Dexamethasone-mediated inhibition of Notch signalling blocks the interaction of leukaemia and mesenchymal stromal cells.

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by a poor prognosis. Bone marrow mesenchymal stromal cells (BM MSCs) support leukaemic cells in preventing chemotherapy-induced apoptosis. This encouraged us to investigate leukaemia-BM niche-associated signalling and to identify signalling cascades supporting the interaction of leukaemic cells and BM MSC. Our study demonstrated functional differences between MSCs originating from leukaemic (AML MSCs) and healthy donors (HD MSCs). The direct interaction of leukaemic and AML MSCs was indispensable in influencing AML cell proliferation. We further identified an important role for Notch expression and its activation in AML MSCs contributing to the enhanced proliferation of AML cells. Supporting this observation, overexpression of the intracellular Notch domain (Notch ICN) in AML MSCs enhanced AML cells' proliferation. From a therapeutic point of view, dexamethasone treatment impeded Notch signalling in AML MSCs resulting in reduced AML cell proliferation. Concurrent with our data, Notch inhibitors had only a marginal effect on leukaemic cells alone but strongly influenced Notch signalling in AML MSCs and abrogated their cytoprotective function on AML cells. In vivo, dexamethasone treatment impeded Notch signalling in AML MSCs leading to a reduced number of AML MSCs and improved survival of leukaemic mice. In summary, targeting the interaction of leukaemic cells and AML MSCs using dexamethasone or Notch inhibitors might further improve treatment outcomes in AML patients.

GSK-3ß inhibitor TWS119 alleviates hypoxic-ischemic brain damage via a crosstalk with Wnt and Notch signaling pathways in neonatal rats.

Preterm infant brain injury is a leading cause of morbidity and disability in survivors of preterm infants. Unfortunately, the effective treatment remains absent. Recent evidence suggests that GSK-3ß inhibitor TWS119 has a neuroprotectiverole in adult brain injury by activation of Wnt/ß-catenin signaling pathway. However, the role on neonatal brain injury is not yet explored. The study aims to evaluate the effect of TWS119 at 7 d after hypoxic-ischemic brain damage and investigate the mechanism that it regulates Wnt and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats. Three-day-old rats were randomly divided into 3 groups: sham group, HI group and TWS119 group. The neonatal rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8.0% O2). A single dose of TWS119 (30 mg/kg) was intraperitoneally injected 20 min prior to hypoxia-ischemia (HI). At 7 d after HI, TWS119 improved the tissue structure, reduced cell apoptosis, up-regulated bcl-2 expression, up-regulated the expression of PSD-95 and Synapsin-1. At 24 h after HI, it activated Wnt/ß-catenin signaling pathway by up-regulation of ß-catenin protein expression and wnt3a/wnt5a/wnt7a mRNA expression. Simultaneously, it suppressed Notch signaling pathway by down-regulation of Notch1 and HES-1 proteins expression. Our study suggested that TWS119 performed a neuroprotective function at 7 d after hypoxic-ischemic brain damage via a crosstalk with Wnt/ß-catenin and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats.

Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells.

Alcohol drinking has been established as a major risk factor for esophageal diseases. Our previous study showed that ethanol exposure inhibited PAX9 expression in human esophageal squamous epithelial cells in vitro and in vivo. In this study, we aimed to investigate the molecular pathways through which alcohol drinking suppresses PAX9 in esophageal squamous epithelial cells. We first demonstrated the inhibition of NOTCH by ethanol exposure in vitro. NOTCH regulated PAX9 expression in KYSE510 and KYSE410 cells in vitro and in vivo. RBPJ and NOTCH intracellular domain (NIC) D1 ChIP-PCR confirmed Pax9 as a direct downstream target of NOTCH signaling in mouse esophagus. NOTCH inhibition by alcohol drinking was further validated in mouse esophagus and human tissue samples. In conclusion, ethanol exposure inhibited NOTCH signaling and thus suppressed PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. Our data support a novel mechanism of alcohol-induced esophageal injury through the inhibition of NOTCH-PAX9 signaling. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Luteolin mediated targeting of protein network and microRNAs in different cancers: Focus on JAK-STAT, NOTCH, mTOR and TRAIL-mediated signaling pathways.

