Ligustilide, a novel SIRT1 agonist, alleviates lipopolysaccharide-induced acute lung injury through deacetylation of NICD.

Development and progression of sepsis-induced acute lung injury (ALI) involve apoptosis and oxidative stress in lung epithelial cells. Ligustilide (LIG) is one of the main bioactive constituents derived from the Angelica sinensis. As a novel SIRT1 agonist, LIG owns powerful anti-inflammatory and antioxidative properties, exerting remarkable therapeutic effects on cancers, neurological disorders, and diabetes mellitus. However, whether LIG could protect against lipopolysaccharide (LPS)-induced ALI by activating SIRT1 remains unclear. Mice underwent intratracheal LPS injection to mimic sepsis-induced ALI while MLE-12 cells were treated with LPS for 6 h to establish an in vitro ALI model. At the same time, mice or MLE-12 cells were treated with different doses of LIG to access its pharmacological effect. The results demonstrated that LIG pretreatment could improve LPS-induced pulmonary dysfunction and pathological injury, apart from increasing 7-day survival rate. In addition, LIG pretreatment also decreased inflammation, oxidative stress and apoptosis during LPS-induced ALI. Mechanically, LPS stimulation decreased the expression and activity of SIRT1 but increased the expression of Notch1 and NICD. And LIG could also enhance the interaction between SIRT1 and NICD, thus deacetylating NICD. In vitro experiments also unveiled that EX-527, a selective SIRT1 inhibitor, could abolish LIG-elicited protection in LPS-treated MLE-12 cells. And in SIRT1 knockout mice with ALI, LIG pretreatment also lost its effects on inflammation, apoptosis, and oxidative stress during ALI.

Tamoxifen Downregulates the Expression of Notch1 and DLL1 Genes in MKN-45 Gastric Cancer Cells.

PURPOSE: Gastric cancer is one of the most prevalent cancers worldwide and the second most common cause for cancer associated mortality. Anti-tumor effects of tamoxifen in breast cancer are well-established. However, no study has so far investigated the effects of tamoxifen on gene expression of Notch1 and DLL1 in gastric cancer cell line. The present study was conducted to explore the effects of tamoxifen, as a repurposed drug, on gene expression of Notch1 and DLL1 in MKN-45, a gastric cancer cell line. METHODS: MKN-45 cells were cultured in DMEM/F12 medium containing 10% FBS. Cytotoxic effects of tamoxifen on these cells at various concentrations were evaluated by trypan blue exclusion assay. For gene expression analysis, the cells were first incubated with 100 µM tamoxifen followed by total RNA extraction from treated and control cells. Then, cDNA was synthesized. Quantitative real-time PCR using specific primers for Notch1 and DLL1 was performed to assess the effect of tamoxifen on the transcript of them. RESULTS: Treatment with tamoxifen decreased viability of MKN-45 cells in a dose-dependent manner. CC50 was estimated to be around 200 µM. Also, tamoxifen at the dose of 100 µM could significantly downregulate mRNA levels of both Notch1 and DLL1 genes as compared with untreated cells by 24% and 92%, respectively. CONCLUSION: Based on these results, tamoxifen interferes with Notch signaling pathway through downregulating the expression of Notch1 and DLL1 genes and this could be regarded as a mechanism for its anti-cancer effects in this malignant disease.

Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions.

CONTEXT: NOTCH signaling is activated in endometriotic lesions, but the exact mechanisms remains unclear. IL-6, which is increased in the peritoneal fluid of women with endometriosis, induces NOTCH1 through E-proteins including E2A and HEB in cancer. OBJECTIVE: To study the role of E-proteins in inducing NOTCH1 expression under the regulation of IL-6 in endometriosis. SETTING AND DESIGN: The expression of E-proteins and NOTCH1 was first investigated in endometrium of women with endometriosis and the baboon model of endometriosis. Regulation of E-proteins and NOTCH1 expression was examined after IL-6 stimulation and siRNA mediated inhibition of E2A or/and HEB in human endometriotic epithelial cells (12Z) in vitro, and subsequently following IL-6 treatment in the mouse model of endometriosis in vivo. RESULTS: E2A, HEB, and NOTCH1 were significantly upregulated in glandular epithelium (GE) of ectopic endometrium compared to eutopic endometrium in both women and the baboon model. IL-6 treatment upregulated the expression of NOTCH1 together with E2A and HEB in 12Z cells. Small interfering RNA inhibition of E2A and HEB or HEB alone decreased NOTCH1 expression. Binding efficiency of both E2A and HEB was significantly higher at the binding sites on the human NOTCH1 promoter after IL-6 treatment. Finally, IL-6 treatment resulted in a significantly increased number of endometriotic lesions along with increased expression of E2A, HEB, and NOTCH1 in GE of the lesions compared with the vehicle group in an endometriosis mouse model. CONCLUSIONS: IL-6 induced NOTCH1 expression is mediated by E-proteins in the ectopic GE cells, which may promote endometriotic lesion development.

