.5. Functional enrichment analysis was performed using BINGO 2.3 plugin for Cytoscape software (http://www.psb.ugent.be/cbd/papers/BiNGO/Home.html), DAVID software  to identify gene ontology (GO) biological processes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways represented by DEGs with statistical significance.Microarray experiments were conducted on HumanHT-12 v4 Sentrix Expression BeadChip . Hybridization of labeled-cRNA to BeadChip, washing, and scanning were performed according to Illumina Bead Station 500X manual. Extraction of mRNA expression data and statistical analysis of raw data were performed using software provided by the manufacturer (Illumina GenomeStudio v2011.1). Expression intensities were normalized by quantile normalization technique; and using normalized intensities, genes differentially expressed in HCT116-WT and HCT116-MT cells were determined by the integrated statistical method previously reportedHCT116-P, HCT116-WT, and HCT116-MT cells were washed with PBS and fixed in 95% ethanol. Cells were then incubated at 4 °C for 24 h, washed again, and stained in a solution of PBS, propidium iodide, and RNase A in the dark at 37 °C for 20 min. All samples were analyzed by flow cytometry using FACS Calibur (BD Biosciences) system.Wnt/β-Catenin signaling activity was measured using TOP/FOP-Flash luciferase activity assay. TOP-Flash contained a wild type LEF/TCF-binding site, while FOP-Flash contained a mutant LEF/TCF-binding site. HCT116 cells were transfected with reporter constructs (TOP-Flash or FOP-Flash). After 2 days, luciferase activity was measured using Dual-Luciferase Reporter Assay System Kit (Promega), according to manufacturer’s instructions. Results are presented as relative luciferase activity.th amino acid using β-catenin wild-type (WT) construct. An EGFP-conjugated E-cadherin expression vector was purchased from Addgene . For gene silencing experiments, cells were transfected with short interfering RNAs (siRNAs) targeting SNAI1, SNAI2, ZEB1, and TWIST1 . The siRNAs sequences are shown in Supplementary Table Coding sequences of β-catenin were cloned into a pLECE3 vector conjugated with EGFP at C-terminus. For generation of a β-catenin Ser45del mutant construct, substitution mutagenesis was performed at 45Representative areas were selected based on microscopic examination of hematoxylin and eosin (H&E)-stained slides, and corresponding spots were marked on the surface of the paraffin block. Using a manual tissue microarrayer, selected areas were removed, and 3-mm tissue cores were placed into 6 × 5 recipient blocks. Tissues from invasive tumors were then extracted; two tissue cores were processed from each sample to minimize extraction bias and ensure each case was represented in the final tissue microarray (TMA) block. Each core was assigned a unique TMA location number that is linked to a database with other clinicopathological data. After microtome sectioning and H&E staining, final TMA blocks contained cores from 101/101 (100%) specimens.15. Samples were then classified as having low (H-score ≤ 200) or high (H-score > 200) protein expression.Paraffin-embedded tissue blocks were cut into 4-µm sections. Immunohistochemical analysis was performed using a Ventana XT automated stainer , with antibodies against β-catenin , E-cadherin , and Claudin-7 . The percentage of nuclear β-catenin staining was evaluated and categorized as negative, weak, or high, based on percentage of tumor cells with nuclear expression . The H-score method, which assigns an immunohistochemistry (IHC) H-score to each case on a continuous scale of 0–300 based on percentage of cells at different staining intensities, was used for the interpretation of Claudin-7 and E-cadherin results. Membranous staining was scored as follows: 0 for “no staining,” 1+ for “weak staining visible at high magnification,” 2+ for “intermediate staining,” and 3+ for “strong staining”. The percentage of cells at different staining intensities was determined by visual assessment, with scores calculated using the following formula: 1(% of 1+ cells) + 2(% of 2+ cells) + 3(% of 3+ cells)6 cells were seeded in 60-mm dishes and cultured until confluent. Using a (yellow) pipette tip, a straight scratch (wound) was generated, keeping the pipette tip at an angle of ~30°. Wound closure was then monitored over 48 h. For invasion assays, QCM 24-Well Cell Invasion assay kit was used , according to manufacturer’s guidelines. Briefly, insert interiors were rehydrated with pre-warmed serum-free media, and 0.3 × 106 cells were added to each insert. Complete media (10% FBS) was added to the lower chambers, and cells were incubated for 48 h in a CO2 incubator. Cells were stained with 0.1% crystal violet solution, and images were obtained using the 4× or 10× objective.For wound-healing assays, 0.5 × 1016. All patients had undergone curative colorectal resection between 2006 and 2012, and none received neo-adjuvant chemotherapy. Specimens were obtained from the archives of Department of Pathology at Yonsei University in Seoul, Korea and from the Liver Cancer Specimen Bank of the National Research Resource Bank Program of the Korean Science and Engineering Foundation of the Ministry of Science and Technology. Patient data were collected retrospectively. All experimental protocols using patient tissues were approved by the Institutional Review Board of Yonsei University College of Medicine  and conducted in accordance with all relevant policies, including obtaining informed consent from all subjects.A total of 101 stage III  CRC tissue samples that were treated with FOLFOX regimen were used in this study. This same cohort was analyzed in our previous study for molecular classification of CRCsP-values < 0.05 were considered statistically significant.Statistical analyses were performed using SPSS software, version 21.0.0.0 for Windows . Mann-Whitney tests, Student’s t-tests, Fisher’s exact tests, or chi-square tests were used, depending on the purpose. Data are expressed as mean ± SD, and one-way analysis of variance (ANOVA) with a post hoc test (Bonferroni) was performed to compare multiple means. Authorization for the use of tissues for research purposes was obtained from the Institutional Review Board of Yonsei University College of Medicine .We utilized three HCT116 cell lines with differential β-catenin mutation status: parental HCT116 (HCT116-P) cells harboring one WT and one mutant allele (Ser45 del), HCT116-WT cells harboring one WT allele, and HCT116-MT cells harboring one mutant allele. As these lines are isogenic and differ only in their β-catenin mutation status, we considered this a proper model for investigating β-catenin mutation-induced phenotypes. Morphological analysis revealed clear differences. Specifically, HCT116-WT cells predominantly show epithelial-like morphology (round and organized sheet pattern), whereas HCT116-MT cells mostly display mesenchymal-like morphology (spindle-shaped and scattered pattern) Fig. . Both moCD44, MMP7, CCND1, CCNE2, BMP4, and AXIN2. qPCR analysis showed that the expression of BMP4, AXIN2, and CCNE2 was strongly up-regulated in HCT116-P and HCT116-MT cells compared to that