Preclinical Animal Study and Pilot Clinical Trial of Using Enriched Peripheral Blood-Derived Mononuclear Cells for Intervertebral Disc Degeneration.

Low back pain (LBP) is a leading cause of long-term disability globally. Intervertebral disk degeneration (IVDD) is mainly responsible for discogenic pain in LBP-affected young patients. There is no effective therapy to reverse disease severity and IVDD progression. This study investigates the effect of human peripheral blood-derived mononuclear cells (PBMCs) on pain relief and life quality improvement in IVDD patients. The enriched monocytes of the PBMCs could differentiate into CD14 and CD206 double-positive M2 macrophages in vitro. Preclinical evidence in rats showed that the transplanted PBMCs exhibited anti-inflammatory and moderate tissue-repair effects on controlling IVDD progress in the rat model. The PBMCs significantly steered the aggrecan and type II collagen expressions and attenuated the pro-inflammatory cytokines in the affected disk. Based on the animal results, 36 patients with chronic low back pain (CLBP) were included in clinical trials. The control group was conservative care only, and the experimental group was platelet-rich plasma (PRP) and PBMCs intradiscal injections. We first confirmed the single lumbar disk causing the discogenic pain by provocative discography or magnetic resonance imaging (MRI). Discogenic LBP participants received one intradiscal injection of autologous PBMCs and followed for 6 months. Our clinical trial showed that patients' LBP and disability were significantly ameliorated after the PBMCs transplantation rather than PRP. These preclinical and pilot clinical studies indicate that intradiscal injection of the enriched PBMCs might be a feasible and potential cell therapy to control pain and disability in IVDD patients.

Mesenchymal Stem Cell-Derived Exosomes Mitigate Acute Murine Liver Injury via Ets-1 and Heme Oxygenase-1 Up-regulation.

BACKGROUND: Mesenchymal stem cells (MSCs)-derived exosomes have been previously demonstrated to promote tissue regeneration in various animal disease models. This study investigated the protective effect of exosome treatment in carbon tetrachloride (CCl4)-induced acute liver injury and delineated possible underlying mechanism. METHODS: Exosomes collected from conditioned media of previously characterized human umbilical cord-derived MSCs were intraperitoneally administered into male CD-1 mice with CCl4-induced acute liver injury. Biochemical, histological and molecular parameters were used to evaluate the severity of liver injury. A rat hepatocyte cell line, Clone-9, was used to validate the molecular changes by exosome treatment. RESULTS: Exosome treatment significantly suppressed plasma levels of AST, ALT, and pro-inflammatory cytokines, including IL-6 and TNF-, in the mice with CCl4-induced acute liver injury. Histological morphometry revealed a significant reduction in the necropoptic area in the injured livers following exosome therapy. Consistently, western blot analysis indicated marked elevations in hepatic expression of PCNA, c-Met, Ets-1, and HO-1 proteins after exosome treatment. Besides, the phosphorylation level of signaling mediator JNK was significantly increased, and that of p38 was restored by exosome therapy. Immunohistochemistry double staining confirmed nuclear Ets-1 expression and cytoplasmic localization of c-Met and HO-1 proteins. In vitro studies demonstrated that exosome treatment increased the proliferation of Clone-9 hepatocytes and protected them from CCl4-induced cytotoxicity. Kinase inhibition experiment indicated that the exosome-driven hepatoprotection might be mediated through the JNK pathway. CONCLUSION: Exosome therapy activates the JNK signaling activation pathway as well as up-regulates Ets-1 and HO-1 expression, thereby protecting hepatocytes against hepatotoxin-induced cell death.

MicroRNA-26a-5p Restoration Ameliorates Unilateral Ureteral Obstruction-Induced Renal Fibrosis in Mice Through Modulating TGF-ß Signaling.

