OBJECTIVETo investigate the expressions and the role of Notch signaling-associated proteins in esophageal squamous cell carcinoma (ESCC).

METHODSFifty patients with ESCC were included in this study. The expressions of Notch signaling-associated protein (4 receptors: Notch1, Notch2, Notch3, Notch4; 5 ligands: Dll1, Dll3, Dll4, Jagged1, Jagged2) in cancer foci and adjacent normal tissues (5 cm distance to cancer) were examined by immunohistochemitry. Correlations of these proteins with cancer cell proliferation(Ki-67 index) and clinicopathologic features were investigated.

RESULTSHigher levels of Notch1 and Notch2 were measured in cancer foci compared with adjacent tissues (all P<0.05). There were no differences in the expressions of Notch3, Dll1 and Dll3 (all P>0.05). Notch4, Dll4 and Jagged2 were not detected in both cancer foci and adjacent tissues. Notch1 expression was negatively correlated with lymph node metastasis and TNM staging (all P<0.01). Jagged 1 expression was positively correlated with TNM staging (P<0.01). Ki-67 index was obviously higher in cancer foci, while it was negatively correlated with Notch1 and Notch3 (all P<0.01) and positively correlated with Dll1 and Jagged 1 (all P<0.01).

CONCLUSIONNotch signaling path may act as tumor suppressive gene in the pathogenesis of esophageal squamous cell cancer, in which Notch1 protein plays an important role.

Carcinoma, Squamous Cell ; metabolism ; Esophageal Neoplasms ; metabolism ; Genes, Tumor Suppressor ; Humans ; Lymphatic Metastasis ; Neoplasm Staging ; Receptor, Notch1 ; metabolism ; Receptor, Notch2 ; metabolism ; Receptor, Notch3 ; Receptors, Notch ; metabolism ; Signal Transduction

OBJECTIVETo investigate the correlation between the expressions of glypican-3 (GPC3) and Notch1 and their roles in the tumorigenesis and progression of hepatocellular carcinoma (HCC).

METHODSImmunohistochemistry and computerized image analysis were utilized to quantitatively detect the expressions of GPC3 and Notch1 in 30 HCC tissue specimens.

RESULTSIn the 30 HCC specimens, GPC3 expression decreased significantly as the grade of tumor differentiation increased (P<0.05 or P<0.01), while Notch1 expression presented with a reverse pattern of changes (P<0.05 or P<0.01). An obvious negative correlation was found between the expressions of GPC3 and Notch1 in HCC tissues (rp=-0.607, P=0.000; r=-0.692, P=0.000).

CONCLUSIONThe expressions of GPC3 and Notch1 show a negative correlation in HCC, suggesting a possible mechanism for mutual regulation between them to contribute to the tumorigenesis and progression of HCC.

Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Glypicans ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Receptor, Notch1 ; metabolism

BACKGROUNDIschemic postconditioning (IPost), able to significantly attenuate myocardial ischemia reperfusion injury, is dependent on RISK signaling. Studies have shown that Notch signaling repairs damaged myocardium, and this study aimed to investigate the effect of Notch signaling in myocardial IPost.

METHODSWe used H9c2 cells to establish the myocardial IPost and Hypoxia/Reoxygenation (H/R) model in vitro. which were randomly divided into control, H/R, IPost, Hepatocyte growth factor (HGF)+IPost and DAPT+IPost, N1ICD+IPost, miRNA+IPost, and Mock treatment groups. The myocardial cell viability was assessed by MTT, the cell apoptosis was detected using Annexin V/PI double staining and flow cytometry analyses. The expression of N1ICD, Hes1, PTEN Phospho-Akt/Akt, Phospho-GSK-3β/GSK-3β were detected by Western blotting. Finally, we assessed the changes in ψm using the potential-sensitive dye JC-1 and measured using flow cytometry analyses.

RESULTSThe Notch1 signaling is activated by HGF and ectopic expression of N1ICD during myocardial IPost, which increased myocardial cell viability, prevented cardiomyocyte apoptosis, and reduced loss of the mitochondrial membrane potential. However, myocardial ischemia reperfusion injury was increased in IPost when Notch1 signaling was inhibited using DAPT or with knockdown by Notch1-miRNA. Western blotting found that PTEN was down-regulated by Hes1 when Notch1 was activated, which consequently promoted Akt and GSK-3β phosphorylation.

CONCLUSIONSNotch1 crosstalk with RISK signaling may be dependent on PTEN, which plays a cardioprotective role during IPost. This mechanism could provide a promising therapeutic target for the treatment of ischemic heart disease.

Cell Line ; Humans ; Ischemic Postconditioning ; Myocardial Reperfusion Injury ; genetics ; metabolism ; prevention & control ; Receptor, Notch1 ; genetics ; metabolism ; Signal Transduction ; physiology

OBJECTIVETo detect the differential expression of Notch1 in the genital tubercle (GT) of fetal male rats with hypospadias induced by maternal exposure to Di-n-butyl phthalate (DBP) and that in normal control fetal rats in order to further explore the role of Notch1 in DBP-induced hypospadias.