of HCT116-WT cells. CCND1 expression was slightly up-regulated in HCT116-MT and HCT116-P cells. On the other hand, CD44 expression was higher in HCT116-WT than that in other cell lines, and MMP7 expression was specifically up-regulated in HCT116-P cells  were also identified  displaying |fold change| >1.5 and ied Fig. . Additioied Fig. . To narried Fig. . Of thesied Fig. .Figure 2To confirm β-catenin activation promotes cell cycle up-regulation, we performed cell proliferation assays and flow cytometric analyses. As expected, HCT116-MT cells proliferated more rapidly than HCT116-WT cells, and HCT116-P cells showed an intermediate proliferation rate phenotype , SNAI2 (SLUG-encoding gene), ZEB1, and TWIST1 by normalizing them to GAPDH expression. All of the four EMT factors were found to be up-regulated in HCT116-MT cells compared to HCT116-WT cells   expression (7.57-fold increase)  under conditions of increasing cell density. Using qRT-PCR, we detected SNAI1 mRNA expression levels that are 2- and 3-fold higher, respectively, in HCT116-P and HCT116-MT cells than in HCT116-WT cells, at all densities . Compared to HCT116-WT cells, ZEB1 mRNA expression was slightly up-regulated in HCT116-P cells, whereas about 2-fold up-regulation was detected in HCT116-MT cells. In contrast, TWIST1 expression was strongly up-regulated only in HCT116-P cells in a cell density-dependent manner. CDH2 expression was slightly higher in HCT116-MT cells, compared to that of HCT116-P and HCT116-WT cells. Notably, Vim expression was selectively and strongly up-regulated in HCT116-MT cells. Similar protein expression patterns were also detected for each EMT marker . Notably, HCT116-P and HCT116-MT cells with repressed β-catenin expression commonly showed epithelial-like morphology Fig. , and theIn order to confirm whether mesenchymal-like features of HCT116-MT and HCT116-P cells directly result from β-catenin activation-mediated down-regulation of cell-cell junction molecules, we generated stable Claudin-7 or E-cadherin knockdown HCT116-WT cells using shRNA. Protein knockdown was confirmed by western blot, and we found that Claudin-7 knockdown promotes decreased E-cadherin expression and vice versa Fig. . CriticaOur data indicate that Claudin-7 is down-regulated in HCT116-P compared to HCT116-WT cells and does not increase with cell density. Furthermore, levels of membranous E-cadherin in HCT116-P cells are similar to those observed in HCT116-WT cells. We therefore tested whether Claudin-7-mediated TJs are indeed impaired in HCT116-P cells, and if additional loss of E-cadherin further up-regulates migratory and invasive activity of these cells. To this end, we generated stable shRNA-knockdowns of Claudin-7 or E-cadherin in HCT116-P knockdown cells. Protein knockdown was confirmed by western blot. We further found that Claudin-7 knockdown promotes a slight decrease in E-cadherin expression and vice versa Fig. . AdditioOur findings suggest that β-catenin mutations promote its nuclear expression, leading to Claudin-7 and E-cadherin down-regulation. We therefore examined the association between nuclear β-catenin expression and expression of Claudin-7/E-cadherin in 101 stage III CRC tissues by IHC. We exclusively detected membrane-associated β-catenin in crypt cells of normal colon mucosa, whereas CRCs showed heterogeneous nuclear accumulation of β-catenin that can be categorized as follows: 1) no nuclear expression, similar to normal tissues, 33/101 samples; 2) low nuclear expression (1–29% of tumor cells), 53/101 samples; and 3) high nuclear expression (≥30% of tumor cells), 15/101 samples  and decreased prevalence of microsatellite instability-high (MSI-H) (P = 0.013). We could not find statistical significance between high nuclear β-catenin expression and expression of Claudin-7 (P = 0.474) and E-cadherin (P = 0.093), probably due to the small case number of CRCs with high nuclear β-catenin expression   of CRC patients with negative and low nuclear β-catenin expression is significantly longer than that of patients with high nuclear β-catenin expression , two CRC cell lines with no Wnt pathway relevant mutations (RKO and HCT8), a hepatocellular carcinoma (HCC) cell line with an AXIN1 mutation (Hep3B), a HCC cell line with a CTNNB1 mutation (HepG2), and a CRC cell line with a CTNNB1 mutation (LS174T). Immunofluorescence microscopy and western blot analysis showed that CRCs with APC mutations more clearly display nuclear β-catenin localization compared to CRC cells with no Wnt pathway relevant mutations and HCC cells with an AXIN1 mutation. Importantly, HCC and CRC cells with a CTNNB1 mutation commonly showed much stronger nuclear β-catenin localization, compared to CRC cells with APC mutations  β-catenin-independent mechanisms for regulation of Claudin-7 and E-cadherin expression; 2) highly heterogeneous membranous and nuclear β-catenin expression in CRCs; or 3) small number of cases with high nuclear β-catenin expression. Therefore, we tested for inverse expressional correlations between β-catenin and Claudin-7/E-cadherin in solid-pseudopapillary neoplasm (SPN) tissues. These have homogenous mutations in exon 3 of β-catenin in nearly all tumor cells, leading to homogenous β-catenin nuclear expression36. Immunohistochemical analysis of 15 SPN samples revealed almost 100% nuclear β-catenin expression, accompanying complete loss of both Claudin-7 and E-cadherin (Supplementary Fig. Results of studies assessing the clinical significance of nuclear β-catenin expression in CRCs had numerous inconsistenciesSupplementary Information


Eye diseases and injuries impose a significant clinical problem worldwide. Safe and effective ocular drug delivery is, however, challenging due to the presence of ocular barriers. Here we report a strategy using an eye patch equipped with an array of detachable microneedles. These microneedles can penetrate the ocular surface tissue, and serve as implanted micro-reservoirs for controlled drug delivery. The biphasic drug release kinetics enabled by the double-layered micro-reservoirs largely enhances therapeutic efficacy. Using corneal neovascularization as the disease model, we show that delivery of an anti-angiogenic monoclonal antibody (DC101) by such eye patch produces ~90% reduction of neovascular area. Furthermore, quick release of an anti-inflammatory compound (diclofenac) followed by a sustained release of DC101 provides synergistic therapeutic outcome. The eye patch application is easy and minimally invasive to ensure good patient compliance. Such intraocular drug delivery strategy promises effective home-based treatment of many eye diseases. Treatment for eye injuries and diseases is most efficient when delivered directly into the eye. Here, the authors developed a patient-friendly eye patch equipped with an array of detachable microneedles, through which drugs can be delivered through the cornea for an extended period of time. However, efficient delivery of drugs into the eye is challenging due to the presence of multiple structural barriers 5. The use of systemic route  requires a large dose to achieve effective local drug concentration, and thus usually produce off-target systemic