There has always been a keen interest of basic and clinical researchers to search for cancer therapeutics having minimum off-target effects and maximum anticancer activities. In accordance with this approach, there has been an explosion in the field of natural products research in the past few decades because of extra-ordinary list of natural extracts and their biologically and pharmacologically active constituents having significant medicinal properties. Apparently, luteolin-mediated anticancer effects have been investigated in different cancers but there is superfluousness of superficial data. Generalized scientific evidence encompassing apoptosis, DNA damage and anti-inflammatory effects has been reported extensively. However, how luteolin modulates deregulated oncogenic pathways in different cancers has not been comprehensively uncovered. In this review we have attempted to focus on cutting-edge research which has unveiled remarkable abilities of luteolin to modulate deregulated oncogenic pathways in different cancers. We have partitioned the review into various sections to separately discuss advancements in therapeutic targeting of oncogenic protein networks. We have provided detailed mechanistic insights related to JAK-STAT signaling and summarized how luteolin inhibited STAT proteins to inhibit STAT-driven gene network. We have also individually analyzed Wnt/ß-catenin and NOTCH pathway and how luteolin effectively targeted these pathways. Mapping of the signaling landscape has revealed that NOTCH pathway can be targeted therapeutically. NOTCH pathway was noted to be targeted by luteolin. We have also conceptually analyzed how luteolin restored TRAIL-induced apoptosis in resistant cancers. Luteolin induced an increase in pro-apoptotic proteins and efficiently inhibited anti-apoptotic proteins to induce apoptosis. Luteolin mediated regulation of non-coding RNAs is an exciting and emerging facet. Excitingly, there is sequential and systematic accumulation of clues which have started to shed light on intricate regulation of microRNAs by luteolin in different cancers. Collectively, sophisticated information will enable us to develop a refined understanding of the multi-layered regulation of signaling pathways and non-coding RNAs by luteolin in different cancers.

Impaired activation of Notch-1 signaling hinders repair processes of bronchial epithelial cells exposed to cigarette smoke.

Notch-1 intervenes in the reparative processes of mucosa by controlling cell proliferation, differentiation and stem cell maintenance. Cigarette smoke alters airway epithelial homeostasis. The present study explored whether: Smokers showed altered Notch-1 expression; and whether in bronchial epithelial cells (16HBE): a) cigarette smoke extracts (CSE) altered the expression of Notch-1, of its ligand Jagged-1 (Jag-1) and the nuclear translocation of Notch-1; b) Notch-1 signaling activation as well as CSE modified Ki67, PCNA, p21, IL-33 expression, cell proliferation and repair processes. Notch-1 expression was assessed in the epithelium from large airway surgical samples from non-smoker and smoker subjects by immunohistochemistry.16HBE were cultured with/without CSE and Jag-1. A Notch-1 inhibitor (DAPT) was used as control. The expression of Notch-1, Jag-1, Ki67, PCNA, p21, IL-33 and cell proliferation (by CFSE) were all assessed by flow cytometry. Notch-1 nuclear expression was evaluated by immunofluorescence and western blot analysis. Repair processes were assessed by wound assay. Smokers had cytoplasmic but not nuclear Notch-1 expression. Although CSE increased Notch-1 expression, it counteracted Notch-1 signaling activation since it reduced Jag-1 expression and Notch-1 nuclear translocation. Notch-1 signaling activation by Jag-1 increased Ki67, PCNA and repair processes but reduced intracellular IL-33 and p21 expression without affecting cell proliferation. DAPT counteracted the effects of Notch-1 activation on PCNA and IL-33. CSE increased Ki67, PCNA, p21 and IL-33 expression but reduced cell proliferation and repair processes. In conclusion, cigarette smoke exposure, limiting Notch-1 signaling activation and hindering repair processes, amplifies injury processes in bronchial epithelial cells.

Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells.

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.

Identification of a Pan-Gamma-Secretase Inhibitor Response Signature for Notch-Driven Cholangiocarcinoma.

