Inhibition Of Notch Pathway Research Articles

The Nerve-Induced Adipose Stem Cells Promote Nerve Repair in Stress Urinary Incontinence by Regulating Schwann Cell Repair Phenotype Conversion Through Activation of the Notch Pathway.

Stress urinary incontinence (SUI) currently lacks effective treatment options, and the restoration of neurological function remains a major challenge, with unmet clinical needs. Research has indicated that adipose-derived stem cells (ADSCs) can be induced to differentiate into neural-induced adipose-derived stem cells (NI-ADSCs) under specific inductive conditions, exhibiting excellent neuroregenerative capabilities. ADSCs were obtained from female SD rats and induced into NI-ADSCs. In vitro, NI-ADSCs were co-cultured with Schwann cells (SCs) to investigate their effects on SC proliferation and repair phenotype transition and further explore its underlying mechanism. In vivo, a rat model of SUI was established using a bilateral pudendal nerve transection method. NI-ADSCs were injected into the urethral sphincter to evaluate their effects on urodynamics, muscle angiogenesis, and neural repair in SUI rats, while also exploring the mechanisms of neural repair. This study used EGF, FGF, and B27 to induce ADSCs into NI-ADSCs expressing neural induction markers (MAP, Nestin, and PAX6). In vitro experiments found no significant difference in the proliferation of L6 and RSC96 between NI-ADSCs and ADSCs (p > 0.05). However, when co-cultured with NI-ADSCs, SCs showed upregulated expression of repair-related phenotypic markers (BDNF, GDNF, and GFAP). In this phenotypic transformation process, the expression of Notch-related pathway proteins (Notch1, NICD, and Hes1) was increased, and the use of DAPT (a Notch pathway inhibitor) could suppress the SC repair phenotype transformation. In vivo, experiments revealed that intraurethral injection of NI-ADSCs significantly promoted the expression of neural marker (S100β) and demyelination markers (GFAP) and urodynamic recovery in SUI rats, while DAPT inhibited its neural repair effect. In summary, our study demonstrates that NI-ADSCs can promote nerve regeneration by promoting and maintaining the repair-related phenotype of SCs. The underlying mechanism may be related to the activation of the Notch signaling pathway.

Cadmium Reduces VE-Cadherin Expression in Endothelial Cells Through Activation of the Notch Signaling Pathway.

Cadmium (Cd) is a toxic heavy metal which induces vascular disorders. Previous studies suggest that Cd in the bloodstream affects vascular endothelial cells (ECs), potentially contributing to vascular-related diseases. However, the molecular mechanisms of effects of Cd on ECs remain poorly understood. Notch signaling pathway abnormalities have been implicated in ECs disruption. The present study aims to investigate the effect of low Cd concentrations on the Notch signaling pathway in ECs. Mice were treated with low concentration of Cd (2.28 mg/kg), and tissues were collected for examination of mRNA and protein levels of Notch pathway components and VE-cadherin, a major junctional protein in ECs. We found that Cd treatment increases expression of NICD1, Hes1, Hey1, Hey2 and decreases expression of VE-cadherin in brain and kidney tissues. In vitro, a low concentration of Cd (1 μM) also induces increase expression of NICD1, Hes1, Hey1, Hey2, and decrease expression of VE-cadherin in human umbilical vein endothelial cells (HUVECs). Low concentration of Cd increased the permeability of HUVECs. We also found that Notch signaling negatively regulates the expression of VE-cadherin. In addition, DAPT, a Notch pathway inhibitor, prevents Cd-induced reduction in VE-cadherin expression in HUVECs. In summary, these findings revealed that Cd exposure decreases VE-cadherin expression through activation of the Notch signaling pathway.

Zuogui pills ameliorate chemotherapy-induced ovarian aging by improving stemness, regulating cell cycle and reducing apoptosis of oogonial stem cells via the Notch1/Nrf2 pathway