side effects7. On the other hand, repetitive drug applications with high dosage are often required for convention topical administration (e.g. eye drops or ointments) due to extremely low bioavailability (<5% can be absorbed by eye) and fast clearance, which may also lead to systemic side-effects 8. Intraocular injection  using conventional hypodermic needles to penetrate the surface barriers (cornea and sclera), however, has poor patient compliance due to pain, need for frequent clinic visit, risk of infection, haemorrhage, even permanent damage9. Similar to topical eye drops, injecting drugs into ocular surface tissues  also has poor drug retention due to back-flow of injected solution and subsequent tear wash-out6. Furthermore, both conventional topical administration and local injection only produce burst release of drug with short effective duration, which is particularly not ideal for treating chronic progressive eye diseases, such as glaucoma10. Although contact lens-like hydrogels have been developed for improved topical delivery, because of prolonged drug residence time with minimal burst effect12, the bioavailability is still poor. Although implanting intraocular drug reservoirs enables sustained release, it requires risky and painful surgical intervention13. Hence, localized, long-lasting and efficient ocular drug delivery with good patient compliance is still an unmet medical need.The increasing prevalence of eye diseases  is correlated to the upsurge in aging population, diabetes mellitus and prolonged wear of contact lens worldwide16. Their patient-friendly feature and effectiveness in transdermal drug release have inspired researchers and clinicians to explore their applications in eye disease treatment. Specifically, drug-coated solid stainless-steel MN have been used for the rapid release of drugs in the cornea18 and hollow glass MN have been employed to infuse drug solution into the sclera19.Microneedle (MN) technology is originally developed for transdermal drug delivery for various therapeutic purposes , with painless, bloodless, high efficiency and ease of administration propertiesHere, we show a flexible polymeric eye patch equipped with an array of biodegradable and detachable MNs for localized, highly efficient and controlled ocular drug delivery Fig. . MNs can20. HA-based MN devices have been employed for transdermal delivery of various hydrophilic or hydrophobic therapeutic compounds, including proteins, peptides and synthetic molecules16. However, because of the fast dissolving nature of HA, HA-MNs cannot maintain their sharp-pointed structural integrity and mechanical strength during penetration into a wet surface like cornea. In addition, HA-MN can only afford burst release of its cargo14. In comparison, crosslinked methacrylated HA (MeHA), which is synthesized by functionalizing HA with methacrylic anhydride  is a non-sulphated glycosaminoglycan distributed abundantly throughout the body in the connective tissues as well as vitreous eye fluid. As a natural biopolymer with unique viscoelastic property and transparency, HA has been widely used in ophthalmology, particularly in artificial tear solution as a lubricant for dry eyesCombining the merits of HA and MeHA, we herein developed an eye-contact patch equipped with double-layered MNs (DL-MN) for controlled ocular drug delivery Fig. , using a25. The eye patches (~2 × 2 mm) with a 3 × 3 MN array were used for mice whose cornea size is ~3 mm in diameter  and optical microscopy  labelled with Alexa Fluor 680 or Alexa Fluor 488 as the model therapeutic compounds were separately loaded in the different layers of MNs Fig. . The conIgG recovered from 3 × 3 DL-MNs dissolved in phosphate buffer saline (PBS) is 0.92 ± 0.21 µg, correlating well with the nominal loading amount (1 µg) . Specifically, >80% was released within 5 min in the former and 30 min in the latter. In contrast, prolonged release profile was observed for IgG(680) loaded in the crosslinked MeHA outer layer (t1/2 of ~2 days in tear fluid and ~3 days in gelatin hydrogel) because IgG molecules can only slowly diffuse through the interwoven meshwork of MeHA. In comparison to the fast release from HA-MNs or slow release from MeHA-MNs, the bi-phasic release profile was realized when a single drug molecule, IgG(680), was loaded in both compartments of DL-MNs  and were continuously monitored under confocal microscope Fig. . Immedia27. In contrast, the mechanical strength of crosslinked MeHA-MNs (~0.15 N per needle) is much weaker. Because MeHA is highly viscose, only a lower polymer concentration can be used to fabricate MNs (50 mg/ml for MeHA-MNs vs. 200 mg/ml for HA-MNs). This compromises the mechanical strength of MeHA. The mechanical strength of DL-MNs is similar to that of HA-MNs (~0.4 N per needle), indicating that the mechanic property of DL-MN is dictated by the inner HA core. In addition, drug loading into DL-MNs (2 µg) does not compromise the mechanical properties of DL-MNs , the force needed to penetrate the cornea can be much lower29.The mechanical strength of MNs was assessed by compression test. Consistent with other studies, the mechanical strength of HA-MNs (~0.4 N per needle) is strong enough for skin penetration Fig. 27. In co30. As demonstrated in Fig. 31. Such elastic deformations make polymeric DL-MNs advantageous over metallic MNs which may cause corneal puncture32. The insertion force into the cornea was estimated  Fig. . Porcineted Fig. . The forted Fig.  and the ted Fig. . The fluted Fig. . Similarted Fig. 33. TakenThe capability of insertion and drug releases of DL-MNs was further investigated in vivo. DL-MN patch loaded with IgG(680) was gently pressed onto the cornea of mouse eye for ~30 s Fig. . MNs wer34. All these observations indicate that MN insertion and implantation into cornea is minimally invasive without causing obvious adverse effects on the eyes, general health state and behaviour.The corneal anatomical structures after MN insertion were monitored over 1 week Fig. . We did 7. In contrast, intra-corneal delivery of IgG(680) at the same dose using MN patch only introduced fluorescence signal in the applied eye (much stronger than that caused by eye-drop). In addition, fluorescence signal in the eye was still observable for over 3 days  have become standard care for vaso-proliferative diseases, including those in the eye38. However for angiogenic ocular diseases like corneal NV, frequent high-dose topical application is required to overcome ocular barrier , which is accompanied with adverse effects 37. On the other hand, conventional intraocular injection may cause infection, bleeding and retinal detachment9. Here, we demonstrate the advantages of DL-MN eye-patches for improving the therapeutic efficacy of the anti-VEGF drug  is a key mediator of corneal NV. VEGF promotes blood vessel formation mainly via VEGF receptor type 2 (VEGFR2)39. Similar to the un-treated eyes, eyes treated with control IgG eye-drop showed substantial corneal NV (1.48 ± 0.45 vs. 1.50 ± 0.24 mm2) (day 7). Similarly, topical delivery of DC101 via eye-drop (with a high dose of 10 µg in 10 µl) had no significant effect on corneal NV as compared to