Cholangiocarcinoma (CCA) mortality rates are increasing as a result of rising incidence and limited curative treatment(s) for patients with advanced disease. NOTCH pathway reactivation has been reported in biliary malignancies to conflicting degrees, hindering prioritization of key therapeutic targets within the network and identification of candidate responder patients for NOTCH-directed therapies. We analyzed genomic data from 341 patients with CCA and identified NOTCH1 significantly increased in a subgroup characterized by distinct stromal infiltration. Network-wide imbalance of the NOTCH pathway was seen in CCA, including correlation of NOTCH1 with NOTCH3 and NOTCH ligands. Given the diversity of observed NOTCH receptor engagement, γ-secretase modulation was rationalized as a therapeutic option. Indeed, subcutaneous transplantation of sensitive and resistant CCA cell lines pretreated with a γ-secretase inhibitor (GSi) cocktail demonstrated the antineoplastic effects of GSi in a subset of CCA and led to the development of a 225-gene responder signature. This signature was validated in an independent cohort of 119 patients. Further, this signature was enriched in liver tumors initiated by hydrodynamic injections of activated-NOTCH as compared with the AKT-RAS-driven tumors. Candidate GSi-responder patients were characterized by distinct transcriptomes overlapping with previous hepatobiliary metastasis and stemness, unique stromal properties, and dysfunctional intratumoral immune infiltration. Pan-cancer analysis identified 41.9% of cancer types to harbor prospective GSi-responder patients, which was adapted into a 20-gene GSi-sensitivity score metric capable of discriminating nanomolar versus micromolar sensitivity to a cell-permeable GSi (Z-LLNle-CHO) across 60 diverse tumor lines (area under the curve = 1). Conclusion: We have established a GSi-responder signature with evidence across several patient cohorts, as well as in vitro and in vivo models, to enable precision medicine application of NOTCH-directed therapy in CCA as well as prospectively across diverse malignancies.

Zerumbone promotes proliferation of endogenous neural stem cells in vascular dementia by regulating Notch signalling.

INTRODUCTION: Present investigation determines the effect of zerumbone on the proliferation of stem cells in vascular dementia (VD) rats. MATERIAL AND METHODS: Vascular dementia was induced by cerebral ischemia and reperfusion through non-invasive clamp. Rats were treated with zerumbone 50 mg/kg and 100 mg/kg intraperitoneally 30 min for four weeks after the surgery. Cognitive functions are determined by the Morris water maze (MWM) test and neurological function score in VD rats. Moreover mediators of inflammation and parameters of oxidative stress were estimated in the brain tissue homogenate of ischemia-induced vascular dementia rats. The expression of proteins and mRNA expressions were determined by western blot assay and RT-PCR methods. Moreover histopathological changes were observed by H&E staining on the brain tissue of vascular dementia rats. RESULTS: There was a significant reduction in the cognitive function and neurological score in the zerumbone-treated group compared to the VD group of rats. Data of the study reveal that treatment with zerumbone attenuates the altered level of cytokines and markers of oxidative stress parameters in the brain tissue of VD rats. The expression of NICD, Hes-1 and Nestin proteins was significantly (p < 0.01) reduced in the brain tissue of the zerumbone-treated group compared to the VD group of rats. There was a significant reduction in the mRNA expression of Notch-1 and Hes-1 in the brain tissue of the zerumbone-treated group compared to the VD group of rats. CONCLUSIONS: This study concludes that treatment with zerumbone protects the neuronal injury and ameliorates the cognitive function by stimulating the proliferation of endogenous neural stem cells. Moreover proliferation of neural stem cells was stimulated in zerumbone-treated rats by regulating the Notch signalling.

Dietary Lipids Modulate Notch Signaling and Influence Adult Intestinal Development and Metabolism in Drosophila.

Tissue homeostasis involves a complex balance of developmental signals and environmental cues that dictate stem cell function. We found that dietary lipids control enteroendocrine cell production from Drosophila posterior midgut stem cells. Dietary cholesterol influences new intestinal cell differentiation in an Hr96-dependent manner by altering the level and duration of Notch signaling. Exogenous lipids modulate Delta ligand and Notch extracellular domain stability and alter their trafficking in endosomal vesicles. Lipid-modulated Notch signaling occurs in other nutrient-dependent tissues, suggesting that Delta trafficking in many cells is sensitive to cellular sterol levels. These diet-mediated alterations in young animals contribute to a metabolic program that persists after the diet changes. A low-sterol diet also slows the proliferation of enteroendocrine tumors initiated by Notch pathway disruption. Thus, a specific dietary nutrient can modify a key intercellular signaling pathway to shift stem cell differentiation and cause lasting changes in tissue structure and physiology.