Ethnopharmacological relevanceZuogui Pills (ZGP) is a classic traditional Chinese herbal formula originating from the Ming Dynasty. It has been widely used in the treatment of kidney deficiency-related diseases, including ovarian aging. Aim of the studyTo investigate the effects and potential mechanisms of ZGP on ovarian aging induced by the chemotherapeutic agent cyclophosphamide (CTX), as well as its impact on the therapeutic target, oogonial stem cells (OSCs), involving the Notch1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Materials and methodsThis study utilized High-Performance Liquid Chromatography (HPLC) to analyze the active components of Zuogui Pills (ZGP). In vivo experiments involved the establishment of an ovarian aging model in female rats through intraperitoneal injection of CTX, followed by an 8-week treatment with ZGP and dehydroepiandrosterone (DHEA). The Notch pathway inhibitor DAPT was administered via intraperitoneal injection, followed by ZGP intervention to validate its therapeutic effects. Transcriptomic sequencing was used to analyze the differential genes before and after ZGP treatment of CTX-induced ovarian aging, and KEGG and GO analyses were applied to assess the changes in relevant signaling pathways and biological processes. In vitro experiments included the extraction, separation, and purification of ovarian germ stem cells, followed by transfection with a Notch1 overexpression plasmid. The CTX active component 4-Hydroxycyclophosphamide (4HC) was used for model intervention, and ZGP, DHEA-containing serum, and DAPT were applied to intervene with the oogonial stem cells. The effects of CTX modeling, the therapeutic efficacy of ZGP, and the general condition of the rats were observed. H&E staining was employed to assess ovarian morphology and follicle counting at various stages. Serum hormone levels were measured using ELISA, while qPCR, Western blot, flow cytometry, immunofluorescence, and IHC were utilized to analyze the expression of the Notch1/Nrf2 pathway, cell cycle proteins, and stemness-related indicators. Flow cytometry, TUNEL fluorescence, and CCK8 assays were conducted to evaluate changes in cell cycle composition, apoptosis, and proliferation. Finally, ChIP-qPCR was employed to validate the transcriptional regulation of the target gene NFE2L2 by Notch1. ResultsZGP improved serum sex hormones in ovarian aging rats, enhanced ovarian index, and optimized ovarian and uterine morphology, as well as follicle quantity composition. After transcriptome sequencing, KEGG analysis enriched the Notch signaling pathway and cell cycle, while GO analysis highlighted enrichment in the Notch pathway and stem cell population maintenance. Various experiments validated that ZGP significantly improved the expression of cell cycle-related proteins Cyclin D1 (CCND1), Cyclin E1 (CCNE1), cyclin-dependent kinase inhibitor 1a (CDKN1A), stemness markers Mouse Vasa Homolog (MVH), Octamer-binding Transcription Factor 4 (Oct4), Fragilis, 5-Bromo-2′-deoxyuridine (BrdU), as well as Notch1 and Nrf2 in aging ovarian tissues and OSCs. Additionally, ZGP promoted the proliferation of 4HC-damaged OSCs, optimized OSCs cell cycle composition, reduced G0/G1 phase arrest, and decreased early and late apoptosis. ZGP could reverse the detrimental effects on stemness and cell cycle of OSCs caused by blocking the Notch pathway. Furthermore, ZGP may activate the regulation of its target gene NFE2L2 by upregulating Notch1 expression in OSCs, thereby exerting therapeutic effects. ConclusionZGP protects ovarian function in CTX-induced ovarian aging rats by regulating the Notch1/Nrf2 pathway. It restores serum sex hormone levels, maintains normal follicle development, promotes the proliferation of aged OSCs, optimizes the cell cycle, reduces apoptosis, and preserves stemness, thereby alleviating ovarian aging.

Evaluating the impact of Xanthoparmelia conspersa extracts on signaling in HeLa cells and exploring their diverse biological activities

Xanthoparmelia conspersa is rich in specific secondary metabolites but an unexplored lichen species. This work determined the chemical composition and biological activities (anti-microbial, anti-protozoal, and cytotoxic) of its methanolic and hexane extracts. Additionally, we evaluated the potential of these extracts in modulating cancer signaling pathways in HeLa cells. The phytochemical analysis revealed that usnic acid was the predominant constituent in the hexane extract, while stictic acid was in the methanolic one. Among tested cell lines (VERO, FaDu, SCC-25, HeLa), cytotoxic selectivity was detected for X. conspersa hexane extract against the FaDu (SI 7.36) and HeLa (SI 2.19) cells. A noticeably better anti-microbial potential was found for hexane extract, however, the overall anti-microbial activity was relatively weak (28, 21, and 20% inhibition of Candida glabrata, Cryptococcus neoformans, and Escherichia coli, respectively). On the contrary, the anti-parasitic action of hexane extract was significant, with an IC50 value of 2.64 µg/mL against Leishmania donovani – amastigote and 7.18 µg/mL against Trypanosoma brucei. The detailed evaluation of the cancer-related signaling pathways in HeLa cells, done by two distinct methodologies (luciferase reporter tests), revealed that especially the hexane extract and usnic acid exhibited selective inhibition of Stat3, Smad, NF-κB, cMYC, and Notch pathways.

VEGF-C propagates 'onward' colorectal cancer metastasis from liver to lung.

