Celastrol alleviates subconjunctival fibrosis induced by silicone implants mimicking glaucoma surgery.

Subconjunctival fibrosis is critical to the outcomes of several ophthalmic conditions or procedures, such as glaucoma filtering surgery. This study aimed to investigate the anti-fibrotic effect of celastrol on subconjunctival fibrosis and to further reveal the underlying mechanisms. We used celastrol-loaded nanomicelles hydrogel hybrid as a sustained-release drug. A rabbit model of subconjunctival fibrosis following silicone implantation was used for in vivo study, and TGF-ß1-induced human pterygium fibroblast (HPF) activation as an in vitro model. The effects of celastrol on inhibiting TGF-ß1-induced migration and proliferation of HPFs were evaluated by scratch wound assay and CCK-8, respectively. Immunofluorescence and western blotting were used to examine the effect of celastrol on the expression of α-SMA, collagen I, fibronectin, and the targets of the Hippo signaling pathway. We found that in vivo celastrol treatment reduced the expression of YAP and TAZ in subconjunctival tissue. Moreover, celastrol alleviated collagen deposition and subconjunctival fibrosis at 8 weeks. No obvious tissue toxicity was observed in the rabbit models. Mechanistically, celastrol significantly inhibited TGF-ß1-induced proliferation and migration of HPFs. Pretreatment of HPFs with celastrol also suppressed the TGF-ß1-induced protein expression of α-SMA, collagen I, fibronectin, TGF-ßRII, phosphorylated Smad2/3, YAP, TAZ, and TEAD1. In conclusion, celastrol effectively prevented subconjunctival fibrosis through inhibiting TGF-ß1/Smad2/3-YAP/TAZ pathway. Celastrol could serve as a promising therapy for subconjunctival fibrosis.

An injectable thermoresponsive-hydrogel for lamellar keratoplasty: In-situ releases celastrol and hampers corneal scars.

Corneal stromal fibrosis is a common cause of visual impairment resulting from corneal injury, inflammation and surgery. Therefore, there is an unmet need for inhibiting corneal stromal fibrosis. However, bioavailability of topical eye drops is very low due to the tear and corneal barriers. In situ delivery offers a unique alternative to improve efficacy and minimize systemic toxicity. Herein, a drug delivery platform based on thermoresponsive injectable hydrogel/nano-micelles composite with in situ drug-controlled release and long-acting features is developed to prevent corneal scarring and reduce corneal stromal fibrosis in lamellar keratoplasty. The in-situ gelation hydrogels enabled direct delivery of celastrol to the corneal stroma. In vivo evaluation with a rabbit anterior lamellar keratoplasty model showed that hydrogel/micelles platform could effectively inhibit corneal stromal fibrosis. This strategy achieves controlled and prolonged release of celastrol in the corneal stroma of rabbit. Following a single corneal interlamellar injection, celastrol effectively alleviated fibrosis via mTORC1 signal promoting autophagy and inhibiting TGF-ß1/Smad2/3 signaling pathway. Overall, this strategy demonstrates promise for the clinical application of celastrol in preventing corneal scarring and reducing corneal stromal fibrosis post-lamellar keratoplasty, highlighting the potential benefits of targeted drug delivery systems in ocular therapeutics.

M2 macrophages promote subconjunctival fibrosis through YAP/TAZ signalling.

