MicroRNA-124a Protects the Myocardium Against Ischemia Reperfusion Injury Through Regulation of the Notch Signaling Pathway.

INTRODUCTION: This study's objective is to investigate the effect of downregulation of micro ribonucleic acid (miR)-124a on myocardial injury after ischemia reperfusion (I/R) in rats. METHODS: Sprague Dawley (SD) rats (n=20) were divided into four groups - sham, I/R, I/R+miR-124a antagomir (I/R+ant-miR-124a), and I/R+ant-normal control (NC). The pathomorphological and infarct size variance of injured myocardial tissues with IR were conducted with hematoxylin (HE) and triphenyltetrazolium chloride (TTC) staining. The expression levels of miR-124a, BAX, nuclear factor kappa B (NF-KB), Notch1, and Hes1 were examined by quantitative real-time polymerase chain reaction or Western blot in myocardium. The inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumor necrosis factor alpha (TNF-α) were detected by the enzyme-linked immunosorbent assay, as well as the activity of lactate dehydrogenase (LDH) and creatine kinase (CK) in serum by colorimetry. RESULTS: The expression of miR-124a was increased in the I/R group. Compared with I/R and I/R+ant-NC groups, after downregulating miR-124a, the expression of IL-6, IL-1ß, TNF-α, BAX, NF-KB, LDH, and CK were decreased, but the expression of Notch1 and Hes1 were increased. In HE staining, myocardial tissue edema, red blood cell exudation, and myocardial fiber arrangement disorder were accompanied by inflammatory cell infiltration and local necrosis in the I/R group. However, the pathological injury of myocardial tissue was alleviated after downregulating miR-124a. Additionally, TTC results showed that the myocardial infarction area was decreased in the I/R+ant-miR-124a group. CONCLUSION: Downregulation of miR-124a expression through Notch pathway can significantly reduce myocardial damage after 24 hours of I/R in SD rats. Therefore, miR-124a may become a potential therapeutic target for I/R injury.

Curcuma as an adjuvant in colorectal cancer treatment.

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide and mostly affects men. Around 20% of its incidence is by familiar disposition due to hereditary syndromes. The CRC treatment involves surgery and chemotherapy; however, the side effects of treatments and the fast emergence of drug resistance evidence the necessity to find more effective drugs. Curcumin is the main polyphenol pigment present in Curcuma longa, a plant widely used as healthy food with antioxidant properties. Curcumin has synergistic effects with antineoplastics such as 5-fluorouracil and oxaliplatin, as well anti-inflammatory drugs by inhibiting cyclooxygenase-2 and the Nuclear factor kappa B. Furthermore, curcumin shows anticancer properties by inhibition of the Wnt/ß-catenin, Hedgehog, Notch, and the phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathways implicated in the progression of CRC. However, the consumption of pure curcumin is less suitable, as the absorption is poor, and the metabolism and excretion are high. Pharmacological formulations and essential oils of the plant improve the curcumin absorption, resulting in therapeutical dosages. Despite the evidence obtained in vitro and in vivo, clinical studies have not yet confirmed the therapeutic potential of curcumin against CRC. Here we reviewed the last scientific information that supports the consumption of curcumin as an adjuvant for CRC therapy.

Cochlea cell-specific marker expression upon in vitro Hes1 knockdown.

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

Inhibition of gamma-secretase activity without interfering in Notch signalling decreases inflammatory response in patients with cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) patients present an exacerbated inflammatory response associated with tissue damage and ulcer development. Increasing numbers of patients have exhibited treatment failure, which remains not well understood. We hypothesized that adjuvant anti-inflammatory therapy would benefit CL patients. The aim of the present study was to investigate the contribution of Notch signalling and gamma-secretase activity to the inflammatory response observed in CL patients. Notch signalling is a molecular signalling pathway conserved among animal species. Gamma-secretase forms a complex of proteins that, among other pathways, modulates Notch signalling and immune response. We found that Notch 1 cell receptor signalling protects against the pathologic inflammatory response, and JLK6, a gamma-secretase inhibitor that does not interfere with Notch signalling, was shown to decrease the in-vitro inflammatory response in CL. Our data suggest that JLK6 may serve as an adjuvant treatment for CL patients.