The formation of lung metastasis as part of the progression of colon cancer is a poorly understood process. Theoretically, liver metastases could seed lung metastases. To assess the contribution of the liver lymphatic vasculature to metastatic spread to the lungs, we generated murine liver-metastasis-derived organoids overexpressing vascular endothelial growth factor (VEGF)-C. The organoids were reimplanted into the mouse liver for tumour generation and onward metastasis. Liver metastases from patients with concomitant lung metastases showed higher expression of VEGF-C, lymphatic vessel hyperplasia, and tumour cell invasion into lymphatic vessels when compared to those without lung metastases. Reimplantation of VEGF-C overexpressing organoids into the mouse liver showed that VEGF-C caused peritumoral lymphatic vessel hyperplasia, lymphatic tumour cell invasion, and lung metastasis formation. This change in metastatic organotropism was accompanied by reduced expression of WNT-driven adult stem cell markers, and increased expression of fetal stem cell markers and NOTCH pathway genes. Further NOTCH pathway inhibition with γ-secretase inhibitor (DAPT) in vivo results in a slight reduction in lung metastases and a decrease in lymphatic hyperplasia and invasion in VEGF-C-overexpressing tumours. Collectively, these data indicate that VEGF-C can drive onward metastasis from the liver to the lung and suggest that targeting VEGF-C/NOTCH pathways may impair the progression of colorectal cancer.

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin.

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the β-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

Activation of the γ-secretase/NICD-PXR/Notch pathway induces Taxol resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is currently the only subtype lacking efficient targeted therapies. Taxol is the primary chemotherapeutic agent for TNBC. However, Taxol resistance often develops in the treatment of TNBC patients, which importantly contributes to high mortality and poor prognosis in TNBC patients. Recent preclinical studies have shown that the inhibition of Notch pathway by γ-secretase inhibitors can slow down the progression of TNBC. Our studies in bioinformatic analysis of breast cancer patients and TNBC/Taxol cells in vitro showed that there was high correlation between the activation of Notch pathway and Taxol resistance in TNBC. Increased γ-secretase activity (by the overexpression of catalytic core PSEN-1) significantly reduced Taxol sensitivity of TNBC cells, and enhanced biological characteristics of malignancy in vitro, and tumour growth in vivo. Mechanistically, increased γ-secretase activity led to the accumulation of NICD in the nucleus, promoting the interaction between NICD and PXR to activate PXR, which triggered the transcription of PXR downstream associated drug resistance genes. Furthermore, we showed that pharmacological inhibition of γ-secretase with γ-secretase inhibitors (Nirogacestat and DAPT) can reverse Taxol resistance in vivo and in vitro. Our results for the first time demonstrate that the activation of γ −secretase/NCD-PXR/Notch pathway is one of important mechanisms to cause Taxol resistance in TNBC, and the blockades of this pathway may represent a new therapeutic strategy for overcoming Taxol resistance in TNBC.

SOX10 mediates glioblastoma cell-state plasticity

Phenotypic plasticity is a cause of glioblastoma therapy failure. We previously showed that suppressing the oligodendrocyte-lineage regulator SOX10 promotes glioblastoma progression. Here, we analyze SOX10-mediated phenotypic plasticity and exploit it for glioblastoma therapy design. We show that low SOX10 expression is linked to neural stem-cell (NSC)-like glioblastoma cell states and is a consequence of temozolomide treatment in animal and cell line models. Single-cell transcriptome profiling of Sox10-KD tumors indicates that Sox10 suppression is sufficient to induce tumor progression to an aggressive NSC/developmental-like phenotype, including a quiescent NSC-like cell population. The quiescent NSC state is induced by temozolomide and Sox10-KD and reduced by Notch pathway inhibition in cell line models. Combination treatment using Notch and HDAC/PI3K inhibitors extends the survival of mice carrying Sox10-KD tumors, validating our experimental therapy approach. In summary, SOX10 suppression mediates glioblastoma progression through NSC/developmental cell-state transition, including the induction of a targetable quiescent NSC state. This work provides a rationale for the design of tumor therapies based on single-cell phenotypic plasticity analysis.