Globotriaosylceramide-induced reduction of KCa1.1 channel activity and activation of the Notch1 signaling pathway in skin fibroblasts of male Fabry patients with pain.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disorder that leads to cellular globotriaosylceramide (Gb3) accumulation due to mutations in the gene encoding α-galactosidase A. Trigger-induced acral burning pain is an early FD symptom of unknown pathophysiology. We aimed at investigating the potential role of skin fibroblasts in nociceptor sensitization. PATIENTS AND METHODS: We enrolled 40 adult FD patients and ten healthy controls, who underwent a 6-mm skin punch biopsy at the lower leg. Dermal fibroblasts were cultivated and analyzed for Gb3 load. Fibroblast electrical activity was assessed using patch-clamp analysis at baseline and upon incubation with agalsidase-α for 24 h. We investigated gene expression of CC motif chemokine ligand 2 (CCL2), Ca2+activated K+-channel 1.1 (KCa1.1), interferone-γ (IFN-γ), transforming growth factor-ß1 (TGF-ß1), and transmembrane receptor notch homolog 1 (Notch1) using quantitative real-time-PCR, and protein levels of KCa1.1 by ELISA. Gene expression was determined at baseline and after fibroblast stimulation with tumor necrosis factor-α (TNF), modeling inflammation as a common pain trigger in FD. RESULTS: Total Gb3 load was higher in FD fibroblasts than in control fibroblasts (p < .01). Upon increase of intracellular Ca2+ concentrations, we detected differential electrical activity of KCa1.1 in fibroblasts obtained from patients with FD. Gene expression (p < .05) and protein levels of KCa1.1 (p < .05) were higher in fibroblasts from FD patients compared to control fibroblasts, whereas electric channel activity was lower in FD fibroblasts. After incubation with agalsidase-α, we observed an over-proportionate increase of KCa1.1 activity in FD fibroblasts reaching 7-fold the currents of control cells (p < .01). Gene expression studies revealed higher mRNA levels of CCL2, INF-γ, and Notch1 in FD fibroblasts compared to controls at baseline and after TNF incubation (p < .05 each), while TGF-ß1 was higher in FD fibroblasts only after incubation with TNF (p < .05). CONCLUSIONS: Gb3 deposition in skin fibroblasts may impair KCa1.1 activity and activate the Notch1 signaling pathway. The resulting increase in pro-inflammatory mediator expression may contribute to cutaneous nociceptor sensitization as a potential mechanism of FD-associated pain.

Intravenous Injection of miR-34a Inhibitor Alleviates Diabetes Mellitus-Induced Vascular Endothelial Dysfunction by Targeting NOTCH1.

BACKGROUND: miR-34a is a multifunctional post-translational modulator, which is involved in several diabetes-related complications. However, miR-34a remains to be fully elucidated in the diabetic endothelium from rats. In this study, the role of miR-34a/NOTCH1 signaling in the progression of hyperglycemia-vascular endothelial dysfunction was investigated. METHODS: In intravenous injection of miR-34a mimics and inhibitors in streptozotocin (STZ)-induced diabetic rats, the biomarkers of endothelial dysfunction was measured. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. The mRNA and protein levels were assayed by qRT-PCR and western blotting, respectively. Immunohistochemical staining was performed to measure NOTCH1 expression in the diabetic endothelium. RESULTS: miR-34a was significantly up-regulated, and NOTCH1 down-regulated, in the thoracic aorta from STZ-induced diabetic rats compared with control group. As compared to model group, the mRNA of NOTCH1 was significantly decreased or increased by miR-34a mimics or inhibitors ex vivo, respectively. Bioinformatics methods further demonstrated that NOTCH1 was a potential target of miR-34a, which was confirmed by dual-luciferase reporter assay. Moreover, both serum ET and NO were significantly increased in diabetic rats as compared to control group. miR-34a inhibitors ex vivo treatment resulted in significant down-regulation ofserum ET and NO levels in diabetic rats as compared to model group. CONCLUSION: These results provide evidence to support the use of miR-34a inhibitors as a therapeutic approach attenuating hyperglycemia-induced vascular endothelial dysfunction.