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear ß-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.

Notch signaling pathway in infectious diseases: role in the regulation of immune response.

BACKGROUND: The Notch signaling pathway is a cell signaling system that is conserved in a variety of eukaryotes. Overall, Notch receptors and their ligands are single-pass transmembrane proteins, which often require cell-cell interactions and proteolytic processing to promote signaling. Since its discovery, it has been the subject of extensive research that revealed its importance in several cellular mechanisms, including cell fate determination, hematopoiesis, tissue self-renewal, proliferation, and apoptosis during embryogenesis. Many studies have described the influence of the Notch pathway in modulating the innate and adaptive immune systems. METHODS: We analyzed the literature on the role of the Notch pathway in regulating immune responses during infections, aiming to discuss the importance of establishing a Notch signaling pathway-based approach for predicting the outcome of infectious diseases. CONCLUSION: In this review, we present an overview of evidence that demonstrates the direct and indirect effects of interaction between the Notch signaling pathway and the immune responses against bacterial, viral, fungal, and parasitic infections, as well as the importance of this pathway to predict the outcome of infectious diseases.

Roads to the strategic targeting of ovarian cancer treatment.

Although ovarian cancer mortality rates have slightly declined in the last 40 years, ovarian cancer continues to be the eighth cause of cancer death in women. Ovarian cancer is characterized by its high response to treatments but also by its high rate of recurrence. Although treatments are limited to cytoreductive surgery and platinum-based chemotherapy, other therapies using antiangiogenic agents and poly (ADP-ribose) polymerase inhibitors are being tested. Nevertheless, these therapeutic strategies have had poor results and new potential targets and approaches are thus needed. The present review focuses on the recent evidence on antiangiogenic strategies in ovarian cancer cells and on the mechanisms governed by Notch and ß-catenin proteins. It also describes the concept of 'vascular normalization' by using the platelet-derived growth factor, PDGFB, molecule as a tool to regulate ovarian tumor angiogenesis and thus improve ovarian tumor treatment. It has been reported that alterations in the Notch system components and changes in the canonical Wnt/ß-catenin signaling, the other pathway of our interest, are relevant to molecular events that contribute to ovarian cancer development. Thus, in this review, we consider these aspects of the ovarian tumor biology as potential new therapeutic strategies for the treatment of this disease.

Cochlea cell-specific marker expression upon in vitro Hes1 knockdown

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

Phosphatidic acid increases Notch signalling by affecting Sanpodo trafficking during Drosophila sensory organ development.

Organ cell diversity depends on binary cell-fate decisions mediated by the Notch signalling pathway during development and tissue homeostasis. A clear example is the series of binary cell-fate decisions that take place during asymmetric cell divisions that give rise to the sensory organs of Drosophila melanogaster. The regulated trafficking of Sanpodo, a transmembrane protein that potentiates receptor activity, plays a pivotal role in this process. Membrane lipids can regulate many signalling pathways by affecting receptor and ligand trafficking. It remains unknown, however, whether phosphatidic acid regulates Notch-mediated binary cell-fate decisions during asymmetric cell divisions, and what are the cellular mechanisms involved. Here we show that increased phosphatidic acid derived from Phospholipase D leads to defects in binary cell-fate decisions that are compatible with ectopic Notch activation in precursor cells, where it is normally inactive. Null mutants of numb or the α-subunit of Adaptor Protein complex-2 enhance dominantly this phenotype while removing a copy of Notch or sanpodo suppresses it. In vivo analyses show that Sanpodo localization decreases at acidic compartments, associated with increased internalization of Notch. We propose that Phospholipase D-derived phosphatidic acid promotes ectopic Notch signalling by increasing receptor endocytosis and inhibiting Sanpodo trafficking towards acidic endosomes.

Regulation of γδT17 cells by Mycobacterium vaccae through interference with Notch/Jagged1 signaling pathway.

The objective of this study was to investigate the effect of Mycobacterium vaccae on Jagged 1 and gamma delta T17 (γδT17) cells in asthmatic mice. An asthma mouse model was established through immunization with ovalbumin (OVA). Gamma-secretase inhibitor (DAPT) was used to block the Notch signaling pathway. M. vaccae was used to treat asthma, and related indicators were measured. Blocking Notch signaling inhibited the production of γδT17 cells and secretion of cytokine interleukin (IL)-17, which was accompanied by a decrease in Jagged1 mRNA and protein expression in the treated asthma group compared with the untreated asthma group. Similarly, treatment with M. vaccae inhibited Jagged1 expression and γδT17 cell production, which was associated with decreased airway inflammation and reactivity. The Notch signaling pathway may play a role in the pathogenesis of asthma through the induction of Jagged1 receptor. On the other hand, the inhibitory effect of M. vaccae on Jagged1 receptor in γδT17 cells could be used for the prevention and treatment of asthma.

Notch signaling and the emergence of hematopoietic stem cells.

The hematopoietic stem cell (HSC) is able to give rise to all blood cell lineages in vertebrates. HSCs are generated in the early embryo after two precedent waves of primitive hematopoiesis. Canonical Notch signaling is at the center of the complex mechanism that controls the development of the definitive HSC. The successful in vitro generation of hematopoietic cells from pluripotent stem cells with the capacity for multilineage hematopoietic reconstitution after transplantation requires the recapitulation of the most important process that takes place in the hemogenic endothelium during definitive hematopoiesis, that is the endothelial-to-hematopoietic transition (EHT). To meet this challenge, it is necessary to thoroughly understand the molecular mechanisms that modulate Notch signaling during the HSC differentiation process considering different temporal and spatial dimensions. In recent years, there have been important advances in this field. Here, we review relevant contributions describing different genes, factors, environmental cues, and signaling cascades that regulate the EHT through Notch interactions at multiple levels. The evolutionary conservation of the hematopoietic program has made possible the use of diverse model systems. We describe the contributions of the zebrafish model and the most relevant ones from transgenic mouse studies and from in vitro differentiated pluripotent cells.

Notch Signaling Pathway Expression in the Skin of Leprosy Patients: Association With Skin and Neural Damage.

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae, a debilitating disease that affects the skin and peripheral nerves. It is possible that tissue changes during infection with leprosy are related to alterations in the activity of the Notch signaling pathway, an innate signaling pathway in the physiology of the skin and peripheral nerves. Methods: This is a descriptive observational study. Thirty skin biopsies from leprosy patients and 15 from individuals with no history of this disease were evaluated. In these samples, gene expressions of cellular components associated with the Notch signaling pathway, Hes-1, Hey-1, Runx-1 Jagged-1, Notch-1, and Numb, were evaluated using q-PCR, and protein expression was evaluated using immunohistochemistry of Runx-1 and Hes-1. Results: Changes were observed in the transcription of Notch signaling pathway components; Hes-1 was downregulated and Runx-1 upregulated in the skin of infected patients. These results were confirmed by immunohistochemistry, where reduction of Hes-1 expression was found in the epidermis, eccrine glands, and hair follicles. Increased expression of Runx-1 was found in inflammatory cells in the dermis of infected patients; however, it is not related to tissue changes. With these results, a multivariate analysis was performed to determine the causes of transcription factor Hes-1 reduction. It was concluded that tissue inflammation was the main cause. Conclusions: The tissue changes found in the skin of infected patients could be associated with a reduction in the expression of Hes-1, a situation that would promote the survival and proliferation of M. leprae in this tissue.

Context-specific functions of Notch in Drosophila blood cell progenitors.

Drosophila Larval hematopoiesis takes place at the lymph gland, where myeloid-like progenitors differentiate into Plasmatocytes and Crystal Cells, under regulation of conserved signaling pathways. It has been established that the Notch pathway plays a specific role in Crystal Cell differentiation and maintenance. In mammalian hematopoiesis, the Notch pathway has been proposed to fulfill broader functions, including Hematopoietic Stem Cell maintenance and cell fate decision in progenitors. In this work we describe different roles that Notch plays in the lymph gland. We show that Notch, activated by its ligand Serrate, expressed at the Posterior Signaling Center, is required to restrain Core Progenitor differentiation. We define a novel population of blood cell progenitors that we name Distal Progenitors, where Notch, activated by Serrate expressed in Lineage Specifying Cells at the Medullary Zone/Cortical Zone boundary, regulates a binary decision between Plasmatocyte and Crystal Cell fates. Thus, Notch plays context-specific functions in different blood cell progenitor populations of the Drosophila lymph gland.

Interaction of Long Noncoding RNAs and Notch Signaling: Implications for Tissue Homeostasis Loss.

The Notch signaling is a crucial pathway involved in cellular development, progression, and differentiation. Deregulation of Notch signaling pathway commonly impacts tissue homeostasis, being highly associated with proliferative disorders. The long noncoding RNAs (lncRNAs), which are transcripts with more than 200 nucleotides that do not code for proteins, were already described as Notch signaling pathway-interacting molecules. Many of them act as important transcriptional and posttranscriptional regulators, affecting gene expression and targeting other regulatory molecules, such as miRNAs. Due to their strong impact on function and gene expression of Notch-related molecules, lncRNAs influence susceptibility to cancer and other diseases, and can be regarded as potential biomarkers and therapeutic targets. Along this chapter, we summarize the cross talk between the Notch signaling pathway and their most important modulating lncRNAs, as well as the pathological consequences of these interactions, in different tissues.

Modulation of the Notch System in Response to Wnt Inhibition Induces Restoration of the Rat Luteal Function.

The aim of this study was to investigate whether the Notch pathway is modulated in response to the downregulation of the Wnt/Β-catenin system in corpora lutea (CLs) from superovulated rats. To this end, we analyzed the effect of in vitro CL Wnt/Β-catenin inhibition on the expression of Notch members and on luteal function. Mechanically isolated rat CLs were cultured with ICG-001, a Wnt/B-catenin inhibitor. In this system, Wnt/B-catenin inhibition reduced progesterone production and decreased StAR protein levels. Besides, Wnt/B-catenin inhibition stimulated the Notch system, evidenced by an increase in Hes1 expression, and promoted the expression of selected Notch family members. At long incubation times, StAR levels and progesterone concentration reached the control values, effects probably mediated by the Notch pathway. These results provide the first evidence of a compensatory mechanism between Wnt/B-catenin signaling and the Notch system, which contributes to the homeostasis of luteal cells.

Linc-OIP5 in the breast cancer cells regulates angiogenesis of human umbilical vein endothelial cells through YAP1/Notch/NRP1 signaling circuit at a tumor microenvironment.

BACKGROUND: LincRNAs have been revealed to be tightly associated with various tumorigeneses and cancer development, but the roles of specific lincRNA on tumor-related angiogenesis was hardly studied. Here, we aimed to investigate whether linc-OIP5 in breast cancer cells affects the angiogenesis of HUVECs and whether the linc-OIP5 regulations are involved in angiogenesis-related Notch and Hippo signaling pathways. METHODS: A trans-well system co-cultured HUVECs with linc-OIP5 knockdown breast cancer cell MDA-MB-231 was utilized to study the proliferation, migration and tube formation abilities of HUVECs and alterations of related signaling indicators in breast cancer cells and their conditioned medium through a series of cell and molecular experiments. RESULTS: Overexpressed linc-OIP5, YAP1, and JAG1 were found in breast cancer cell lines MCF7 and MDA-MB-231 and the expression levels of YAP1 and JAG1 were proportional to the breast cancer tissue grades. MDA-MB-231 cells with linc-OIP5 knockdown led to weakened proliferation, migration, and tube formation capacity of co-cultured HUVECs. Besides, linc-OIP5 knockdown in co-cultured MDA-MB-231 cells showed downregulated YAP1 and JAG1 expression, combined with a reduced JAG1 level in conditioned medium. Furthermore, a disrupted DLL4/Notch/NRP1 signaling in co-cultured HUVECs were also discovered under this condition. CONCLUSION: Hence, linc-OIP5 in MDA-MB-231 breast cancer cells may act on the upstream of the YAP1/Notch/NRP1 signaling circuit to affect proliferation, migration, and tube formation of co-cultured HUVECs in a non-cellular direct contact way through JAG1 in conditioned medium. These findings at least partially provide a new angiogenic signaling circuit in breast cancers and suggest linc-OIP5 could be considered as a therapeutic target in angiogenesis of breast cancers.

Notch pathway in ependymoma RELA-fused subgroup: upregulation and association with cancer stem cells markers expression.

RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.

Regulation of γδT17 cells by Mycobacterium vaccae through interference with Notch/Jagged1 signaling pathway

The objective of this study was to investigate the effect of Mycobacterium vaccae on Jagged 1 and gamma delta T17 (γδT17) cells in asthmatic mice. An asthma mouse model was established through immunization with ovalbumin (OVA). Gamma-secretase inhibitor (DAPT) was used to block the Notch signaling pathway. M. vaccae was used to treat asthma, and related indicators were measured. Blocking Notch signaling inhibited the production of γδT17 cells and secretion of cytokine interleukin (IL)-17, which was accompanied by a decrease in Jagged1 mRNA and protein expression in the treated asthma group compared with the untreated asthma group. Similarly, treatment with M. vaccae inhibited Jagged1 expression and γδT17 cell production, which was associated with decreased airway inflammation and reactivity. The Notch signaling pathway may play a role in the pathogenesis of asthma through the induction of Jagged1 receptor. On the other hand, the inhibitory effect of M. vaccae on Jagged1 receptor in γδT17 cells could be used for the prevention and treatment of asthma.

Linc-OIP5 in the breast cancer cells regulates angiogenesis of human umbilical vein endothelial cells through YAP1/Notch/NRP1 signaling circuit at a tumor microenvironment

BACKGROUND: LincRNAs have been revealed to be tightly associated with various tumorigeneses and cancer development, but the roles of specific lincRNA on tumor-related angiogenesis was hardly studied. Here, we aimed to investigate whether linc-OIP5 in breast cancer cells affects the angiogenesis of HUVECs and whether the linc-OIP5 regulations are involved in angiogenesis-related Notch and Hippo signaling pathways. METHODS: A trans-well system co-cultured HUVECs with linc-OIP5 knockdown breast cancer cell MDA-MB-231 was utilized to study the proliferation, migration and tube formation abilities of HUVECs and alterations of related signaling indicators in breast cancer cells and their conditioned medium through a series of cell and molecular experiments. RESULTS: Overexpressed linc-OIP5, YAP1, and JAG1 were found in breast cancer cell lines MCF7 and MDA-MB-231 and the expression levels of YAP1 and JAG1 were proportional to the breast cancer tissue grades. MDA-MB-231 cells with linc-OIP5 knockdown led to weakened proliferation, migration, and tube formation capacity of co-cultured HUVECs. Besides, linc-OIP5 knockdown in co-cultured MDA-MB-231 cells showed downregulated YAP1 and JAG1 expression, combined with a reduced JAG1 level in conditioned medium. Furthermore, a disrupted DLL4/Notch/NRP1 signaling in co-cultured HUVECs were also discovered under this condition. CONCLUSION: Hence, linc-OIP5 in MDA-MB-231 breast cancer cells may act on the upstream of the YAP1/Notch/NRP1 signaling circuit to affect proliferation, migration, and tube formation of co-cultured HUVECs in a non-cellular direct contact way through JAG1 in conditioned medium. These findings at least partially provide a new angiogenic signaling circuit in breast cancers and suggest linc-OIP5 could be considered as a therapeutic target in angiogenesis of breast cancers.