The new pattern for dual NOTCH pathway involving nuclear transcription and mitochondrial regulation supports therapeutic mechanism of 4-butyl benzophenone derivatives against SIRS

The systemic inflammatory response syndrome (SIRS) represents a self-amplifying cascade of inflammatory reactions and pathophysiological states triggered by infectious or non-infectious factors. The identification of disease targets and differential proteins in the liver (the unique and important immune organ) of SIRS mice treated with the lead compound D1 was conducted using the Genecards database and proteomic analysis, respectively. Subsequently, NOTCH1 was identified as the potential hub target via an intersection analysis between the aforementioned differentially expressed proteins and disease targets. Based on our previous research on the structure-activity relationship, we designed and synthesized a series of SIRS-related derivatives, wherein butyl, halogen, and ester groups were incorporated into benzophenone, aiming at exploring the anti-inflammatory protective action from the perspective of macrophage polarization. Notably, these derivatives exhibited a direct binding capability to the O-glucosylation site (SER496) or its vicinities (such as SER492, VAL485) of NOTCH1 using docking, SPR, DARTS, and CETSA techniques. Mechanistically, derivative D6 exerted anti-inflammatory effects via the dual NOTCH pathway. Firstly, it could inhibit NOTCH1 nuclear transcriptional activity, attenuate the interaction between NICD and RBPJK, concurrently suppress NF-κB and NLRP3 inflammasome (NLRP3, ASC, and cleaved CASP1) activation, and promote NICD (NOTCH1 active fragments) ubiquitination metabolism (the nuclear transcriptional pathway). Secondly, it might possess the ability to increase PGC1α level, subsequently, enhance ATP and MMP levels, mitigate ROS production, increase mitochondrial numbers, and ameliorate mitochondrial inflammatory damage (the mitochondrial pathway). Importantly, the activator Jagged1 could effectively reverse the aforementioned effects, while the inhibitor DAPT exhibited a synergistic effect, suggesting that the nuclear transcriptional regulation and mitochondrial regulation were both in a NOTCH1-dependent manner. Subsequently, it effectively alleviated the inflammatory response and preserved organ function as evidenced by up-regulating M2-type macrophage-related anti-inflammatory cytokines (IL10, TGFβ, CD206, and ARG1) and down-regulating M1-type macrophage-related pro-inflammatory cytokines (NO, IL6, IL18, iNOS, TNFα, CD86, and IL1β). In a word, derivative D6 modulated macrophage polarization and effectively mitigated SIRS by targeting inhibition of the dual NOTCH pathway.

Dibenzazepine, a γ-Secretase Enzyme Inhibitor, Protects Against Doxorubicin-Induced Cardiotoxicity by Suppressing NF-κB, iNOS, and Hes1/Hey1 Expression.

Doxorubicin (DOX) is an effective chemotherapeutic drug; however, its cardiotoxicity and resistance compromise its therapeutic index. The Notch pathway was reported to contribute to DOX cancer resistance. The role of Notch pathway in DOX cardiotoxicity has not been identified yet. Notch receptors are characterized by their extracellular (NECD) and intracellular(NICD) domains (NICD). The γ-secretase enzyme helps in the release of NICD. Dibenzazepine (DBZ) is a γ-secretase inhibitor. The present study investigated the effect of Notch pathway inhibition on DOX cardiotoxicity. Twenty-four male Wistar rats were divided into four groups: control group, DOX group, acute cardiotoxicity was induced by a single dose of DOX (20 mg/kg) i.p., DOX (20 mg/kg) plus DBZ group, and DBZ group. The third and fourth groups received i.p. injection of DBZ daily for 14 days at 2 mg/kg dose. DOX cardiotoxicity increased the level of serum creatine kinase-MB and cardiac troponin I, and it was confirmed by the histopathological examination. Moreover, the antioxidants glutathione peroxidase and superoxide dismutase levels were markedly decreased, and the inflammatory markers, inducible nitric oxide synthase, nuclear factor-ķB, and tumor necrosis factor-α were markedly increased. Furthermore, DOX increased BAX protein and downregulated BCL-2. In addition, DOX upregulated Notch pathway-related parameters: Hes1 and Hey1 mRNA levels, and increased Hes1 protein levels. DBZ ameliorated DOX-induced cardiotoxicity, evidenced by reducing the cardiac injury biomarkers, improving cardiac histopathological changes, correcting antioxidant levels, and reducing inflammatory and apoptotic proteins. Our study indicates the protective effect of Notch inhibitor against DOX-induced cardiotoxicity.

Abstract 3146: HRA00130-C004, a novel anti-DLL3 ADC with bystander effect, high DAR and favorable safety profiles

Abstract Small Cell Lung Cancer (SCLC) is a highly aggressive neuroendocrine tumor, accounting for ~15% of all new lung cancer cases1. It is estimated that there are 250,000 new SCLC cases and at least 200,000 deaths globally each year2. SCLC exhibits high recurrence after first-line treatment, poor response to subsequent therapies, and rare survival3. Lacking of recommended treatments for prolonging survival remains a problem over decades4. DLL3 is an inhibitory ligand of the Notch receptor. It binds to its Notch receptor through a cis-interaction, thus, mediates inhibition of Notch pathway to facilitate neuroendocrine tumorigenesis5. DLL3 is highly upregulated and aberrantly expressed on the cell surface in SCLC and other high-grade neuroendocrine tumors with limited expression in normal tissues6.Here we presented a DLL3-directed ADC, HRA00130-C004, which features a differentiated topoisomerase I inhibitor payload DXh conjugated via a cleavable linker to a humanized IgG1 antibody. HRA00130-C004 strongly inhibited the proliferation of cell lines with different DLL3 expression. In the bystander killing assays, HRA00130-C004 was able to kill both DMS53 cells (DLL3 over-expressed) and U-2OS (DLL3-negative) cells when they are co-cultured. The in vivo treatment of HRA00130-C004 resulted in a dramatic and sustained inhibition of tumor growth in H1184 (DLL3 high) and DMS53 (DLL3 low) xenograft models. No obvious weight loss was observed during the experiment. Furthermore, HRA00130-C004 demonstrated a favorable PK profile and satisfactory molecular integrity in rats with 3mg/kg dosing and cynomolgus monkeys with 10 mg/kg dosing. The exposure of total antibody and intact ADC was consistent. Moreover, HRA00130-C004 was well tolerated in rats and cynomolgus monkeys with no related adverse findings, which indicated its favorable safety profiles. In summary, taking advantage of Hengrui’s DXh platform, HRA00130-C004 has demonstrated great potency and good safety profiles. These data support the future clinical development of HRA00130-C004. 1.CA Cancer J. Clin., 2018, 68, 7; 2.J. Natl. Compr. Cancer Netw. 2018, 16, 1171; 3.Mol. Ther. Oncolytics, 2021, 20, 470; 4.Journal of Cancer, 2022, 13, 2945; 5.Cellular Oncology, 2019, 42, 261; 6.Journal of Hematology and Oncology 2019, 12, 61; Citation Format: Qingqing Yao, Changding Xue, Zhibin Xu, Lingfeng You, Zhendong Xue, Yuchang Mao, Sophie Lin, Jun Feng, Zhe Zhang, Xin Ye, Min Hu, Feng He. HRA00130-C004, a novel anti-DLL3 ADC with bystander effect, high DAR and favorable safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3146.

Androgens and Notch signaling cooperate in seminiferous epithelium to regulate genes related to germ cell development and apoptosis

It was reported previously that in adult males disruption of both androgen and Notch signaling impairs spermatid development and germ cell survival in rodent seminiferous epithelium. To explain the molecular mechanisms of these effects, we focused on the interaction between Notch signaling and androgen receptor (AR) in Sertoli cells and investigate its role in the control of proteins involved in apical ectoplasmic specializations, actin remodeling during spermiogenesis, and induction of germ cell apoptosis. First, it was revealed that in rat testicular explants ex vivo both testosterone and Notch signaling modulate AR expression and cooperate in the regulation of spermiogenesis-related genes (Nectin2, Afdn, Arp2, Eps8) and apoptosis-related genes (Fasl, Fas, Bax, Bcl2). Further, altered expression of these genes was found following exposure of Sertoli cells (TM4 cell line) and germ cells (GC-2 cell line) to ligands for Notch receptors (Delta-like1, Delta-like4, and Jagged1) and/or Notch pathway inhibition. Finally, direct interactions of Notch effector, Hairy/enhancer-of-split related with YRPW motif protein 1, and the promoter of Ar gene or AR protein were revealed in TM4 Sertoli cells. In conclusion, Notch pathway activity in Sertoli and germ cells regulates genes related to germ cell development and apoptosis acting both directly and indirectly by influencing androgen signaling in Sertoli cells.

Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of murine juvenile myogenic progenitors

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accretion as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.