Renal fibrosis is a hallmark of chronic and progressive renal diseases characterized by excessive fibroblast proliferation, extracellular matrix accumulation, and a loss of renal function, eventually leading to end-stage renal diseases. MicroRNA-26a-5p (miR-26a-5p) downregulation has been previously noted in the sera of unilateral ureteral occlusion (UUO)-injured mice, and exosome-mediated miR-26a-5p reportedly attenuated experimental pulmonary and cardiac fibrosis. This study evaluated the expression patterns of miR-26a in a human tissue microarray with kidney fibrosis and in tissues from a mouse model of UUO-induced renal fibrosis. Histologic analyses showed that miR-26a-5p was downregulated in human and mouse tissues with renal interstitial nephritis and fibrosis. Moreover, miR-26a-5p restoration by intravenous injection of a mimic agent prominently suppressed the expression of transforming growth factor ß1 (TGF-ß1) and its cognate receptors, the inflammatory transcription factor NF-κB, epithelial-mesenchymal transition, and inflammatory markers in UUO-injured kidney tissues. In vitro, miR-26a-5p mimic delivery significantly inhibited TGF-ß1-induced activation of cultured normal rat kidney NRK-49F cells, in terms of downregulation of TGF-ß1 receptors, restoration of the epithelial marker E-cadherin, and suppression of mesenchymal markers, including vimentin, fibronectin, and α-smooth muscle actin, as well as TGF-ß1/SMAD3 signaling activity. Our findings identified miR-26a-5p downregulation in kidney tissues with human interstitial nephritis and UUO-induced mouse kidney fibrosis. MiR-26a-5p restoration may exhibit an antifibrotic effect through the blockade of both TGF-ß and NF-κB signaling axes and is considered a novel therapeutic target for treating obstruction-induced renal fibrosis.

Genetic Interference of FGFR3 Impedes Invasion of Upper Tract Urothelial Carcinoma Cells by Alleviating RAS/MAPK Signal Activity.

Upper tract urothelial cancer (UTUC) is a less common disease in Western countries but has a high level of prevalence in Asian populations. Compared to bladder cancer, unique etiologic and genomic factors are involved in UTUC. Fibroblast growth factor receptor 3 (FGFR3) up-regulation has been proposed as a promising target for bladder cancer therapy. In this study, we aimed to profile the expression of FGFR3 in Asian and Caucasian UTUC tissues and to evaluate the in vitro therapeutic efficacy of small interference RNA (siRNA)-mediated FGFR3 silencing in UTUC treatment. The FGFR3 expression levels in renal pelvis tissues and microarray sections from Asian and Caucasian patients with UTUC, respectively, were measured via immunohistochemistry. The BFTC-909 and UM-UC-14 UTUC cell lines were used to examine the effects of FGFR3 silencing on proliferation, migration, epithelial-mesenchymal transition (EMT) marker expression, and signaling machinery. FGFR3 expression increased as the TNM stage increased in both Asian and Caucasian UTUC tumors, and no statistical difference was identified between the two groups. In vitro studies demonstrated that FGFR3 siRNA delivery significantly inhibited proliferation and migration and suppressed the expression of EMT markers and transcription factors in UTUC cells. Mechanistically, FGFR3 silencing alleviated the constitutive expression of RAS and the phosphorylation of MAPK signaling mediators, including ERK1/2 and JNK1/2. FGFR3 silencing elicited an apoptosis-inducing effect similar to that of FGFR inhibition. Conclusion: siRNA-targeted FGFR3 expression may impede the expansion and invasion of UTUC cells by alleviating the RAS/MAPK signaling pathway. The genetic interference of FGFR3 expression via siRNA in UTUC cells may constitute a useful therapeutic strategy.

Chaperonin counteracts diet-induced non-alcoholic fatty liver disease by aiding sirtuin 3 in the control of fatty acid oxidation.