the untreated eyes (1.24 ± 0.21 mm2). In contrast, eyes treated with DC101 (~1 µg) delivered through fast-dissolving HA-only MNs led to ~44% reduction in neovascular area (0.84 ± 0.43 mm2) . At day 2 when neovascular growth was started to appear from the limbus (the border between cornea and sclera), mice corneas were treated once with non-specific control IgG or anti-VEGFR2 IgG (DC101) delivered through topical eye-drop or MN patch application Fig. . DC101 wmm2) day . Similar2)  offered much improved therapeutic effect with 90% reduction of neovascular area . The therapy combining both drugs was much more effective (0.16 ± 0.24 mm2). We further show in Supplementary Fig. Ocular delivery of multiple drugs at different stages of the disease progression can offer a more effective treatment outcome due to their synergistic effects. It is well known that the initial inflammatory response is a key factor to trigger ocular NV . Under chronic inflammatory condition, inflammatory cells (e.g. macrophages) produce a large number of pro-inflammatory cytokines  and angiogenic growth factors (particularly VEGF), which creates a vicious circle of persistent inflammation and NV. To tackle this issue, DL-MNs were loaded with two drugs, nonsteroidal anti-inflammatory drug (1 µg diclofenac) in its fast-dissolving HA core and anti-VEGFR2 drug (0.5 µg DC101) in slow-dissolving crosslinked MeHA shell. As shown in Fig. We further analysed the corneal inflammation by immunofluorescence staining Fig. , and dem41. As MeHA-based patch is highly swellable24, it was used to collect mouse tear film for analysis of the concentration of inflammatory and angiogenic cytokines  in tear film after treatment ent Fig. . As showilm Fig. . And con4. Herein, we demonstrate an eye patch with micro-drug-reservoirs self-implantable into the ocular surface tissue for controlled drug release. The flexible patch can be readily applied by gentle and brief thumb pressing on the ocular surface, which is as easy as wearing a disposable contact lens without causing discomfort or requiring high skills. As the micro-drug-reservoirs comprise multiple compartments, they allow the release of the same drug with biphasic kinetics or sequentially release of different drugs for synergistic therapy. Comparing to conventional topical eye drop application which requires repeated high-dose instillation and often associates with systemic side-effects, and comparing to painful and risky intraocular injections in clinics, the demonstrated eye patches offer a unique opportunity for patients to conveniently and effectively manage their eye disorders at home.Effective ocular drug delivery for the treatment of vision-threatening diseases  remains challenging due to the presence of various anatomical and physiological barriers39. This is expected as the major challenge faced by ocular drug delivery is the limited ability of drug molecules to penetrate the ocular tissue efficiently, due to the presence of ocular barriers. It has been reported that only 0.1% of bevacizumab  in eye drop can reach aqueous humour42. It has been shown that eye drop application of 10 µg axitinib  failed to show significant effect on corneal NV of mice43. Clinical studies in human suggested that repeated high-dosage of topical administration is required for the treatment of corneal NV disease. But still only ~41% or ~48% reduction of neovascular area can be achieved using ranibizumab eye drop 36 or bevacizumab eye drop 44, respectively. Based on our mouse experiments and considering that human cornea is ~20 times larger than mice cornea, single treatment with ~20 µg should be effective for human corneal NV using our approach. As the human cornea  is thicker and larger than mice cornea 45, a larger patch (e.g. 10 × 10 MN array) with longer MNs (e.g. 800 µm) may be suitable for human. With the ability to overcome the ocular surface barriers for localized delivery with high bioavailability, and achieve multi-phasic release kinetics or synergistic therapy from sequential release of multiple drugs, our MN approach can realize low effective dosage and application frequency. This is important to relieve the patient’s burden and enhance patient compliance.Here, corneal NV in mice was used as the disease model to demonstrate the effectiveness of our approach. We demonstrate that intra-corneal delivery of DC101  using micro-implants can achieve ~90% reduction of neovascular area with a single treatment of 1 µg dosage. In comparison, eye drop application of DC101 even at a much higher dosage (10 µg) failed to show significant therapeutic effect. In a previous study, systemic intraperitoneal injection of 1 mg DC101 (every 2nd day for 1 week) only led to marginal effect (~20% reduction of neovascular area)46. Two weeks of sustained release can be attained when HA-IgG conjugates are loaded in MeHA matrix  can be readily tailored14.Although DL-MNs were used in our experiments, triple compartmentalization can also be fabricated  for anterior uveitis48, fluconazole for fungal keratitis49. It may also be used for intra-corneal delivery of riboflavin to patients with keratoconus, without the need of corneal epithelial scraping and debridement whereby avoiding post-operative pain, infection and permanent damage usually associated with the traditional surgical methods50. In summary, the demonstrated MN eye patch, which implants micro-drug-reservoirs for localized, controlled and efficient ocular drug delivery in a convenient, safe and painless manner, provides a cost-effective and home-based solution for many ocular diseases.Our MN approach promises for other eye diseases as well, for example, delivery of β-adrenergic receptor blockers or prostaglandin analogues for glaucomaSodium hyaluronate (HA) with different molecular weights were purchased from Bloomage Freda Biopharm. IgG, IgGs conjugated with Alexa Fluor dyes, alamarBlue cell viability reagent, Medium 200, low serum growth supplements, Geltrex LDEV-free reduced growth factor basement membrane matrix, bovine serum albumin (BSA), PBS, penicillin–streptomycin and trypsin-EDTA were obtained from ThermoFisher Scientific. VEGFR2 antibodies  were obtained from GeneTex. Anti-F4/80 (MAB5580) was from R&D Systems. All other reagents and solvents were purchased from Sigma-Aldrich. All reagents were of analytical grade and used without further purification.g, 5 min). After air-drying at room temperature in a fume hood (~12 h), un-modified HA solution  was applied and centrifuged  to fill the MN cavities. After air-drying again for ~12 h, HA solution  was added to produce a robust supporting substrate, and dried overnight. Finally, MN patches were gently peeled off from the micromolds, and exposed to low-intensity ultraviolet light for 3 min .MN patches were prepared via simple micromolding method. Briefly, PDMS  micromolds were created by pouring PDMS solution into the custom-designed stainless steel master-molds , which was followed by degassing (10 min in vacuum oven) and curing (70 °C for 2 h). To fabricate eye patches with DL-MN, MeHA aqueous solution (50 mg/ml MeHA together with 0.5 mg/ml Irgacure 2959 in DI water) was casted into the plasma-treated