Allergen immunotherapy combined with Notch pathway inhibitors improves HDM-induced allergic airway inflammation and inhibits ILC2 activation.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in house dust mite (HDM)-induced allergic inflammation, and allergen immunotherapy (AIT) holds promise for treating the disease by reducing the frequency of ILC2s. Despite significant progress in AIT for allergic diseases, there remains a need to improve the control of allergic symptoms. We investigated the synergistic effect of the Notch signaling pathway and subcutaneous immunotherapy (SCIT) in treating allergic airway inflammation in mice and their impact on the ratio of ILC2s in lung tissues. This was achieved by establishing the HDM-induced airway allergic disorders (HAAD) model and SCIT model. Additionally, we conducted in vitro investigations into the effect of the Notch signaling pathway on the secretory function of activated ILC2s using fluorescence-activated cell sorting. Furthermore, we explored the coactivation of the Notch signaling pathway with SCIT in vitro by sorting ILC2s from the lung tissues of mice after SCIT modeling. Previously, our group demonstrated that Notch signaling pathway inhibitors can reduce allergic airway inflammation in mice. Notch signaling induces lineage plasticity of mature ILC2s. In this study, we showed that AIT alleviates allergic airway inflammation and suppresses the frequency of ILC2s induced by HDM. Interestingly, AIT combined with a γ-secretase inhibitor (GSI), an inhibitor of the Notch signaling pathway, significantly inhibited the frequency of ILC2s, reduced airway inflammation, and suppressed Th2-type responses in a mouse model. Furthermore, lung ILC2s from HDM-challenged mice with or without AIT were treated with GSI in vitro, and we found that GSI dramatically reduced the secretion of type 2 inflammatory factors in ILC2s. These findings suggest that Notch signaling pathway inhibitors can be used as adjuvant therapy for AIT and may hold potential treatment value in the cooperative control of allergic airway inflammation during early AIT.

Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway.

Vitamin D/Vitamin D receptor (VD/VDR) signaling and the Notch pathway are involved in intestinal barrier restoration in colitis; however, their relationship and underlying mechanism are largely unknown. Therefore, this study aimed to investigate the role and mechanism of VD/VDR and the Notch pathways in intestinal barrier protection. Genetic Vdr knockout (VDR KO) and VD deficient (VDd) mice were established, and colitis was induced by feeding 2.5% dextran sodium sulfate (DSS) water. Mechanistic studies, including real-time PCR, immunofluorescence, Western blotting and dual-luciferase reporter assays, were performed on cultured Caco-2 cells and intestinal organoids. VD deficiency and VDR genetical KO increased the severity of DSS-induced colitis in mice, which presented a higher disease activity index score, increased intestinal permeability, and more severe intestinal histological damage than controls, accompanied by decreased and disrupted claudin-1 and claudin-3. Moreover, inhibition of Notch pathway by LY411,575 aggravated the severity of DSS-induced colitis and intestinal injury. In Caco-2 cells and intestinal organoids, the expression of Notch-1, N1ICD and Hes1 decreased upon downregulation or KO of VDR but increased upon paricalcitol (PAR, a VDR agonist) treatment. Meanwhile, PAR rescued claudin-1 and claudin-3 impairments that resulted from TNF-α exposure but failed to restore claudin-3 upon Notch inhibition. The dual-luciferase reporter assay further suggested that VD/VDR positively regulated the Notch signaling pathway by modulating Notch-1 transcription. VD/VDR positively modulates Notch activation by promoting Notch-1 transcription to maintain intestinal tight junction integrity and barrier function. This highlights the VD/VDR-Notch pathway as a potential new therapeutic target for protecting the intestinal barrier against ulcerative colitis.

LINC00460 promotes neuroblastoma tumorigenesis and cisplatin resistance by targeting miR-149-5p/DLL1 axis and activating Notch pathway invitro and in vivo.

Long noncoding RNAs (lncRNAs) have been demonstrated to participate in neuroblastoma cisplatin resistance and tumorigenesis. LncRNA LINC00460 was previously reported to play a critical regulatory role in many cancer development. Nevertheless, its role in modulating neuroblastoma cisplatin resistance has not been explored till now. Cisplatin-resistant neuroblastoma cell lines were established by exposing neuroblastoma cell lines to progressively increasing concentrations of cisplatin for 6months. LINC00460, microRNA (miR)-149-5p, and delta-like ligand 1 (DLL1) mRNA expression was measured through RT-qPCR. The protein levels of DLL1, epithelial-to-mesenchymal transition (EMT) markers, and the Notch signaling-related molecules were measured via western blotting. The IC50 value for cisplatin, cell growth, metastasis and apoptosis were analyzed in cisplatin-resistant neuroblastoma cells. The binding between LINC00460 (or DLL1) and miR-149-5p was validated through dual-luciferase reporter assay. The murine xenograft model was established to perform in vivo assays. LINC00460 and DLL1 levels were elevated, while miR-149-5p level was reduced in cisplatin-resistant neuroblastoma cells. LINC00460 depletion attenuated IC50 values for cisplatin, weakened cell growth, metastasis, and EMT, and enhanced apoptosis in cisplatin-resistant neuroblastoma cells. Mechanically, LINC00460 sponged miR-338-3p to increase DLL1 level, thereby activating Notch signaling pathway. DLL1 overexpression antagonized LINC00460 silencing-induced suppression on neuroblastoma cell cisplatin resistance and malignant behaviors, while such effects were further reversed by treatment with DAPT, the inhibitor of Notch pathway. Additionally, LINC00460 knockdown further augmented cisplatin-induced impairment on tumor growth in vivo. LINC00460 contributes to neuroblastoma cisplatin resistance and tumorigenesis through miR-149-5p/DLL1/Notch pathway, providing new directions to improve the therapeutic efficacy of chemotherapy drugs applied in patients with neuroblastoma.