AIMS/HYPOTHESIS: The mitochondrial chaperonin heat shock protein (HSP) 60 is indispensable in protein folding and the mitochondrial stress response; however, its role in nutrient metabolism remains uncertain. This study investigated the role of HSP60 in diet-induced non-alcoholic fatty liver disease (NAFLD). METHODS: We studied human biopsies from individuals with NAFLD, murine high-fat-diet (HFD; a diet with 60% energy from fat)-induced obesity (DIO), transgenic (Tg) mice overexpressing Hsp60 (Hsp60-Tg), and human HepG2 cells transfected with HSP60 cDNA or with HSP60 siRNA. Histomorphometry was used to assess hepatic steatosis, biochemistry kits were used to measure insulin resistance and glucose tolerance, and an automated home cage phenotyping system was used to assess energy expenditure. Body fat was assessed using MRI. Macrophage infiltration, the lipid oxidation marker 4-hydroxy-2-nonenal (4-HNE) and the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were detected using immunohistochemistry. Intracellular lipid droplets were evaluated by Nile red staining. Expression of HSP60, and markers of lipogenesis and fatty acid oxidation were quantified using RT-PCR and immunoblotting. Investigations were analysed using the two-way ANOVA test. RESULTS: Decreased HSP60 expression correlated with severe steatosis in human NAFLD biopsies and murine DIO. Hsp60-Tg mice developed less body fat, had reduced serum triglyceride levels, lower levels of insulin resistance and higher serum adiponectin levels than wild-type mice upon HFD feeding. Respiratory quotient profile indicated that fat in Hsp60-Tg mice may be metabolised to meet energy demands. Hsp60-Tg mice showed amelioration of HFD-mediated hepatic steatosis, M1/M2 macrophage dysregulation, and 4-HNE and 8-OHdG overproduction. Forced HSP60 expression reduced the mitochondrial unfolded protein response, while preserving mitochondrial respiratory complex activity and enhancing fatty acid oxidation. Furthermore, HSP60 knockdown enhanced intracellular lipid formation and loss of sirtuin 3 (SIRT3) signalling in HepG2 cells upon incubation with palmitic acid (PA). Forced HSP60 expression improved SIRT3 signalling and repressed PA-mediated intracellular lipid formation. SIRT3 inhibition compromised HSP60-induced promotion of AMP-activated protein kinase (AMPK) phosphorylation and peroxisome proliferator-activated receptor α (PPARα levels), while also decreasing levels of fatty acid oxidation markers. CONCLUSION/INTERPRETATION: Mitochondrial HSP60 promotes fatty acid oxidation while repressing mitochondrial stress and inflammation to ameliorate the development of NAFLD by preserving SIRT3 signalling. This study reveals the hepatoprotective effects of HSP60 and indicates that HSP60 could play a fundamental role in the development of therapeutics for NAFLD or type 2 diabetes.

MiR-26a-5p as a useful therapeutic target for upper tract urothelial carcinoma by regulating WNT5A/ß-catenin signaling.

The role of miRNAs in cancer and their possible function as therapeutic agents are interesting and needed further investigation. The miR-26a-5p had been demonstrated as a tumor suppressor in various cancers. However, the importance of miR-26a-5p regulation in upper tract urothelial carcinoma (UTUC) remains unclear. Here, we aimed to explore the miR-26a-5p expression in UTUC tissues and to identify its regulatory targets and signal network involved in UTUC tumorigenesis. The miR-26a-5p expression was validated by quantitative real-time polymerase chain reaction (qPCR) using renal pelvis tissue samples from 22 patients who were diagnosed with UTUC and 64 cases of renal pelvis tissue microarray using in situ hybridization staining. BFTC-909 UTUC cells were used to examine the effects of miR-26a-5p genetic delivery on proliferation, migration and expression of epithelial-to-mesenchymal transition (EMT) markers. MiR-26a-5p was significantly down-regulated in UTUC tumors compared to adjacent normal tissue and was decreased with histological grades. Moreover, restoration of miR-26a-5p showed inhibition effects on proliferation and migration of BFTC-909 cells. In addition, miR-26a-5p delivery regulated the EMT marker expression and inhibited WNT5A/ß-catenin signaling and expression of downstream molecules including NF-κB and MMP-9 in BFTC-909 cells. This study demonstrated that miR-26a-5p restoration may reverse EMT process and regulate WNT5A/ß-catenin signaling in UTUC cells. Further studies warranted to explore the potential roles in biomarkers for diagnostics and prognosis, as well as novel therapeutics targets for UTUC treatment.

Triamcinolone acetonide modulates TGF­ß2­induced angiogenic and tissue­remodeling effects in cultured human retinal pigment epithelial cells.

