Notch Receptors Research Articles

Siah1 promotes the proliferation of NSCLC cells through ubiquitinating and stabilizing Notch1

Seven in absentia homolog 1 (Siah1) has been shown plays important roles in the pathogenesis and development of multiple cancers. However, the functions and mechanisms of Siah1 in non-small cell lung cancer (NSCLC) remain unclear. In our study, we found that knock down of Siah1 could inhibit the proliferation of NSCLC cells, while over-expression of Siah1 had the opposite effects. Molecularly, the bioinformatics analysis determined that notch receptor 1 (Notch1) might be the potential target of Siah1. Subsequently, we identified that Siah1 acted as an E3 ligase to promote the ubiquitination and stabilization of Notch1 through the proteasome pathway. Furthermore, the results showed that the Siah1 expression was directly correlated with CTR9 in human NSCLC tissues. Finally, Siah1 could promote Akt phosphorylation through regulating Notch1, thus promoting the proliferation of NSCLC cells. In conclusion, our study demonstrated that Siah1 acts as an oncogene, can ubiquitinate and stabilize Notch1 by proteasome pathway, which promotes Akt phosphorylation and ultimately leads to NSCLC cell proliferation.

Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease.

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

AL101, a gamma-secretase inhibitor, has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.

Abstract P3136: Notch3 Deficiency Impairs Vascular Smooth Muscle Cell Contractility And Glymphatic Function In The Brain

While most cases of vascular dementia represent complex interactions between host genetics and environmental factors, mendelian forms of vascular dementia also exist. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian disease characterized by progressive vascular deterioration, cognitive deficits, and strokes. Mutations in the NOTCH3 receptor underlies the pathologies in CADASIL. NOTCH3 is primarily expressed in vascular smooth muscle cells (vSMCs) and its’ expression is critical for differentiation and functional integrity of arterial vSMCs, albeit through unclear mechanism(s). To elucidate the contribution of NOTCH3 in the maintenance of cerebral vascular architecture and function, we performed micro-computed tomography (micro-CT) on the brains of aged Notch3 -deficient animals. Micro-CT assessment of the cerebral vasculature architecture showed significant abnormalities including severe vessel dilation and tortuosity (dolicoectasia) of the middle cerebral artery and its branches in the Notch3 -/- compared to aged-match controls. To identify the molecular pathway from NOTCH3 dysregulation to the observed cerebral vascular dysfunction, we performed single-cell RNASeq on cerebral arteries isolated from young (4w) and old (104w) Notch3 -/- animals. Evaluation of the vSMC-specific transcriptomes indicated significant loss of proteins associated with muscle contraction and increased extracellular matrix production in animals that lack NOTCH3. Using a combination of immunofluorescence microscopy and in vitro functional assays, we confirmed that continued expression of Notch3 is a critical requirement for maintenance of vSMC contractile function. Impaired contractility also affected flow of cerebrospinal fluid in the parenchyma of Notch3 -/- . MRI and behavioral assessments were performed in the Notch3 -/- animals to elucidate the relationship between impaired vascular contractility to cognitive function. Taken together these findings link the molecular dysfunction of NOTCH3 through its regulation of vascular contractility and cerebral vessel architecture to altered neurological function and clarify the molecular pathways to cellular pathology of Notch3 driven dementias.

Notch1 and Notch2 Signaling Exclusively but Cooperatively Maintain Fetal Myogenic Progenitors.

Myogenic progenitors (MPs) generate myocytes that fuse to form myofibers during skeletal muscle development while maintaining the progenitor pool, which is crucial for generating sufficient muscle. Notch signaling has been known to reserve a population of embryonic MPs during primary myogenesis by promoting cell cycle exit and suppressing premature differentiation. However, the roles of individual Notch receptors (Notch1-4) during embryonic/fetal myogenesis are still elusive. In this study, we found that Notch1 and Notch2, which exhibit the highest structural similarity among Notch receptors, maintain the MP population by distinct mechanisms: Notch1 induces cell cycle exit and Notch2 suppresses premature differentiation. Moreover, genetic and cell culture studies showed that Notch1 and Notch2 signaling in MPs are distinctively activated by interacting with Notch ligand-expressing myofibers and MP-lineage cells, respectively. These results suggest that through different activation modes, Notch1 and Notch2 distinctively and cooperatively maintain MP population during fetal myogenesis for proper muscle development.

