Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification / 中国骨伤

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.

Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1 / 中国医学科学院学报

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.

Regulation of Notch1 and Foxp1 by MiR-34a in psoriasis vulgaris / Regulación de Notch1 y Foxp1 por MiR-34ª en psoriasis vulgaris

SUMMARY: This study is to investigate the regulation of Notch1 and Foxp1 by miR-34a in the development of psoriasis vulgaris. RT-PCR was used to compare the levels of miR-34a in the skin lesions of 20 patients with psoriasis vulgaris and 20 normal skin tissues. Immunohistochemistry was used to detect the expression of Notch1 and Foxp1 in 51 patients with psoriasis vulgaris, which were further compared with that in 29 normal control tissues. In addition, in HaCaT cells, we used miR-34a mimics and inhibitors to overexpress and inhibit miR-34a, respectively, and detected the mRNA and protein levels of miR-34a, Notch1, and Foxp1. The level of miR-34a in the skin lesions of patients with psoriasis vulgaris was significantly higher than that in normal skin tissues (t=2.192, P<0.05). The positive rate of Notch1 in the skin lesions of patients with psoriasis vulgaris was 76.47 %, which was significantly higher than that in normal skin tissues (13.79 %) (t=29.215, P<0.01). The positive rate of FOXP1 in the psoriasis vulgaris group was 92.16 %, which was also significantly higher than that in the normal skin group (65.52 %) (t=9.087, P<0.01). In addition, overexpression of miR-34a significantly promoted the expression of Notch1 and Foxp1. However, inhibition of miR-34a significantly reduced Notch1 and Foxp1 levels. miR- 34a is highly expressed in the skin tissues of patients with psoriasis vulgaris, and may participate in the development of psoriasis vulgaris by regulating Notch1 and Foxp1.

RESUMEN: El objetivo de este estudio fue investigar la regulación de Notch1 y Foxp1 por miR-34a en el desarrollo de la psoriasis vulgar. Se utilizó RT-PCR con el fin de comparar los niveles de miR-34a en las lesiones cutáneas de 20 pacientes con psoriasis vulgar y 20 tejidos de piel normales. Se utilizó inmunohistoquímica para detectar la expresión de Notch1 y Foxp1 en 51 pacientes con psoriasis vulgar, que se compararon además con la de 29 tejidos normales control. Además, en las células HaCaT, usamos miméticos e inhibidores de miR-34a para sobreexpresar e inhibir miR-34a, respectivamente, y detectamos los niveles de ARNm y proteína de miR-34a, Notch1 y Foxp1. El nivel de miR- 34a en las lesiones cutáneas de pacientes con psoriasis vulgar fue significativamente mayor que en los tejidos normales de la piel (t=2,192, P<0,05). La tasa de positividad de Notch1 en las lesiones cutáneas de pacientes con psoriasis vulgar fue del 76,47 %, que fue significativamente mayor que la de los tejidos normales de la piel (13,79 %) (t=29,215, P<0,01). La tasa positiva de FOXP1 en el grupo de psoriasis vulgar fue del 92,16 %, que también fue significativamente mayor que la del grupo de piel normal (65,52 %) (t=9,087, P<0,01). Además, la sobreexpresión de miR-34a promovió significativamente la expresión de Notch1 y Foxp1. Sin embargo, la inhibición de miR-34a redujo de manera importante los niveles de Notch1 y Foxp1. miR-34a se expresa en gran medida en los tejidos de la piel en pacientes con psoriasis vulgar y puede participar en el desarrollo de la psoriasis vulgar mediante la regulación de Notch1 y Foxp1.

Chidamide inhibits the NOTCH1-MYC signaling axis in T-cell acute lymphoblastic leukemia / 医学前沿

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.

Effect of Notch1 on extracellular matrix deposition in the renal tubulointerstitium of diabetes / 生理学报

The aim of the present study was to observe the effects of Notch1 and autophagy on extracellular matrix deposition in renal tubulointerstitium of diabetes and to explore the mechanism. The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice). After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. Rat renal tubular epithelial cells NRK52E were cultured under normal glucose (NG) and high glucose (HG) respectively, and the expression of Notch1 and LC3 proteins were detected by Western blotting. Autophagosomes in NRK52E cells with overexpressed and knockdown Notch1 under NG and HG conditions were observed by confocal microscope, and the expression changes of Notch1, Collagen-I and III protein were detected by immunofluorescence. The results showed that the Notch1 and Collagen-III expressions were increased (P < 0.01) and the LC3 expression was decreased (P < 0.05) in db/db mice compared with db/m mice. In vitro, the Notch1 was increased (P < 0.01) and the LC3 expression was decreased significantly (P < 0.01) in NRK52E cells of HG group compared with NG group. There was no significant change of Notch1 and LC3 expression between the mannitol (MA) group and the NG group. Autophagy was decreased and extracellular matrix deposition was aggravated when Notch1 was overexpressed. In contrast, autophagy was increased and extracellular matrix deposition was relieved by knockdown of Notch1 under HG conditions. In conclusion, Notch1 protein expression was increased and autophagy was reduced in renal tissue of diabetes and renal tubular epithelial cells under HG. The extracellular matrix deposition in the renal tubulointerstitium was relieved by regulating autophagy after the knockdown of Notch1.