Transforming growth factor­ß2 (TGF­ß2) has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), due to its ability to stimulate the overproduction of pro­angiogenic factors, such as vascular endothelial growth factor (VEGF), and remodeling of the extracellular matrix (ECM). Although intravitreal triamcinolone acetonide (TA) is clinically useful in the treatment of PVR and PDR, its molecular mechanism has yet to be fully elucidated. The present study investigated whether TA treatment altered TGF­ß2­driven biological effects on the behavior of cultured human retinal pigment epithelial (RPE) cells, in order to determine which signaling pathway may be essential for the pharmacological action of TA. The R­50 human RPE cell line was treated with TA in the presence of TGF­ß2, followed by analyses of cell viability and contraction using cell viability and collagen gel contraction assays. VEGF mRNA expression and protein production were measured using reverse transcription­quantitative PCR and ELISA, respectively. The phosphorylation status of signaling mediators and the protein expression of type I collagen (COL1A1), α­smooth muscle actin (α­SMA), and ECM­remodeling enzymes, including MMP­2 and MMP­9, were analyzed using western blotting. The gelatinolytic activity of MMPs was detected using gelatin zymography. TA treatment exhibited no prominent cytotoxicity but markedly antagonized TGF­ß2­induced cytostatic effects on RPE cell viability and TGF­ß2­enhanced contractility in collagen gels. In the context of TGF­ß2­related signaling, TA significantly attenuated TGF­ß2­elicited Smad2, extracellular­regulated kinase (ERK)1/2 and p38 mitogen­activated protein kinase (MAPK) phosphorylation. Moreover, TA markedly mitigated TGF­ß2­induced VEGF upregulation through ablation of p38 signaling activity. TA also partially attenuated TGF­ß2­elicted expression of COL1A1, α­SMA, MMP­2, and MMP­9, but only suppressed TGF­ß2­induced MMP­9 gelatinolytic activity. Mechanistically, the MEK/ERK signaling pathway may have a critical role in the TGF­ß2­induced upregulation of COL1A1, α­SMA and MMP­9. In conclusion, TA may be considered a useful therapeutic agent for treating TGF­ß2­associated intraocular angiogenesis and tissue remodeling, the underlying mechanism of which may involve the ERK and p38 MAPK signaling pathways.

Oxidized low-density lipoprotein induced hepatoma-derived growth factor upregulation mediates foam cell formation of cultured rat aortic vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) are important vascular components that are essential for the regulation of vascular functions during vascular atherosclerogenesis and vascular injury. Oxidized low-density lipoprotein (oxLDL) is known to induce SMC activation and foam cell transformation. This study characterized the role of hepatoma-derived growth factor (HDGF) in oxLDL-induced foam cell formation in cultured primary rat aortic SMCs. OxLDL exposure significantly increased HDGF expression and extracellular release. It also upregulated atherogenic regulators in SMCs, including TLR4, MyD88, LOX-1, and CD36. Exogenous HDGF stimulation not only increased the expression of cognate receptor nucleolin, but also the innate immunity regulators TLR4/MyD88 and lipid metabolism regulators, including LOX-1 and CD36. Oil red O staining showed that HDGF did not initiate, but enhanced oxLDL-driven foam cell formation in SMCs. Further signaling characterization demonstrated that oxLDL evoked activation of PI3K/Akt and p38 MAPK signaling pathways, both of which were involved in the upregulation of HDGF, LOX-1, and CD36 induced by oxLDL. Gene knockdown experiments using LOX-1 targeted siRNA demonstrated that LOX-1 expression was critical for oxLDL-induced HDGF upregulation, while HDGF gene depletion completely abolished oxLDL-triggered TLR4, LOX-1, and CD36 overexpression and foam cell formation in SMCs. These findings strongly suggest that oxLDL-induced HDGF upregulation participates in subsequent LOX-1 and CD36 expression in aortic SMCs and mechanistically contributes to the formation of SMC-derived foam cells. The oxLDL/LOX-1/HDGF axis may serve as a target for anti-atherogenesis therapy.

Targeting mTOR-CCL20 Signaling May Improve Response to Docetaxel in Head and Neck Squamous Cell Carcinoma.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) usually show a dismal prognosis. It is this worthwhile to develop new, effective therapeutic regimens for these patients, such as molecular targeted therapy, which is promising as an alternative or combination treatment for HNSCC. The mammalian target of rapamycin (mTOR) pathway, which plays an important role in the carcinogenesis of HNSCC, is the most frequently activated, and is thus worthy of further investigation. In this study, two human HNSCC cell lines, FaDu and SAS, were evaluated for cell growth with trypan blue staining and tumor growth using an orthotopic xenograft model. The immunohistochemical expression of mTOR in the subcutaneous xenograft model and the inhibitory effects of docetaxel on the growth and state of activation of the PI3K/mTOR pathway were also evaluated and examined by colony formation and Western blot, respectively. Cell proliferation and migration were measured by water-soluble tetrazolium salt (WST-1) and OrisTM cell migration assay, respectively. Furthermore, the effects of rapamycin and BEZ235, a phosphatidylinositol 3-kinases (PI3K) and mTOR inhibitor in combination with docetaxel or CCL20 were evaluated in the FaDu and SAS cells. The results showed that the expression of mTOR was significantly higher in the SAS and FaDu xenograft models than in the control. Docetaxel treatment significantly suppressed HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. Additionally, when administered in a dose-dependent fashion, mTOR inhibitors inhibited the growth and migration of the HNSCC cells. This combination was synergistic with docetaxel, resulting in almost complete cell growth and migration arrest. In conclusion, docetaxel significantly inhibited HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. The synergistic and additive activity of mTOR inhibitors combined with docetaxel shows potential as a new treatment strategy for HNSCC.