Identification of 8-gene risk prediction signature associated with NOTCH1 in stomach adenocarcinoma based on bioinformatics analysis.

BackgroundStomach adenocarcinoma (STAD), is the most common histological type of gastric cancer (GC) with high mortality and poor prognosis. We sought to investigate the contribution of Notch receptor 1 (NOTCH1) to STAD immunity.MethodsThe profiles of immune cells in STAD cohorts were compared, and a correlation analysis between the NOTCH1 gene and tumor immune cell infiltration was then conducted. The immune-related genes (IRGs) associated with the NOTCH1 gene were identified. Based on the NOTCH1-associated IRGs, multiple-gene risk prediction signatures were established. The relationship between the expression levels of the selected IRGs and overall survival (OS) was analyzed by a univariate analysis. The risk score was calculated using the formula of β1x1 + β2x2 +... + βixi. A prognostic nomogram was constructed to predict individuals’ survival probabilities.ResultsIn STAD, NOTCH1 expression levels were significantly negatively associated with tumor-infiltrating lymphocyte (TIL) Act dendritic cells (DCs) (r=−0.196, P value =6.24e-05), TIL cluster of differentiation (CD) 56 bright cells (r=−0.115, P value =0.0193), TIL immature DCs (r=−0.293, P value =1.16e-09), TIL monocyte cells (r=−0.185, P value =0.000149), TIL central memory T CD4 cells (r=−0.126, P value =0.0103), and TIL gamma delta T cells (r=−0.149, P value =0.00229). The resulting risk scores of the 8-gene risk prediction signature (corticotrophin releasing hormone receptor 2 (CRHR2) (HR =1.858, P value =0.048), fms related receptor tyrosine kinase 1 (FLT1) (HR =1.268, P value =0.048), fms related receptor tyrosine kinase 4 (FLT4) (HR =1.334, P value =0.031), glial fibrillary acidic protein (GFAP) (HR =2.739, P value =0.008), platelet-derived growth factor receptor beta (PDGFRB) (HR =1.192, P value =0.02), prostaglandin D2 receptor (PTGDR) (HR =1.564, P value =0.049), semaphorin 5B (SEMA5B) (HR =1.154, P value =0.029), and tyrosine kinase 2 (TYK2) (HR =0.734, P value =0.041) were independent prognostic predictors for STAD patients.ConclusionsNOTCH1 could be a potential target for STAD. The mechanisms underpinning NOTCH1-medicated prognostic values of immune signatures should be further explored.

NOTCH1 mutation associates with impaired immune response and decreased relapse-free survival in patients with resected T1-2N0 laryngeal cancer.