METHODSTwenty pregnant SD rats were equally and randomly divided into an experimental and a control group, the former given DBP and the latter soybean oil intragastrically at 800 mg/(kg x d) and 2 ml/d respectively from gestation day (GD) 14 to GD 18. On GD 19, the birth weight (BW), anogenital distance (AGD) and hypospadias incidence were recorded, GTs of the fetal male rats collected, and the expression of Notch1 analyzed by Western blot and immunohistochemistry.

RESULTSThe BW of the fetal male rats was (4.40 +/- 0.30) g in the experimental group, significantly lower than (6.11 +/- 0.40) g in the control (P <0.05), and the AGD was (2.17 +/- 0.18) mm in the former, markedly shorter than (3.28 +/- 0.16) mm in the latter (P<0.05). The incidence of hypospadias was 42.9%. The relative expression of Notch1 was remarkably lower in the hypospadiac rats than in the normal controls (0.671 +/- 0.021 vs 1.327 +/- 0.031, P<0.05), and it was mainly located in the epithelial cells of the GT. The staining intensity was obviously weaker in the hypospadias than in the normal control group.

CONCLUSIONDBP has an obvious toxic effect on fetal male rats and can change the expression of Notch1 in the GT. It possibly affects cell proliferation and apoptosis and epithelial-to-mesenchymal transition (EMT), resulting in the occurrence of hypospadias.

Animals ; Dibutyl Phthalate ; toxicity ; Female ; Fetus ; Hypospadias ; chemically induced ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; metabolism

Cadherins ; metabolism ; Female ; Humans ; MCF-7 Cells ; Neoplastic Stem Cells ; metabolism ; Receptor, Notch1 ; metabolism ; Receptors, CXCR4 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism

This study was aimed at exploring the expression pattern of P2X family receptors (P2XR) in peritoneal macrophages and their relationship with the activation states of macrophages in Notch1-induced mouse T-ALL model. After establishment of the leukemia model, F4/80(+) peritoneal macrophages, F4/80(+)CD206(+) M2-like and F4/80(+)CD206(-) M1-like peritoneal macrophages were sorted by flow cytometry based on F4/80 and CD206 surface markers. The expression of P2XR in each cell population was detected by real time RT-PCR. The results showed that macrophages,M1-like and M2-like macrophages moderately expressed P2XR except for P2X5R. The expression of P2XR varied with the development of leukemia. The expression of P2X1R and P2X7R in peritoneal macrophages increased steadily; the expression of P2X2R and P2X3R decreased at late stage of leukemia;the expression of P2X4R slightly decreased at intermediate stage;the expression of P2X6R kept unchanged. At intermediate stage of leukemia, the expression of P2XR in M1-like and M2-like peritoneal macrophages varied. M1-like macrophages expressed higher level of P2X1R than M2-like macrophages, whereas M2-like macrophages expressed higher level of P2X7R than M1-like macrophages, which suggested that the expression of P2XR were related to the activation states. It is concluded that the expression of P2XR in peritoneal macrophages from leukemia mice is related to the progression of leukemia and the activation states of macrophages, which lay a foundation for further studying the role of macrophages in the development of leukemia.
Animals ; Cytokines ; metabolism ; Disease Models, Animal ; Macrophages, Peritoneal ; metabolism ; Mice ; Mice, Inbred C57BL ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Receptor, Notch1 ; metabolism ; Receptors, Purinergic P2X ; metabolism ; Signal Transduction

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most dangerous hematological malignancies, with high tumor heterogeneity and poor prognosis. More than 60% of T-ALL patients carry NOTCH1 gene mutations, leading to abnormal expression of downstream target genes and aberrant activation of various signaling pathways. We found that chidamide, an HDAC inhibitor, exerts an antitumor effect on T-ALL cell lines and primary cells including an anti-NOTCH1 activity. In particular, chidamide inhibits the NOTCH1-MYC signaling axis by down-regulating the level of the intracellular form of NOTCH1 (NICD1) as well as MYC, partly through their ubiquitination and degradation by the proteasome pathway. We also report here the preliminary results of our clinical trial supporting that a treatment by chidamide reduces minimal residual disease (MRD) in patients and is well tolerated. Our results highlight the effectiveness and safety of chidamide in the treatment of T-ALL patients, including those with NOTCH1 mutations and open the way to a new therapeutic strategy for these patients.
Aminopyridines ; Benzamides ; Cell Line, Tumor ; Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism* ; Proto-Oncogene Proteins c-myc/metabolism* ; Receptor, Notch1/metabolism* ; Signal Transduction ; T-Lymphocytes/metabolism*

OBJECTIVETo detect the differential expression of Notch1 and Notch2 in human astrocytoma and medulloblastoma; and to study the role of Notch1 and Notch2 in the development of both tumors.

METHODSImmunohistochemical staining (SP method) and Western blot analysis were used to detect Notch1 and Notch2 expression in tissue arrays and freshly resected samples of normal brain tissue, astrocytoma and medulloblastoma.

