MT1-MMP Cooperates with TGF-β Receptor-Mediated Signaling to Trigger SNAIL and Induce Epithelial-to-Mesenchymal-like Transition in U87 Glioblastoma Cells

Epithelial-to-mesenchymal transition (EMT) recapitulates metastasis and can be induced in vitro through transforming growth factor (TGF)-β signaling. A role for MMP activity in glioblastoma multiforme has been ascribed to EMT, but the molecular crosstalk between TGF-β signaling and membrane type 1 MMP (MT1-MMP) remains poorly understood. Here, the expression of common EMT biomarkers, induced through TGF-β and the MT1-MMP inducer concanavalin A (ConA), was explored using RNA-seq analysis and differential gene arrays in human U87 glioblastoma cells. TGF-β triggered SNAIL and fibronectin expressions in 2D-adherent and 3D-spheroid U87 glioblastoma cell models. Those inductions were antagonized by the TGF-β receptor kinase inhibitor galunisertib, the JAK/STAT inhibitors AG490 and tofacitinib, and by the diet-derived epigallocatechin gallate (EGCG). Transient gene silencing of MT1-MMP prevented the induction of SNAIL by ConA and abrogated TGF-β-induced cell chemotaxis. Moreover, ConA induced STAT3 and Src phosphorylation, suggesting these pathways to be involved in the MT1-MMP-mediated signaling axis that led to SNAIL induction. Our findings highlight a new signaling axis linking MT1-MMP to TGF-β-mediated EMT-like induction in glioblastoma cells, the process of which can be prevented by the diet-derived EGCG.     

Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-β1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.     

Somatostatin Receptor Splicing Variant sst5TMD4 Overexpression in Glioblastoma Is Associated with Poor Survival,Increased Aggressiveness Features,and Somatostatin Analogs Resistance

Glioblastoma (GBM) is the most malignant and lethal brain tumor. Current standard treatment consists of surgery followed by radiotherapy/chemotherapy; however, this is only a palliative approach with a mean post-operative survival of scarcely ~12–15 months. Thus, the identification of novel therapeutic targets to treat this devastating pathology is urgently needed. In this context, the truncated splicing variant of the somatostatin receptor subtype 5 (sst5TMD4), which is produced by aberrant alternative splicing, has been demonstrated to be overexpressed and associated with increased aggressiveness features in several tumors. However, the presence, functional role, and associated molecular mechanisms of sst5TMD4 in GBM have not been yet explored. Therefore, we performed a comprehensive analysis to characterize the expression and pathophysiological role of sst5TMD4 in human GBM. sst5TMD4 was significantly overexpressed (at mRNA and protein levels) in human GBM tissue compared to non-tumor (control) brain tissue. Remarkably, sst5TMD4 expression was significantly associated with poor overall survival and recurrent tumors in GBM patients. Moreover, in vitro sst5TMD4 overexpression (by specific plasmid) increased, whereas sst5TMD4 silencing (by specific siRNA) decreased, key malignant features (i.e., proliferation and migration capacity) of GBM cells (U-87 MG/U-118 MG models). Furthermore, sst5TMD4 overexpression in GBM cells altered the activity of multiple key signaling pathways associated with tumor aggressiveness/progression (AKT/JAK-STAT/NF-κB/TGF-β), and its silencing sensitized GBM cells to the antitumor effect of pasireotide (a somatostatin analog). Altogether, these results demonstrate that sst5TMD4 is overexpressed and associated with enhanced malignancy features in human GBMs and reveal its potential utility as a novel diagnostic/prognostic biomarker and putative therapeutic target in GBMs.     