BackgroundPatients with early-stage laryngeal cancer, even stage T1-2N0, are at considerable risk of recurrence and death. The genetic and immunologic characteristics of recurrent laryngeal cancer remain unclear.MethodsA total of 52 T1-2N0 laryngeal cancer patients were enrolled. Of these, 42 tissue samples were performed by targeted DNA sequencing, and 21 cases were performed by NanoString immuno-oncology targeted RNA sequencing to identify the distinct molecular bases and immunologic features associated with relapse in patients with early laryngeal cancer, respectively.ResultsTo the best to our knowledge, we present for the first time an overview of the genomic mutation spectrum of early-stage laryngeal cancers. A total of 469 genomic alterations were detected in 211 distinct cancer-relevant genes, and the genes found to be mutated in more than five patients (>10%) included tumor protein p53 (TP53, 78.5%), FAT atypical cadherin 1 (FAT1, 26%), LDL receptor related protein 1B (LRP1B, 19%), cyclin dependent kinase inhibitor 2A (CDKN2A, 17%), tet methylcytosine dioxygenase 2 (TET2, 17%), notch receptor 1 (NOTCH1, 12%) and neuregulin 1 (NRG1, 12%). Recurrent laryngeal cancer demonstrated a higher tumor mutation burden (TMB), as well as higher LRP1B mutation and NOTCH1 mutation rates. Univariate and multivariate analyses revealed that high TMB (TMB-H) and NOTCH1 mutation are independent genetic factors that are significantly associated with shorter relapse-free survival (RFS). Simultaneously, the results of the transcriptome analysis presented recurrent tumors with NOTCH1 mutation displayed upregulation of the cell cycle pathway, along with decreased B cells score, T cells score, immune signature score and tumor-infiltrating lymphocytes (TILs) score. The Cancer Genome Atlas (TCGA)-laryngeal cancer dataset also revealed weakened immune response and impaired adhesion functions in NOTCH1-mutant patients.ConclusionsGenomic instability and impaired immune response are key features of the immunosurveillance escape and recurrence of early laryngeal cancer after surgery. These findings revealed immunophenotypic attenuation in recurrent tumors and provided valuable information for improving the management of these high-risk patients. Due to the small number of patients in this study, these differences need to be further validated in a larger cohort.

Notch-dependent and -independent functions of transcription factor RBPJ.

Signal transduction pathways often involve transcription factors that promote activation of defined target gene sets. The transcription factor RBPJ is the central player in Notch signaling and either forms an activator complex with the Notch intracellular domain (NICD) or a repressor complex with corepressors like KYOT2/FHL1. The balance between these two antagonizing RBPJ-complexes depends on the activation state of the Notch receptor regulated by cell-to-cell interaction, ligand binding and proteolytic cleavage events. Here, we depleted RBPJ in mature T-cells lacking active Notch signaling and performed RNA-Seq, ChIP-Seq and ATAC-seq analyses. RBPJ depletion leads to upregulation of many Notch target genes. Ectopic expression of NICD1 activates several Notch target genes and enhances RBPJ occupancy. Based on gene expression changes and RBPJ occupancy we define four different clusters, either RBPJ- and/or Notch-regulated genes. Importantly, we identify early (Hes1 and Hey1) and late Notch-responsive genes (IL2ra). Similarly, to RBPJ depletion, interfering with transcriptional repression by squelching with cofactor KYOT2/FHL1, leads to upregulation of Notch target genes. Taken together, RBPJ is not only an essential part of the Notch co-activator complex but also functions as a repressor in a Notch-independent manner.

DNMT1-mediated demethylation of lncRNA MEG3 promoter suppressed breast cancer progression by repressing Notch1 signaling pathway

ABSTRACT Breast carcinoma is one of the common causes of cancer-related mortality in women. Maternally expressed gene 3 (MEG3), a lncRNA located at 14q32, can be involved in carcinogenesis. In this study, we discovered that MEG3 was downregulated by CpG hypermethylation within its gene promoter. Functionally, treatment of breast cancer cells with the DNA methylation inhibitor 5-AzadC as well as silencing of DNA methyltransferase-1 (DNMT1) could decrease the abnormal hypermethylation of the MEG3 promoter, reverse MEG3 expression, inhibit cell proliferation and promote cell apoptosis. In addition, we found that MEG3 expression was negatively correlated with DNMT1. Mechanistically, MEG3 knockdown combined with 5-AzadC or sh-DNMT1 treatment restored the expression of Notch1 receptor, leading to the Notch1 pathway activation, and promoted the progression of epithelial mesenchymal transformation (EMT). Finally, the mice tumor model experiments showed that DNMT1 knockdown can increase MEG3 expression and inhibit tumor growth. Collectively, our findings uncovered that DNMT1-mediated MEG3 demethylation leads to MEG3 upregulation, which in turn inhibits the Notch1 pathway and EMT process in breast cancer.

Effects of the interaction of Notch and TLR4 pathways on inflammation and heart function in septic heart.