Endogenous Notch Signaling in Adult Kidneys Maintains Segment-Specific Epithelial Cell Types of the Distal Tubules and Collecting Ducts to Ensure Water Homeostasis.

BACKGROUND: Notch signaling is required during kidney development for nephron formation and principal cell fate selection within the collecting ducts. Whether Notch signaling is required in the adult kidney to maintain epithelial diversity, or whether its loss can trigger principal cell transdifferentiation (which could explain acquired diabetes insipidus in patients receiving lithium) is unclear. METHODS: To investigate whether loss of Notch signaling can trigger principal cells to lose their identity, we genetically inactivated Notch1 and Notch2, inactivated the Notch signaling target Hes1, or induced expression of a Notch signaling inhibitor in all of the nephron segments and collecting ducts in mice after kidney development. We examined renal function and cell type composition of control littermates and mice with conditional Notch signaling inactivation in adult renal epithelia. In addition, we traced the fate of genetically labeled adult kidney collecting duct principal cells after Hes1 inactivation or lithium treatment. RESULTS: Notch signaling was required for maintenance of Aqp2-expressing cells in distal nephron and collecting duct segments in adult kidneys. Fate tracing revealed mature principal cells in the inner stripe of the outer medulla converted to intercalated cells after genetic inactivation of Hes1 and, to a lesser extent, lithium treatment. Hes1 ensured repression of Foxi1 to prevent the intercalated cell program from turning on in mature Aqp2+ cell types. CONCLUSIONS: Notch signaling viaHes1 regulates maintenance of mature renal epithelial cell states. Loss of Notch signaling or use of lithium can trigger transdifferentiation of mature principal cells to intercalated cells in adult kidneys.

High-Dose Tanshinone IIA Suppresses Migration and Proliferation While Promoting Apoptosis of Astrocytoma Cells Via Notch-1 Pathway.

Malignant astrocytoma is the most common malignant tumor with strong invasion in the central nervous system. Tanshinone IIA is an effective compound to suppress cell proliferation and promote cell apoptosis. However, there is little research about the role of tanshinone IIA in the treatment of astrocytoma. This study aimed to investigate the effect of tanshinone IIA on migration, proliferation and apoptosis of astrocytoma cells. The efficacy of tanshinone IIA on migration, proliferation and apoptosis of astrocytoma cells were evaluated by flow cytometry and the assays of plate clone formation, CCK-8, wound healing and transwell migration. The protein molecule and signaling pathway were detected by western blot. High-dose tanshinone IIA suppressed migration and proliferation of astrocytoma cells while promoting apoptosis of astrocytoma cells. The western blot results showed that there were high Notch-1 protein expression and low c-Myc, MMP-9 and Bcl-2 activation in the high-dose tanshinone IIA group compared with the control group. High-dose tanshinone IIA suppresses astrocytoma cell proliferation, migration while promoting apoptosis through Notch-1 pathway. Tanshinone IIA may be used to develop new drugs for the treatment of astrocytoma.

MiR-92a Inhibits the Progress of Osteosarcoma Cells and Increases the Cisplatin Sensitivity by Targeting Notch1.

BACKGROUND: MicroRNAs (miRs) have been implicated in the development and progression of osteosarcoma. Here, we aimed to illustrate the important role of miR-92a on the regulation of OS development which may help to establish a novel strategy for OS diagnosis and treatment. MATERIALS AND METHODS: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle and apoptosis were assessed by flow cytometry with PI and PI/Annexin-V stain, respectively. The expression of proteins was examined by western blot. qPCR was used to detect the expression of RNA. Cell migration was assayed with transwell assay. RESULTS: MiR-92a inhibited the proliferation and the migration of OS in vitro and reduced the volume of the tumour in vivo. Further, miR-92a enhanced cisplatin sensitivity of OS. MiR-92a directly targeted Notch1. CONCLUSION: Together, our results indicate that miR-92a inhibited cell growth, migration, and enhanced cisplatin sensitivity of OS cell by targeting Notch1.