Pregabalin and lacosamide ameliorate paclitaxel-induced peripheral neuropathy via inhibition of JAK/STAT signaling pathway and Notch-1 receptor.

Anticonvulsant drugs such as pregabalin (PGB) and lacosamide (LCM), exhibit potent analgesic effects in diabetic neuropathy; however, their possible role/mechanisms in paclitaxel (PTX)-induced peripheral neuropathy have not been elucidated, which is the aim of the present study. Neuropathic pain was induced in rats by injecting PTX (2 mg/kg, i. p) on days 0, 2, 4 and 6. Forty eight hours after the last dose of PTX, rats were treated orally with 30 mg/kg/day of either PGB or LCM for 21 days. Both therapies improved thermal hyperalgesia and cold allodynia induced by PTX. Interestingly, LCM therapy showed no motor impairment that was observed upon using PGB, as demonstrated using rotarod test. Treatment with PGB or LCM restored the sciatic nerve content of the depleted total antioxidant capacity (TAC) and nerve growth factor (NGF), and lessened the elevated contents of nuclear factor kappa B p65 (NF-kB p65), tumor necrosis factor-α (TNF-α), and active caspase-3. On the molecular level, the drugs reduced the protein expression of Notch1 receptor, phosphorylated p38 mitogen-activated protein kinase (p-p38-MAPK), and the trajectory interleukin-6/phosphorylated janus kinase 2/phosphorylated signal transducer and activator of transcription 3 (IL-6/p-JAK2/p-STAT3). Therefore, the current study demonstrated a pivotal role for LCM in the management of PTX-induced peripheral neuropathy similar to PGB, but without motor adverse effects via the inhibition of oxidative stress, inflammation and apoptosis, as well as IL-6/JAK/STAT pathway and Notch1 receptor over-expression.

Cervical Cancer Cell Growth, Drug Resistance, and Epithelial-Mesenchymal Transition Are Suppressed by y-Secretase Inhibitor RO4929097.

BACKGROUND The Notch signaling pathway has been reported to play a pivotal role in tumorigenesis. Emerging evidence has demonstrated that the Notch signaling pathway regulates several cellular processes. The present study investigated the effect of the Notch signaling pathway on cell growth, invasiveness, and drug resistance, as well as epithelial-mesenchymal transition (EMT), of cervical cancer cells. MATERIAL AND METHODS We used quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis to measure the expression level of Notch2. CCK-8, clonality, wound healing, and Transwell assays were used to evaluate the effect of γ-secretase inhibitor (GSI) RO4929097 on cervical cancer cell lines HeLa and Caski. To explore the role of the Notch signaling pathway in EMT, the epithelial and mesenchymal markers were detected by qRT-PCR and Western blot after cervical cancer cell lines were treated with GSI RO4929097. RESULTS The expression of Notch2 was found to increase in cervical cancer cell lines compared with the normal immortalized human cervical epithelial cells. GSI RO4929097 was confirmed to inhibit the Notch signaling pathway and impaired the proliferation, drug resistance, migration, and invasion abilities of cervical cancer cells. The protein expression levels of the mesenchymal biomarkers Snail, Twist, and neural cadherin (N-cadherin) decreased; however, the expression of the epithelial biomarker epithelial cadherin (E-cadherin) increased in the cervical cancer cells treated with GSI RO4929097. CONCLUSIONS Notch signaling pathway plays an important role in the development and progression of cervical cancer. Blockade of the Notch pathway using GSI RO4929097 inhibited cell growth and reduced chemoresistance, invasion, metastasis, and EMT in cervical cancer cells.

Gremlin activates the Notch pathway linked to renal inflammation.