PDMS micromolds through centrifugation (322027. The transmittance of the MNs (loaded with or without 2 µg IgG), isolated porcine cornea and aqueous humour (in PBS) was measured in a spectrophotometer (Shimadzu UV-1800).MN patches were examined using a field-emission scanning electron microscope , and digital microscope (Leica DVM6). MN patches loaded with different IgGs—IgG(405), IgG(488) and IgG(680)—were visualized with a confocal laser scanning microscope . The mechanical property of MNs was tested using an Instron 5543 Tensile Tester (Instron). A vertical force was applied to MNs using a flat-headed stainless steel cylindrical probe (at a constant speed of 0.5 mm/min). The force was continuously recorded until a displacement of 450 μm was reached. MN insertion test was performed on the isolated porcine cornea. Briefly, MNs  were mounted onto the cylindrical probe, and pressed perpendicular to the cornea at a rate of 5 mm/min until a pre-set maximum load of 4 N was reached. Force exerted on the cornea by the MN as a function of its displacement into cornea was recorded. The insertion force was estimated when the force against the cornea showed discontinuity followed by a steep slopeTo evaluate the in vitro biocompatibility of MNs, primary human corneal epithelial cells  grown with the EpiGRO ocular complete media  were exposed to different types of MNs for 2 days, before analysing cell morphology using an inverted microscope  and cytotoxicity test using an almarBlue cell viability assay. The absorbance of the incubated media containing 10% alamarBlue (~3 h) was measured at 570 nm using a plate reader .The protein bands of IgGs being released from MNs (on 12% polyacrylamide gel) were stained with InstantBlue solution (Expedeon) and imaged with a G:BOX Chemi XT4 imaging system (Syngene). The ability of released IgGs to bind IgG-specific proteins on human umbilical vein endothelial cells  was confirmed by immunofluorescence staining. Briefly, cells were fixed with 4% formaldehyde solution (15 min) before washing with PBS . After being blocked with 1% BSA in PBST (PBS with 0.1% tween20) (1 h), the cells were incubated overnight in PBST containing 1% BSA and anti-VEGFR2 IgG (free IgG or IgG released from MNs). After washing with PBS, the cells were then incubated with the secondary antibody tagged with Alexa Fluor 680 (2 h), before washing again, and imaging using a confocal microscope.To evaluate the in vitro bioactivity of IgGs loaded in MNs, HUVECs exposed to 10 ng/ml VEGF (Santa Cruz Biotechnology) were treated with different doses of anti-VEGFR2 IgG (free IgG or IgG released from MNs) for ~18 h. Tube formation of HUVECs grown on the Geltrex matrix (ThermoFisher) was then recorded using an inverted microscope, and tube lengths were measured using the ImageJ (NIH.gov).To determine the in vitro insertion capability, MN patches (3 × 3 MN array) were applied briefly to the porcine cornea (~30 s), before the supporting base was removed. The corneas were then excised, washed with PBS and analysed for the presence of fluorescence spots produced from IgG(680) loaded in MNs using confocal microscopy. For histological analyses, the corneal tissues were fixed with 4% formaldehyde solution (24 h), and cryoprotected with 30% sucrose solution (24 h), before embedding in FSC22 Frozen Section Media (Leica Microsystem) for cryosectioning (5 µm thick) , and haematoxylin and eosin staining (Sigma-Aldrich).3 0.22 g, KCl 0.14 g, CaCl2·2H2O 0.008 g, in 100 ml DI water, pH 7.4), gelatin hydrogel  or PBS (pH 7.4), and placed in an incubator shaker . IgG molecules (conjugated with Alexa Fluor dyes) released from MNs were measured using a fluorescence spectrometer . The real-time visualization of IgG releases from DL-MNs in agarose hydrogel  or porcine cornea was analysed using a confocal microscopy.To evaluate the release profiles of MNs, MNs were immersed in 0.5 ml of simulated tear fluid . Specifically, under anesthetized condition, a MN patch with 3 × 3 array of IgG(680)-loaded DL-MNs was gently applied on one cornea (one eye only) for 30 s. The mice were then imaged immediately (day 0) or at day 3 using an in vivo imaging system . In some experiments, MN-treated mice were euthanized before collecting the eyeballs and incising the cornea to identify the presence of fluorescence spots produced by MNs using confocal microscopy.The in vivo bio-distribution of IgG(680) released from MNs was also analysed. Briefly, mice were divided into 4 groups, with the treatment of 10 µg IgG(680) through intraperitoneal injection, topical eye-drop instillation or intra-corneal delivery using MN patch, or without treatment as a control. At 2 h post-treatment, the mice were euthanized and the eyes and the major organs  were dissected and visualized by IVIS imaging system.All the animal experiments were approved by the Nanyang Technological University, Institutional Animal Care and Use Committee (NTU-IACUC) under protocol ARF-A0350. The mice were housed in light and temperature controlled facility , and allowed free access to water and normal diet. DL-MN patches (3 × 3 MN array) were applied onto the central cornea area of anesthetized mice for 30 s. After removing the supporting base, corneas were imaged by a bright-field microscope. After receiving MN insertion, mice were immediately returned to their cages, allowing recovery from anaesthesia. After 10 min, mouse behaviour was monitored to access their pain . The body weight and food intake were also recorded . In some tested mice groups, mice were euthanized immediately after MN insertion or later , and corneas were collected to examine histological changes.π × VL × CH51. In some experiments, mice were euthanized and corneas were collected for immunohistochemistry analyses of macrophage infiltration in corneal stromal layer. F4/80 as a major macrophage marker was stained with a specific antibody. Tear films from differently treated mice were collected by simply placing MeHA patch without MNs on their ocular surface for 1 min, before centrifugation of the patch at 16,000g for 5 min. Subsequently, interleukin-6 (IL6) and VEGF concentrations were determined by IL6 and VEGF ELISA kits (Invitrogen).Chemical-burn injury to the mouse eye was inflicted by placing a sterilized Whatman filter paper (2 mm) soaked with 1 N NaOH solution for 30 s under the anesthetized condition (day 0). Eyes were then extensively flushed with sterilized PBS solution (~10 ml) using a syringe. At day 2, corneal neovascular outgrowths were imaged by a microscope. Mice were then randomly divided into 6 groups, treated only once with eye-drop instillation of either non-specific control IgG or anti-VEGFR2-IgG (DC101) (10 µg in 20 µl PBS), application of HA-only MN loaded with either control IgG or DC101 (1 µg), or application of DL-MN loaded with either control IgG or DC101 . For testing the ocular delivery of two drugs, mice were randomly divided into different groups, treated only once with DC101-loaded DL-MN (0.5 µg in MeHA layer), diclofenac-loaded DL-MN (1 µg in HA core), 2-drug-loaded DL-MN (DC101 in MeHA and diclofenac in HA core), or eye-drop instillation of both drugs. For all the interventions, anaesthesia was maintained throughout the procedure with 2% isoflurane. After 5–6 days, corneas were imaged and corneal NV was analysed using ImageJ. Briefly, the vessel lengths (VL) were measured from the limbus to their leading edges, sectoral circumference was expressed as clock hours , and the vessel area (VA) was calculated using the formula, VA = 0.2p value of <0.05 was considered to be statistically significant.Quantitative data were represented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A Supplementary InformationPeer Review FileReporting Summary