RESULTSNotch1 and Notch2 were negative in normal human brain tissue. Notch1 was highly expressed (total positive rate 80.0%, 48/60) while Notch2 was not detected in grade IV astrocytomas and sporadically observed in lower grade astrocytomas (total positive rate 10.0%, 6/60). The percentage of positive tumor cells and expression level of Notch1 increased with higher histologic grade (r = 0.859, P < 0.05). On the other hand, overexpression of Notch2 was detected in medulloblastoma (9/10) in contrast with lower expression of Notch1 (2/10).

CONCLUSIONSNotch1 and Notch2 show differential expression in astrocytoma and medulloblastoma. This may be related to their different functional activities during the process of brain development.

Adolescent ; Adult ; Aged ; Astrocytoma ; metabolism ; Biomarkers, Tumor ; metabolism ; Brain ; metabolism ; Brain Neoplasms ; metabolism ; Child ; Child, Preschool ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Medulloblastoma ; metabolism ; Middle Aged ; Receptor, Notch1 ; metabolism ; physiology ; Receptor, Notch2 ; metabolism ; physiology ; Young Adult

This study was aimed to explore the effect of DLL4/Notch1 ligand on cell growth in leukemia cell line K562 and its relevant mechanism. The pBudCE4.1-DLL4 plasmid was transfected into K562 cells by lipofectamine 2 000, RT-PCR and Western blot were applied to monitor the mRNA and the protein expression of exogenous DLL4 gene, as well as the expression of Notch1-ICD and target gene Hes1. Expression levels of Rb, YY1 and C-MYC protein in K562 cells were also detected by Western blot. Cell counting Kit-8 was used to detect the proliferation of K562 cells, and flow cytometry with Annexin V staining was used to detect the cell apoptosis. The results showed that the mRNA and protein expression levels of DLL4, Notch1-ICD and Hes1 in cells of experimental group were significantly higher than those of control groups (P < 0.05), indicating the successful activation of the Notch1 signaling pathway. The protein expression levels of Rb, YY1 and C-MYC in cells of experimental group significantly increased when compared with that of control group cells (P < 0.05). After transfection, the proliferation of K562 cells was obviously inhibited, and apoptosis rate in DLL4-transfected cells was significantly enhanced. DLL4 transfection significantly increased the number of cells in G1 phase and decreased that in S phase. It is concluded that the over-expression of DLL4 ligand gene in K562 cells results in successful activation of the Notch1 signaling pathway, increases expression of Rb, YY1 and C-MYC genes, which induces apoptosis and reduces proliferation.
Apoptosis ; Cell Cycle ; Cell Proliferation ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; K562 Cells ; Ligands ; Plasmids ; Proto-Oncogene Proteins c-myc ; RNA, Messenger ; Receptor, Notch1 ; metabolism ; Signal Transduction ; Transfection

OBJECTIVE: To characterize the expression of Toll-like receptor 4 (TLR4) in monocytes of diabetic nephropathy (DN) patients and the response of TLR4 to lipopolysaccharide (LPS), and, further, to explore the potential effects of inflammatory immune response in DN. METHODS: Thirty DN patients with uremia, ten early-type 2 DN patients, and twenty healthy volunteers were enrolled for the determination of TLR4 expression in monocytes by using peripheral blood flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and subjected to 1 μg/mL LPS for 24 h. Monocytes were collected to assay NF-κB p65 and Notch1 expression by Western blot, with immuneofluorescence detection. Serum and supernatants were sampled for the determination of interleukin-6 (IL-6) concentration by using ELISA. Serum C-reactive protein (CRP) level was determined by using the immunoturbidimetry. RESULTS: Compared with the normal control, type 2 DN uremic patients had a significantly higher TLR4 fluorescence-blot intensities (FI), and serum CRP and IL-6 levels [TLR4 FI: DN uremia patients 2.8±0.9; early type 2 DN patients 3.4 ±0.7; healthy subjects 1.6±0.7. IL-6 concentration: DN uremia patients (84.8±20.7) pg/mL; early type 2 DN patients (63.20±14.4) pg/mL; healthy subjects (11.0±2.0) pg/mL. CRP concentraton: DN uremia patients (5.4±2.8) mg/L; early type 2 DN patients (3.7±1.7) mg/L; healthy subjects (1.7±0.7) mg/L. P<0.01 for any DN-group vs control]. In early type 2 DN patients, following exposure to LPS, PBMCs showed a significant upregulation in TLR4 and NF-κB p65 expression and a remarked increase in serum IL-6 level (all P<0.05), and NF-κB p65 transfer to the nucleus is enhanced. Notch1 protein expression was not significantly altered in any group. CONCLUSION A disturbance in proinflammatory CD14(+)CD16(+) monocytes occurs in type 2 DN patients. Such immunological dysfunction may be related to activation in NF-κB/TLR4 signaling pathways, and have nothing to do with the Notch1 signaling pathway.
Adult ; Aged ; Diabetes Mellitus, Type 2 ; blood ; Diabetic Nephropathies ; blood ; Female ; Humans ; Lipopolysaccharides ; pharmacology ; Male ; Middle Aged ; Monocytes ; metabolism ; Receptor, Notch1 ; genetics ; metabolism ; Signal Transduction ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism