Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.
Humans ; bcl-2-Associated X Protein/metabolism* ; Carcinoma ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Drug Resistance ; Forkhead Box Protein M1/metabolism* ; Gene Expression Regulation, Neoplastic ; MicroRNAs/genetics* ; Proliferating Cell Nuclear Antigen/metabolism* ; Receptor, Notch1/metabolism* ; RNA, Small Interfering/genetics* ; Stomach Neoplasms/genetics*

OBJECTIVE: To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification. METHODS: Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins. RESULTS: The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01). CONCLUSION Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.
Animals ; Rats ; Bone Morphogenetic Protein 2/metabolism* ; Calcinosis ; Cell Differentiation ; Cells, Cultured ; Lipopolysaccharides ; Osteogenesis ; Rats, Sprague-Dawley ; Receptor, Notch1/genetics* ; 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*

Alveolar Epithelial Cells/cytology* ; Animals ; Bleomycin ; Epithelial-Mesenchymal Transition ; Lung ; Pulmonary Fibrosis/chemically induced* ; Rats ; Receptor, Notch1/metabolism* ; Signal Transduction ; Transforming Growth Factor beta1 ; Twist-Related Protein 1/metabolism*

The present study was aimed to investigate the effects and mechanisms of electro-acupuncture (EA) on proliferation and differentiation of neural stem cells in the hippocampus of C57 mice exposed to different doses of X-ray radiation. Thirty-day-old C57BL/6J mice were randomly divided into control, irradiation, and EA groups. The control group was not treated with irradiation. The irradiation groups were exposed to different doses of X-ray (4, 8 or 16 Gy) for 10 min. The EA groups were electro-acupunctured at Baihui, Fengfu and bilateral Shenyu for 3 courses of treatment after X-ray radiation. Immunohistochemistry was used to evaluate proliferation and differentiation of the hippocampal neural stem cell. RT-PCR and Western blot were used to detect mRNA and protein expressions of Notch1 and Mash1 in the hippocampus, respectively. The results showed that, compared with the control group, the numbers of BrdU positive cells (4, 8 Gy subgroup) and BrdU/NeuN double-labeling positive cells (3 dose subgroups) were decreased significantly in the irradiation group, but the above changes could be reversed by EA. Compared with the control group, the number of BrdU/GFAP double-labeling positive cells in each dose subgroup of irradiation group was decreased significantly, while EA could reverse the change of 4 and 8 Gy dose subgroups. In addition, compared with the control group, the expression levels of Notch1 mRNA and protein in hippocampus were up-regulated, and the expression levels of Mash1 mRNA and protein were significantly decreased in each dose subgroup of irradiation group. Compared with irradiation group, the expression levels of Notch1 mRNA and protein in hippocampus of EA group were decreased significantly in each dose subgroup, and the expression levels of Mash1 mRNA and protein were increased significantly in 4 and 8 Gy subgroups. These results suggest that irradiation affects the proliferation and differentiation of neural stem cells in hippocampus of mice, whereas EA may significantly increase the proliferation and differentiation of hippocampal neural stem cells via the regulation of Notch signaling pathway.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Differentiation ; Cell Proliferation ; Electroacupuncture ; Hippocampus ; cytology ; radiation effects ; Mice, Inbred C57BL ; Neural Stem Cells ; cytology ; radiation effects ; Random Allocation ; Receptor, Notch1 ; metabolism ; X-Rays ; adverse effects

Valproic acid (VPA), an agent that is used to treat epileptic seizures, can cause spatial memory impairment in adults and children. This effect is thought to be due to the ability of VPA to inhibit neurogenesis in the hippocampus, which is required for learning. We have previously used an animal model to show that VPA significantly impairs hippocampal-spatial working memory and inhibits neuronal generation in the sub-granular zone of the dentate gyrus. As there are patient reports of improvements in memory after discontinuing VPA treatment, the present study investigated the recovery of both spatial memory and hippocampal neurogenesis at two time points after withdrawal of VPA. Male Wistar rats were given intraperitoneal injections of 0.9% normal saline or VPA (300 mg/kg) twice a day for 10 d. At 1, 30, or 45 d after the drug treatment, the novel object location (NOL) test was used to examine spatial memory; hippocampal cell division was counted using Ki67 immunohistochemistry, and levels of brain-derived neurotrophic factor (BDNF) and Notch1 were measured using western immunoblotting. Spatial working memory was impaired 1 and 30 d after the final administration, but was restored to control levels by 45 d. Cell proliferation had increased to control levels at 30 and 45 d. Both markers of neurogenesis (BDNF and Notch1 levels) had returned to control levels at 45 d. These results demonstrate that memory recovery occurs over a period of six weeks after discontinuing VPA treatment and is preceded by a return of hippocampal neurogenesis to control levels.
Animals ; Brain-Derived Neurotrophic Factor/metabolism* ; Cell Proliferation ; Cognition/drug effects* ; Dentate Gyrus/drug effects* ; Enzyme Inhibitors/pharmacology* ; Hippocampus/metabolism* ; Immunohistochemistry ; Male ; Memory Disorders/therapy* ; Memory, Short-Term/drug effects* ; Neurogenesis/drug effects* ; Neurons/metabolism* ; Rats ; Rats, Wistar ; Receptor, Notch1/metabolism* ; Spatial Memory/drug effects* ; Valproic Acid/pharmacology*

OBJECTIVETo examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis.

METHODSThe osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL (50 ng/mL) and macrophage-colony stimulating factor (50 ng/mL) were added to this system, followed by treatment with brucine (0.02, 0.04 and 0.08 mmol/L), or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1 (TGF-β1), nuclear factor-kappa B (NF-κB) and Hes1 were determined by enzyme-linked immunosorbent assay.