Cardiovascular diseases have a high prevalence worldwide and constitute the leading causes of mortality. Recently, malfunctioning of β-catenin signaling has been addressed in hypertensive heart condition. Ang-II is an important mediator of cardiovascular remodeling processes which not only regulates blood pressure but also leads to pathological cardiac changes. However, the contribution of Ang-II/β-catenin axis in hypertrophied hearts is ill-defined. Employing in vitro H9c2 cells and in vivo spontaneously hypertensive rats (SHR) cardiac tissue samples, western blot analysis, luciferase assays, nuclear-cytosolic protein extracts, and immunoprecipitation assays, we found that under hypertensive condition β-catenin gets abnormally induced that co-activated LEF1 and lead to cardiac hypertrophy changes by up-regulating the IGF-IIR signaling pathway. We identified putative LEF1 consensus binding site on IGF-IIR promoter that could be regulated by β-catenin/LEF1 which in turn modulate the expression of cardiac hypertrophy agents. This study suggested that suppression of β-catenin expression under hypertensive condition could be exploited as a clinical strategy for cardiac pathological remodeling processes. Global prevalence of cardiovascular diseases has been on the rise and constitutes the leading causes of death worldwide . CardiacTo elucidate the contribution of β-catenin/LEF1 signaling axis in cardiac hypertrophy, Ang-II treatment was used as a stimulus in a dose-dependent manner as it is known to induce hypertrophic responses under cardiac stresses. We found that Ang-II treatment could induce protein expressions of IGF-IIR, Gαq, PKC-α, and cardiac hypertrophy markers ANP, BNP. Interestingly, the β-catenin expression was increased upon Ang-II treatment in a dose-dependent manner A. HoweveTo further confirm the contribution of β-catenin in hypertrophied hearts, gain-of-function, and loss-of-function approaches were utilized in H9c2 cells. Upon β-catenin overexpression, IGF-IIR, Gαq, and PKC-α were upregulated dose-dependently A. HowevePrevious researches have implicated GATA-4 and NFATc3 as primary transcription factors in cardiac hypertrophy responses. Therefore, we pursued to determine the effect of Ang-II treatment on nucleo-cytoplasmic enrichment of β-catenin. Our results indicated that Ang-II treatment in a dose-dependent manner induced nuclear mobilization of β-catenin co-enriched with GATA-4 and NFATc3. These data indicate that β-catenin has a partial contribution to Ang-II induced hypertrophy responses during cardiac stresses A.β-catenin is known to act as a transcriptional co-activator of TCF/LEF1 target genes. Since β-catenin nuclear translocation of was increased by Ang-II, we questioned whether β-catenin could modulate IGF-IIR transcription via β-catenin/LEF1. A conserved LEF1 binding sequence 603 bp upstream of the ATG transcription start site was identified on IGF-IIR promoter using bioinformatics analysis B. Next, To further assure the function of β-catenin/LEF1 in Ang-II-induced hypertrophy, either β-catenin or LEF1 or both were silenced using si-RNA with/without Ang-II treatment. Protein expression analysis revealed Ang-II induced cardiac hypertrophy agents such as IGF-IIR, Gαq, and PKCα, were downregulated upon silencing of either β-catenin, or LEF1, or both A.Further, immunoprecipitation and protein expression analysis in the SHRs revealed β-catenin protein level was increased in the nuclear fraction of hypertensive myocardial tissues and directly bound to LEF1 B.2) at 37 °C. H9c2 cells at 60–70% confluence were used for experimental treatments. pCMV-β-catenin plasmid or siRNAs against β-catenin or LEF1  were delivered into the cells using PureFectionTM Reagent  as per the instruction manual. XAV939   was used at varying concentrations and time durations as indicated.H9c2 cells were procured from American Type Culture Collection  and grown in DMEM medium supplied with 10% fetal bovine serum, glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 mg/mL), and pyruvate (1 mM) in humidified air (5% COSpontaneously hypertensive rats (SHR) 8-week-old (approx. 200 g), were acquired from the National Laboratory Animal Center  and sustained following our previous protocol . All aniCellular protein extracts were prepared following previous publication with minor modifications as described . Lysis bH9c2 cells were fixed with 4% paraformaldehyde at room temperature (RT) for 30 min, washed with ice-cold PBS and permeabilized with 0.1% Triton X-100 for 10 min at 4 °C. Blocking was done in PBS containing 2% BSA at RT for 30 min. Actin filaments were stained using rhodamine- phalloidin and images were captured under a fluorescence microscope (magnification X 200). Cell surface area was determined using Image J software (NIH).PureProteome Protein G Magnetic Bead System (Millipore) was used for IP as per instruction manual. 300 µg of protein extracts were used for processing and next, incubated with 2 μg of LEF1 antibody. Next, to each IP reaction, Protein G Magnetic Bead (50 µL) was added and then incubated overnight at 4 °C on a rotor. Elution was done at 95 °C for 5 min and then separated by SDS-PAGE. After transferring proteins to PVDF membrane, they were probed with specific primary antibodies.Dual-Glo Luciferase Assay  was used to assess IGF-IIR promoter activity in H9c2 cells. Briefly, cell lysates at the end of various treatments were collected and luminescence was detected by Luminometer .Cytosolic and nuclear protein extracts were prepared using the Nuclear/Cytosol Fractionation Kit  as per instruction manual. Isolated proteins were quantified using Bradford assay (Bio-Rad), and subjected to further analysis.p-value < 0.05 was considered to be statistically significant. The data shown represent mean ± SD from at least three experimental replicates.Student’s t-test was used to assess the significance using SPSS10.0 software and a Through this study, we examined the impact of β-catenin in Ang-II induced hypertrophy responses and demonstrated that IGF-IIR expression could be regulated by β-catenin/LEF1 leading to pathological cardiac hypertrophy signaling. Our earlier studies have shown that abnormal activation of IGF-IIR signaling pathway during cardiac stresses such as elevated Ang II levels , or highIn summary, Ang-II induced cardiac hypertrophy responses to involve partial contribution through increased β-catenin expression which, in turn, upregulate IGF-IIR expression via a consensus TCF/LEF1 binding sequence on IGF-IIR promoter and, thereafter, IGF-IIR signaling activates the downstream cardiac hypertrophy cascade.