MiR-199-3p replacement affects E-cadherin expression through Notch1 targeting in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, due to a high recurrence rate after curative treatments and a drug resistance phenotype. In this scenario, the identification of innovative and effective therapeutic strategies is an unmet clinical need. The safety and efficacy of microRNA (miRNA) mediated approaches in preclinical models and clinical trials have been widely described in cancer. MicroRNA-199a downregulation is a common feature of HCC where its reduced expression contributes to mTOR and c-Met pathways activation. Notch1 activation is also a common event in HCC, influencing epithelial-to-mesenchymal transition, tumor invasion and recurrence at least in part through E-cadherin regulation. Here we identified a negative correlation between miR-199a-3p and Notch1 or E-cadherin protein levels in HCC patients and demonstrated that miR-199a-3p regulates E-cadherin expression through Notch1 direct targeting in in vitro models. Moreover, we showed that a strong correlation exists between miR-199a-5p and miR-199a-3p in HCC specimens and that miR-199a-5p contributes to E-cadherin regulation as well, underlying the complex network of interaction carried out by miR-199a and its influence on tumor aggressiveness. In conclusion, our findings suggest the restoration of miR-199a-3p physiologic levels as a possible therapeutic strategy for the treatment of HCC.

Extrinsic Calcitonin Gene-Related Peptide Inhibits Hyperoxia-Induced Alveolar Epithelial Type II Cells Apoptosis, Oxidative Stress, and Reactive Oxygen Species (ROS) Production by Enhancing Notch 1 and Homocysteine-Induced Endoplasmic Reticulum Protein (HERP) Expression.

BACKGROUND Lung alveolar epithelial type II cells (AEC II) are the most important stem cells in lung tissues, which are critical for wound repair of bronchopulmonary dysplasia (BPD). This study investigated the effects of calcitonin gene-related peptide (CGRP) on AEC II cells exposed to hyperoxia. MATERIAL AND METHODS Neonatal rat AEC II cells were isolated and identified by detecting surfactant protein C (SP-C). Three small interfering RNAs targeting Notch 1 were synthesized and transfected into AEC II. A hyperoxia-exposed AEC II cell injury model was established and was divided into 8 groups. MDA levels and SOD activity were examined using lipid peroxidation assay kits. Apoptosis and reactive oxygen species (ROS) production were evaluated using flow cytometry. Notch 1 mRNA expression was examined using RT-PCR. Homocysteine-induced endoplasmic reticulum protein (HERP) was examined using Western blot analysis. RESULTS CGRP treatment significantly enhanced MDA levels and decreased SOD activity compared to hyperoxia-treated AEC II cells (P<0.05). CGRP treatment significantly inhibited hyperoxia-induced AEC II cell apoptosis, and significantly suppressed hyperoxia-induced ROS production compared to hyperoxia-treated AEC II cells (P<0.05) either undergoing g secretase inhibitor or Notch RNA interference. CGRP significantly triggered Notch 1 mRNA expression and significantly enhanced HERP expression compared to hyperoxia-treated AEC II cells (P<0.05) either undergoing g secretase inhibitor or Notch RNA interference. CONCLUSIONS In AEC II cells, extrinsic peptide CGRP suppressed hyperoxia-induced apoptosis, oxidative stress, and ROS production, which may be triggered by Notch 1 and HERP signaling pathway.

Developmental changes in Notch1 and NLE1 expression in a genetic model of absence epilepsy.

Childhood absence epilepsy (CAE) is an epilepsy syndrome with seizures occurring in the early childhood, highlighting that seizures susceptibility in CAE is dependent on brain development. The Notch 1 signalling pathway is important in brain development, yet the role of the Notch1 signalling pathway in CAE remains elusive. We here explored Notch1 and its modulator notchless homologue 1 (NLE1) expression in WAG/Rij and control rats using immunohistochemistry. Functional Notch 1 effects were assessed in WAG/Rij rats in vivo. WAG/Rij rats lack the developmental increase in cortical Notch1 and NLE 1 mRNA expression seen in controls, and Notch 1 and NLE1 mRNA and protein expression were lower in somatosensory cortices of WAG/Rij rats when compared to controls. This coincided with an overall decreased cortical GFAP expression in the early development in WAG/Rij rats. These effects were region-specific as they were not observed in thalamic tissues. Neuron-to-glia ratio as a marker of the impact of Notch signalling on differentiation was higher in layer 4 of somatosensory cortex of WAG/Rij rats. Acute application of Notch 1 agonist Jagged 1 suppressed, whereas DAPT, a Notch antagonist, facilitated spike and wave discharges (SWDs) in WAG/Rij rats. These findings point to Notch1 as an important signalling pathway in CAE which likely shapes architectural organization of the somatosensory cortex, a region critically involved in developmental epileptogenesis in CAE. More immediate effects of Notch 1 signalling are seen on in vivo SWDs in CAE, pointing to the Notch 1 pathway as a possible treatment target in CAE.