TGF-β Induced CTGF Expression in Human Lung Epithelial Cells through ERK,ADAM17, RSK1, and C/EBPβ Pathways

Background: Lung epithelial cells play critical roles in idiopathic pulmonary fibrosis. Methods: In the present study, we investigated whether transforming growth factor-β (TGF-β)-induced expression of connective tissue growth factor (CTGF) was regulated by the extracellular signal-regulated kinase (ERK)/a disintegrin and metalloproteinase 17 (ADAM17)/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ) signaling pathway in human lung epithelial cells (A549). Results: Our results revealed that TGF-β-induced CTGF expression was weakened by ADAM17 small interfering RNA (ADAM17 siRNA), TNF-α processing inhibitor-0 (TAPI-0, an ADAM17 inhibitor), U0126 (an ERK inhibitor), RSK1 siRNA, and C/EBPβ siRNA. TGF-β-induced ERK phosphorylation as well as ADAM17 phosphorylation was attenuated by U0126. The TGF-β-induced increase in RSK1 phosphorylation was inhibited by TAPI-0 and U0126. TGF-β-induced C/EBPβ phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. In addition, TGF-β increased the recruitment of C/EBPβ to the CTGF promoter. Furthermore, TGF-β enhanced fibronectin (FN), an epithelial–mesenchymal transition (EMT) marker, and CTGF mRNA levels and reduced E-cadherin mRNA levels. Moreover, TGF-β-stimulated FN protein expression was reduced by ADAM17 siRNA and CTGF siRNA. Conclusion: The results suggested that TGF-β induces CTGF expression through the ERK/ADAM17/RSK1/C/EBPβ signaling pathway. Moreover, ADAM17 and CTGF participate in TGF-β-induced FN expression in human lung epithelial cells.     

Chrysin Ameliorates Cyclosporine-A-Induced Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial–Mesenchymal Transition

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK₁ cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β₁ signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β₁-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.     

The Wnt11 Signaling Pathway in Potential Cellular EMT and Osteochondral Differentiation Progression in Nephrolithiasis Formation

The molecular events leading to nephrolithiasis are extremely complex. Previous studies demonstrated that calcium and transforming growth factor-β1 (TGF-β1) may participate in the pathogenesis of stone formation, but the explicit mechanism has not been defined. Using a self-created genetic hypercalciuric stone-forming (GHS) rat model, we observed that the increased level of serous/uric TGF-β1 and elevated intracellular calcium in primary renal tubular epithelial cells (PRECs) was associated with nephrolithiasis progression in vivo. In the setting of high calcium plus high TGF-β1 in vitro, PRECs showed great potential epithelial to mesenchymal transition (EMT) progression and osteochondral differentiation properties, representing the multifarious increased mesenchymal and osteochondral phenotypes (Zeb1, Snail1, Col2A1, OPN, Sox9, Runx2) and decreased epithelial phenotypes (E-cadherin, CK19) bythe detection of mRNAs and corresponding proteins. Moreover, TGF-β-dependent Wnt11 knockdown and L-type Ca²⁺ channel blocker could greatly reverse EMT progression and osteochondral differentiation in PRECs. TGF-β1 alone could effectively promote EMT, but it had no effect on osteochondral differentiation in NRK cells (Rat kidney epithelial cell line). Stimulation with Ca²⁺ alone did not accelerate differentiation of NRK. Co-incubation of extracellular Ca²⁺ and TGF-β1 synergistically promotes EMT and osteochondral differentiation in NRK control cells. Our data supplied a novel view that the pathogenesis of calcium stone development may be associated with synergic effects of TGF-β1 and Ca²⁺, which promote EMT and osteochondral differentiation via Wnt11 and the L-type calcium channel.     

Molecular Imaging and Preclinical Studies of Radiolabeled Long-Term RGD Peptides in U-87 MG Tumor-Bearing Mice

The Arg–Gly–Asp (RGD) peptide shows a high affinity for α_vβ₃ integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(¹¹¹In) radiolabeled DOTA-EB-cRGDfK in α_vβ₃ integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that ¹¹¹In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC₅₀ = 71.7 nM). NanoSPECT/CT imaging showed ¹¹¹In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (¹¹¹In-DOTA-cRGDfK and ¹¹¹In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the ¹¹¹In-DOTA-EB-cRGDfK peptide has a long-term half-life (T_1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.     