We investigated the role of the interaction between the Notch and Toll-like receptor 4 (TLR4) pathways in septic myocardial injury. The sepsis model was induced in rats with lipopolysaccharide (LPS). Rats were divided into control, LPS, LPS + TAK242 ((6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate) and LPS + DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycinetbutylester) groups. Heart function was evaluated with a Cardiac Doppler ultrasound. Myocardial morphological changes were detected by hematoxylin-eosin staining (H&E). Apoptosis was assessed by a TUNEL assay. The mRNA and protein levels were detected with real-time PCR, Western blot, and immunohistochemistry analysis. We found that heart function in the LPS + TAK242 group was significantly improved, but not in the LPS + DAPT group. LPS + TAK242 had a lower level of degeneration and necrosis of cardiomyocytes and inflammatory cell infiltration, as well as lower apoptosis and caspase-3 expression than the LPS group. Compared with the LPS group, the inflammatory cell infiltration was reduced in the LPS + DAPT group, while the degeneration and necrosis of cardiomyocytes were not obviously improved. Additionally, the expression levels of tumor necrosis factor-α and Interleukin-6, the protein contents of Notch intracellular domain and Hes1, and the P65 nuclear factor kappa-B (NF-κB) to P-P65 NF-κB ratio in LPS + TAK242 group and LPS + DAPT group were significantly lower than those in LPS group. Conclusively, the interaction between TLR4 and Notch signaling pathways enhances the inflammatory response in the septic heart by activating NF-κB. Blocking the TLR4 pathway with TAK242 can improve heart dysfunction and myocardial damage in sepsis, while blocking the Notch pathway with DAPT cannot effectively prevent heart dysfunction and myocardial damage in sepsis.

Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2–/– mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone–like B cells when transferred into Rag2–/– lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.

Notch signaling determines cell-fate specification of the two main types of vomeronasal neurons of rodents.

ABSTRACTThe ability of terrestrial vertebrates to find food and mating partners, and to avoid predators, relies on the detection of chemosensory information. Semiochemicals responsible for social and sexual behaviors are detected by chemosensory neurons of the vomeronasal organ (VNO), which transmits information to the accessory olfactory bulb. The vomeronasal sensory epithelium of most mammalian species contains a uniform vomeronasal system; however, rodents and marsupials have developed a more complex binary vomeronasal system, containing vomeronasal sensory neurons (VSNs) expressing receptors of either the V1R or V2R family. In rodents, V1R/apical and V2R/basal VSNs originate from a common pool of progenitors. Using single cell RNA-sequencing, we identified differential expression of Notch1 receptor and Dll4 ligand between the neuronal precursors at the VSN differentiation dichotomy. Our experiments show that Notch signaling is required for effective differentiation of V2R/basal VSNs. In fact, Notch1 loss of function in neuronal progenitors diverts them to the V1R/apical fate, whereas Notch1 gain of function redirects precursors to V2R/basal. Our results indicate that Notch signaling plays a pivotal role in triggering the binary differentiation dichotomy in the VNO of rodents.

O-217 Associations between endometrial stem cells quantities, serum levels of progesterone and estradiol and expression of their respective receptors in endometrial tissue during the mid-luteal phase

