S-phenylglycine T-butyl Ester Research Articles

Signaling pathway inhibitors target breast cancer stem cells in triple-negative breast cancer.

The present study aimed to investigate the efficacy of five signaling pathway inhibitors, N-[N-(3,5‑difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, vismodegib, salinomycin, ruxolitinib and stattic, as novel therapeutic agents that target breast cancer stem cells (BCSCs) in triple-negative breast cancer (TNBC). The invitro anti-proliferative, anti-invasive, pro-apoptotic and inhibitory effects on BCSC self-renewal of these signaling pathway inhibitors on the TNBC stem cell line HCC38 were examined by MTT assays, Matrigel invasion assays, flow cytometry and suspension mammosphere assays, respectively. For the invivo study, another TNBC stem cell line, HCC1806, pretreated with these signaling pathway inhibitors, was inoculated into female nonobese diabetic/severe combined immunodeficient mice, and the tumor volumes were measured following tumor formation. Treatment of HCC38 cells with each signaling pathway inhibitor significantly decreased TNBC cell proliferation, cell invasion and mammosphere formation while inducing cell apoptosis by inhibiting the protein expression or phosphorylation of downstream signaling molecules. In the xenograft mouse models, tumor formation and growth of HCC1806 cells pretreated with each signaling pathway inhibitor were effectively suppressed. Treatment with these signaling pathway inhibitors may provide a novel therapeutic strategy against TNBC by targeting BCSCs, thus providing promising insight for clinical applications in patients with TNBC.

Aberrant activation of Notch-1 signaling inhibits podocyte restoration after islet transplantation in a rat model of diabetic nephropathy

Signaling abnormalities play important roles during podocyte injury and have been indicated as crucial events for triggering many glomerular diseases. There is emerging evidence demonstrating significant improvements in preventing renal injury and restoring podocytes after islet transplantation. However, whether signaling abnormalities affect the therapeutic efficacy of islet transplantation remain unclear. This study was established to investigate the impact of Notch-1 signaling activation on renal injury and podocyte restoration after islet transplantation. Experiments were performed in vivo and in vitro under conditions of diabetic nephropathy and high-glucose medium, respectively. Podocyte injury in vitro was induced by high-glucose concentration, and expression levels of genes associated with the Notch-1 pathway were also regulated by Jagged-1/FC and N-[N-(3,5-Difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT). Podocytes were co-cultured with islets to investigate the protective effect of islets in high-glucose conditions. Histopathological staining and transmission electron microscopy were performed to assess pathological changes in podocytes in glomeruli. The results from this study showed that Notch-1 signaling in podocytes was significantly decreased by functional islet cells in vivo and in vitro. Compared with the co-cultured group and transplanted group, highly activated Notch-1 signaling significantly moderated the effect of islets in affecting podocyte restoration and renal injury. Renal damage and podocyte injury were alleviated after DAPT treatment. Furthermore, the balance between apoptosis and autophagy was diverse under different treatments. All the data in this study showed that highly activated Notch-1 signaling could affect the therapeutic efficacy of islet transplantation on renal injury and podocyte restoration in high-glucose conditions. The balance between apoptosis and autophagy was also closely associated with the degree of podocyte restoration. This finding may suggest that the in vivo microenvironment plays a critical role in podocyte restoration after islet transplantation, which provides a promising and individual assessment and targeting treatment for different diabetic nephropathy patients after islet transplantation into the future.

ICBP90 mediates Notch signaling to facilitate human hepatocellular carcinoma growth.

The Notch signaling pathway plays a key role in cell proliferation and development that is closely related to an inverted CCAAT box binding protein (ICBP90), but little is known about whether there is a correlation between Notch signaling and ICBP90. The aim of the current study was to elucidate this. MTT assay and flow cytometry were used to determine the proliferation, cell cycle and apoptosis of HepG2 or Hepa1-6 cells treated by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a specific inhibitor of the Notch pathway. RT-PCR, Western Blot and in situ immunofluorescence staining were employed to examine expression of ICBP90 in the cells. DAPT caused inhibition of the activation of the Notch signaling pathway, followed by preventing the cells at the G0/G1 phases to enter S and G2/M phases. ICBP90 and Hes-1 proteins were highly expressed in the untreated cells. The reduced levels of Notch intracellular domain (NICD) protein were observed in the DAPT-treated cells, thereby bringing about the down-regulation of ICBP90 with the increment of the DAPT dose. Consistent with this, knockdown of the Hes-1 gene, which encodes a critical transcriptional factor in the Notch pathway, also led to the attenuation of ICBP90. On the contrary, Jagged-1, a Notch ligand, facilitated ICBP90 production. Adriamycin could result in the reduction of ICBP90, which was not accompanied with the alteration of Hes-1. ICBP90 was almost fully distributed within the nuclei, but Hes-1 was visible within both the cytoplasm and nuclei. Our novel findings strongly indicate that inactivation of the Notch signaling pathway impedes hepatocellular carcinoma progress via reduction of ICBP90.

Inhibition of Notch1 Signaling at the Subacute Stage of Stroke Promotes Endogenous Neurogenesis and Motor Recovery After Stroke.

Background and Purpose: It is still not clear whether Notch1 signaling inhibition can promote functional outcomes after stroke, given that it plays time-dependent roles in the sequential process of endogenous neurogenesis. The purpose of this study was to identify the appropriate time frame for Notch1 signaling inhibition according to the temporal evolution of Notch1 signaling activation and the responses of neural stem cells (NSCs), in order to target it for therapeutic intervention and stimulate neurorestorative strategies after stroke.Methods: Sprague-Dawley (SD) rats were subjected to 90-min of middle cerebral artery occlusion (MCAO). Rats were sacrificed before, and at day 1, day 2, day 3, day 4, and day 7 after ischemia for immunohistochemical analysis of the Notch intracellular domain (NICD), Nestin and doublecortin (Dcx). Next, MCAO rats were treated with the γ-secretase inhibitor N-[N-(3,5-di uorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester (DAPT) or with saline at day 4 after ischemia, and subsequently evaluated with behavioral test analysis and magnetic resonance imaging (MRI). The rat brains were then harvested for immunohistochemical analysis of Dcx, NeuN and myelin basic protein (MBP) at 2, 3, 4, and 8 weeks.Results: Notch1 signaling was maximally activated at day 3 after ischemia in parallel with the temporal evolution of NSCs. Inhibiting Notch1 signaling at day 4 after reperfusion with DAPT further promoted recovery of MRI parameters of the corticospinal tract (CST) and the functional outcomes, concomitantly with an increase in neuroblasts, their migration to the ischemic boundary, and potential differentiation to mature neurons, as well as the amelioration of axonal bundle integrity.Conclusion: Inhibition of Notch1 signaling at the subacute stage of stroke could maximally promote endogenous neurogenesis and axonal reorganization.

Neuroprotective Effects of Cerebral Ischemic Preconditioning in a Rat Middle Cerebral Artery Occlusion Model: The Role of the Notch Signaling Pathway.

Cerebral ischemia-reperfusion (I/R) injury is a major problem worldwide. The Notch signaling pathway plays an important role in neural progenitor cell differentiation and in the inflammatory response after central nervous system injury. This study evaluated whether the neuroprotective effect of cerebral ischemic preconditioning (cIPC) is mediated by the preactivation of the Notch signaling pathway. A rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and glucose deprivation/reoxygenation (OGD/R) cell model were constructed to detect the neuroprotective effects of cIPC. In in vivo experiments, cIPC reduces the neurological functional deficit, cerebral infarction, and cellular apoptosis in the hippocampus induced by middle cerebral artery occlusion/reperfusion (MCAO/R), thus indicating that cIPC can improve neurologic function. Moreover, cIPC can reveal the expression peak of Jagged1, Notch1, NICD, and Hes1 protein, thereby indicating that cIPC can preactivate Notch signaling. However, cIPC-induced improvements in neurologic function are compromised by the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-1-alanyl)-S-phenylglycine t-butyl ester (DAPT). In in vitro experiments, OGD preconditioning (OGDPC) can clearly upregulate Notch1 expression in the OGD/R-treated neuron and neural stem cell. Notch1 pre-overexpression can decrease neuron death and apoptosis under OGD/R treatment. Notch1 pre-overexpression can decrease the percentage of G1 stage cells and increase the percentage of S stage cells in OGD/R-treated neural stem cell. Furthermore, Notch1 pre-knockdown has the opposite effect on cell survival, apoptosis, and cycle in both OGD/R-treated neuron and neural stem cell. In conclusion, our results demonstrate that the neuroprotective effects of cIPC in a rat MCAO/R model are mediated by the preactivation of the Notch signaling pathway.