Protective effect of melatonin on soluble Aß1-42-induced memory impairment, astrogliosis, and synaptic dysfunction via the Musashi1/Notch1/Hes1 signaling pathway in the rat hippocampus.

BACKGROUND: Amyloid-beta (Aß) plays a key role in Alzheimer's disease (AD) pathogenesis, and soluble Aß oligomers are more cytotoxic than Aß fibrils. Recent evidence suggests that Notch signaling is affected by AD and other brain diseases. Melatonin exerts beneficial effects on many aspects of AD and may protect against myocardial ischemia via Notch1 signaling regulation. Therefore, we hypothesized that the Notch1 signaling pathway is involved in the neuroprotective role of melatonin against soluble Aß1-42. METHODS: An AD rat model was established via repeated intracerebroventricular administration of soluble Aß1-42. Melatonin treatment was administered 24 hours prior to Aß1-42 administration via an intraperitoneal injection. The effects of melatonin on spatial learning and memory, synaptic plasticity, and astrogliosis were investigated. The expression of several Notch1 signaling components, including Notch1, the Notch1 intracellular domain (NICD), Hairy and enhancer of split 1 (Hes1, a downstream effector of Notch), and Musashi1 (a positive regulator of Notch), were examined using immunohistochemistry, western blotting, and quantitative real-time PCR. In vitro studies were conducted to determine whether the melatonin-mediated protection against Aß1-42 was inhibited by DAPT, an inhibitor of Notch signaling. RESULTS: Melatonin improved the Aß1-42-induced impairment in spatial learning and memory, attenuated synaptic dysfunction, and reduced astrogliosis. Melatonin also ameliorated the effects of Aß1-42 on Notch1, NICD, Hes1, and Musashi1. The in vitro studies demonstrated that DAPT effectively blocked the neuroprotective effect of melatonin against Aß1-42. CONCLUSIONS: These findings suggest that melatonin may improve the soluble Aß1-42-induced impairment of spatial learning and memory, synaptic plasticity, and astrogliosis via the Musashi1/Notch1/Hes1 signaling pathway.

TQ inhibits hepatocellular carcinoma growth in vitro and in vivo via repression of Notch signaling.

Thymoquinone (TQ) has been reported to possess anti-tumor activity in various types of cancer. However, its effects and molecular mechanism of action in hepatocellular carcinoma (HCC) are still not completely understood. We observed that TQ inhibited tumor cell growth in vitro, where treatment with TQ arrested the cell cycle in G1 by upregulating p21 and downregulating cyclinD1 and CDK2 expression; moreover, TQ induced apoptosis by decreasing expression of Bcl-2 and increasing expression of Bax. Simultaneously, TQ demonstrated a suppressive impact on the Notch pathway, where overexpression of NICD1 reversed the inhibitory effect of TQ on cell proliferation, thereby attenuating the repressive effects of TQ on the Notch pathway, cyclinD1, CDK2 and Bcl-2, and also diminishing upregulation of p21 and Bax. In a xenograft model, TQ inhibited HCC growth in nude mice; this inhibitory effect in vivo, as well as of HCC cell growth in vitro, was associated with a discernible decline in NICD1 and Bcl-2 levels and a dramatic rise in p21 expression. In conclusion, TQ inhibits HCC cell growth by inducing cell cycle arrest and apoptosis, achieving these effects by repression of the Notch signaling pathway, suggesting that TQ represents a potential preventive or therapeutic agent in HCC patients.

Notch1 targeting siRNA delivery nanoparticles for rheumatoid arthritis therapy.

Notch pathway plays a pivotal role in synoviocytes involved in progression of rheumatoid arthritis (RA). Herein, we designed the Notch1 targeting siRNA delivery nanoparticles (siRNA-NPs) in order to confirm the anti-inflammatory effect in collagen-induced arthritis (CIA) model. The siRNA-NPs were successfully produced by encapsulating polymerized siRNA (poly-siRNA) into thiolated glycol chitosan (tGC) nanoparticles in aqueous condition. The in vitro Notch1 inhibition of siRNA-NPs in murine macrophage cell (RAW 264.7) was confirmed using confocal microscopy and real time PCR. Fluorescently labeled siRNA-NPs were successfully transfected in RAW 264.7 and modulated the expression of Notch1 in mRNA level. For in vivo study, siRNA-NPs exhibited the higher targeting efficiency in the arthritic joins of CIA mice, confirmed by the near-infrared fluorescence (NIRF) imaging. Furthermore, inhibition of Notch1 with siRNA-NPs resulted in retarded progression of inflammation, bone erosion, and cartilage damage in CIA mice. Novel Notch1 targeting siRNA delivery system of siRNA-NPs showed effective RA treatment by suppressing Notch1 signaling pathway without undesirable severe toxicity. Thus, Notch1 inhibiting siRNA-NPs demonstrated the great potential in RA therapeutics that was hard to be achieved using conventional drugs.

Lyso-Gb3 activates Notch1 in human podocytes.

Podocyte injury is an early feature of Fabry nephropathy, but the molecular mechanisms of podocyte injury are poorly understood. Lyso-Gb3 accumulates in serum in Fabry disease and increases extracellular matrix synthesis in podocytes. We explored the contribution of Notch1 signaling, a mediator of podocyte injury, to lyso-Gb3-elicited responses in cultured human podocytes. At clinically relevant concentrations, lyso-Gb3 activates podocyte Notch1 signaling, resulting in increased active Notch1 and HES1, a canonical Notch transcriptional target. A γ-secretase inhibitor or specific Notch1 small interfering RNA (siRNA) inhibited HES1 upregulation in response to lyso-Gb3. Notch1 siRNA or γ-secretase inhibition also prevented the lyso-Gb3-induced upregulation of Notch1, Notch ligand Jagged1 and chemokine (MCP1, RANTES) expression. Notch siRNA prevented the activation of nuclear factor kappa B (NFκB), and NFκB activation contributed to Notch1-mediated inflammatory responses as the NFκB inhibitor, parthenolide, prevented lyso-Gb3-induced chemokine upregulation. Notch1 also mediates fibrogenic responses in podocytes as Notch siRNA prevented lyso-Gb3 upregulation of fibronectin mRNA. Supporting the clinical relevance of cell culture findings, active Notch1, Jagged1 and HES1 were observed in Fabry kidney biopsies. Lyso-Gb3 elicited similar responses in mouse kidney. In conclusion, lyso-Gb3 promotes Notch1-mediated inflammatory and fibrogenic responses in podocytes that may contribute to Fabry nephropathy.

Overexpression of miR-34c inhibits high glucose-induced apoptosis in podocytes by targeting Notch signaling pathways.

Recent findings have shown that microRNAs play critical roles in the pathogenesis of diabetic nephropathy. miR-34c has been found to inhibit fibrosis and the epithelial-mesenchymal transition of kidney cells. However, the role of miR-34c in diabetic nephropathy has not been well studied. The current study was designed to investigate the role and potential underlying mechanism of miR-34c in regulating diabetic nephropathy. After treating podocytes with high glucose (HG) in vitro, we found that miR-34c was downregulated and that overexpression of miR-34c inhibited HG-induced podocyte apoptosis. The direct interaction between miR-34c and the 3'-untranslated region (UTR) of Notch1 and Jagged1 was validated by dual-luciferase reporter assay. Moreover, Notch1 and Jagged1 as putative targets of miR-34c were downregulated by miR-34c overexpression in HG-treated podocytes. Overexpression of miR-34c inhibited HG-induced Notch signaling pathway activation, as indicated by decreased expression of the Notch intracellular domain (NICD) and downstream genes including Hes1 and Hey1. Furthermore, miR-34c overexpression increased the expression of the anti-apoptotic gene Bcl-2, and decreased the expression of the pro-apoptotic protein Bax and cleaved Caspase-3. Additionally, the phosphorylation of p53 was also downregulated by miR-34c overexpression. Taken together, our findings suggest that miR-34c overexpression inhibits the Notch signaling pathway by targeting Notch1 and Jaggged1 in HG-treated podocytes, representing a novel and potential therapeutic target for the treatment of diabetic nephropathy.

Inhibition of gamma-secretase activity impedes uterine serous carcinoma growth in a human xenograft model.