MMP9 Differentially Regulates Proteins Involved in Actin Polymerization and Cell Migration during TGF-β-Induced EMT in the Lens

Fibrotic cataracts have been attributed to transforming growth factor-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT). Using mouse knockout (KO) models, our laboratory has identified MMP9 as a crucial protein in the TGF-β-induced EMT process. In this study, we further revealed an absence of alpha-smooth muscle actin (αSMA) and filamentous-actin (F-actin) stress fibers in MMP9KO mouse lens epithelial cell explants (LECs). Expression analysis using NanoString revealed no marked differences in αSMA (ACTA2) and beta-actin (β-actin) (ACTB) mRNA between the lenses of TGF-β-overexpressing (TGF-βtg) mice and TGF-βtg mice on a MMP9KO background. We subsequently conducted a protein array that revealed differential regulation of proteins known to be involved in actin polymerization and cell migration in TGF-β-treated MMP9KO mouse LECs when compared to untreated controls. Immunofluorescence analyses using rat LECs and the novel MMP9-specific inhibitor, JNJ0966, revealed similar differential regulation of cortactin, FAK, LIMK1 and MLC2 as observed in the array. Finally, a reduction in the nuclear localization of MRTF-A, a master regulator of cytoskeletal remodeling during EMT, was observed in rat LECs co-treated with JNJ0966 and TGF-β. In conclusion, MMP9 deficiency results in differential regulation of proteins involved in actin polymerization and cell migration, and this in turn prevents TGF-β-induced EMT in the lens.     

miR-29b Regulates TGF-β1-Induced Epithelial–Mesenchymal Transition by Inhibiting Heat Shock Protein 47 Expression in Airway Epithelial Cells

Tissue remodeling contributes to ongoing inflammation and refractoriness of chronic rhinosinusitis (CRS). During this process, epithelial-mesenchymal transition (EMT) plays an important role in dysregulated remodeling and both microRNA (miR)-29b and heat shock protein 47 (HSP47) may be engaged in the pathophysiology of CRS. This study aimed to determine the role of miR-29b and HSP47 in modulating transforming growth factor (TGF)-β1-induced EMT and migration in airway epithelial cells. Expression levels of miR-29b, HSP47, E-cadherin, α-smooth muscle actin (α-SMA), vimentin and fibronectin were assessed through real-time PCR, Western blotting, and immunofluorescence staining. Small interfering RNA (siRNA) targeted against miR-29b and HSP47 were transfected to regulate the expression of EMT-related markers. Cell migration was evaluated with wound scratch and transwell migration assay. miR-29b mimic significantly inhibited the expression of HSP47 and TGF-β1-induced EMT-related markers in A549 cells. However, the miR-29b inhibitor more greatly induced the expression of them. HSP47 knockout suppressed TGF-β1-induced EMT marker levels. Functional studies indicated that TGF-β1-induced EMT was regulated by miR-29b and HSP47 in A549 cells. These findings were further verified in primary nasal epithelial cells. miR-29b modulated TGF-β1-induced EMT-related markers and migration via HSP47 expression modulation in A549 and primary nasal epithelial cells. These results suggested the importance of miR-29b and HSP47 in pathologic tissue remodeling progression in CRS.     

Conditioned Media of Adipose-Derived Stem Cells Suppresses Sidestream Cigarette Smoke Extract Induced Cell Death and Epithelial-Mesenchymal Transition in Lung Epithelial Cells

The role of the epithelial–mesenchymal transition (EMT) in lung epithelial cells is increasingly being recognized as a key stage in the development of COPD, fibrosis, and lung cancers, which are all highly associated with cigarette smoking and with exposure to second-hand smoke. Using the exposure of human lung cancer epithelial A549 cells and non-cancerous Beas-2B cells to sidestream cigarette smoke extract (CSE) as a model, we studied the protective effects of adipose-derived stem cell-conditioned medium (ADSC-CM) against CSE-induced cell death and EMT. CSE dose-dependently induced cell death, decreased epithelial markers, and increased the expression of mesenchymal markers. Upstream regulator analysis of differentially expressed genes after CSE exposure revealed similar pathways as those observed in typical EMT induced by TGF-β1. CSE-induced cell death was clearly attenuated by ADSC-CM but not by other control media, such as a pass-through fraction of ADSC-CM or A549-CM. ADSC-CM effectively inhibited CSE-induced EMT and was able to reverse the gradual loss of epithelial marker expression associated with TGF-β1 treatment. CSE or TGF-β1 enhanced the speed of A549 migration by 2- to 3-fold, and ADSC-CM was effective in blocking the cell migration induced by either agent. Future work will build on the results of this in vitro study by defining the molecular mechanisms through which ADSC-CM protects lung epithelial cells from EMT induced by toxicants in second-hand smoke.     