Notch-1 Signaling Activation and Progesterone Receptor Expression in Ectopic Lesions of Women With Endometriosis.

ContextProgesterone (P) resistance is a hallmark of endometriosis, but the underlying mechanism(s) for loss of P sensitivity leading to lesion establishment remains poorly understood.ObjectiveTo evaluate the association between Notch-1 signaling activation and P resistance in the progression of endometriosis.DesignCase control study; archived formalin-fixed, paraffin-embedded tissues.SettingUniversity hospitals (United States, Taiwan).PatientsWomen with endometriosis; human endometrial stromal cell line (HESC).InterventionEutopic endometria (EU) and ectopic lesions (ECs) were collected from surgically diagnosed patients. Archived tissue sections of EU and ECs were identified. HESCs were treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and valproic acid (VPA) to, respectively, suppress and induce Notch-1 activation.Outcome MeasuresTissues were analyzed for Notch Intra-Cellular Domain 1 (NICD1) and progesterone receptor (PGR) protein expression by immunohistochemistry and for transcript levels of NICD1 target genes HES1, PGR, and PGR-B by quantitative reverse transcription polymerase chain reaction. DAPT- or VPA-treated HESCs with and without P cotreatment were evaluated for cell numbers and for PGR, HES1, and PGR target gene DKK1 transcript levels.ResultsNuclear-localized stromal NICD1 protein levels were inversely associated with those of total PGR in EU and ECs. Stromal ECs displayed higher HES1 and lower total PGR and PGR-B transcript levels than EU. In HESCs, DAPT reduction of NICD1 decreased cell numbers and increased PGR transcript and nuclear PGR protein levels and, with P cotreatment, maintained P sensitivity. Conversely, VPA induction of NICD1 decreased PGR transcript levels and, with P cotreatment, abrogated P-induced DKK1 and maintained HES1 transcript levels.ConclusionsAberrant Notch-1 activation is associated with decreased PGR that contributes to P resistance in endometriosis.

Highly Tumorigenic Diffuse Large B Cell Lymphoma Cells Are Produced by Coculture with Stromal Cells

Background/Aims: Diffuse large B cell lymphoma (DLBCL) is heterogeneous. We aimed to explore how tumor microenvironment promotes lymphoma cell aggressiveness and heterogeneity. Methods: We created a coculture system using human DLBCL cells and mouse bone marrow stromal cells. Proliferative capacity, drug resistance, clonogenicity, and tumorigenicity were compared in lymphoma cells from the coculture system and lymphoma cells cultured alone. Expression of Notch signaling associated genes was evaluated using real-time reverse transcriptase PCR and Western blot. Results: Lymphoma cells in the coculture system differentiated into a suspended cell group and an adherent cell group. They acquired a stronger proliferative capacity and drug resistance than lymphoma cells cultured alone, and differences existed between the adherent cell and suspended cell groups. The suspended cell group acquired the most powerful clonogenic and tumorigenic potential. However, Notch3 was exclusively expressed in the adherent lymphoma cell group and the use of N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, an inhibitor of Notch pathway, could abolish the emergence of highly aggressive lymphoma cells. Conclusion: Highly tumorigenic lymphoma cells could be generated by coculture with stromal cells, and it was dependent on Notch3 expression in the adjacent lymphoma cells through interaction with stromal cells.

Notch1 Mediates Preconditioning Protection Induced by GPER in Normotensive and Hypertensive Female Rat Hearts.