RESULTSCompared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells (P<0.01). Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1 (P<0.05 or P<0.01).

CONCLUSIONBrucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

Animals ; Bone Neoplasms ; metabolism ; prevention & control ; secondary ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Humans ; Jagged-1 Protein ; metabolism ; Macrophages ; drug effects ; physiology ; Mice ; Osteoclasts ; drug effects ; physiology ; Receptor, Notch1 ; metabolism ; Signal Transduction ; drug effects ; Strychnine ; analogs & derivatives ; pharmacology ; therapeutic use

BACKGROUNDThe addition of anti-human epidermal growth factor receptor 2 (HER2)-targeted drugs, such as trastuzumab, lapatinib, and trastuzumab emtansine (T-DM1), to chemotherapy significantly improved prognosis of HER2-positive breast cancer patients. However, it was confused that metastatic patients vary in the response of targeted drug. Therefore, methods of accurately predicting drug response were really needed. To overcome the spatial and temporal limitations of biopsies, we aimed to develop a more sensitive and less invasive method of detecting mutations associated with anti-HER2 therapeutic response through circulating-free DNA (cfDNA).

METHODSFrom March 6, 2014 to December 10, 2014, 24 plasma samples from 20 patients with HER2-positive metastatic breast cancer who received systemic therapy were eligible. We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes, and then used targeted next-generation sequencing (NGS) to identify somatic mutation of these samples in those 50 genes. Samples taken before their first trastuzumab administration and subsequently proven with clinical benefit were grouped into sensitive group. The others were collected after disease progression of the trastuzumab-based therapy and were grouped into the resistant group.

RESULTSA total of 486 single-nucleotide variants from 46 genes were detected. Of these 46 genes, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), proto-oncogene c-Kit (KIT), and tumor protein p53 (TP53) were the most common mutated genes. Seven genes, including epidermal growth factor receptor (EGFR), G protein subunit alpha S (GNAS), HRas proto-oncogene (HRAS), mutL homolog 1 (MLH1), cadherin 1 (CDH1), neuroblastoma RAS viral oncogene homolog (NRAS), and NOTCH1, that only occurred m utations in the resistant group were associated with the resistance of targeted therapy. In addition, we detected a HER2 S855I mutation in two patients who had persistent benefits from anti-HER2 therapy.

CONCLUSIONTargeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.

Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; metabolism ; Cadherins ; genetics ; Chromogranins ; genetics ; Class I Phosphatidylinositol 3-Kinases ; Drug Resistance, Neoplasm ; genetics ; Female ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Phosphatidylinositol 3-Kinases ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptor, ErbB-2 ; metabolism ; Receptor, Notch1 ; genetics ; Tumor Suppressor Protein p53 ; genetics ; Young Adult

OBJECTIVETo explore the mRNA expression level of Notch1 and ASB2 in bone marrow mononuclear cells of patients with P210(+) chronic myeloid leukemia and the correlation between Notch signaling pathway and ubiquitination in chronic myeloid leukemia, so as to provide the valuable information for investigating the pathogenesis of chronic myeloid leukemia and clinical treatment.

METHODSBone marrow was collected from 32 patients with newly diagnosed chronic myeloid leukemia and 34 non-hematopathic and healthy individuals (control group), respectively. The mRNA expression levels of Notch1 and ASB2 were detected by real-time quantitative PCR.

RESULTSThe expression of Notch1 mRNA in CML patients were significantly different from that of healthy individuals group (t=36.3, P<0.01), which was 337.8 times of the healthy individuals. Moreover, the expression level of ASB2 mRNA in CML group was significantly higher than that in healthy individuals (t=19.4, P<0.01). The mRNA expression of Notch1 and ASB2 gene was positive correlation (r=0.504, P<0.01).

CONCLUSIONThe aberrant expression of Notch1 and Asb2 exists in patients with P210 positive CML, which may be involved in incidence and development of CML.

Bone Marrow ; metabolism ; Case-Control Studies ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptor, Notch1 ; genetics ; metabolism ; Signal Transduction ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism

OBJECTIVETo explore the role of PDK1 in T-ALL development through establishing the Notch1-induced T-ALL mouse model by using Mx1-cre; LoxP system to knock-out PDK1.

METHODSCell cycle and apoptosis of leukemic cells were detected by flow cytometry, and relative expression of tumor-related genes and transcription factors of leukemic cells were determined by quantitative real-time PCR.

RESULTSNotch1-induced T-ALL mouse model with inducible knock-out of PDK1 was established successfully. Compared to T-ALL control mouse model, PDK1 knock-out mice showed a significant longer survival time (P<0.01). There was no difference of cell cycle between control and PDK1 knock-out mice, and the apoptosis rate of leukemic cells in PDK1 knock-out mice was higher than that of control mice (P<0.001). PDK1 knock-out resulted in decreased expression of tumor-related genes and transcription factors, such as c-Myc and NF-κB (P<0.01), and increased expression level of P53 (P<0.01).

CONCLUSIONPDK1 knock-out can inhibit the development of T-ALL, and its mechanism may be the leukemia progression inhibited by regulating the apoptosis and expression of multiple related genes and transcription factors.

Animals ; Apoptosis ; Cell Cycle ; Disease Models, Animal ; Gene Expression Regulation, Leukemic ; Mice ; Mice, Knockout ; NF-kappa B ; genetics ; metabolism ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Protein-Serine-Threonine Kinases ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptor, Notch1 ; genetics ; Tumor Suppressor Protein p53 ; genetics ; metabolism