PURPOSE: To evaluate the role of M2 macrophages in subconjunctival fibrosis after silicone implantation (SI) and investigate the underlying mechanisms. MATERIALS AND METHODS: A model of subconjunctival fibrosis was established by SI surgery in rabbit eyes. M2 distribution and collagen deposition were evaluated by histopathology. The effects of M2 cells on the migration (using wound-scratch assay) and activation (by immunofluorescence and western blotting) of human Tenon's fibroblasts (HTFs) were investigated. RESULTS: There were more M2 macrophages (CD68+/CD206+ cells) occurring in tissue samples around silicone implant at 2 weeks postoperatively. Dense collagen deposition was observed at 8 weeks after SI. In vitro experiment showed M2 expressed high level of CD206 and transforming growth factor-ß1 (TGF-ß1). The M2-conditioned medium promoted HTFs migration and the synthesis of collagen I and fibronectin. Meanwhile, M2-conditioned medium increased the protein levels of TGF-ß1, TGF-ßR II, p-Smad2/3, yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ). Verteporfin, a YAP inhibitor, suppressedTGF-ß1/Smad2/3-YAP/TAZ pathway and attenuated M2-induced extracellular matrix deposition by HTFs. CONCLUSIONS: TGF-ß1/Smad2/3-YAP/TAZ signalling may be involved in M2-induced fibrotic activities in HTFs. M2 plays a key role in promoting subconjunctival fibrosis and can serve as an attractive target for anti-fibrotic therapeutics.

Excess folic acid supplementation before and during pregnancy and lactation activates ß-catenin in the brain of male mouse offspring.

Folic acid (FA) supplementation in early pregnancy is recommended to protect against birth defects. But excess FA has exhibited neurodevelopmental toxicity. We previously reported that the mice treated with 2.5-fold the dietary requirement of FA one week before mating and throughout pregnancy and lactation displayed abnormal behaviors in the offspring. Here we found the levels of non-phosphorylated ß-catenin (active) were increased in the brains of weaning and adult FA-exposed offspring. Meanwhile, demethylation of protein phosphatase 2 A catalytic subunit (PP2Ac), which suppresses its enzyme activity in regulatory subunit dependent manner, was significantly inhibited. Among the upstream regulators of ß-catenin, PI3K/Akt/GSK-3ß but not Wnt signaling was stimulated in FA-exposed brains only at weaning. In mouse neuroblastoma N2a cells, knockdown of PP2Ac or leucine carboxyl methyltransferase-1 (LCMT-1), or overexpression of PP2Ac methylation-deficient mutant decreased ß-catenin dephosphorylation. These results suggest that excess FA may activate ß-catenin via suppressing PP2Ac demethylation, providing a novel mechanism for the influence of FA on neurodevelopment.

Celastrol-based nanomedicine promotes corneal allograft survival.

Effectively promoting corneal allograft survival remains a challenge in corneal transplantation. The emerging therapeutic agents with high pharmacological activities and their appropriate administration routes provide attractive solutions. In the present study, a celastrol-loaded positive nanomedicine (CPNM) was developed to enhance corneal penetration and to promote corneal allograft survival. The in vitro, in vivo and ex vivo results demonstrated the good performance of CPNM prolonging the retention time on ocular surface and opening the tight junction in cornea, which resulted in enhanced corneal permeability of celastrol. Both in vitro and in vivo results demonstrated that celastrol inhibited the recruitment of M1 macrophage and the expression of TLR4 in corneal allografts through the TLR4/MyD88/NF-κB pathway, thereby significantly decreasing secretion of multiple pro-inflammatory cytokines to promote corneal allograft survival. This is the first celastrol-based topical instillation against corneal allograft rejection to provide treatment more potent than conventional eye drops for ocular anterior segment diseases.

Hypoxia-Responsive Polymeric Micelles for Enhancing Cancer Treatment.

Polymeric drug vectors have shown great potentials in cancer therapy. However, intelligent controlled release of drugs has become a major challenge in nanomedicine research. Hypoxia-responsive polymeric micelles have received widespread attention in recent years due to the inherent hypoxic state of tumor tissue. In this study, a novel diblock polymer consisting of polyethylene glycol and poly[glutamic acid (3-(2-nitro-imidazolyl)-propyl)] was synthesized and self-assembled into hypoxia-responsive polymeric micelles for the controlled release of doxorubicin (DOX). The cell experiments demonstrated that DOX-loaded micelles had a stronger killing capacity on tumor cells under hypoxic conditions, while the blank micelles had good biocompatibility. All the experiments indicate that our hypoxia-responsive polymeric micelles have a great potential for enhanced cancer treatment.