Abstract Study question Is the percentage of endometrial stem cells linked to serum progesterone, serum estradiol, and their receptors expression in human endometrium during the mid-luteal phase? Summary answer We found an association between CD117+ endometrial stem cells and serum progesterone, estradiol, as well as with the expression of their receptors in the endometrium. What is known already Various types of stem cells including NOTCH1+ and CD117+ stromal cells have been found in the endometrium. Their quantities have been hypothesised to fluctuate with the menstrual cycle and to have a link with endometrial receptivity. It has been well established that progesterone and estradiol play a crucial role in the preparation of the endometrium for implantation and the expression of their receptors also varies with the menstrual cycle. Nonetheless, the relationship between stem cells and other established markers of endometrial receptivity, such as the sex hormones signaling system, constitutes a novel field of research. Study design, size, duration The present observational study included 87 women aged 26-56 (mean 39.5 years old) undergoing in-vitro fertilisation and for whom a peripheral blood sample and an endometrial biopsy were obtained during the mid-luteal phase (LH + 7) of a natural cycle. Immunohistochemical (IHC) markers for NOTCH1 (E-AB-12815, Elabscience), CD117 (CD117/c-Kit/SCF-Receptor RB-9038-RQ, Epredia), progesterone (PR) and estrogen receptors (ER) (1-PR026-07, 1-ES006-07, Quartett) were used to stain endometrial tissue. The investigation was carried out between March 2020 and January 2022. Participants/materials, setting, methods Serum progesterone and estradiol were measured via electrochemiluminescence using Cobas e411 analyser (Roche Diagnostics, Germany) on the day of obtaining the endometrial biopsy. Stem cells (count and percentage) and the expression of hormone receptors (6-point scale) were determined using ImageJ. Stem cells percentage was compared between groups with high and low progesterone (analogously for estradiol) using Wilcoxon signed-rank test. To assess the link between stem cell percentages and receptor expression, Spearman correlation was used. Main results and the role of chance The mean percentage of positively stained NOTCH1 endometrial stromal stem cells was 0.13 ± 0.29% and CD117+ cells was 0.034 ± 0.039%. The hormone serum levels ranged from 0.2 to 60 ng/mL (median of 14.9 ng/mL) for progesterone and from 12 to 1985 pg/mL (median of 135.6 pg/mL) for estradiol. While the quantities of the two stem cell types demonstrated a positive correlation with each other (R = 0.379, p < 0.001), the percentage of NOTCH1+ stem cells showed no difference between the studied serum hormone levels (p > 0.05), nor revealed a relationship with the expression of their receptors (p > 0.05). In contrast, the amount of CD117+ cells differed significantly between patients with high and low serum progesterone (cutoff 14.9 ng/mL, p = 0.001) and estradiol levels (cutoff 135.6 pg/mL, p = 0.019). Furthermore, the quantity of stem cells positive for CD117 correlated positively with the two hormone receptor levels in the endometrium – PR (R = 0.277, p = 0.019) and ER (R = 0.318, p = 0.007). Limitations, reasons for caution A limitation of the study was the sample size. These findings should be confirmed using a larger population. In addition, confounding variables such as the immunological profile of the tissue should also be taken into account. Wider implications of the findings This study shows that certain endometrial stem cell types (CD117+) but not others (NOTCH1+) are associated with sex hormone signaling during the mid-luteal phase. This relationship highlights the role these cells play in preparing the endometrium for embryo implantation and their quantity might be an indirect indicator of endometrial receptivity. Trial registration number not applicable

Computational analysis of the role of mechanosensitive Notch signaling in arterial adaptation to hypertension

Arteries grow and remodel in response to mechanical stimuli. Hypertension, for example, results in arterial wall thickening. Cell-cell Notch signaling between vascular smooth muscle cells (VSMCs) is known to be involved in this process, but the underlying mechanisms are still unclear. Here, we investigated whether Notch mechanosensitivity to strain may regulate arterial thickening in hypertension. We developed a multiscale computational framework by coupling a finite element model of arterial mechanics, including residual stress, to an agent-based model of mechanosensitive Notch signaling, to predict VSMC phenotypes as an indicator of growth and remodeling. Our simulations revealed that the sensitivity of Notch to strain at mean blood pressure may be a key mediator of arterial thickening in hypertensive arteries. Further simulations showed that loss of residual stress can have synergistic effects with hypertension, and that changes in the expression of Notch receptors, but not Jagged ligands, may be used to control arterial growth and remodeling and to intensify or counteract hypertensive thickening. Overall, we identify Notch mechanosensitivity as a potential mediator of vascular adaptation, and we present a computational framework that can facilitate the testing of new therapeutic and regenerative strategies.

