Upregulation Of Notch1 Research Articles

Role of oleanolic acid in relieving psoriasis and its underlying mechanism of action

The present study aimed to determine whether oleanolic acid (Ole) could be used to treat psoriasis and its related underlying mechanism of action via in vitro analysis. HaCaT cells were stimulated with IL-22 to established an in vitro psoriasis cell model. MTT, flow cytometry and TUNEL assays, respectively. Transmission electron microscopy was used to observe the cell ultrastructure. LC3B protein expression levels were analyzed using immunofluorescence, other protein expression levels were determined using western blotting. Cell viability was significantly increased, while the apoptotic rate was significantly decreased in the model group (P < 0.001). In addition, Notch1, Hes1, beclin 1 and LC3B protein expression levels were significantly downregulated, while P62 protein expression levels were significantly upregulated in the model group compared with the control group (P < 0.001). Supplementation of Ole, the increased levels of proliferation were significantly suppressed, while cell apoptosis was significantly increased (P < 0.05) in a dose-dependent manner, which was discovered to occur via Notch1 upregulation. Notably, the transfection with small interfering RNA-Notch1 significantly reversed the effect of Ole treatment (P < 0.001) and the levels of autophagy were also decreased. In conclusion, the findings of the current study suggested that Ole may relieve psoriasis via upregulating Notch1, which subsequently regulates cell autophagy.

JARID2 promotes stemness and cisplatin resistance in non-small cell lung cancer via upregulation of Notch1

Increased stemness is causally linked to development of drug resistance in cancers. JARID2 is a member of the Jumonji family of proteins and regulates differentiation of embryonic stem cells. However, the role of JARID2 in lung cancer stemness and drug resistance is still unclear. In this study, we investigated the expression of JARID2 in parental and cisplatin (CDDP) resistant non-small cell lung cancer (NSCLC) cells. The function of JARID2 in modulating CDDP sensitivity of NSCLC cells was determined. It was found that JARID2 is upregulated in CDDP resistant NSCLC cells, which depends on SOX2 expression. JARID2 overexpression promotes CDDP resistance in NSCLC cells, whereas JARID2 depletion restores CDDP sensitivity in CDDP resistant NSCLC cells. Moreover, JARID2 overexpression enhances cancer stem cell-like properties in NSCLC cells, which is coupled with increased expression of cancer stem cell markers. Mechanistically, JARID2-induced stemness and CDDP resistance is mediated by upregulation of Notch1. In clinical settings, high expression of JARID2 is significantly associated with advanced TNM stage, shorter overall survival, and poor chemotherapeutic response. These findings point toward an important role of JARID2 in CDDP resistance and stemness of NSCLC and provide a promising target for overcoming CDDP resistance.

Long Non-Coding RNA SNHG6 Supports Glioma Progression Through Upregulation of Notch1, Sox2, and EMT.

Gliomas, particularly the advanced grade glioblastomas, have poor 5-year survival rates and worse outcomes. lncRNAs and EMT have been extensively studied in gliomas but the disease progression remains poorly understood. SNHG6 has been shown to affect glioma cell proliferation but its effect on EMT of glioma cells along with its effect on disease progression is not known. We screened four glioma cell lines; H4, A172, U87MG, and SW088 and grouped them based on high vs. low SNHG6 expression. Transfections with SNHG6 specific siRNA resulted in induction of apoptosis of high SNHG6 expressing A172 and U87MG cells. This was accompanied by inhibition of EMT and downregulation of EMT-modulating factor Notch1, β-catenin activity and the cancer stem cell marker Sox2. The regulation was not found to be reciprocal as silencing of Notch1 and Sox2 failed to affect SNHG6 levels. The levels of SNHG6 and Notch1 were also found elevated in Grade IV glioma patients (n = 4) relative to Grade II glioma patients (n = 5). These results identify SNHG6 and Notch1 as valid targets for glioma therapy.

Roles of the Notch Signaling Pathway in Ovarian Functioning.