Effectively suppressed angiogenesis-mediated retinoblastoma growth using celastrol nanomicelles.

Celastrol, a Chinese herbal medicine, has already shown an inhibition effect on retinoblastoma growth activity in our previous research, but its mechanism is not well understood. Angiogenesis is a main driving force in many tumors. Here, we studied whether celastrol could inhibit angiogenesis-mediated retinoblastoma growth, if so, through what mechanism. In this work, we developed celastrol-loaded polymeric nanomicelles to improve the poor water solubility of celastrol. When given an intraperitoneal injection to mice bearing human retinoblastoma xenografts, celastrol nanomicelles (CNMs, 27.2 mg/kg/2 days) significantly reduced the weight and the volume of tumors and decreased tumor angiogenesis. We found that CNMs suppressed hypoxia-induced proliferation, migration, and invasion by human umbilical vascular endothelial cells (EA.hy 926) in a dose-dependent manner. Furthermore, CNMs inhibited SO-Rb 50 cells-induced sprouting of the vessels and vascular formation in chick embryo chorioallantoic membrane assay in vitro. To understand the molecular mechanism of these activities, we assessed the signaling pathways in CoCl2 treated EA.hy 926. CNMs inhibited the hypoxia-induced HIF-1α and VEGF. In conclusion, our results reveal that CNMs target the HIF-1α/VEGF pathway, which may be an important reason for the suppression of retinoblastoma growth and angiogenesis.

Glycogen synthase kinase-3ß suppresses the expression of protein phosphatase methylesterase-1 through ß-catenin.

Protein phosphatase 2A (PP2A) is the major tau phosphatase. Its activity toward tau is regulated by the methylation of PP2A catalytic subunit (PP2Ac) at Leu309. Protein phosphatase methylesterase-1 (PME-1) demethylates PP2Ac and suppresses its activity. We previously found that glycogen synthase kinase-3ß (GSK-3ß) suppresses PME-1 expression. However, the underlying molecular mechanism is unknown. In the present study, we analyzed the promoter of PME-1 gene and found that human PME-1 promoter contains two lymphoid enhancer binding factor-1/T-cell factor (LEF1/TCF) cis-elements in which ß-catenin serves as a co-activator. ß-catenin acted on these two cis-elements and promoted PME-1 expression. GSK-3ß phosphorylated ß-catenin and suppressed its function in promoting PME-1 expression. Inhibition and activation of GSK-3ß by PI3K-AKT pathway promoted and suppressed, respectively, PME-1 expression in primary cultured neurons, SH-SY5Y cells and in the mouse brain. These findings suggest that GSK-3ß phosphorylates ß-catenin and suppresses its function on PME-1 expression, resulting in an increase of PP2Ac methylation.

Cyclic AMP-Dependent Protein Kinase Phosphorylates TDP-43 and Modulates Its Function in Tau mRNA Processing.

Trans-active response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved and ubiquitously expressed nuclear protein. As a member of heterogeneous ribonucleoproteins, TDP-43 plays pivotal roles in mRNA processing. We recently found that TDP-43 promoted tau mRNA instability via acting on the 3'-untranslated region of its mRNA and enhanced tau exon 10 inclusion. TDP-43 is a phospho-protein. The function and the pathological aggregation of TDP-43 are regulated by the phosphorylation. In the present study, we determined phosphorylation of TDP-43 by cyclic AMP-dependent protein kinase (PKA). We found that TDP-43 was co-immunoprecipitated by and co-localized with PKA in the nucleus. PKA phosphorylated TDP-43 at Ser379, Ser403/404, and Ser409/410 in vitro and in cultured cells. Phosphorylation of TDP-43 at these sites enhanced mutually their phosphorylation by PKA in vitro and in cultured cells. Overexpression of PKA suppressed TDP-43's activity in promoting tau mRNA instability and tau exon 10 inclusion. These findings shed light on the role of PKA in phosphorylation and function of TDP-43. Downregulation of PKA signaling in AD brain may attenuate the impact of TDP-43 pathology in tau pathogenesis.