Preclinical studies suggest that Gremlin participates in renal damage and could be a potential therapeutic target for human chronic kidney diseases. Inflammation is a common characteristic of progressive renal disease, and therefore novel anti-inflammatory therapeutic targets should be investigated. The Notch signaling pathway is involved in kidney development and is activated in human chronic kidney disease, but whether Gremlin regulates the Notch pathway has not been investigated. In cultured tubular cells, Gremlin up-regulated gene expression of several Notch pathway components, increased the production of the canonical ligand Jagged-1, and caused the nuclear translocation of active Notch-1 (N1ICD). In vivo administration of Gremlin into murine kidneys elicited Jagged-1 production, increased N1ICD nuclear levels, and up-regulated the gene expression of the Notch effectors hes-1 and hey-1 All these data clearly demonstrate that Gremlin activates the Notch pathway in the kidney. Notch inhibition using the γ-secretase inhibitor DAPT impaired renal inflammatory cell infiltration and proinflammatory cytokines overexpression in Gremlin-injected mice and in experimental models of renal injury. Moreover, Notch inhibition blocked Gremlin-induced activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathway, identifying an important mechanism involved in the anti-inflammatory actions of Notch inhibition. In conclusion, Gremlin activates the Notch pathway in the kidney and this is linked to NF-κB-mediated inflammation, supporting the hypothesis that Notch inhibition could be a potential anti-inflammatory strategy for renal diseases.

Arsenic trioxide induces apoptosis and inhibits the growth of human liver cancer cells.

AIMS: As a fifth most common cancer type, Hepatocellularcarcinoma (HCC) ranked third leading cause of cancer deaths worldwide. Arsenic trioxide (As2O3) is known as chemotherapeutic agent against few cancer including Acute promyelocyticleukemia and solid tumors. But its effect and possible associated mechanism in HCC is meager. Present study aimed to assess As2O3 modulatory effect on liver cancer by assessing cell growth and viability. METHODS: Liver normal (Chang liver) and cancerous cells (Hep3B) were exposed to different concentration's (0, 1, 5, 10 & 15 µM) of As2O3 at different intervals (24, 48 & 72 h). Cell growth was assessed microscopically, and Cytotoxicity assays were done through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Water-soluble tetrazolium salt (WST) growth inhibition assays. Cell viability was studied by trypan blue staining. Apoptosis was analyzed by Annexin V/PI assay, and expression of genes (Notch and anti-apoptotic) were determined through western blotting and Q-PCR method. KEY FINDINGS: A significant reduction in cell growth and viability was reported in liver cancerous cells as compare to normal cells at 5 µM As2O3. Consistently, As2O3 induced apoptosis along with down-regulation of anti-apoptotic protein Bcl-xL, and up regulates expression of Notch that leads towards apoptosis. SIGNIFICANCE: Results clearly suggest that As2O3 restricted growth and induces apoptosis more in liver cancer cells as compared to normal cells. This finding suggests that it could be a promising potential therapeutic agent against liver cancer which need further testing by in-vivo investigations.

Notch activation promotes osteoblast mineralization by inhibition of apoptosis.

Notch activator Jagged1 (JAG1) plays a critical role in the regulation of osteoblast differentiation and bone metabolism. In this study, JAG1-induced osteoblast proliferation, differentiation, and mineralization has been analyzed in primary osteoblasts for up to 7 days. Alkaline phosphatase and Alizarin red staining showed an enhanced osteoblast maturation and mineralization in JAG1 treated cells, as well as higher mRNA levels of late osteoblast differentiation markers. In contrast, Notch inhibitor DAPT and deletion of Runx2 totally blocked JAG1 effects on osteoblast mineralization. Flow cytometry data further showed a significantly higher cell proliferation in early stages of culture at day 3, and lower levels of osteoblast apoptosis in late stages of culture at day 7. More importantly, activation of anti-apoptotic factor BCL-2 was enhanced, while pro-apoptotic factor Caspase3 was reduced in JAG1 treated osteoblasts. Therefore, we conclude that cell mineralization is enhanced via anti-apoptotic actions of Notch signaling within the osteoblast cells.

Regulation of Gene Expression by Thyroid Hormone in Primary Astrocytes: Factors Influencing the Genomic Response.