Combinación de bardoxolona y DMF Mejora la isquemia cerebral/reperfusión Lesión en ratas macho / Combination of Bardoxolone and DMF Ameliorates Cerebral Ischemia / Reperfusion Injury in Male Rats

Background: Ischemic stroke has been ranked as the second cause of death in patients worldwide. Inflammation which is activated during cerebral ischemia/reperfusion (I/R) is an important mechanism leading to brain injury. The present study aimed to investigate the effect of Berberine on cerebral I/R injury and the role of inflammation in this process. Material and Methods: The study was carried out on 36 Wistar-albino rats, divided into four groups including: Sham group, I/R group, I/R+ (control-vehicle DMSO) and I/R+ Berberine 5 mg/kg injected intraperitoneally 1 hour before induction of ischemia. Measurement of brain tissue IL-1ß, ICAM1, caspase-3, Notch 1 and Jagged 1 was done after one hour of reperfusion in addition to assessment of the brain infracted area and histopathological analysis. Results: Berberine attenuates cerebral I/R injury induced increase in inflammatory cytokine (IL-1ß), adhesion molecule (ICAM-1) and proapoptotic enzyme (caspase-3). Additionally, it reduces the size of infracted area and histopathological damage; such protective effect could be mediated by Notch 1 signaling pathway since Berberine further unregulated the increased levels of Notch 1 and Jagged 1 seen in brain with I/R injury. Conclusions: Berberine has a neurocytoprotective outcome against cerebral I/R injury which is manifested as anti-inflammatory anti-apoptotic effect that preserved cell structure and viability, in the meantime this effect could be mediated by Notch 1 signaling pathway.

Gene Mutation in Acute Lymphoblastic Leukemia by DNA Sequencing / 中国实验血液学杂志

OBJECTIVE@#To analyze the characteristics of gene mutation in adult ALL and its clinical significance.@*METHODS@#Clinical data of 134 primary adult ALL patients and DNA sequencing results of 16 kinds of gene mutation were collected. The characteristic of gene mutation and clinical significances were statistically analyzed.@*RESULTS@#In 31 cases of 134 ALL cases (23.13%) the gene mutations were detected as follows: 19 cases of 114 B-ALL cases (16.67%), 11 cases of 19 T-ALL cases (57.89%) and 1 case of T/B-ALL. The incidence of T-ALL gene mutation was significantly higher than that of B-ALL (χ@*CONCLUSION@#There may be multiple gene mutations in adult ALL patients. IL7R and NOTCH1 are the most common gene mutations and NOTCH1 mutation may indicate poor prognosis. Detection of gene mutations is helpful to understand the pathogenesis of ALL and evaluate the prognosis of adult ALL patients.

Mutaciones del gen NOTCH1 en la leucemia linfocítica crónica / NOTCH1 gene mutations in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. The disease has\r\na highly variable clinical course, ranging from very indolent cases to patients with aggressive and rapidly\r\nprogressing outcome. Genetic studies are useful tools in analyzing this pathology, and have been incorporated in international risk classifications. The analysis of genomic rearrangements and the mutational status of immunoglobulin heavy chain variable have allowed risk groups of high prognostic value to be established. More recently, next generation sequencing studies have identified novel somatic mutations that could explain the wide clinical variability of this pathology. Among them, the analysis of NOTCH1 (neurogenic locus notch homolog protein 1) gene mutations are of interest, as deregulation is associated with tumorigenesis. NOTCH11 mutations are mostly located at exon 34 (80% of cases) and 3´UTR (untranslated region). They produce premature stop codons that produce a constitutively active and stable NOTCH1 protein. NOTCH1 mutations are associated with adverse prognosis and refractoriness to treatment. The aim of this study was to analyze NOTCH1 mutations in CLL patients by ASO-PCR and sequencing. Our results found 4.4% of cases with NOTCH1 mutated values concordant with international observations (5%-10%). Including them in the genetic status of CLL patients allows the characterization of risk groups, an aspect of great importance in clinical practice and therapeutic decisions, to be refined.

La leucemia linfocítica crónica (LLC) es la leucemia más frecuente en adultos de Occidente. Presenta\r\nun curso clínico altamente variable, con pacientes que requieren tratamiento inmediato y otros con un curso indolente de la enfermedad. Los estudios genéticos constituyen herramientas de suma utilidad en esta enfermedad, encontrándose incorporados a las clasificaciones de riesgo internacionales. El análisis de los rearreglos genómicos y del estado mutacional de los genes IGHV (immunoglobulin heavy chain variable region) ha hecho factible establecer grupos de riesgo de alto valor pronóstico. Más recientemente, estudios de secuenciación de última generación permitieron la detección de mutaciones\r\nsomáticas previamente desconocidas en esta afección, que podrían explicar la amplia variabilidad clínica\r\nobservada en la LLC. Entre ellas, resultan de interés las observadas en el gen NOTCH1 (neurogenic locus notch homolog protein 1), cuya desregulación se asocia con el desarrollo tumoral. Estas mutaciones se acumulan en mayor medida en el exón 34 (80% de los casos) y en la región 3´UTR (untraslated region), lo que genera codones de terminación prematuros que originan una proteína NOTCH1 constitutivamente activa y más estable, los cuales se asocian con pronóstico adverso y refractariedad al tratamiento. Nuestro objetivo fue evaluar mutaciones de NOTCH1 en nuestros pacientes mediante ASO-PCR y secuenciación. Se detectaron mutaciones en el 4.4% de los casos, valor concordante con los datos internacionales (5% a 10%). Su inclusión en la caracterización genética de los pacientes con LLC permitirá refinar la categorización de los grupos de riesgo, aspecto de suma importancia tanto en el seguimiento clínico como en la toma de decisiones terapéuticas.

HtrA1 suppresses the growth of pancreatic cancer cells by modulating Notch-1 expression

Pancreatic cancer is well known to be the most deadly malignancy with the worst survival rate of all cancers. High temperature requirement factor A1 (HtrA1) plays an important role in cancer cell proliferation, migration, apoptosis, and differentiation. This study aimed to explore the function of HtrA1 in pancreatic cancer cell growth and its underlying mechanism. We found that the expression of HtrA1 was lower in pancreatic cancer tissue compared to the adjacent normal tissue. Consistently, HtrA1 levels were also decreased in two human pancreatic cancer cell lines, PANC-1 and BXPC-3. Moreover, enforced expression of HtrA1 inhibited cell viability and colony formation of PANC-1 and BXPC-3 cells. Overexpression of HtrA1 promoted apoptosis and suppressed migratory ability of tumor cells. On the contrary, siRNA-mediated knockdown of HtrA1 promoted the growth potential of pancreatic cancer cells. In addition, we found that up-regulation of HtrA1 reduced the expression of Notch-1 in pancreatic cancer cells. On the contrary, knockdown of HtrA1 increased the expression levels of Notch-1. Furthermore, overexpression of Notch-1 abolished the anti-proliferative effect of HtrA1 on pancreatic cancer cells. Taken together, our findings demonstrated that HtrA1 could inhibit pancreatic cancer cell growth via regulating Notch-1 expression, which implied that HtrA1 might be developed as a novel molecular target for pancreatic cancer therapy.

Curcumin Increases the Chemosensitivity of Multiple Myeloma to Bortezomib by Inhibiting the Notch1 Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To evaluated the effect of curcumin on the bortezomib-resistant myeloma cells and the expression of Notch1 signaling pathway, in order to further explore its potential mechanism.@*METHODS@#Curcumin, bortezomib, and curcumin combined bortezomib were added into RPMI 8266, U266, 5 nmol/L bortezomib-resistant RPMI 8266 (RPMI 8226-V5R), 5 nmol/L bortezomib-resistant U 266 (U266-V5R) and CD138+ plasma cells respectively. The cell proliferation was measured by MTT assay. the apoptotic rate was determined by flow cytometry, and the Western blot was used to detect the expression of apoptosis-related proteins. Then, the expression of Notch1 in cells was inhibited by notch1 inhibitor DAPT and RNA interference, the above-motioned experiments should be repeated.@*RESULTS@#Compared with single drug-treated groups, the treatment with 2 drugs could further inhibit cell proliferation, induce apoptosis and enhance the inhibition effect on notch1 signaling pathway (P<0.05), while the inhibiting Notch1 signaling pathway could reduce cell proliferation and increase the expression of cleaved caspase-3.@*CONCLUSION@#Curcumin can increase chemosensitivity of myeloma cells to bortezomib, this effect may be related to the inhibition of Notch1.

Notch1 in Tumor Microvascular Endothelial Cells and Tumoral miR-34a as Prognostic Markers in Locally Advanced Triple-Negative Breast Cancer / 한국유방암학회지

Effects and mechanisms of electro-acupuncture on proliferation and differentiation of neural stem cells in C57 mice exposed to different doses of X-ray radiation / 生理学报

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.