G protein-coupled estrogen receptor (GPER) is an estrogen receptor expressed in the cardiovascular system. G1, a selective GPER ligand, exerts cardiovascular effects through activation of the PI3K-Akt pathway and Notch signaling in normotensive animals. Here, we investigated whether the G1/GPER interaction is involved in the limitation of infarct size, and improvement of post-ischemic contractile function in female spontaneous hypertensive rat (SHR) hearts. In this model, we also studied Notch signaling and key components of survival pathway, namely PI3K-Akt, nitric oxide synthase (NOS) and mitochondrial K+-ATP (MitoKATP) channels. Rat hearts isolated from female SHR underwent 30 min of global, normothermic ischemia and 120 min of reperfusion. G1 (10 nM) alone or specific inhibitors of GPER, PI3K/NOS and MitoKATP channels co-infused with G1, just before I/R, were studied. The involvement of Notch1 was studied by Western blotting. Infarct size and left ventricular pressure were measured. To confirm endothelial-independent G1-induced protection by Notch signaling, H9c2 cells were studied with specific inhibitor, N-[N-(3,5 difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 5 μM), of this signaling. Using DAPT, we confirmed the involvement of G1/Notch signaling in limiting infarct size in heart of normotensive animals. In the hypertensive model, G1-induced reduction in infarct size and improvement of cardiac function were prevented by the inhibition of GPER, PI3K/NOS, and MitoKATP channels. The involvement of Notch was confirmed by western blot in the hypertensive model and by the specific inhibitor in the normotensive model and cardiac cell line. Our results suggest that GPERs play a pivotal role in mediating preconditioning cardioprotection in normotensive and hypertensive conditions. The G1-induced protection involves Notch1 and is able to activate the survival pathway in the presence of comorbidity. Several pathological conditions, including hypertension, reduce the efficacy of ischemic conditioning strategies. However, G1-induced protection can result in significant reduction of I/R injury also female in hypertensive animals. Further studies may ascertain the clinical translation of the present results.

Abstract 312: Upregulation of Notch3 Contributes to Aneurysm Formation in a Murine Model of Marfan Syndrome

Notch3 functions as a receptor for membrane-bound ligands (Jag1, DLL1) in signaling pathways for cell-cell communication, gene regulation and control of cell differentiation during embryonic and adult vascular development. However, the role of Notch3 in Marfan Syndrome (MFS), which the main cardiovascular complication is thoracic aneurysms that results in high mortality, has yet to be fully elucidated. Previously we have shown that during aneurysm development, aortic smooth muscle cells (SMCs) in mgR mice, a murine model of MFS, display premature switch from a synthetic to a more contractile phenotype. Therefore we hypothesized that Notch3 could be playing a key role in this aberrant differentiation and aneurysm formation. To test this hypothesis we examined Notch3 mRNA and protein levels in the aortic tissue and SMCs from 1, 4, and 8 weeks-old mgR mice and wild type controls by real time-PCR, Western blot, and immunohistochemistry. Aortic histological changes were examined by Verhoeff-Van Gieson (VVG) elastin staining. In addition, mice were treated with DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), a known γ-secretase inhibitor. The effects of notch signaling inhibition on aneurysm rupture were evaluated. We found that aortic mRNA and protein levels of Notch3 in mgR mice was upregulated ~2 fold by 1 week, and 4-fold at 4 and 8 weeks compared to WT littermate controls. The expression levels of upstream and downstream targets of Notch3, Jagged1 and p21, respectively, were significantly increased in the aorta of mgR mice compared to wild type controls. DAPT treatment prolonged the median lifespan of mgR mice from 16 weeks (untreated) to 32 weeks (DAPT treated). The results from VVG staining of aortic tissue showed that DAPT treatment inhibited aortic elastic degradation in mgR mice. Taken together, our findings indicate that Notch3 is upregulated in the early aortic development and continues to increase during the course of the aneurysm formation in mgR mice. The increase in Notch3 expression may contribute to aneurysm development in MFS. This study lays the groundwork to further examine Notch3 as a novel potential target for treatment.

Concurrent Treatment with Anti-DLL4 Enhances Antitumor and Proapoptotic Efficacy of a γ-Secretase Inhibitor in Gastric Cancer