Notch signaling activation contributes to paclitaxel-induced neuropathic pain via activation of A1 astrocytes

Paclitaxel-induced neuropathic pain (PINP) is a progressive and refractory side effect of chemotherapy with few effective treatments at present. It is well-established that astrocytes activation contributes to the development of PINP. Recent reports showed astrocytes can be divided into A1 and A2 phenotypes. However, whether the transformation of astrocytes participates in PINP and the underlying mechanisms remain unknown. As Notch signaling pathway have shown to be involved in neuropathic pain, we aimed to investigate the relationship between Notch signaling pathway and A1 astrocytes in PINP. Herein we found that both A1 astrocytes and Notch signaling were markedly activated in the spinal cord of PINP rats and the downstream molecules of Notch signaling were colocalized with A1 astrocytes. DAPT (an inhibitor of Notch signaling) not only suppressed the mechanical allodynia of PINP rats, but also inhibited the activation of Notch signaling pathway and A1 astrocytes. Furthermore, Jagged1 (a ligand of Notch1 receptors) dose-dependently induced mechanical hyperalgesia in naïve rats and simultaneously led to Notch signaling activation and A1 astrocytes transformation, all of which were inhibited by DAPT. Taken together, these results demonstrate Notch signaling activation contributes to PINP via A1 astrocytes activation, which provides a promising therapeutic target for PINP.

Multiple Mechanisms of NOTCH1 Activation in Chronic Lymphocytic Leukemia: NOTCH1 Mutations and Beyond.

Simple SummaryMutations of the NOTCH1 gene are a validated prognostic marker in chronic lymphocytic leukemia and a potential predictive marker for anti-CD20-based therapies. At present, the most frequent pathological alteration of the NOTCH1 gene is due to somatic genetic mutations, which have a multifaceted functional impact. However, beside NOTCH1 mutations, other factors may lead to activation of the NOTCH1 pathway, and these include mutations of FBXW7, MED12, SPEN, SF3B1 as well as other B-cell pathways. Understanding the preferential strategies though which CLL cells hijack NOTCH1 signaling may present important clues for designing targeted treatment strategies for the management of CLL.The Notch signaling pathway plays a fundamental role for the terminal differentiation of multiple cell types, including B and T lymphocytes. The Notch receptors are transmembrane proteins that, upon ligand engagement, undergo multiple processing steps that ultimately release their intracytoplasmic portion. The activated protein ultimately operates as a nuclear transcriptional co-factor, whose stability is finely regulated. The Notch pathway has gained growing attention in chronic lymphocytic leukemia (CLL) because of the high rate of somatic mutations of the NOTCH1 gene. In CLL, NOTCH1 mutations represent a validated prognostic marker and a potential predictive marker for anti-CD20-based therapies, as pathological alterations of the Notch pathway can provide significant growth and survival advantage to neoplastic clone. However, beside NOTCH1 mutation, other events have been demonstrated to perturb the Notch pathway, namely somatic mutations of upstream, or even apparently unrelated, proteins such as FBXW7, MED12, SPEN, SF3B1, as well as physiological signals from other pathways such as the B-cell receptor. Here we review these mechanisms of activation of the NOTCH1 pathway in the context of CLL; the resulting picture highlights how multiple different mechanisms, that might occur under specific genomic, phenotypic and microenvironmental contexts, ultimately result in the same search for proliferative and survival advantages (through activation of MYC), as well as immune escape and therapy evasion (from anti-CD20 biological therapies). Understanding the preferential strategies through which CLL cells hijack NOTCH1 signaling may present important clues for designing targeted treatment strategies for the management of CLL.

Role of Microbial Metabolites of Histidine in the Development of Colitis.

Colitis is a chronic relapsing inflammatory disease of colon. Clinical studies show that meat-rich diet plays a critical role in the relapse of colitis. However, it is unclear whether the microbial metabolites of histidine, which is an amino acid widely found in meat, have an impact on the health of the intestine. Six metabolites of histidine are given to IEC-6 cells. The cell activity measurement shows that imidazole propionate (IMP) is the most detrimental metabolite. Then, IMP is injected to mice by rectal administration, with blood and colon tissues collected for the measurement of colitis related parameters. The results show that treatment with IMP significantly increased NF-κB, iNOS, and IL-6, decreased number of goblet cell, and inhibited expressions of miR-146b. However, overexpression of miR-146b in mice rescues the decline of the physical condition. Additionally, Notch receptor 1 (Notch1) is identified as a target gene of miR-146b. Further analysis shows that miR-146b restored the abundance of goblet cells by regulating Notch1 signaling pathway. IMP is able to induce intestinal inflammation, impairs the intestinal barrier, and affects the proliferation of goblet cells. The underlined mechanism may partially contribute to the dysregulation of miR-146b/Notch1 axis.