Fabrication and Biocompatibility of Core-Shell Structured Magnetic Fibrous Scaffold.

In recent years, various magnetic bio-materials with physical and morphological cues have been used in biomedical area due to their advantageous characteristics. As one class of them, magnetic fibrous scaffolds have attracted many researchers' interests because they have an important positive impact on cellular growth behavior. They provide various physical cues to regulate the regeneration and repair of damaged tissue, and promote new tissue formation. In this study, we developed core-shell structured magnetic fibers (3D CS-MFs) using cooperative assembly method the combination with electrospinning technology. The obtained magnetic 3D CS-MFs displayed excellent magnetic performance, biocompatibility, and provided a desirable microenvironment for model cells (bone marrow mesenchymal stem cells, BMSCs) growth. More importantly, BMSCs exhibited excellent viability and 3D growth. The novel preparation method will greatly enhance the potential application of magnetic fibrous scaffold in the biomedical area, such as drug release, and tissue engineering.

Quantitative proteomic analysis of the miR-148a-associated mechanisms of metastasis in non-small cell lung cancer.

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression and protein synthesis. Our previous study demonstrated that miR-148a suppressed the metastasis of non-small cell lung cancer (NSCLC) in vitro and in vivo. However, the modulatory mechanism of this effect remains unclear. In the present study, quantitative proteomic technology was used to study the protein expression profile of SPC-A-1 cells subsequent to the downregulation of miR-148a expression, in order to elucidate the molecular mechanism of the suppression of NSCLC metastasis by miR-148a. The differentially expressed proteins identified were analyzed using bioinformatics tools, including the Database for Annotation, Visualization and Integrated Discovery and the Search Tool for the Retrieval of Interacting Genes/proteins. In two experiments, 4,048 and 4,083 proteins were identified, and 4,014 and 4,039 proteins were quantified, respectively. In total, 44 proteins were upregulated and 40 proteins were downregulated. This was verified at the protein and mRNA levels by western blotting and reverse transcription-quantitative polymerase chain reaction, respectively. Bioinformatics analysis was used to identify potential interactions and signaling networks for the differentially expressed proteins. This may have provided an appropriate perspective for the comprehensive analysis of the modulatory mechanism underlying the metastasis-suppressive effects of miR-148a in NSCLC. In conclusion, quantitative proteomic technology revealed that miR-148a may regulate a panel of tumor-associated proteins to suppress metastasis in NSCLC.

Calpain I Activation Causes GLUT3 Proteolysis and Downregulation of O-GlcNAcylation in Alzheimer's Disease Brain.

Impairment of cerebral glucose uptake/metabolism in individuals with Alzheimer's disease (AD) is believed to lead to downregulation of protein O-GlcNAcylation, which contributes to tau pathogenesis through tau hyperphosphorylation. Level of glucose transporter 3 (GLUT3), a neuronal specific glucose transporter, is decreased in AD brain, which may contribute to impaired brain glucose uptake/metabolism. However, what causes the reduction of GLUT3 in AD brain is not fully understood. Here, we report 1) that decrease of GLUT3 is associated with the reduction of protein O-GlcNAcylation in AD brain, 2) that GLUT3 level is negatively correlated with calpain I activation in human brain, 3) that calpain I proteolyzes GLUT3 at the N-terminus in vitro, and 4) that activation of calpain I is negatively correlated with protein O-GlcNAcylation in AD brain. Furthermore, we found that overexpression of GLUT3 enhances protein O-GlcNAcylation in N2a cells. Overexpression of calpain I suppresses protein O-GlcNAcylation in these cells. These findings suggest a novel mechanism by which calpain I overactivation leads to GLUT3 degradation and the consequent down-regulation of protein O-GlcNAcylation in AD brain.

O-GlcNAcylation modulates PKA-CREB signaling in a manner specific to PKA catalytic subunit isoforms.