The Notch signaling pathway has been identified as a therapeutic target for cancers. γ-Secretase inhibitors (GSIs) have been progressively recognized as potential anticancer drugs. The present study aimed to investigate the effects of anti-delta like legend 4 (anti-DLL4) treatment on the anticancer efficacy of GSIs in gastric cancer. SGC-7901-GFP human gastric cancer cells were tested for DLL4 expression by rosette formation test and immunofluorescence, and then were treated with anti-DLL4 antibody N-[N-(3,5-difluorophenacetyl)-L-ananyl]-S-phenylglycine t-butyl ester (DAPT, a type of GSI), or a combination of anti-DLL4 antibody and DAPT. The effects of in vitro treatments on cell apoptosis, cell cycle, and cell invasion were analyzed. For in vivo study, an orthotopic mouse model of gastric cancer was established with green fluorescence expressing SGC-7901. Ultrasound targeted microbubble destruction was used to treat tumor-bearing mice with anti-DLL4 antibody conjugated microbubbles, DAPT, and a combination of the two. Real-time fluorescence imaging was performed to assess tumor cell inhibition in each group. Following in vivo treatments, apoptosis of tumor cells and the expression of apoptosis-related genes BAX, Bcl-2, and P53 were detected by TUNEL and immunohistochemical staining. In vivo combined treatment of anti-DLL4 and DAPT led to a higher rate of cell apoptosis and greater inhibition of cell invasion than that observed with DAPT treatment alone. DAPT and anti-DLL4 combination therapy resulted in decreased cell distribution at G1 phase and increased cell distribution at S phase, compared to the untreated control group (P < .01). In vivo combined therapy with anti-DLL4 and DAPT significantly increased tumor growth inhibition and tumor cell apoptosis when compared to DAPT therapy alone (P < .05). In addition, combined treatment significantly increased expression of BAX and P53 and reduced Bcl-2 expression (P < .05). Conversely, treatment with DAPT alone only increased expression of BAX and P53 (P < .05), suggesting that the reduction of Bcl-2 expression may play an important role in the synergetic antitumor and proapoptosis effects of the combined treatment. Concurrent treatment with anti-DLL4 enhances the antitumor and proapoptotic efficacy of the γ-secretase inhibitor in gastric cancer both in vitro and in vivo.

Inhibition of nuclear translocation of notch intracellular domain (NICD) by diosgenin prevented atherosclerotic disease progression

Notch signaling plays a pivotal role in homeostasis and cardiovascular development. The role of notch signaling in atherosclerosis cannot be complete without analysing the key role of notch in macrophages, which trigger the inflammatory response and subsequent plaque formation in atherosclerosis. Diosgenin showed its anti-atherosclerotic property by the unifying mechanism of suppressing the expression of notch signaling pathway, particularly the nuclear translocation of notch intracellular domain (NICD) in aorta and in differentiated macrophage cells. It is further confirmed by the inhibition of NICD by DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), which also restricted the differentiation of macrophage. Hence, inhibition of nuclear translocation of NICD by diosgenin aids in preventing atherosclerosis.

Combination treatment of all-trans retinoic acid (ATRA) and γ-secretase inhibitor (DAPT) cause growth inhibition and apoptosis induction in the human gastric cancer cell line.

Current medication for gastric cancer patients has a low success rate with resistance and side effects. According to recent studies, γ-secretase inhibitors is used as therapeutic drugs in cancer. Moreover, all-trans retinoic acid (ATRA) is a natural compound proposed for the treatment/chemo-prevention of cancers. The aim of this study was to explore the effects of ATRA in combination with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester(DAPT) as γ-secretase inhibitor on viability and apoptosis of the AGS and MKN-45 derived from human gastric cancer. AGS and MKN-45 gastric cancer cell lines were treated with different concentrations of ATRA or DAPT alone or ATRA plus DAPT. The viability, death detection and apoptosis of cells was examined by MTT assay and Ethidium bromide/acridine orange staining. The distribution of cells in different phases of cell cycle was also evaluated through flow cytometry analyses. In addition, caspase 3/7 activity and the expression of caspase-3 and bcl-2 were examined. DAPT and ATRA alone decreased gastric cancer cells viability in a concentration dependent manner. The combination of DAPT and ATRA exhibited significant synergistic inhibitory effects. The greater percentage of cells were accumulated in G0/G1 phase of cell cycle in combination treatment. The combination of DAPT and ATRA effectively increased the proportion of apoptotic cells and the level of caspase 3/7 activities compared to single treatment. Moreover, augmented caspase-3 up-regulation and bcl-2 down-regulation were found following combined application of DAPT and ATRA. The combination of DAPT and ATRA led to more reduction in viability and apoptosis in respect to DAPT or ATRA alone in the investigated cell lines.