Astrocytes mediate the action of thyroid hormone in the brain on other neural cells through the production of the active hormone triiodothyronine (T3) from its precursor thyroxine. T3 has also many effects on the astrocytes in vivo and in culture, but whether these actions are directly mediated by transcriptional regulation is not clear. In this work, we have analyzed the genomic response to T3 of cultured astrocytes isolated from the postnatal mouse cerebral cortex using RNA sequencing. Cultured astrocytes express relevant genes of thyroid hormone metabolism and action encoding type 2 deiodinase (Dio2), Mct8 transporter (Slc16a2), T3 receptors (Thra1 and Thrb), and nuclear corepressor (Ncor1) and coactivator (Ncoa1). T3 changed the expression of 668 genes (4.5% of expressed genes), of which 117 were responsive to T3 in the presence of cycloheximide. The Wnt and Notch pathways were downregulated at the posttranscriptional level. Comparison with the effect of T3 on astrocyte-enriched genes in mixed cerebrocortical cultures isolated from fetal cortex revealed that the response to T3 is influenced by the degree of astrocyte maturation and that, in agreement with its physiological effects, T3 promotes the transition between the fetal and adult patterns of gene expression.

Tumor Architecture and Notch Signaling Modulate Drug Response in Basal Cell Carcinoma.

Hedgehog (Hh) pathway inhibitors such as vismodegib are highly effective for treating basal cell carcinoma (BCC); however, residual tumor cells frequently persist and regenerate the primary tumor upon drug discontinuation. Here, we show that BCCs are organized into two molecularly and functionally distinct compartments. Whereas interior Hh+/Notch+ suprabasal cells undergo apoptosis in response to vismodegib, peripheral Hh+++/Notch- basal cells survive throughout treatment. Inhibiting Notch specifically promotes tumor persistence without causing drug resistance, while activating Notch is sufficient to regress already established lesions. Altogether, these findings suggest that the three-dimensional architecture of BCCs establishes a natural hierarchy of drug response in the tumor and that this hierarchy can be overcome, for better or worse, by modulating Notch.

Natural Products Inspired Modulators of Cancer Stem Cells-specific Signaling Pathways Notch and Hedgehog.

It is nowadays widely accepted that some tumors have a niche of cells endowed with stemness features, which may cause resistance to conventional anticancer therapies and relapse/recurrence of the malignancy. These cells are usually referred to as cancer stem cells (CSCs) and, different from normal cancer cells, are rather quiescent. Targeting CSCs is thus a highly challenging but promising strategy to counteract tumor growth, and to develop innovative anticancer agents. Here, we review the chemical, biological and multidisciplinary efforts that have been spent in targeting CSCsspecific signaling pathways Notch and Hedgehog (Hh) for anticancer drug discovery. In particular, the use of natural products as a valuable source of lead compounds or chemical biology tools is emphasized. Examples of natural products functionalization through semi-synthetic transformations or total syntheses, aimed at improving pharmacokinetics and/or pharmacodynamics properties of natural products in Notch or Hh inhibition, are provided as well.

Parathyroid hormone inhibits Notch signaling in osteoblasts and osteocytes.

Parathyroid hormone (PTH) and Notch receptors regulate bone formation by governing the function of osteoblastic cells. To determine whether PTH interacts with Notch signaling as a way to control osteoblast function, we tested the effects of PTH on Notch activity in osteoblast- and osteocyte-enriched cultures. Notch signaling was activated in osteoblast-enriched cells from wild-type C57BL/6J mice following exposure to the Notch ligand Delta-like (Dll)1 or by the transient transfection of the Notch intracellular domain (NICD), the transcriptionally active fragment of Notch1. To induce Notch signaling in osteocyte-enriched cultures, a murine model of Notch2 gain-of-function was used. PTH opposed the stimulatory effects of Dll1 on Hey1, Hey2 and HeyL mRNA levels in osteoblast-enriched cells and suppressed the expression of selected Notch target genes in osteocyte-enriched cultures, either under basal conditions or in the context of Notch2 gain-of-function. Induction of Notch signaling in osteocytes did not alter the inhibitory effect of PTH on Sost expression, but reduced the stimulation of Tnfsf11 mRNA levels by PTH. In agreement with these in vitro observations, male mice administered with PTH displayed suppressed Hey1 and HeyL expression in parietal bones. Transactivation experiments with a Notch reporter construct and electrophoretic mobility shift assays in osteoblast-enriched cells suggest that PTH acts by decreasing the capacity of Rbpjκ to bind to DNA. In conclusion, downregulation of Notch in osteoblasts and osteocytes may represent a mechanism contributing to the anabolic effects of PTH in bone.