The Notch signaling pathway regulates cell invasion, adhesion, proliferation, apoptosis, and differentiation via cell-to-cell interactions and plays important physiological roles in the ovary. This review summarizes current knowledge about the Notch signaling pathway in relation to ovarian functions and reveals the potential underlying mechanisms. We conducted an in-depth review of relevant literature to determine the current status of research into the Notch signaling pathway in relation to ovarian functioning and reveal potential underlying mechanisms. The activation of different Notch receptors promotes the formation of primordial follicles and proliferation of granulosa cells and inhibits steroid secretion. Abnormal regulation of the Notch signaling pathway or direct mutations might lead to over-activation or under-activation of the receptors, resulting in Notch upregulation or downregulation. It can also disrupt the normal physiological functions of the ovary. The lncRNA HOTAIR and growth hormones improved premature ovarian failure (POF) and promoted follicle maturation in a mouse model of POF by upregulating Notch1 expression. They also stimulated the Notch1 signaling pathway, increased the level of plasma estradiol, and decreased the level of plasma follicle-stimulating hormone. Thus, Notch1 could serve as a novel therapeutic target for POF. Several studies have reported multiple roles of Notch in regulating female primordial follicle formation and follicle maturation. Direct mutations in Notch-related molecules or abnormal gene regulation in the signaling pathway can lead to ovarian dysfunction. However, the underlying mechanisms are not fully understood.

A Reversible Shift of Driver Dependence from EGFR to Notch1 in Non-Small Cell Lung Cancer as a Cause of Resistance to Tyrosine Kinase Inhibitors.

Simple SummaryNotch1 and EGFR are two surface receptors activating different cellular processes in cancer cells. EGFR, harboring activating mutations, drives unlimited cell proliferation in non-small cell lung cancer (NSCLC), and treatment of tumors with tyrosine kinase inhibitors (TKIs) results in growth arrest and cell death. On the other hand, Notch1 plays a key role in the loss of epithelial characteristics and the concomitant acquisition of mesenchymal traits and invasive potential of tumor cells. Interestingly, high levels of Notch1 are associated with the resistance to EGFR TKIs. Here, we evaluated the mechanisms by which Notch1 causes resistance of NSCLC to EGFR TKIs, and provided evidence that high levels of activated Notch1 induce a decrease of EGFR, by modulating the activity of the promoter of the EGFR gene. Therefore, blocking the Notch1 pathway in tumors treated with EGFR inhibitors would prevent EGFR downregulation, maintaining drug sensitivity.Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.

The Cell-specific Engagement of Notch and Wnt Pathways in Calcific Aortic Valve Disease

Objective: The Notch and Wnt pathways participate in calcific aortic valve disease (CAVD), but with conflicting findings to date. We hypothesize that Notch and Wnt pathway engagement during CAVD initiation are cell-type specific. Methods: Primary porcine aortic valvular endothelial (PAVEC) and/or interstitial (PAVIC) cells were cultured in a 3D spring mechanical constraint system or in 2D culture and challenged with osteogenic media (OGM) to induce calcification. DAPT and IMR-1 were used as Notch1 inhibitors, and lentiviral transduction was used to create constitutively-active b-catenin (B-cat+) PAVIC and doxycycline-inducible Notch1 intracellular domain (NICD+) PAVEC and PAVIC. Calcification was evaluated with alizarin red staining. Results: PAVIC treatment with Notch inhibitors DAPT and IMR-1 resulted in opposite calcific responses, and this correlated with active-form b-catenin signaling. Further interrogation using B-cat+ PAVIC showed increased calcification compared to wild-type (WT) cells. Treatment of B-cat+ PAVIC with DAPT, but not IMR-1, was able to reduce OGM-induced calcification to different extents in B-cat+ and WT cells. NICD overexpression was investigated in PAVIC and PAVEC. Culture of NICD+ PAVIC in 3D decreased calcification compared to controls, while NICD+ PAVIC and PAVEC co-culture increased calcification. Gene expression data in 2D NICD+ VEC found no difference in EndMT SNAI1/2 expression, and an increase in osteogenic genes Runx2, BMP2, SPARC, and SPP1 vs. WT controls. Conclusions: We demonstrate a divergence in Notch1 regulation of calcification with PAVIC DAPT and IMR-1 treatment with and without constitutively active b-catenin, and with NICD+ PAVIC monoculture vs. NICD+ PAVIC and PAVEC co-culture. Given our previous findings demonstrating early upregulation and later downregulation of Notch1 in VEC with TNFa-stimulus, our results are consistent with the hypothesis that Notch1 signaling has a dynamic and cell-specific influence over CAVD progression. Nuances in Notch1 and Wnt signaling could be vital to development of a CAVD therapeutic.

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia.

The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in >65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both invitro and invivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.

Combination of baicalein and miR-106a-5p mimics significantly alleviates IL-1β-induced inflammatory injury in CHON-001 cells.

Osteoarthritis (OA) induces inflammation and degeneration of all joint components, and as such, is a considerable source of disability, pain and socioeconomic burden worldwide. Baicalein (BAI) and microRNA (miR)-106a-5p suppress the progression of OA; however, the effects of BAI and miR-106a-5p for the combined treatment of OA are not completely understood. An in vitro OA model was established by treating CHON-001 cells with 20 ng/ml interleukin (IL)-1β. Cell Counting Kit-8 and flow cytometry assays were conducted to evaluate cell viability and apoptosis, respectively. Western blotting was performed to determine the expression levels of Bax, active caspase-3, Bcl-2, collagen I, collagen III, aggrecan, matrix metallopeptidase (MMP)-13, MMP-9, active Notch1 and transcription factor hes family bHLH transcription factor 1 (Hes1). The levels of IL-6 and tumor necrosis factor-α in the cell culture medium were quantified via ELISA. The present study revealed that treatment with BAI or miR-106a-5p mimic alleviated IL-1β-induced apoptosis, and BAI + miR-106a-5p combination treatment exerted enhanced anti-inflammatory effects compared with monotherapy. Furthermore, IL-1β-induced accumulation of collagen, collagen III, MMP-13 and MMP-9 in CHON-001 cells was reversed to a greater degree following combination treatment compared with monotherapy. Likewise, IL-1β-induced aggrecan degradation was markedly reversed by combination treatment. IL-1β-induced upregulation of active Notch1 and Hes1 in CHON-001 cells was also significantly attenuated by combined BAI + miR-106a-5p treatment. In conclusion, the results of the present study revealed that the combination of BAI and miR-106a-5p mimic significantly decreased IL-1β-induced inflammatory injury in CHON-001 cells, which may serve as a novel therapeutic strategy for OA.

Dibenzazepine Attenuates Against Cisplatin-Induced Nephrotoxicity in Rats: Involvement of NOTCH Pathway.

Cisplatin is one of the standard anti-cancer agents that are used to treat variety of solid tumors. Nevertheless, due to the accumulation of cisplatin in the renal epithelial cells, nephrotoxicity was found to be the main side effect that limits its clinical use. The current study was conducted to assess the potential nephroprotective effect of dibenzazepine, a Notch inhibitor, against cisplatin-induced nephrotoxicity in rats as well as the possible mechanisms underlying this nephroprotection. The rats were pre-treated with 2 mg/kg dibenzazepine for 7 days before giving a single nephrotoxic dose of cisplatin (7 mg/kg). Cisplatin induced acute nephrotoxicity, where blood urea nitrogen and serum creatinine levels were significantly increased. Besides, lipid peroxidation was markedly elevated and the levels of reduced glutathione and catalase were significantly reduced. Also, the tissue levels of the pro-inflammatory mediators; IL-1β, TNF-α, and NF-kB, were significantly increased in the cisplatin group. The pre-treatment with dibenzazepine significantly mitigated the nephrotoxic effects of cisplatin, the oxidative stress and inflammatory status as well as decreased caspase-3 expression, as compared to the cisplatin group. Furthermore, the up-regulation of Notch-1 and Hes-1 was found to be involved in cisplatin-induced nephrotoxicity and their expression was significantly reduced by dibenzazepine. The nephroprotective effect of dibenzazepine was further confirmed by the histopathological assessment. Moreover, dibenzazepine pre-treatment of hela and PC3 cells in vitro did not antagonize the cisplatin anti-cancer activity. In conclusion, these findings show that dibenzazepine provides protection against cisplatin-induced nephrotoxicity. Moreover, the up-regulation of the Notch pathway was shown to play a role in the pathogenesis of cisplatin-induced renal injury.

Notch expression profile and satellite cell stimulation in masseter muscle before and after orthognathic surgery

The aim of this prospective study was to compare the expression of the Notch receptor family with the biomarker for stimulation of satellite cells (SC), which are responsible for functional adaptation.Tissue samples from the masseter muscle were taken presurgically and 7 months later. Samples from controls came from the extraction of third molars. The expression of Notch 1 to 4 and the satellite cell markers CD34, Pax7, and MyoD1 were investigated. PCR was used for relative quantification of gene expression, which was calculated with the ΔΔCT method.The study involved 38 white patients — 10 prognathic, 18 retrognathic, and 10 orthognathic controls. The median value for Notch 1 was significantly reduced presurgically for prognathic (0.46, SD 0.45) and retrognathic (0.57, SD 0.35) patients compared with the controls. Postsurgically, Notch 2 was significantly upregulated in the prognathic group (0.55, SD 0.28/1.37, SD 0.85). Similarly, there was upregulation of Notch 3 in the prognathic group (0.33, SD 0.42/0.59, SD 1.37) and downregulation in retrognathic patients (0.59, SD 0.79/0.52, SD 0.97). Upregulations for the satellite cell markers CD34 and Pax7 were also found in prognathic patients.The significant upregulation of Notch 1–3 and CD34 in prognathics, but unchanged MyoD expression, signals high stimulation for SC and maintenance of the regeneration cell pool. A lower expression of Notch and SC in retrognathic patients could be responsible for weak functional adaptation.

MicroRNA-27 Inhibits Autophagy and Promotes Proliferation of Multiple Myeloma Cells by Targeting the NEDD4/Notch1 Axis.

Multiple myeloma (MM) is a malignant tumor disease that seriously affects the health of patients. Previous studies have shown the crucial role of autophagy in the development of MM. Therefore, the study aimed to study the effect of miR-27 on autophagy in MM via NEDD4/Notch1 axis. RT-qPCR or western blot analysis was used to detect the expression of miR-27, NEDD4, and Notch1 in bone marrow tissues and CD138+ plasma cells of patients and MM cells. After gain- and loss-of-function assays in MM cells, proliferation and invasion were assessed by clone formation and Transwell assays. Meanwhile, expression of autophagy-related proteins was measured by western blot analysis, followed by evaluation of autophagosomes and autophagic flow. The targeting relationship was evaluated by luciferase reporter assay, whereas the binding of NEDD4 to Notch1 protein was analyzed by co-immunoprecipitation. The ubiquitination level of Notch1 protein was detected. A nude mouse tumor model was established to determine the role of miR-27 in MM in vivo. miR-27 and Notch1 upregulation and NEDD4 downregulation were observed in bone marrow tissues and CD138+ plasma cells of patients and MM cells. miR-27 negatively targeted NEDD4, while NEDD4 could specifically bind to Notch1 protein to increase Notch1 ubiquitin degradation in MM cells. miR-27 or Notch1 overexpression or NEDD4 silencing diminished autophagy but enhanced proliferation and invasion of MM cells. miR-27 upregulation promoted the formation of subcutaneous tumor in nude mice. Collectively, miR-27 elevated Notch1 expression by targeting NEDD4 and promoted the development of MM by inhibiting cell autophagy, which provides a new idea and basis for MM treatment.

Long noncoding RNA HCG18 inhibits the differentiation of human bone marrow-derived mesenchymal stem cells in osteoporosis by targeting miR-30a-5p/NOTCH1 axis

BackgroundRecent studies have demonstrated that long non-coding RNAs (LncRNAs) can influence bone cell differentiation and formation. However, it is unclear whether lncRNA HCG18 is involved in osteoporosis (OP). This study was conducted to investigate the regulation of HCG18 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsBMSCs were isolated and cultured from mouse pathological models and osteoporosis patients. RT-qPCR was performed to detect the expression of HCG18 and miR-30a-5p in BMSCs. The interaction between HCG18 and miR-30a-5p was analyzed by dual luciferase assay and RNA pulldown assay. The interaction between miR-30a-5p and NOTCH1 3′-UTR was analyzed by dual luciferase assay. RT-qPCR and Western blotting were used to detect the expression of osteogenic genes Runx2, OCN and OPN. Hindlimb-unloaded (HU) mice model was established, and HCG18 was knocked down on bone-formation surfaces by using lentivirus mediated shRNA transfection.ResultsThe expression of HCG18 was increased in BMSCs of OP patients, while the expression of miR-30a-5p was decreased. The expression of HCG18 and miR-30a-5p was negatively correlated in BMSCs. During the differentiation from BMSCs to osteoblasts, the expression of HCG18 was significantly downregulated, and the expression of miR-30a-5p was significantly upregulated. Overexpression of HCG18 was able to reverse the osteogenic-induced upregulation of miR-30a-5p expression, and knockdown of HCG18 further promoted the expression of miR-30a-5p. In addition, miR-30a-5p partially abolished the effect of HCG18 on osteogenic differentiation of BMSCs. NOTCH1 was a target protein of miR-30a-5p, and upregulation of NOTCH1 reversed the effect of miR-30a-5p on osteogenic differentiation of BMSCs. Furthermore, this study found that lentivirus mediated HCG18 knockdown on the bone-formation surfaces of hindlimb-unloaded (HU) mice partially alleviated unloading-induced bone lossConclusionsHCG18 inhibited osteogenic differentiation of BMSCs induced by OP via the miR-30a-5p/NOTCH1 axis. HCG18 can be identified as a regulator of osteogenic differentiation of BMSCs.

NBS1 interacts with Notch signaling in neuronal homeostasis.

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

Osthole inhibits proliferation of kainic acid‑activated BV‑2 cells by modulating the Notch signaling pathway.

Epilepsy is a syndrome involving chronic recurrent transient brain dysfunction. Activation and proliferation of microglia serve important roles in epilepsy pathogenesis and may be targets for treatment. Although osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has been demonstrated to improve epilepsy in rats, its underlying mechanism remains to be elucidated. The present study investigated the effect of osthole on proliferation of kainic acid (KA)-activated BV-2 cells and explored the molecular mechanism by which it inhibited their proliferation. Using Cell Counting Kit-8, enzyme-linked immunosorbent assay, reverse transcription-quantitative PCR, western blot analysis and immunofluorescence staining, it was identified that following exposure of KA-activated BV-2 cells to 131.2 µM osthole for 24 h, cell proliferation and release of tumor necrosis factor α, interleukin 6 and nitric oxide synthase/induced nitric oxide synthase were significantly inhibited (P<0.05). Further experiments revealed that osthole significantly downregulated mRNA and protein levels of Notch signaling components in KA-activated BV-2 cells (P<0.05). Therefore, it was hypothesized that osthole inhibited the proliferation of microglia by modulating the Notch signaling pathway, which may be useful for the treatment of epilepsy and other neurodegenerative diseases characterized by Notch upregulation.

AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer.

Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack of animal models that faithfully recapitulate the different molecular subtypes impedes adequate preclinical testing of stratified therapeutic concepts. Here, we demonstrate that constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis in Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with the carcinogen azoxymethane. Gene-expression profiling indicates that Trp53ΔIECAktE17K tumors resemble the human mesenchymal colorectal cancer subtype (CMS4), which is characterized by the poorest survival rate among the four CMSs. Trp53ΔIECAktE17K tumor cells are characterized by Notch3 up-regulation, and treatment of Trp53ΔIECAktE17K mice with a NOTCH3-inhibiting antibody reduces invasion and metastasis. In CRC patients, NOTCH3 expression correlates positively with tumor grading and the presence of lymph node as well as distant metastases and is specifically up-regulated in CMS4 tumors. Therefore, we suggest NOTCH3 as a putative target for advanced CMS4 CRC patients.

A Gli inhibitor GANT61 suppresses cell proliferation, promotes cell apoptosis and induces G1/G0 cycle retardation with a dose- and time-dependent manner through inhibiting Notch pathway in multiple myeloma

ABSTRACT Purpose This study aimed to explore the effect of GANT61 on regulating cell proliferation, cell apoptosis and cell cycle, and to investigate whether GANT61 would function in multiple myeloma (MM) via inhibiting Notch pathway. Methods: RPMI-8226 and U266 cells were treated by GANT61 (0, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 μmol/L) for 18, 24 and 36 hours (h), and cell proliferation was detected by Cell Counting Kit 8. Then these cells were treated by GANT61 at 0, 2.5, 5.0, 10.0 μmol/L for 24 h or treated by 10.0 μmol/L GANT61 for 0, 18, 24 and 36 h, and cell apoptosis rate, apoptosis markers and cell cycle were detected by AV/PI, Western blot, and PI staining. Notch1, Jagged1, Jagged2 and Hes1 expressions were detected by qPCR and Western blot. Further rescue experiments were conducted by upregulating Notch1. Results: In RPMI-8226 and U266 cells, GANT61 inhibited cell proliferation, increased cell apoptosis rate and cell percentage of G1/G0 phase while decreased cell percentage of S phase in a dose- and time-dependent manner. Besides, GANT61 inhibited Notch1, Jagged1, Jagged2 and Hes1 expressions in a dose- and time-dependent manner as well. In rescue experiments, Notch1 upregulation attenuated the inhibition of cell proliferation, promotion of cell apoptosis, induction of G1/G0 cycle retardation and repression of Notch signaling pathway induced by GANT61 treatment in RPMI-8226 and U266 cells. Conclusions: GANT61 suppresses cell proliferation, promotes cell apoptosis and induces G1/G0 cycle retardation with a dose- and time-dependent manner through inhibiting Notch pathway in MM. Abbreviations MM: Multiple myeloma; Hh: Hedgehog; EMT: epithelial mesenchymal transition; AML: acute myeloid leukemia; GANT61: GLI antagonist; DMSO: dimethyl sulfoxide; CCK-8: Cell Counting Kit 8; C-Caspase 3: Cleaved Caspase 3; Bcl-2: B-cell lymphoma-2; RT-qPCR: real-time quantitative polymerase chain reaction; OD: optical density; PTCH1: Patched1

Up-regulation of miR-34c-5p inhibits nasopharyngeal carcinoma cells by mediating NOTCH1.

Objective: To explore the correlation between miR-34c-5p and NOTCH1 in nasopharyngeal carcinoma (NPC). Materials and methods: qPCR was employed to quantify miR-34c-5p and NOTCH1 mRNA in NPC, and Western blot to detect NOTCH1. MiR-34c-5p mimics/inhibitor and NOTCH1 siRNA were constructed to analyze the role of miR-34c-5p/NOTCH1 on the biological function of NPC cells. Results: NPC cells showed lower miR-34c-5p expression and higher NOTCH1 expression than normal cells, and up-regulating miR-34c-5p or inhibiting NOTCH1 could strongly suppress the epithelial–mesenchymal transition (EMT), proliferation, invasion and migration of NPC cells, and induce apoptosis in them. Up-regulating miR-34c-5p could inhibit NOTCH1, and miR-34c-5p was negatively correlated with NOTCH1. Rescue experiment results revealed that NOTCH1 up-regulation could counteract the changes of cell process induced by increased miR-34c-5p. Conclusion: MiR-34c-5p inhibits the growth of NPC by down-regulating NOTCH1, so up-regulating miR-34c-5p or down-regulating NOTCH1 may become the potential direction of NPC treatment.

Leptin and Notch Signaling Cooperate in Sustaining Glioblastoma Multiforme Progression.