O-GlcNAcylation is a post-translational modification of proteins. Protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling plays critical roles in multiple biological processes. Isoforms α and ß of PKA catalytic subunit (PKAc) and CREB are modified by O-GlcNAcylation. In the present study, we determined the role of O-GlcNAcylation in PKAc isoform-specific CREB signaling. We found that up-regulation of O-GlcNAcylation enhanced CREB phosphorylation, but suppressed CREB expression in exogenous PKAc isoform-unspecific manner. PKAc isoforms affected exogenous expression of OGT or OGA and protein O-GlcNAcylation differently. Up-regulation of O-GlcNAcylation did not significantly affect net PKAcα-CREB signaling, but enhanced PKAcß-CREB signaling. The role of O-GlcNAcylation in PKA-CREB signaling was desensitized by insulin treatment. This study suggests a role of O-GlcNAcylation in PKA-CREB signaling by affecting phosphorylation of CREB in a PKAc isoform-specific manner.

A DOX-loaded polymer micelle for effectively inhibiting cancer cells.

A novel triblock polymer is synthesized and self-assembled with doxorubicin to form DOX-loaded micelles. The synthetic process involves the ring-opening polymerization, carboxylation and amidation reactions, and the structures are characterized. The drug release test indicated that the micelles have the ability to control the release of drugs. The cell uptake results indicated that the DOX-loaded micelles could enter cancer cells easily, and the cytotoxicity and apoptosis test confirmed that DOX-loaded micelles have a strong killing effect on tumor cells, while the blank micelles do not have cytotoxicity. Therefore, the novel polymer micelles are a promising carrier for delivery of anticancer drugs to enhance cancer treatment.

MetaLnc9 Facilitates Lung Cancer Metastasis via a PGK1-Activated AKT/mTOR Pathway.

Long noncoding RNAs (lncRNA) participate in carcinogenesis and tumor progression in lung cancer. Here, we report the identification of a lncRNA signature associated with metastasis of non-small cell lung cancer (NSCLC). In particular, elevated expression of LINC00963 (MetaLnc9) in human NSCLC specimens correlated with poor prognosis, promoted migration and invasion of NSCLC cells in vitro, and enhanced lung metastasis formation in vivo Mechanistic investigations showed that MetaLnc9 interacted with the glycolytic kinase PGK1 and prevented its ubiquitination in NSCLC cells, leading to activation of the oncogenic AKT/mTOR signaling pathway. MetaLnc9 also interacted with P54nrb/NonO (NONO) to help mediate the activity of CRTC, a coactivator for the transcription factor CREB, reinforcing a positive feedback loop for metastasis. Taken together, our results establish MetaLnc9 as a driver of metastasis and a candidate therapeutic target for treating advanced NSCLC. Cancer Res; 77(21); 5782-94. ©2017 AACR.

Systematic analysis of mRNA expression profiles in NSCLC cell lines to screen metastasis-related genes.

Lung cancer is the most prevalent cancer in humans and has the lowest survival outcomes due to its high metastatic potential. The aim of the present study was to screen for metastasis­related genes (MRGs) by investigating the differential expression genes (DEGs) identified by the mRNA expression profiles in SPC­A­1sci (highly metastatic) and SPC-A-1 (parental) cells. DEGs were screened using Genespring software. Gene Ontology and pathway enrichment analyses of these DEGs were performed. Interaction networks between the proteins encoded by the DEGs were identified using the database BioGRID and were visualized by Cytoscape. Modular analysis of the protein­protein interaction network was performed in CFinder. Among these DEGs, the expression levels of 18 genes were examined in SPC­A­1sci and SPC­A­1 cell lines with reverse transcription­quantitative polymerase chain reaction, and 10 of the 18 genes were assessed by western blotting to validate the results of the microarray. Furthermore, the role of metallothionein 1X (MT1X) in non­small cell lung cancer was explored in functional assays and 72 pairs of clinical samples in vitro. Finally, 4,838 DEGs were screened, including 798 upregulated and 4,040 downregulated genes. The significantly enriched functions included gene expression, cytosol and poly­(A) RNA binding, and the most enriched pathway was biosynthesis of antibiotics. Furthermore, MT1X was revealed to promote the migration and invasion ability in SPC­A­1sci and PC­9 lung cancer cell lines. Therefore, MT1X was identified as a candidate MRG through systematic analysis in the present microarray, which was demonstrated to offer potential reference value in screening MRGs.