Notch signaling molecule is involved in the invasion of MiaPaCa2 cells induced by CoCl2 via regulating epithelial‑mesenchymal transition.

Pancreatic cancer exhibits a high mortality rate resulting from metastasis and there is currently no effective treatment strategy. Hypoxia serves an important role in cancer cells, where cellular metabolic rate is high. The underlying mechanisms that trigger hypoxia and the invasion of pancreatic cancer cells remain unknown. Investigation of the importance of hypoxia in the invasion of pancreatic cancer cells for potential, novel treatment strategies is of primary concern. Cell Counting Kit-8 assay, invasion assay, western blotting and reverse transcription-quantitative polymerase chain reaction were used to investigate invasion and epithelial mesenchymal transition (EMT) and the expression of Notch1 in MiaPaCa2 cells treated with cobalt II chloride (CoCl2). Hypoxia-inducible factor 1α (HIF-1α) small interfering (si)RNA and Notch1 inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were also selected to investigate these mechanisms. Data indicated that CoCl2 increased the invasion ability and altered EMT in MiaPaCa2 cells. CoCl2 regulated the expression of HIF-1α and Notch1 in MiaPaCa2 cells. In addition, HIF-1α siRNA inhibited the effects of CoCl2 on the expression of Notch1 and decreased Snail, EMT and invasion in MiaPaCa2 cells. DAPT increased the expression of epithelial-cadherin and decreased the content of neural-cadherin, Snail and invasion in MiaPaCa2 cells in the presence or absence of CoCl2. CoCl2 promoted invasion by stimulating the expression of HIF-1α and regulating the expression of Notch1 and EMT in MiaPaCa2 cells. Targeting the Notch1 signaling molecule may be a novel treatment strategy for the prevention and treatment of pancreatic cancer.

Shear Stress and VE-Cadherin.

Objective- Vascular fusion represents an important mechanism of vessel enlargement during development; however, its significance in postnatal vessel enlargement is still unknown. During fusion, 2 adjoining vessels merge to share 1 larger lumen. The aim of this research was to identify the molecular mechanism responsible for vascular fusion. Approach and Results- We previously showed that both low shear stress and DAPT ( N-[ N-(3,5-difluorophenacetyl)-L-alanyl]- S-phenylglycine t-butyl ester) treatment in the embryo result in a hyperfused vascular plexus and that increasing shear stress levels could prevent DAPT-induced fusion. We, therefore, investigated vascular endothelial-cadherin (VEC) phosphorylation because this is a common downstream target of low shear stress and DAPT treatment. VEC phosphorylation increases after DAPT treatment and decreased shear stress. The increased phosphorylation occurred independent of the cleavage of the Notch intracellular domain. Increasing shear stress rescues hyperfusion by DAPT treatment by causing the association of the phosphatase vascular endothelial-protein tyrosine phosphatase with VEC, counteracting VEC phosphorylation. Finally, Src (proto-oncogene tyrosine-protein kinase Src) inhibition prevents VEC phosphorylation in endothelial cells and can rescue hyperfusion induced by low shear stress and DAPT treatment. Moesin, a VEC target that was previously reported to mediate endothelial cell rearrangement during lumenization, relocalizes to cell membranes in vascular beds undergoing hyperfusion. Conclusions- This study provides the first evidence that VEC phosphorylation, induced by DAPT treatment and low shear stress, is involved in the process of fusion during vascular remodeling.

Dynamic Evaluation of Notch Signaling-Mediated Angiogenesis in Ischemic Rats Using Magnetic Resonance Imaging.

Objective The Notch signaling pathway is involved in angiogenesis induced by brain ischemia and can be efficiently inhibited by the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT). The aim of the present study was to noninvasively investigate the effect of DAPT treatment on angiogenesis in brain repair after stroke using magnetic resonance imaging (MRI). Methods Sprague-Dawley rats (n = 40) were subjected to 90 minutes of transient middle cerebral artery (MCA) occlusion and treated with PBS (n = 20) or DAPT (n = 20) at 72 hours after the onset of ischemia. MRI measurements including T2-weighted imaging (T2WI), susceptibility-weighted imaging (SWI), and cerebral blood flow (CBF) were performed at 24 hours after reperfusion and weekly up to 4 weeks using a 3-Tesla system. Histological measurements were obtained at each time point after MRI scans. Results SWI showed that DAPT treatment significantly enhanced angiogenesis in the ischemic boundary zone (IBZ) with respect to the control group, with local CBF in the angiogenic area elevated, along with increases in vascular density confirmed by histology. Conclusion Treatment of ischemic stroke with DAPT significantly augments angiogenesis, which promotes poststroke brain remodeling by elevating CBF level, and these processes can be dynamically monitored and evaluated by MRI.