The biological process known as post-translational modification (PTM) is a condition whereby proteomes are modified that affects normal cell biology, and hence the pathogenesis. A number of PTMs have been discovered in the recent years and lysine phosphoglycerylation is one of the fairly recent developments. Even with a large number of proteins being sequenced in the post-genomic era, the identification of phosphoglycerylation remains a big challenge due to factors such as cost, time consumption and inefficiency involved in the experimental efforts. To overcome this issue, computational techniques have emerged to accurately identify phosphoglycerylated lysine residues. However, the computational techniques proposed so far hold limitations to correctly predict this covalent modification.We propose a new predictor in this paper called Bigram-PGK which uses evolutionary information of amino acids to try and predict phosphoglycerylated sites. The benchmark dataset which contains experimentally labelled sites is employed for this purpose and profile bigram occurrences is calculated from position specific scoring matrices of amino acids in the protein sequences. The statistical measures of this work, such as sensitivity, specificity, precision, accuracy, Mathews correlation coefficient and area under ROC curve have been reported to be 0.9642, 0.8973, 0.8253, 0.9193, 0.8330, 0.9306, respectively.https://github.com/abelavit/Bigram-PGK.The proposed predictor, based on the feature of evolutionary information and support vector machine classifier, has shown great potential to effectively predict phosphoglycerylated and non-phosphoglycerylated lysine residues when compared against the existing predictors. The data and software of this work can be acquired from  The biological process of enzymatic change in proteins brought about after the translation in the ribosome is known as post-translational modification (PTM). The high interest in PTM for various organisms have emerged as a result of efforts in high-throughput proteomics for the study of site-specific PTM as well as enzymes which cause these modifications . The genPhosphoglycerylation, which is a non-enzymatic lysine modification, is a type of PTM that has been recently discovered in human cells and mouse liver , 17. CarThe method of computational techniques to identify phosphoglycerylation sites is getting popular nowadays –34 as thIn the recent years, there have been a number of studies involved to identify phosphoglycerylation using computational technique. Phogly-PseAAC was the earliest work to be carried out where it utilized a KNN-based predictor to predict phosphoglycerylation using a feature set of pseudo amino acid composition . The secThe proposed predictors of phosphoglycerylation in the literature are still limited in terms of their performance. In this regard, we are introducing a novel predictor called Bigram-PGK which employs evolutionary information to predict phosphoglycerylated and non-phosphoglycerylated lysine residues. A total of 91 protein sequences were used in this work which contained experimentally confirmed phosphoglycerylated sites and their profile bigram was obtained from the position-specific scoring matrix (PSSM). With the evolutionary information of the sequences, different segment sizes for each lysine residue was analyzed in terms of the performance. The residue window of ±32 performed the best on Mathews correlation coefficient (MCC) metric when the size of ±1 to ±32 was considered  when they are one of the k neighbors of a positive instance (phosphoglycerylated site) , 42, 43.In proposing any new predictor, it is crucial to assess its performance. In this work, we have employed five statistical measures including sensitivity, specificity, precision, accuracy, and Mathews correlation coefficient , 44–47. The first metric, sensitivity, determines the ability of the classifier to correctly predict phosphoglycerylated lysine sites. The measure ranges from 0 to 1 where a higher value indicates the better the predictor is in classifying the phosphoglycerylated sites. Specificity is the second metric and it measures the ability of the classifier to correctly predict non-phosphoglycerylated lysine sites. This metric also takes on the 0 to 1 range of values where a high value indicates that the predictor is effective at predicting non-phosphoglycerylated sites. The third and fourth metrics are precision and accuracy, respectively, and they take on the same range of values as sensitivity and specificity. Precision metric assesses capability of the predictor to correctly classify phosphoglycerylated sites from all the phosphoglycerylated sites predicted. The accuracy metric evaluates how correctly the predictor distinguishes between phosphoglycerylated sites and non-phosphoglycerylated sites. Mathews correlation metric, which is the fifth measure, assesses the quality of the predictor. The range of values of MCC metric is − 1 to + 1 where − 1 signifies a completely negative correlation, while + 1 indicates a highly positive correlation. These five statistical measures can be written as equations as shown below:In the equations above, FN, FP, TN, and TP represents false negatives, false positives, true negatives and true positives, respectively. False negatives represents instances which are phosphoglycerylated sites but predicted as non-phosphoglycerylated. False positives are those that are non-phosphoglycerylated sites yet predicted as phosphoglycerylated. The true negatives are instances correctly predicted as non-phosphoglycerylated sites and finally, true positives are instances correctly predicted as phosphoglycerylated sites. It is desirable for the best predictor to have high scores in all of the statistical measures. Nevertheless, the proposed predictor should at least have higher sensitivity measure compared to the existing predictors.Step 1: Divide the dataset into 10 equal partsStep 2: Train predictor by combining the 9 parts and test it using the part left outStep 3: Adjust the classifier parameters with training setStep 4: Obtain the statistical measures with the test setStep 5: Repeat steps 2 to 4 until all the folds have been used as test sets and average the statistical measuresThe statistical measures outlined in the previous section to assess the predictor’s performance was carried out using the 10-fold cross-validation scheme. In the literature, there are three common ways of determining the effectiveness of a predictor and these are n-fold cross-validation test, independent dataset test, and the jackknife test , 49. ThoThe result of 10-fold cross-validation scheme on Bigram-PGK is presented in the following section.There are three predictors in the literature which carry out the classification of phosphoglycerylated and non-phosphoglycerylated sites, and these are Phogly_PseAAC , CKSAAP_The comparison result of Phogly_PseAAC , CKSAAP_The promising result in Table In the Additional file This paper presents a novel predictor Bigram-PGK, which utilizes the feature PSSM + bigram to predict phosphoglycerylation. The underlying evolutionary information in PSSM of protein sequences and its transformation to bigram occurrences appears to be a crucial property in detecting the lysine modification. The use of studied feature in this work and the SVM classifier with polynomial kernel to obtain a decent hyperplane separation was effective to distinguish between the modified and unmodified lysine sites.http://cplm.biocuckoo.org) which has now been upgraded to Protein Lysine Modification Database (PLMD). PLMD contains a number of different protein lysine modifications that have been experimentally identified. Phosphoglycerylation dataset obtained was initially prepared by removing sequences which had 40% or higher sequential similarities, which is a widely used level in the literature [The benchmark dataset used in this work was obtained from the Compendium of Protein Lysine Modifications (CPLM) repository, accessed 1 March 2017  resulting in the matrix of size 20 × 20. Each lysine was then represented by transforming this matrix to a 400 dimensional row vector capturing evolutionary information of the segment P.From eq. , the dowij, can be said to be the transitional probability of j-th amino acid at i-th location within the given protein sequence. The segment P, consisting of 65 amino acids (a fraction of the protein sequence), is hence represented by a 65 × 20 feature matrix in which 20 denotes the amino acids of the genetic code. Therefore the PSSM was calculated based on the substitution probabilities of each amino acid in the segment to the 20 amino acids. For the matrix M, its profile bigram is calculated by.The profile bigrams technique has displayed promising results in dealing with discriminatory information –79. For From the above equation, the resulting dimension of matrix B representing the PSSM + bigram is 20 × 20. Finally, matrix B is converted to a 400 dimensional row vector indicated by Eq.  which reSupport vector machine is one of the supervised learning model listed under the topic of machine learning. The algorithm is commonly used in classification and regression applications. It is a discriminative classifier that works by defining a separating hyperplane. Usage of SVM is not only popular in protein problems , 80–83, Additional file 1.  MCC values for different segment sizes.Additional file 2. Number of Phosphoglycerylation sites detected by each predictor.