GSK-3ß is Dephosphorylated by PP2A in a Leu309 Methylation-Independent Manner.

Hyperphosphorylation of tau is pivotally involved in the pathogenesis of Alzheimer's disease (AD) and related tauopathies. Glycogen synthase kinase-3ß (GSK-3ß) and protein phosphate 2A (PP2A) are crucial enzymes to regulate tau phosphorylation. GSK-3ß activity is regulated by its inhibitory phosphorylation at Ser9. We previously reported the cross-talk between GSK-3ß and PP2A signaling and showed that PP2A could dephosphorylate GSK-3ß at Ser9. Here, we investigated the dephosphorylation of GSK-3ß in brain extracts in the presence of phosphatase inhibitors and found that a PP2A-like phosphatase activity was required for dephosphorylation of GSK-3ß at Ser9. PP2A interacted with GSK-3ß and suppressed its Ser9 phosphorylation in vitro and in HEK-293FT cells. Activity of PP2A negatively correlated to the level of phosphorylated GSK-3ß in kainic acid-induced excitotoxic mouse brain. Alteration of methylation of the catalytic subunit of PP2A (PP2Ac) at Leu309 did not affect GSK-3ß phosphorylation. These findings suggest that Leu309 methylation is not required for PP2A to dephosphorylate GSK-3ß at Ser9.

MicroRNA-148a Suppresses Invasion and Metastasis of Human Non-Small-Cell Lung Cancer.

BACKGROUND/AIMS: microRNAs (miRNAs) are noncoding RNAs that regulate multiple targets through either the degradation of mRNAs or the inhibition of protein translation, thereby altering several functions simultaneously. Growing evidence indicates that miRNAs are involved in carcinogenesis and tumor progression in non-small-cell lung cancer (NSCLC). METHODS: In this study, the mRNA expression levels of miR-148a were examined in NSCLC cell lines and patient specimens using quantitative reverse transcription-PCR. The functions of miR-148a in migration/invasion and lung metastasis formation were determined by using transwell and tail vein injection assays, respectively. RESULTS: We demonstrated that miR-148a was down-regulated in NSCLC metastatic samples, and its expression was suppressed in NSCLC compared with the corresponding nonmalignant lung tissues. Clinical analysis indicated that miR-148a expression was lower in NSCLC patients compared with nonmalignant lung tissues . Decreased miR-148a was significantly associated with tumor node metastasis stage and lymph node metastasis. Furthermore, functional assays showed that miR-148a expression suppressed NSCLC cell invasive and migratory abilities in vitro and suppressed cancer metastasis in vivo, while inhibition of miR-148a enhanced NSCLC cell invasion and lung metastasis formation in a mouse model. CONCLUSIONS: Evidence from this study demonstrated that miR-148a exerts tumor-suppressive effects in NSCLC and suggests a new therapeutic option for NSCLC.

MiRNA-10a is upregulated in NSCLC and may promote cancer by targeting PTEN.

MicroRNAs (miRNAs) are involved in human cancer including non-small cell lung cancer (NSCLC). In this study, we compared miRNA expression microarray of SPC-A-1sci (high metastatic) and SPC-A-1 (weakly metastatic) cells. We found that miRNA-10a was up-regulated in NSCLC compared with corresponding normal tissues. High expression of miR-10a was associated with tumor node metastasis and lymph node metastasis. Furthermore, overexpression of miR-10a promoted NSCLC cell proliferation, migration and invasion in vitro. We found that PTEN was a direct target of miR-10a in NSCLC. Also miR-10a activated the PTEN/AKT/ERK pathway. We suggest that miR-10a contributes to NSCLC by targeting PTEN.