Notch signaling negatively regulates BMP9-induced osteogenic differentiation of mesenchymal progenitor cells by inhibiting JunB expression.

Although interaction between BMP and Notch signaling has been demonstrated to be crucial for osteogenic differentiation of mesenchymal stem cells (MSCs), the precise molecular mechanism remains unknown. Here, we show that Notch intracellular domain (NICD) overexpression inhibits BMP9-induced C3H10T1/2 cell osteogenesis in vivo and in vitro. Our results show that activated Notch signaling results in down-regulation of Runx2 and early osteogenesis differentiation factors, without affecting p-Smad1/5/8 expression, and that blocking Notch signaling with DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) significantly increases p-Smad1/5/8 expression. Interestingly, Notch signaling also regulates the cell cycle by increasing PCNA (proliferation cell nuclear antigen) and CyclinD1 expression. Furthermore, similar results were obtained by ectopic bone formation and histological analyses, indicating that Notch signaling activation significantly inhibits BMP9-induced MSC osteogenic, cartilage and adipogenic differentiation. Moreover, we are the first to show that Notch regulates by suppressing JunB synthesis and that the negative effect of Notch is partially reversed by treatment with the JunB activator TPA (12-O-tetradeca-noylphorbol-13-acetate). Our findings demonstrate that Notch signaling significantly enhances cell proliferation but inhibits MSC osteogenic differentiation induced by BMP9 via JunB protein suppression rather than by BMP/Smad signaling regulation.

DNER modulates the length, polarity and synaptogenesis of spiral ganglion neurons via the Notch signaling pathway.

The Delta/Notch-like epidermal growth factor-related receptor (DNER) serves an important role in the developing central nervous system. However, the actions of DNER in the development of the spiral ganglion in the inner ear have yet to be elucidated. Wild-type C57BL/6 mice were housed and time-mated for use in the present study. Primary neuronal cultures were prepared using spiral ganglion progenitors isolated from the modiolus of postnatal day 1 (P1) mice. DNER recombinant lentiviral vectors were constructed and transfected into the cultured primary neurons. The relative proportion of differentiated neurons and the length of their neurites were evaluated using microscopy. The results of the present study demonstrated that DNER was expressed in spiral ganglion neurons (SGNs) that exhibited significant polarity in the early differentiation stages; DNER expression gradually decreased until the polarity was lost on week 35. The in vitro expression of DNER was revealed to be similar to that in vivo. When DNER expression was silenced using RNA interference, the polarity of the differentiated neurons was altered and they exhibited significantly reduced dendritic length. In addition, the proportion of bipolar neurons was decreased compared with the control group. Furthermore, the expression of α-synuclein and the GluR2/3 subunits of the α-amin-o-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor were also reduced in cultured neurons in which DNER was silenced. Notch1 was co-expressed with DNER in SGNs isolated from P1 mice. The indirect Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester also affected the polarity and the formation of protrusions, and reduced the expression of DNER and glial fibrillary acidic protein in SGNs. In conclusion, the present study demonstrated that DNER was expressed in SGNs and appeared to be involved in the mechanisms underlying neuronal polarity and neuritogenesis, via a Notch-dependent signaling pathway.

Notch/Wnt cross-signalling regulates stemness of dental pulp stem cells through expression of neural crest and core pluripotency factors.