Cooperation of Wnt/β-catenin and Dll1-mediated Notch pathway in Lgr5-positive intestinal stem cells regulates the mucosal injury and repair in DSS-induced colitis mice model.

Lgr5-positive cells located in the basal layer of crypts have self-regenerative and proliferative differentiation potentials of intestinal stem cells (ISCs), maintaining a balance of regeneration-repair in mucosal epithelium. However, the mechanisms of mucosal repair that are regulated by ISCs in ulcerative colitis (UC) remain unclear. Colon tissues from patients with UC were collected to test β-catenin and Notch1 expression by using Western blot and quantitative real-time polymerase chain reaction (PCR). β-cateninfl/fl mice, β-cateninTg mice, and Dll1tm1 Gos mice were used to cross with Lgr5-EGFP-IRES-creERT2 mice to generate mice of different genotypes, altering the activation of Wnt/β-catenin and Dll1-mediated Notch signaling in ISCs in vivo. Dextran sulfate sodium (DSS) was used to induce a colitis mice model. Intestinal organoids were isolated and cultured to observe the proliferation and differentiation levels of ISCs. β-catenin and Notch1 expression were significantly increased in the inflamed colon tissues from patients with UC. Wnt/β-catenin activation and Dll1-mediated Notch pathway inhibition in Lgr5-positive stem cells promoted the expressions of E-cadherin, CK20, and CHGA in colonic organoids and epithelium, implying the promotion of colonic epithelial integrity. Activation of Wnt/β-catenin and suppression of Dll1-mediated Notch pathway in Lgr5-positive ISCs alleviated the DSS-induced intestinal mucosal inflammation in mice. Lgr5-positive ISCs are characterized by self-renewal and high dividend potential, which play an important role in the injury and repair of intestinal mucosa. More importantly, the Wnt/β-catenin signaling pathway cooperates with the Notch signaling pathway to maintain the function of the Lgr5-positive ISCs.

The role of estrogen receptors (ERs)-Notch pathway in thyroid toxicity induced by Di-2-ethylhexyl phthalate (DEHP) exposure: Population data and in vitro studies

BackgroundThis study aimed to assess the exposure level and risk of Di-2-ethylhexyl Phthalate (DEHP) among adults in Jilin Province, China, clarify the impact of DEHP on human thyroid function, and to explore the role of estrogen receptors (ERs)-Notch signaling pathway in the effect of DEHP metabolites on thyroid hormones based on population data and in vitro experiments. Methods312 adults participated in this study. Urinary DEHP metabolites were determined by high performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS). Two pharmacokinetic models were used to evaluate the estimated daily intake (EDI) and hazard quotient (HQ) of the adults. Multiple linear regression and mediating effect models were used to evaluate the target associations. In cell experiments, thyroid follicular epithelial (Nthy-ori3–1) cells were exposed to mono (2-ethylhexyl) phthalate (MEHP) for testing. The inhibitions of ERα and Notch pathway were conducted by siRNA and Notch pathway inhibitor DAPT. ResultsThe detection rate of five DEHP metabolites was 97.1∼100.0%. The HQ value of 0.3% of adults was higher than 1. The levels of urinary DEHP metabolites were significantly correlated with thyrotropin (TSH), thyrotropin-releasing hormone (TRH), total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3) and free thyroxine (FT4) and gene (estrogen receptor α (ERα), Notch1, Dll4) levels. The ERα-Notch pathway played a mediating role in the association between DEHP metabolite levels and FT4. The cell results showed, the levels of FT3 and FT4 in cell supernatant decreased after MEHP exposure, and the downward trend was reversed after ERα and notch pathways were inhibited, notch pathway genes also decreased after ERα inhibition. ConclusionAdults in the Jilin Province of China were widely exposed to DEHP. ERs-Notch pathway played an important role in the effect of DEHP metabolites on thyroid hormones.