MicroRNA-100 Mediates Hydrogen Peroxide-Induced Apoptosis of Human Retinal Pigment Epithelium ARPE-19 Cells.

This study investigated the regulatory role of microRNA 100 (miR-100) in hydrogen peroxide (H2O2)-induced apoptosis of human retinal pigment epithelial ARPE-19 cells. H2O2 induced oxidative cell death of cultured ARPE-19 cells was measured by cytotoxicity assay. qRT-PCR was used to quantify cytosolic and extracellular contents of miR-100. Kinase and miR-100 inhibition treatments were applied to determine the regulatory signaling pathways involved in cell death regulation. H2O2 dose-dependently reduced viability of ARPE-19 cells and simultaneously upregulated miR-100 levels in both cytosolic and extracellular compartments. Western blotting detection indicated that H2O2 elicited hyperphosphorylation of PI3K/Akt, ERK1/2, JNK, p38 MAPK, and p65 NF-κB. Further kinase inhibition experiments demonstrated that PI3K, p38 MAPK, and NF-κB activities were involved in oxidative-stress-induced miR-100 upregulation in ARPE-19 cells, while blockade of PI3K, JNK, and NF-κB signaling significantly attenuated the oxidative cell death. Intriguingly, MiR-100 antagomir treatment exerted a cytoprotective effect against the H2O2-induced oxidative cell death through attenuating the oxidation-induced AMPK hyperphosphorylation, restoring cellular mTOR and p62/SQSTM1 levels and upregulating heme oxygenase-1 expression. These findings support that miR-100 at least in part mediates H2O2-induced cell death of ARPE-19 cells and can be regarded as a preventive and therapeutic target for retinal degenerative disease.

Infusion of Human Mesenchymal Stem Cells Improves Regenerative Niche in Thioacetamide-Injured Mouse Liver.

BACKGROUND: This study investigated whether xenotransplantation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) reduces thioacetamide (TAA)-induced mouse liver fibrosis and the underlying molecular mechanism. METHODS: Recipient NOD/SCID mice were injected intraperitoneally with TAA twice weekly for 6 weeks before initial administration of WJ-MSCs. Expression of regenerative and pro-fibrogenic markers in mouse fibrotic livers were monitored post cytotherapy. A hepatic stallate cell line HSC-T6 and isolated WJ-MSCs were used for in vitro adhesion, migration and mechanistic studies. RESULTS: WJ-MSCs were isolated from human umbilical cords by an explant method and characterized by flow cytometry. A single infusion of WJ-MSCs to TAA-treated mice significantly reduced collagen deposition and ameliorated liver fibrosis after 2-week therapy. In addition to enhanced expression of hepatic regenerative factor, hepatocyte growth factor, and PCNA proliferative marker, WJ-MSC therapy significantly blunted pro-fibrogenic signals, including Smad2, RhoA, ERK. Intriguingly, reduction of plasma fibronectin (pFN) in fibrotic livers was noted in MSC-treated mice. In vitro studies further demonstrated that suspending MSCs triggered pFN degradation, soluble pFN conversely retarded adhesion of suspending MSCs onto type I collagen-coated surface, whereas pFN coating enhanced WJ-MSC migration across mimicked wound bed. Moreover, pretreatment with soluble pFN and conditioned medium from MSCs with pFN strikingly attenuated the response of HSC-T6 cells to TGF-ß1-stimulation in Smad2 phosphorylation and RhoA upregulation. CONCLUSION: These findings suggest that cytotherapy using WJ-MSCs may modulate hepatic pFN deposition for a better regenerative niche in the fibrotic livers and may constitute a useful anti-fibrogenic intervention in chronic liver diseases.