OBJECTIVE: Uterine serous carcinoma (USC) represents an aggressive subtype of endometrial cancer. We sought to understand Notch pathway activity in USC and determine if pathway inhibition has anti-tumor activity. METHODS: Patient USC tissue blocks were obtained and used to correlate clinical outcomes with Notch1 expression. Three established USC cell lines were treated with gamma-secretase inhibitor (GSI) in vitro. Mice harboring cell line derived or patient derived USC xenografts (PDXs) were treated with vehicle, GSI, paclitaxel and carboplatin (P/C), or combination GSI and P/C. Levels of cleaved Notch1 protein and Hes1 mRNA were determined in GSI treated samples. Statistical analysis was performed using the Wilcoxon rank sum and Kaplan-Meier methods. RESULTS: High nuclear Notch1 protein expression was observed in 58% of USC samples with no correlation with overall survival. GSI induced dose-dependent reductions in cell number and decreased levels of cleaved Notch1 protein and Hes1 mRNA in vitro. Treatment of mice with GSI led to decreased Hes1 mRNA expression in USC xenografts. In addition, GSI impeded tumor growth of cell line xenografts as well as UT1 USC PDXs. When GSI and P/C were combined, synergistic anti-tumor activity was observed in UT1 xenografts. CONCLUSIONS: Notch1 is expressed in a large subset of USC. GSI-mediated Notch pathway inhibition led to both reduced cell numbers in vitro and decreased tumor growth of USC some xenograft models. When combined with conventional chemotherapy, GSI augmented anti-tumor activity in one USC PDX line suggesting that targeting of the Notch signaling pathway is a potential therapeutic strategy for future investigation.

Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFßR1.

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-ß1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-ß1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-ß1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-ß1 signaling pathway, including the TGF-ß receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-ß receptor type 1 and the response to TGF-ß1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-ß1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

New promising molecular targets in head and neck squamous cell carcinoma.

PURPOSE OF REVIEW: Despite advances in multimodality therapy, the overall 5-year survival rate is 40-50% in patients with head and neck squamous cell carcinoma (HNSCC) and current multimodality approaches impart significant toxicities. This review highlights promising targets with the potential to improve clinical outcomes in HNSCC. RECENT FINDINGS: In addition to mutagenic exposure to tobacco and alcohol as risk factors, recent studies have shown that human papillomavirus is one of the main causes of HNSCC and as such is being investigated as a therapeutic target. Furthermore, recent data generated from whole exome sequencing of HNSCC, new insights into the biology of DNA damage repair, and increased understanding of tumor hypoxia responses are pointing to new therapeutic possibilities for treating HNSCC. SUMMARY: HNSCC is a heterogeneous disease. Improved treatment will require a rapid translation of basic science research, and the simultaneous development of novel therapeutics and corresponding biomarkers to guide their application.

Intraperitoneal injection of JNK-specific inhibitor SP600125 inhibits the expression of presenilin-1 and Notch signaling in mouse brain without induction of apoptosis.

Presenilin-1 (PS1) is a multifunctional protein involved in many cellular functions including the processing of type 1 membrane proteins such as ß-amyloid precursor protein (APP) and Notch 1 receptor. PS1 acts as the catalytic subunit of the γ-secretase complex, and participates in Notch 1 processing to release Notch intracellular domain (NICD) in the cytoplasm. NICD subsequently migrates to the nucleus and causes Notch signaling by increasing the expression of the Hes1 gene. We have previously shown that inhibition of basal activity of c-jun-NH2-terminal kinase (JNK) with JNK-specific inhibitor SP600125 represses the expression of PS1 and γ-secretase activity by increasing p53 level in SK-N-SH cell line in vitro (Lee and Das, 2008, 2010). However, it is largely unknown whether PS1 can be effectively suppressed in vivo in adult mouse brains. In this report we showed that intraperitoneal (i.p) injection of JNK-specific inhibitor SP600125 decreased p-JNK level, and reduced PS1 expression by increasing p53 level in adult mouse brains. We also showed that suppression of PS1 expression by SP600125 reduced γ-secretase activity which decreased Notch 1 processing to reduce NICD in mouse brains. Furthermore, inhibition of Notch 1 processing by SP600125 decreased Notch 1 signaling by reducing the expression of the NICD target Hes1 gene in mouse brains without induction of apoptosis. These results provide insights for further study on PS1-mediated reduction of Notch 1 and APP processing for the treatment of Alzheimer's disease.