Glioblastoma multiforme (GBM) is the most malignant form of glioma, which represents one of the commonly occurring tumors of the central nervous system. Despite the continuous development of new clinical therapies against this malignancy, it still remains a deadly disease with very poor prognosis. Here, we demonstrated the existence of a biologically active interaction between leptin and Notch signaling pathways that sustains GBM development and progression. We found that the expression of leptin and its receptors was significantly higher in human glioblastoma cells, U-87 MG and T98G, than in a normal human glial cell line, SVG p12, and that activation of leptin signaling induced growth and motility in GBM cells. Interestingly, flow cytometry and real-time RT-PCR assays revealed that GBM cells, grown as neurospheres, displayed stem cell-like properties (CD133+) along with an enhanced expression of leptin receptors. Leptin treatment significantly increased the neurosphere forming efficiency, self-renewal capacity, and mRNA expression levels of the stemness markers CD133, Nestin, SOX2, and GFAP. Mechanistically, we evidenced a leptin-mediated upregulation of Notch 1 receptor and the activation of its downstream effectors and target molecules. Leptin-induced effects on U-87 MG and T98G cells were abrogated by the selective leptin antagonist, the peptide LDFI (Leu-Asp-Phe-Ile), as well as by the specific Notch signaling inhibitor, GSI (Gamma Secretase Inhibitor) and in the presence of a dominant-negative of mastermind-like-1. Overall, these findings demonstrate, for the first time, a functional interaction between leptin and Notch signaling in GBM, highlighting leptin/Notch crosstalk as a potential novel therapeutic target for GBM treatment.

IMPAD1 functions as mitochondrial electron transport inhibitor that prevents ROS production and promotes lung cancer metastasis through the AMPK-Notch1-HEY1 pathway.

The tumor microenvironment (TME) and metabolic reprogramming have been implicated in cancer development and progression. However, the link between TME, metabolism, and cancer progression in lung cancer is unclear. In the present study, we identified IMPAD1 from the conditioned medium of highly invasive CL1-5. High expression of IMPAD1 was associated with a poorer clinical phenotype in lung cancer patients, with reduced survival and increased lymph node metastasis. Knockdown of IMPAD1 significantly inhibited migration/invasion abilities and metastasis in vitro and in vivo. Upregulation of IMPAD1 and subsequent accumulation of AMP in cells increased the pAMPK, leading to Notch1 and HEY1 upregulation. As AMP is an ADORA1 agonist, treatment with ADORA1 inhibitor reduced the expression of pAMPK and HEY1 expression in IMPAD1-overexpressing cells. IMPAD1 caused mitochondria dysfunction by inhibiting mitochondrial Complex I activity, which reduced mitochondrial ROS levels and activated the AMPK-HEY1 pathway. Collectively this study supports the multipotent role of IMPAD1 in promotion of lung cancer metastasis by simultaneously increasing AMP levels, inhibition of Complex I activity to decrease ROS levels, thereby activating AMPK-Notch1-HEY1 signaling, and providing an alternative metabolic pathway in energy stress conditions.

LncRNA FEZF1-AS1 promotes non-small lung cancer cell migration and invasion through the up-regulation of NOTCH1 by serving as a sponge of miR-34a

BackgroundThe involvement of lncRNA FEZF1-AS1 has been analyzed in many types of cancers, while its roles in non-small cell lung cancer (NSCLC) remains unclear. We then explored the role of FEZF1-AS1 in NSCLC.MethodsqPCR and western blot were performed to measure gene expression. FEZF1-AS1, miR-34a, and NOTCH-1 were overexpressed to analyze the relationship between them. Transwell assays were performed to analyze the effects of transfections on cell invasion and migration.ResultsFEZF1-AS1 was up-regulated in NSCLC patients. Increased expression levels of FEZF1-AS1 were observed with the increase in clinical stages. Bioinformatics analysis showed that miR-34a can bind with FEZF1-AS1. In NSCLC tissues, NOTCH-1 and FEZF1-AS1 were positively correlated. In NSCLC cells, over-expression of FEZF1-AS1 resulted in up-regulated expressions of NOTCH-1, while miR-34a over-expression mediated down-regulated expressions of NOTCH-1. In addition, FEZF1-AS1 and miR-34a did not alter each other, while bioinformatics analysis showed that miR-34a can bind FEZF1-AS1. Analysis of cell migration and invasion showed increased cell invasion and migration rates after FEZF1-AS1 and NOTCH-1 over-expression. MiR-34a played the opposite role and reduced the effects of FEZF1-AS1 over-expression.ConclusionsFEZF1-AS1 promoted NSCLC cell migration and invasion through the up-regulation of NOTCH1 by serving as a sponge of miR-34a.