Monounsaturated oleic acid modulates autophagy flux and upregulates angiogenic factor production in human retinal pigment epithelial ARPE-19 cells.

AIMS: Dyslipidemia-associated diabetic retinopathy is featured by macular edema and retinal angiogenesis. This study investigated the in vitro lipotoxicity of free fatty acids and their modulatory roles in regulation of autophagy and angiogenic factor production in cultured human retinal pigment epithelium (RPE) ARPE-19 cells. MAIN METHODS: ARPE-19 cells were exposed to monounsaturated oleic acid (OA), saturated palmitic acid (PA), or both. Cell viability, cell cycle distribution, migration, and autophagy of the treated cells were monitored. Angiogenic factor production was determined by RT-qPCR and ELISA. KEY FINDINGS: OA, but not PA, at doses higher than 500 µM significantly induced cytostasis and lipotoxicity in ARPE-19 cells. OA exposure not only markedly enhanced autophagy flux, but also enhanced cell migration, while PA suppressed motility of RPE cells. Meanwhile, OA stimulated de novo synthesis of angiogenic factors including VEGF and bFGF in ARPE-19 cells. Mechanistically, OA treatment stimulated not only AMPK/mTOR/p70S6K signaling, but also induced hyperphosphorylation of MAPK pathway mediators, including ERK, JNK and p38 MAPK, as well as NF-κB activation. Kinase inhibition assays showed that blockade of PI3K/Akt, MAPK and NF-κB pathways prevented the OA-upregulated VEGF transcription and its peptide release. Comparatively, only NF-κB inhibition significantly suppressed bFGF peptide release from ARPE-19 cells. SIGNIFICANCE: Out findings support the OA-exhibited cytostasis, autophagy modulation and angiogenic factor production in RPE cells. This study sheds light on the interrelationship between metabolic disorder and retinopathy and provides molecular strategies for preventing and treating choroidal neovascularization in diabetic retinopathy.

Hepatoma-derived growth factor enhances osteoblastic transformation of rat aortic vascular smooth muscle cells in vitro.

AIMS: Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. MATERIALS AND METHODS: Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. KEY FINDINGS: Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SIGNIFICANCE: In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. CHEMICAL COMPOUNDS STUDIED IN THIS STUDY: HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).

Nerve growth factor induced farnesoid X receptor upregulation modulates autophagy flux and protects hepatocytes in cholestatic livers.

Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.

DNA Repair Protein Rad51 Induces Tumor Growth and Metastasis in Esophageal Squamous Cell Carcinoma via a p38/Akt-Dependent Pathway.

BACKGROUND: Rad51 is a protein which plays a vital role in DNA double-strand break repair and maintenance of telomeres. However, the underlying mechanism for its action in esophageal squamous cell carcinoma (ESCC) remains unclear. PATIENTS AND METHODS: Eighty-seven patients with ESCC were enrolled in this study. Expression of Rad51 in ESCC was determined by immunohistochemistry and correlated with clinicopathological variables by Chi square test. The role of Rad51 in patient survival was determined by Kaplan-Meier estimates. The effects of Rad51 knockdown and overexpression on esophageal cancer growth, migration, and invasion were examined using TE8, CE81T, and KYSE70 cells. The mechanisms involved were also analyzed. Nude mice models were used for assessment of tumor growth. RESULTS: Rad51 staining was predominantly observed in ESCC patients. ESCC patients with high Rad51 expression had significantly decreased survival (P < 0.001) combined with increased tumor size (P = 0.034) and lymph node metastasis (P = 0.039). Rad51 overexpression promoted, while its knockdown attenuated, esophageal cancer cell viability through cell cycle entry and migration/invasion via epithelial-mesenchymal transition. Moreover, Rad51 overexpression increased colony formation in vitro and tumor growth in vivo. In addition, high Rad51 expression increased cancer progression through the p38/Akt/Snail signaling pathway. CONCLUSIONS: This study indicates a new biological role for Rad51 in ESCC progression. Rad51 may serve as a potential prognostic biomarker and therapeutic target for ESCC patients.

Ets-1 enhances tumor migration through regulation of CCR7 expression.

Ets-1 is a prototype of the ETS protein family. Members of the ETS protein family contain a unique ETS domain. Ets-1 is associated with cancer progression and metastasis in many types of cancer. Many studies have shown a link between elevated expression of Ets-1 in cancer biopsies and poor survival. CCR7 is a chemokine that binds to specific ligand CCL21/CCL19. CCR7 expression is associated with tumor metastasis and infiltration into lymph nodes. The objective of this study was to test whether Ets-1 could regulate CCR7 expression and enhance tumor metastasis. Our data showed that CCR7 expression was downregulated in Ets-1-deficient T cells upon T-cell stimulation. Overexpression of Ets-1 increased CCR7 expression in breast cancer cell lines. In contrast, knockdown of Ets-1 reduced CCR7 expression. Ets-1 could directly bind to CCR7 promoter and mediate CCR7 expression in luciferase reporter assays and chromatin immunoprecipitation assays. Transactivation activity of Ets-1 was independent of the Pointed domain of Ets-1. Ets-1 could also enhance NF-κB and CBP transactivation of CCR7 promoter. Our results also showed that Ets-1 could modulate cancer cell transmigration by altering CCR7 expression in transwell assay and wound healing assay. Taken together, our data suggest that Ets-1 can enhance CCR7 expression and contribute to tumor cell migration. [BMB Reports 2019; 52(9): 548-553].

Butein induces apoptotic cell death of human cervical cancer cells.

Butein is a chalcone, a flavonoid that is widely biosynthesized in plants. Butein has been identified to possess varied pharmacological activity and is extractable from traditional Chinese medicinal herbs, therefore applicable for disease treatment. Recently, in vitro and in vivo studies have shown that butein may induce apoptotic cell death in various human cancer cells. In this study we investigated the apoptotic effect of butein and the underlying mechanisms in human cervical cancer cells. Two cell lines, C-33A and SiHa cells, were treated with butein at different dosages for different durations. The effect of butein on cell viability was assessed by MTT assay, which revealed that butein exerted cytotoxicity in both cervical cancer cells in a dose- and time-dependent fashion. Apoptotic pathway-related factors in the butein-treated cervical cancer cells were then examined. JC-1 flow cytometry, cytochrome c assay, and caspase activity assays demonstrated that butein disturbed mitochondrial transmembrane potential, and increased cytosolic cytochrome c levels and caspase activities in both cervical cancer cells. Western blot analysis revealed that butein downregulated anti-apoptotic protein Bcl-xL and led to proteolytic cleavage of poly (ADP-ribose) polymerase. In addition, butein decreased expressions of the inhibitor of apoptosis (IAP) proteins, including X-linked IAP, survivin, and cellular IAP-1. The findings of this study suggest that butein can decrease cervical cancer cell viability via a pro-apoptotic effect, which involves inhibition of the IAP proteins and activation of both extrinsic and intrinsic pro-apoptotic pathways. Therefore, butein may be applicable for cervical cancer treatment.

BMP-2 restoration aids in recovery from liver fibrosis by attenuating TGF-ß1 signaling.

Transforming growth factor-ß (TGF-ß) plays a central role in hepatic fibrogenesis. This study investigated the function and mechanism of bone morphogenetic protein-2 (BMP-2) in regulation of hepatic fibrogenesis. BMP-2 expression in fibrotic liver was measured in human tissue microarray and mouse models of liver fibrosis induced by bile duct ligation surgery or carbon tetrachloride administration. Adenovirus-mediated BMP-2 gene delivery was used to test the prophylactic effect on liver fibrosis. Primary hepatic stellate cells (HSC), HSC-T6 and clone-9 cell lines were used to study the interplay between BMP-2 and TGF-ß1. Hepatic BMP-2 was localized in parenchymal hepatocytes and activated HSCs and significantly decreased in human and mouse fibrotic livers, showing an opposite pattern of hepatic TGF-ß1 contents. BMP-2 gene delivery alleviated the elevations of serum hepatic enzymes, cholangiocyte marker CK19, HSC activation markers, and liver fibrosis in both models. Mechanistically, exogenous TGF-ß1 dose dependently reduced BMP-2 expression, whereas BMP-2 significantly suppressed expression of TGF-ß and its cognate type I and II receptor peptides, as well as the induced Smad3 phosphorylation levels in primary mouse HSCs. Aside from its suppressive effects on cell proliferation and migration, BMP-2 treatment prominently attenuated the TGF-ß1-stimulated α-SMA and fibronectin expression, and reversed the TGF-ß1-modulated epithelial-to-mesenchymal transition marker expression in mouse HSCs. The mutual regulation between BMP-2 and TGF-ß1 signaling axes may constitute the anti-fibrogenic mechanism of BMP-2 in the pathogenesis of liver fibrosis. BMP-2 may potentially serve as a novel therapeutic target for treatment of liver fibrosis.