Nox4 is involved in high glucose-induced apoptosis in renal tubular epithelial cells via Notch pathway.

It has previously been demonstrated that nicotinamide adenine dinucleotide phosphate­oxidase (NADPH) oxidase 4 (Nox4), is important in the pathogenesis of diabetic nephropathy (DN), however the exact mechanisms remain to be elucidated. The present study aimed to examine the effect of Nox4 on the alteration of the Notch pathway and cell apoptosis in the renal tubular epithelial cell line, HKC, under conditions of high glucose (HG; 30 mmol/l glucose). Nox4 and the Notch pathway were inhibited by N­acetylcysteine (NAC), diphenylene iodonium (DPI) or γ­secretase inhibitor (DAPT). The protein levels of Nox4, Notch1, Notch intracellular domain 1 (NICD1), phosphorylated (p) Ras­related C3 botulinum toxin substrate 1 (Rac1), Rac1, B­cell lymphoma 2 apoptosis regulator (Bcl­2), Bcl­2 associated protein X apoptosis regulator (Bax) and cleaved caspase­3 were determined by western blotting. The Nox4 and Notch1 mRNA levels were detected by reverse transcription­quantitative polymerase chain reaction. Intracellular reactive oxygen species (ROS) levels were detected via chloromethyl­2',7'­dichlorodihydrofluorescein diacetate. Apoptotic cells were determined using an Annexin V/propidium iodide apoptosis detection kit. HG upregulated Nox4, Notch1, NICD1, p­Rac1, Bax and cleaved caspase­3 expression levels and downregulated Bcl­2 expression in cultured HKC cells, compared with cells cultured in normal glucose levels. Inhibition of the Notch pathway via DAPT increased Bcl­2 expression, decreased Bax and cleaved caspase­3 levels and prevented HKC cell apoptosis. Inhibition of Nox4 by NAC and DPI inhibited the Notch signaling pathway and ROS generation, which prevented HKC cell apoptosis. These findings indicated that Nox4 potentially mediates HG­induced HKC cell apoptosis via the Notch pathway, and may be involved in renal tubular epithelial cell injury in DN.

Enhancement of vascular endothelial growth factor's angiogenic capacity by the therapeutic modulation of notch signalling improves tram flap survival in rats submitted to nicotine.

BACKGROUND: Smoke of cigarettes, and specifically nicotine, has been shown to diminish pedicled transverse rectus abdominis musculocutaneous (TRAM) flap survival. Considering that Notch signalling through its ligand Delta-like 4 (Dll4) functions as anti-angiogenic factor by inhibiting the pro-angiogenic effects of vascular endothelial growth factor (VEGF), it is hypothesised that inhibition of the Notch would promote angiogenesis and increase TRAM flap survival in rats submitted to nicotine. METHODS: Twenty rats were treated with nicotine for 28 days preoperatively. Thereafter, a pedicled TRAM flap was created in all animals. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine-t-butyl-ester was administered in animals of the treatment group. Animals in the control group were given the same amount of solvent. Five days after the surgery, viable flap areas were determined. Skin samples were evaluated for VEGF and Dll4 mRNA levels. Immunohistochemical analysis was used for the assessment of endothelial Dll4 expression. Vascular density was determined histologically. Plasma levels of VEGF and Dll4 were measured. RESULTS: A significant improvement in TRAM flap surviving area was observed in the treatment group (53.50 ± 14.25%) compared with the controls (32.20 ± 9.15%). Immunohistochemical analysis revealed a significant increase in the number of Dll4 stained vessels in animals of the treatment group (9.2 ± 1.6) in comparison with the controls (5.7 ± 1.9). VEGF mRNA levels (0.22 ± 0.08) in the treatment group were significantly lower than those in the control group (0.36 ± 0.09). CONCLUSION: Notch inhibition significantly improved TRAM flap survival in animals exposed to nicotine by promoting VEGF-induced angiogenesis.