FOXG1) belong to a distinct clinical entity, termed “FOXG1-related encephalopathy”. There are two clinical phenotypes/syndromes identified in FOXG1-related encephalopathy, duplications and deletions/intragenic mutations. In children with deletions or intragenic mutations of FOXG1, the recognized clinical features include microcephaly, developmental delay, severe cognitive disabilities, early-onset dyskinesia and hyperkinetic movements, stereotypies, epilepsy, and cerebral malformation. In contrast, children with duplications of FOXG1 are typically normocephalic and have normal brain magnetic resonance imaging. They also have different clinical characteristics in terms of epilepsy, movement disorders, and neurodevelopment compared with children with deletions or intragenic mutations. FOXG1 is a transcriptional factor. It is expressed mainly in the telencephalon and plays a pleiotropic role in the development of the brain. It is a key player in development and territorial specification of the anterior brain. In addition, it maintains the expansion of the neural proliferating pool, and also regulates the pace of neocortical neuronogenic progression. It also facilitates cortical layer and corpus callosum formation. Furthermore, it promotes dendrite elongation and maintains neural plasticity, including dendritic arborization and spine densities in mature neurons. In this review, we summarize the clinical features, molecular genetics, and possible pathogenesis of FOXG1-related syndrome.Individuals with mutations in forkhead box G1 ( MECP2) mutations. In addition to MECP2 mutations, other genetic mutations, such as forkhead box G1 (FOXG1) and cyclin-dependent kinase-like 5 (CDKL5), have been identified in atypical RTT variants ,66FOXG1 FOXG1 also interacts with FAST2 and inhibits the expression of transforming growth factor-beta (TGF-β). FOXG1 interacts with FAST2 to inhibit the FAST2-SMAD2-SMAD4 complex formation, thereby repressing TGF-β-responsive transcriptions and allowing cell proliferation. B 67]. F. F67]. FIn addition to the involvement in neurodevelopmental disorders, FOXG1 is also shown to be associated with tumor formation. The overexpression or loss-of-function of FOXG1 can lead to both malignant transformation and tumorigenesis. FOXG1 in brain tumor-initiating cells might result in downregulation of neural progenitor and proliferation markers and upregulation of astroglial differentiation genes [Foxg1 in mouse hair cells has also shown the interaction of Foxg1 with the IGF1, Wnt, and Hippo pathways, etc. [Previous studies have shown the potential role for FOXG1 expression and activated Notch signaling in impeding proneural differentiation in tumors, and also the interaction of the FOXG1/Notch pathway during neurogenesis . More reon genes . A studyys, etc. . These iFOXG1 mutations, initiating cell apoptosis. This indicates that FOXG1 expression may have a direct regulation on the caspase family and may act as a negative regulator of apoptosis of tumor cells, inhibiting cell apoptosis.Previous studies also showed that the expression of FOXG1 was high in embryonal and small cell components in some tumors ,75, and In contrast, FOXG1 expression is low with an increased expression of the oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) in breast cancer cells . In thesFOXG1 is evolutionarily conserved, and is required in vertebrate neurodevelopment [elopment . The amielopment , whereaselopment . In miceelopment ,80,81. Ielopment . In addielopment ,83,84.FOXG1 plays a pleiotropic role in the development of the brain  and is aIn addition, it maintains the expansion of the neural proliferating pool , properlBeyond its complex impacts in cortical arealization, lamination, and neurogenesis, FOXG1 is crucial in dendritogenesis. It induces neurite outgrowth , promoteFurthermore, Foxg1 promotes optic fissure closure, regulates retinal axon pathfinding, and facilitates the crossing of retinal ganglion cell axon during development ,84,90. IFoxg1 mutations that resemble human phenotypes, which could partially explain the underlying pathogenic mechanism of FOXG1-related syndrome. However, there remains much to explore on the pathogenesis leading to FOXG1-related syndrome.The functions of Foxg1 have been progressively delineated via in vivo and in vitro studies. There are some manifestations in animal models of Foxg1 in in vivo studies that could explain the possible pathogenic mechanisms underlying FOXG1-related syndrome.Herein, we summarize the important findings regarding abnormal phenotypes caused by alteration of Foxg1 knockout mice. In morpholino knockdown of Foxg1 in embryos of zebrafish, there was an absence of subpallial  cells, which was caused by both their transformation into pallial progenitors and partial displacement into the hypothalamic territory [Foxg1 was initially suppressed and later reactivated in order for the neuron to migrate to cortical plates. When multipolar cells failed to reexpress Foxg1, they permanently lost their ability to enter into the cortical plate [FOXG1 deletion/intragenic mutations. In these patients, they have significant microcephalies and hypoplastic forebrain, ranging from mildly simplified or normal gyration to frontal lobe predominant-pachygyria [Foxg1 in pioneer neurons is mandatory for their differentiation as Cajal–Retzius cells, and the suppression of Foxg1 is required during the early process of neuronal migration [FOXG1 duplication are typically normocephalic with normal brain or nonspecific brain structural anomalies. There are probably other possible mechanisms leading to brain development, particularly neuron migration in the context of FOXG1 duplication.The role of Foxg1 as a key regulator in development and specification has been well demonstrated in animal studies. A knockout mice research performed by Xuan et al. first showed the fundamental role of Foxg1 in brain development . There werritory , indicatal plate . These fchygyria . Intriguigration , patientFOXG1 deletion/intragenic mutations, in which corpus callosum agenesis/dysgenesis is one of the hallmarks [Further, in mouse models, Foxg1 has been shown to be highly expressed in postmigratory projector neurons. It is essential for cortical layer and corpus callosum formation. There were significantly thinner cortices, enlarged ventricles, and absent corpus callosum in conditional knockout mice . This fiallmarks .FOXG1-related encephalopathy [Foxg1 in mice [In addition, hippocampus atrophy had also been reported in alopathy ,51. Hipp in mice . There w in mice .FOXG1+/− patients and Foxg1+/− mouse brain, there were increased inhibitory synaptic markers and decreased levels of excitatory synaptic markers [MECP2 and CDKL5 [FOXG1-, CDKL5- and MECP2-related syndromes.A proper level of FOXG1 has been found to be crucial for the balance of inhibitory/excitatory neurons and their markers during neuronal differentiation ,33. Patr markers . Interesnd CDKL5 . This shFoxg1+/Cre mice had a significant reduction in response amplitude and visual acuity of visually-evoked potentials compared with wild-type littermates. The morphological investigation showed abnormalities in the organization of excitatory/inhibitory circuits in the visual cortex with no alterations in the retinal structure. This finding could well correlate to the high-level visual dysfunctions in individuals with FOXG1 intragenic mutations.Moreover, in a study using a mouse model that focused on visual function , the FoxFOXG1-related syndrome patients.However, there remains much to be clarified. On top of all is that as the overexcitation may contribute to the pathogenesis of epilepsy in children, increased inhibition of neurons in such situations seems to be conflicting with the early onset of epilepsy in Foxg1 also exerts a critical impact on dendritogenesis and maintains the neural plasticity [MECP2 mutations [asticity . Dendritasticity , as wellutations .Foxg1 leads to dendrite elongation, whereas the knockdown or knockout of Foxg1 leads to reduced axon and dendrite length, as well as dendrite branching and spine densities [Foxg1 dosage could probably contribute to distinctive clinical characteristics between FOXG1 duplication and deletion/intragenic mutation. In particular, in West syndrome patients with FOXG1 duplication, hypsarrhythmia in EEG might be related to the eccentric enlargement of the dendritic trees. Furthermore, the study on the conditional knockout of Foxg1 in mice showed defects in social and cognitive behaviors, which could be correlated to the clinical symptoms of patients with FOXG1-related encephalopathy [FOXG1 in dendritogenesis of human remains to be explored and delineated.In vitro and animal studies have shown that the overexpression of ensities ,36. The alopathy . HoweverFOXG1-related encephalopathy in humans. However, there is still much to be explored to further delineate the pathogenesis of FOXG1-related encephalopathy.As shown in in vitro and animal studies, Foxg1 is an essential transcription factor in neurodevelopment. It regulates different cellular and molecular mechanisms, which are crucial for cell and neuronal survival. A fine balance of Foxg1 is necessary for the proliferation, specification, and pattern formation of the nervous system. Therefore, changes in the expression pattern of Foxg1 may lead to numerous cellular abnormalities. These may link to underlying pathogenesis of