Dental pulp stem cells (DPSCs) from adult teeth express neural crest (NC) markers together with core transcriptional factors associated with stem cell pluripotency, such as Oct4a, Sox2, c-Myc, Rex1, Stella/Dppa3, Ssea1/Fut4, Lin28 and Nanog. The possibility to boost the natural stemness features of DPSCs by mild methods, that do not involve gene and/or chromatin modification or gene transfection, is highly desirable for cell therapy. Canonical Wnt and Notch are two highly conserved developmental signalling pathways that are involved in NC emergence and stem cell self-renewal. We determined that both pathways coordinate to regulate the expression of core pluripotency and NC factors in DPSCs. Pharmacological inhibition of the Notch pathway for 48 h, by the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), abolished the expression of NC and core factors. In addition, it induced a silencing of the canonical Wnt signalling and a clear reduction in the stemness potential of DPSCs, as shown by a reduced ability to generate mature, fully differentiated osteoblasts and adipocytes. Conversely, pharmacological activation of the Wnt pathway for 48 h, by either the glycogen synthase kinase 3 beta (GSK3-β) inhibitor 6-bromoindirubin-3´-oxime (BIO) or the human recombinant protein Wnt-3a, not only largely increased the expression of NC and core factors, but also increased the efficiency of DPSCs to differentiate into mature osteoblasts and adipocytes. These results showed that a short preconditioning activation of Wnt/Notch signalling by small molecules and/or recombinant proteins enhanced the stemness and potency of DPSCs in culture, which could be useful for optimising the therapeutic use of these and other tissue-specific stem cells.

Activation of the Notch-1 signaling pathway may be involved in intracerebral hemorrhage-induced reactive astrogliosis in rats.

OBJECTIVE Reactive astrogliosis, a key feature that is characterized by glial proliferation, has been observed in rat brains after intracerebral hemorrhage (ICH). However, the mechanisms that control reactive astrogliosis formation remain unknown. Notch-1 signaling plays a critical role in modulating reactive astrogliosis. The purpose of this paper was to establish whether Notch-1 signaling is involved in reactive astrogliosis after ICH. METHODS ICH was induced in adult male Sprague-Dawley rats via stereotactic injection of autologous blood into the right globus pallidus. N-[ N-(3,5-difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT) was injected into the lateral ventricle to block Notch-1 signaling. The rats' brains were perfused to identify proliferating cell nuclear antigen (PCNA)-positive/GFAP-positive nuclei. The expression of GFAP, Notch-1, and the activated form of Notch-1 (Notch intracellular domain [NICD]) and its ligand Jagged-1 was assessed using immunohistochemical and Western blot analyses, respectively. RESULTS Notch-1 signaling was upregulated and activated after ICH as confirmed by an increase in the expression of Notch-1 and NICD and its ligand Jagged-1. Remarkably, blockade of Notch-1 signaling with the specific inhibitor DAPT suppressed astrocytic proliferation and GFAP levels caused by ICH. In addition, DAPT improved neurological outcome after ICH. CONCLUSIONS Notch-1 signaling is a critical regulator of ICH-induced reactive astrogliosis, and its blockage may be a potential therapeutic strategy for hemorrhagic injury.

Lidamycin decreases CD133 expression in hepatocellular carcinoma via the Notch signaling pathway.

Cluster of differentiation (CD)133 is considered a molecular marker of cancer stem cells in hepatocellular carcinoma. In the present study, the effect of lidamycin (LDM) on CD133 expression in hepatocellular carcinoma (Huh7 cells) was evaluated and the potential molecular mechanism was investigated. Flow cytometry analysis, as well as sorting, sphere formation and western-blot assays, were performed in vitro to explore the effects of LDM on CD133 expression. A subcutaneous tumor model in nude mice was used to observe the effects of LDM on tumor volume and CD133 protein in vivo. To investigate the potential underlying molecular mechanism, Notch signaling pathway activity was detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction. The proportion of CD133+ cells and the expression of CD133 protein were revealed to be downregulated by LDM. Sphere formation of sorted CD133+ cells was suppressed 7 days after LDM treatment. In addition, LDM inhibited tumor volume formed from sorted CD133+ cells and CD133 protein level in vivo. LDM decreased the mRNA level of NOTCH1, Hes1 (Hes family BHLH transcription factor 1) and Hey1 (Hes-related family BHLH transcription factor with YRPW motif 1) genes; consequently, the protein expression of NOTCH1, Notch intracellular domain, Hes1 and Hey1 was decreased by LDM. Downregulation of the Notch signaling pathway by LDM was enhanced through combination with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. In brief, these data suggest that LDM suppresses CD133 expression via the Notch signaling pathway, indicating the potential mechanism of LDM on CD133 and the benefits for further clinical application.