Gamma Secretase Inhibition Sensitizes Pancreatic Adenocarcinoma Tumors to RT In Vivo

Pancreatic adenocarcinoma (PDAC) is an extremely aggressive cancer that lacks curative treatment options. Almost half of patients present with unresectable disease limiting treatment to non-curative options. Patients treated with neoadjuvant radiation therapy (RT) exhibit increases in fibrosis and epithelial-to-mesenchymal transition (EMT). ADAM10, an extracellular sheddase, can stimulate stromal fibrosis, EMT, and radioresistance. ADAM10 also mediates EMT through Notch signaling by cleaving its extracellular domain. Further cleavage by gamma secretase produces the Notch intracellular domain (NICD), which translocates to the nucleus and activates downstream transcriptional targets. Here, we explore whether inhibition of Notch cleavage by gamma secretase radiosensitizes PDAC tumors. Bilateral flank subcutaneous PDAC isografts were produced in 40 mice using PK5L1940 KPC cells. Intraperitoneal injections of the gamma secretase inhibitor, DAPT (5 mg/kg), were delivered daily for 7 days, starting 3 days prior to RT. A single dose of 20 Gy was administered to each flank tumor, and volumes were measured twice weekly. Colony formation assays of KPC cells were performed after RT, in the presence or absence of DAPT. Since stromal fibrosis can mediate radio-resistance in the tumor microenvironment (TME), the effect of tumor cells on Notch pathway activation in mouse fibroblasts (3T3 cells) was investigated using a luciferase reporter assay. Thus, 3T3 cells transfected with a Notch pathway luciferase reporter were incubated with PDAC cells for 48 h, followed by measurement of luciferase activity. In vivo, the combination of DAPT and RT significantly delayed tumor growth, and some tumors were completely eradicated. Mean tumor size for the combination at 21 days was 21 mm3 (range = 0-53, p = 0.005), while tumor size was 577 mm3 (range = 217-955, p = 0.69) for DAPT alone, 435 mm3 for RT alone (range = 51-932, p = 0.79), and 367 mm3 for untreated vehicle (range = 97-1144). Surprisingly, DAPT did not reduce clonogenic survival in vitro. Both ADAM10 knockout and DAPT decreased NICD cleavage and transcription of the downstream target Hes1 in vivo and in vitro. Co-culture with PDAC cells increased Notch luciferase reporter activity in fibroblasts. This effect was not mimicked by PDAC-conditioned media, suggesting a requirement for intercellular contact. Notch pathway inhibition sensitizes PDAC tumors to RT in vivo, but not in vitro, suggesting involvement of the TME. Indeed, co-culture with PDAC cells stimulates notch signaling in fibroblasts, suggesting non-cell autonomous mechanisms mediating fibrosis in the TME driving radioresistance. Future studies will determine if ADAM10 inhibition targeting PDAC cells and/or gamma secretase inhibition targeting the TME enhances radiation sensitivity in vivo by blocking fibroblast Notch signaling.

Characterization of human stem cell-derived hepatic stellate cells and liver sinusoidal endothelial cells during extended in vitro culture.

Background: There is a significant need for predictive and stable in vitro human liver representations for disease modeling and drug testing. Hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) are important non-parenchymal cell components of the liver and are hence of relevance in a variety of disease models, including hepatic fibrosis. Pluripotent stem cell- (PSC-) derived HSCs (scHSCs) and LSECs (scLSECs) offer an attractive alternative to primary human material; yet, the suitability of scHSCs and scLSECs for extended in vitro modeling has not been characterized. Methods: In this study, we describe the phenotypic and functional development of scHSCs and scLSECs during 14 days of 2D in vitro culture. Cell-specific phenotypes were evaluated by cell morphology, immunofluorescence, and gene- and protein expression. Functionality was assessed in scHSCs by their capacity for intracellular storage of vitamin A and response to pro-fibrotic stimuli induced by TGF-β. scLSECs were evaluated by nitric oxide- and factor VIII secretion as well as endocytic uptake of bioparticles and acetylated low-density lipoprotein. Notch pathway inhibition and co-culturing scHSCs and scLSECs were separately tested as options for enhancing long-term stability and maturation of the cells. Results and Conclusion: Both scHSCs and scLSECs exhibited a post-differentiation cell type-specific phenotype and functionality but deteriorated during extended culture with PSC line-dependent variability. Therefore, the choice of PSC line and experimental timeframe is crucial when designing in vitro platforms involving scHSCs and scLSECs. Notch inhibition modestly improved long-term monoculture in a cell line-dependent manner, while co-culturing scHSCs and scLSECs provides a strategy to enhance phenotypic and functional stability.