Propolin G-Suppressed Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer Cells via Glycogen Synthase Kinase 3β-Mediated Snail and HDAC6-Regulated Vimentin Degradation

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer with a poor prognosis. The incidence and mortality rate of TNBC are frequently found in younger women. Due to the absence of a good therapeutic strategy, effective remedies for inhibiting TNBC have been developed for improving the cure rate. Epithelial-to-mesenchymal transition (EMT) is a critical mechanism to regulate cancer cell motility and invasion. Furthermore, ectopic expression of EMT molecules correlates with the metastasis and poor prognosis of TNBC. Targeting EMT might be a strategy for the therapy and prevention of TNBC. Propolin G, an active c-prenylflavanone in Taiwanese propolis, has been shown to possess anti-cancer activity in many cancers. However, the anti-metastasis activity of propolin G on TNBC is still unclear. The present study showed that the migration and invasion activities of TNBC cells was suppressed by propolin G. Down-regulated expression of Snail and vimentin and up-regulated expression of E-cadherin were dose- and time-dependently observed in propolin G-treated MDA-MB-231 cells. Propolin G inhibited Snail and vimentin expressions via the signaling pathways associated with post-translational modification. The activation of glycogen synthase kinase 3β (GSK-3β) by propolin G resulted in increasing GSK-3β interaction with Snail. Consequently, the nuclear localization and stability of Snail was disrupted resulting in promoting the degradation. Propolin G-inhibited Snail expression and the activities of migration and invasion were reversed by GSK-3β inhibitor pretreatment. Meanwhile, the outcomes also revealed that histone deacetylase 6 (HDAC6) activity was dose-dependently suppressed by propolin G. Correspondently, the amounts of acetyl-α-tubulin, a down-stream substrate of HDAC6, were increased. Dissociation of HDAC6/Hsp90 with vimentin leading to increased vimentin acetylation and degradation was perceived in the cells with the addition of propolin G. Moreover, up-regulated expression of acetyl-α-tubulin by propolin G was attenuated by HDAC6 overexpression. On the contrary, down-regulated expression of vimentin, cell migration and invasion by propolin G were overturned by HDAC6 overexpression. Conclusively, restraint cell migration and invasion of TNBC by propolin G were activated by the expression of GSK-3β-suppressed Snail and the interruption of HDAC6-mediated vimentin protein stability. Aiming at EMT, propolin G might be a potential candidate for TNBC therapy.     

Hypoxia Differently Affects TGF-β2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells

The hypoxia associated with the transforming growth factor-β2 (TGF-β2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O₂ gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-β2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-β2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-β2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-β2 increased proton leakage. The findings reported herein indicate that the TGF-β2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.     

Antitumor Effects of 5-Aminolevulinic Acid on Human Malignant Glioblastoma Cells

5-Aminolevulinic acid (5-ALA) is a naturally occurring non-proteinogenic amino acid, which contributes to the diagnosis and therapeutic approaches of various cancers, including glioblastoma (GBM). In the present study, we aimed to investigate whether 5-ALA exerted cytotoxic effects on GBM cells. We assessed cell viability, apoptosis rate, mRNA expressions of various apoptosis-related genes, generation of reactive oxygen species (ROS), and migration ability of the human U-87 malignant GBM cell line (U87MG) treated with 5-ALA at different doses. The half-maximal inhibitory concentration of 5-ALA on U87MG cells was 500 μg/mL after 7 days; 5-ALA was not toxic for human optic cells and NIH-3T3 cells at this concentration. The application of 5-ALA led to a significant increase in apoptotic cells, enhancement of Bax and p53 expressions, reduction in Bcl-2 expression, and an increase in ROS generation. Furthermore, the application of 5-ALA increased the accumulation of U87MG cells in the SUB-G1 population, decreased the expression of cyclin D1, and reduced the migration ability of U87MG cells. Our data indicate the potential cytotoxic effects of 5-ALA on U87MG cells. Further studies are required to determine the spectrum of the antitumor activity of 5-ALA on GBM.