Investigation of the Anticancer Effect of S-Allyl-L Cysteine on the C6 Rat Glioma Cell Line in vitro in 2D- and 3D-Cell Culture Models.

In our study, we aimed to investigate the effects of SAC on C6 glioblastoma cells and its antiproliferative and apoptotic effects on two- and three-dimensional cell culture systems. The rat glioma cell line C6 was separated into 2D-Control, 2D-SAC, 3D-CMC-Control and 3D-CMC-SAC groups. While control cells were incubated under standard culture conditions, cells in the SAC groups were incubated with the IC50 dose (50 µM for both the 2D-SAC C6 and 3D-CMC-SAC groups) of SAC in culture medium for 24 and 48 hours. Thereafter, all groups were stained with antibodies recognizing NOTCH1 and JAGGED1, and the mRNA expression levels of NOTCH1 and JAGGED1 were evaluated by qRT-PCR. Increasing doses of SAC were administered for 24 hours in the C6 glioma cell line. The concentration of 50 μM was chosen as the most suitable dose for administration. The gene expression profiles were different in the two forms of culture. NOTCH1 receptor mRNA expression levels were significantly decreased in cells treated with 50 μM SAC for 24 hours compared to control cells in both the two-dimensional (2D) and three-dimensional (3D) cell cultures. The immunoreactivities of both of these markers (JAGGED1 and NOTCH1) were significantly decreased in the glioma cells in the SAC group compared to the control group. This study shows that SAC has potential as a drug for human use, as indicated by its nontoxic nature. The present study confirmed the anticancer activity of SAC by modulating NOTCH1 and JAGGED1 signaling in glioma cancer cells.

Amelioration of high-fat diet (HFD) + CCl4 induced NASH/NAFLD in CF-1 mice by activation of SIRT-1 using cinnamoyl sulfonamide hydroxamate derivatives: in-silico molecular modelling and in-vivo prediction

Non-alcoholic fatty liver disease (NAFLD) is one of the major hepatic metabolic disorders that occurs because of the accumulation of lipids in hepatocytes in the form of free fatty acids (FFA) and triglycerides (TG) which become non-alcoholic steatohepatitis (NASH). NOTCH-1 receptors act as novel targets for the development of NAFLD/NASH, where overexpression of NOTCH-1 receptor alters the lipid metabolism in hepatocytes leading to NAFLD. SIRT-1 deacetylates the NOTCH-1 receptor and inhibits NAFLD. Hence, computer-aided drug design (CADD) was used to check the SIRT-1 activation ability of cinnamic sulfonyl hydroxamate derivatives (NMJ 1–8), resveratrol, and vorinostat. SIRT-1 (PDB ID: 5BTR) was docked with eight hydroxamate derivatives and vorinostat using Schrödinger software. Based on binding energy obtained (– 26.31 to – 47.34 kcal/mol), vorinostat, NMJ-2, NMJ-3, NMJ-5 were selected for induced-fit docking (IFD) and results were within – 750.70 to – 753.22 kcal/mol. Qikprop tool was used to analyse the pre pharmacokinetic parameters (ADME analysis) of all hydroxamate compounds. As observed in the molecular dynamic (MD) study, NMJ-2, NMJ-3 were showing acceptable results for activation of SIRT-1. Based on these predictions, in-vivo studies were conducted in CF1 mice, where NMJ-3 showed significant (p < 0.05) changes in lipid profile and anti-oxidant parameters (Catalase, SOD, GSH, nitrite, and LPO) and plasma insulin levels. NMJ-3 treatment also reduced inflammation, fibrosis, and necrosis in liver samples.