Dexamethasone restores transforming growth factor-ß activated kinase 1 expression and phagocytosis activity of Kupffer cells in cholestatic liver injury.

The role of transforming growth factor-ß activated kinase 1 (TAK1) in modulating the function of Kupffer cells (KCs) within cholestatic livers remains unclear. This study examined the immunopharmacological action of dexamethasone (DEX) in modulating hepatic TAK1 expression and related signaling activity in a rat model of bile duct ligation-mimicked obstructive jaundice. The in vitro effects of DEX on porcine biliary extract (PBE)-modulated gene expression and phagocytosis of KCs were examined using a rat alveolar macrophage cell line (NR8383 cells). Although DEX therapy did not restore the downregulated TAK1 expression and phosphorylation, it significantly attenuated the upregulation of high-mobility group box 1 expression and caspase-3 activation in whole liver extracts of cholestatic rats, possibly via enhancing extracellular signal-regulated kinase-mediated signaling. Dual immunofluorescence staining of cholestatic livers and western detection on primary KCs isolated from cholestatic livers identified that DEX treatment indeed increased both the expression and phosphorylation levels of TAK1 in the KCs of cholestatic livers. In vitro studies using alveolar NR8383 macrophages with KC-characteristic gene expression further demonstrated that DEX not only repressed the pro-inflammatory cytokine production including with respect to interleukin (IL)-1ß and IL-6, but also enhanced gene expression of TAK1 and a phagocytic marker, natural-resistance-associated macrophage protein 1, under PBE-mimicked cholestatic conditions. However, WST-1 assay showed that DEX did not protect NR8383 macrophages against the PBE-induced cytotoxicity. Immunofluorescence visualization of cellular F-actin by phalloidin suggested that DEX sustained the PBE-induced phagocytosis morphology of NR8383 macrophages. In conclusion, DEX treatment may pharmacologically restore the expression and activity of TAK1 in KCs, and sustain the phagocytic phenotype of KCs in cholestatic livers.

Cyclic Mechanical Stretch Up-regulates Hepatoma-Derived Growth Factor Expression in Cultured Rat Aortic Smooth Muscle Cells.

Hepatoma-derived growth factor (HDGF) is a potent mitogen for vascular smooth muscle cells (SMCs) during embryogenesis and injury repair of vessel walls. Whether mechanical stimuli modulate HDGF expression remains unknown. This study aimed at investigating whether cyclic mechanical stretch plays a regulatory role in HDGF expression and regenerative cytokine production in aortic SMCs. A SMC cell line was grown on a silicone-based elastomer chamber with extracellular matrix coatings (either type I collagen or fibronectin) and received cyclic and uni-axial mechanical stretches with 10% deformation at frequency 1 Hz. Morphological observation showed that fibronectin coating provided better cell adhesion and spreading and that consecutive 6 hours of cyclic mechanical stretch remarkably induced reorientation and realignment of SMCs. Western blotting detection demonstrated that continuous mechanical stimuli elicited up-regulation of HDGF and PCNA, a cell proliferative marker. Signal kinetic profiling study indicated that cyclic mechanical stretch induced signaling activity in RhoA/ROCK and PI3K/Akt cascades. Kinase inhibition study further showed that blockade of PI3K activity suppressed the stretch-induced TNF-a, whereas RhoA/ROCK inhibition significantly blunted the IL-6 production and HDGF over-expression. Moreover, siRNA-mediated HDGF gene silencing significantly suppressed constitutive expression of IL-6, but not TNF-α, in SMCs. These findings support the role of HDGF in maintaining vascular expression of IL-6, which has been regarded a crucial regenerative factor for acute vascular injury. In conclusion, cyclic mechanical stretch may maintain constitutive expression of HDGF in vascular walls and be regarded an important biophysical regulator in vascular regeneration.