Valproic acid withdrawal ameliorates impairments of hippocampal-spatial working memory and neurogenesis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Nerve Growth Factor Stimulates Glioblastoma Proliferation through Notch1 Receptor Signaling

OBJECTIVE: Notch receptors are heterodimeric transmembrane proteins that regulate cell fate, such as differentiation, proliferation, and apoptosis. Dysregulated Notch pathway signaling has been observed in glioblastomas, as well as in other human malignancies. Nerve growth factor (NGF) is essential for cell growth and differentiation in the nervous system. Recent reports suggest that NGF stimulates glioblastoma proliferation. However, the relationship between NGF and Notch1 in glioblastomas remains unknown. Therefore, we investigated expression of Notch1 in a glioblastoma cell line (U87-MG), and examined the relationship between NGF and Notch1 signaling.METHODS: We evaluated expression of Notch1 in human glioblastomas and normal brain tissues by immunohistochemical staining. The effect of NGF on glioblastoma cell line (U87-MG) was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. To evaluate the relationship between NGF and Notch1 signaling, Notch1 and Hes1 expression were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To confirm the effects of NGF on Notch1 signaling, Notch1 and Hes1 small interfering RNAs (siRNAs) were used.RESULTS: In immunohistochemistry, Notch1 expression was higher in glioblastoma than in normal brain tissue. MTT assay showed that NGF stimulates U87-MG cells in a dose-dependent manner. RT-PCR and Western blot analysis demonstrated that Notch1 and Hes1 expression were increased by NGF in a dose-dependent manner. After transfection with Notch1 and Hes1 siRNAs, there was no significant difference between controls and 100 nM NGF-β, which means that U87-MG cell proliferation was suppressed by Notch1 and Hes1 siRNAs.CONCLUSION: These results indicate that NGF stimulates glioblastoma cell proliferation via Notch1 signaling through Hes 1.

Effects of irbesartan on Notch1 signaling pathway in diabetic rats with myocardial injury / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects and mechanisms of irbesartan on myocardial injury in diabetic rats, and to analyze the changes of Notch1 signaling pathway in it.@*METHODS@#Thirty rats were randomly divided into four groups:normal control group (CON, =6), high calorie group (HC, =6) and diabetes mellitus group (DM, =9), irbesartan + diabetes group (Ir + DM, =9). After modeling 8 weeks later, the body weight ratio and left ventricular weight index were measured and the serum levels of triglyceride (TG) and total cholesterol (TC) were measured by automatic biochemical analyzer. The changes of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in myocardium of rats were determined by the kit and the expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 assaciated X protein (Bax) protein in myocardium were detected by immunohistochemistry. The expressions of Notch1, Hes-1 and jagged-1 in myocardium of rats were detected by Western blot.@*RESULTS@#Compared with CON group, the levels of heart weight/body weight (H/B), left ventricular weight index(LVWI) and fasting blood glucose(FBG) in HC group were not significantly changed, while the levels of blood lipids, MDA and Bax were increased significantly, and the expressions of SOD, Bcl-2 and Notch1, Hes-1 and Jagged-1 were decreased. Compared with HC group, the levels of H/B, LVWI, FBG, MDA and Bax in DM group were increased significantly, and the levels of SOD, Bcl-2 and Notch1, Hes-1 and Jagged-1 were decreased. The expression of H/B, LVWI, Notch1, Hes-1 and Jagged-1 in Ir+DM group were increased, but there was no significant difference between the other indexes. The H/B and LVWI in Ir + DM group were significantly lower than those in DM group, the levels of blood lipid and blood glucose did not change significantly, but the incidence of oxidative stress and apoptosis was reduced. While Notch1, Hes-1, Jagged -1 protein expressions were increased.@*CONCLUSIONS@#Diabetes can induce myocardial injury, and irbesartan has myocardial protective effects through activation of Notch1.

Nerve Growth Factor Stimulates Glioblastoma Proliferation through Notch1 Receptor Signaling

OBJECTIVE: Notch receptors are heterodimeric transmembrane proteins that regulate cell fate, such as differentiation, proliferation, and apoptosis. Dysregulated Notch pathway signaling has been observed in glioblastomas, as well as in other human malignancies. Nerve growth factor (NGF) is essential for cell growth and differentiation in the nervous system. Recent reports suggest that NGF stimulates glioblastoma proliferation. However, the relationship between NGF and Notch1 in glioblastomas remains unknown. Therefore, we investigated expression of Notch1 in a glioblastoma cell line (U87-MG), and examined the relationship between NGF and Notch1 signaling. METHODS: We evaluated expression of Notch1 in human glioblastomas and normal brain tissues by immunohistochemical staining. The effect of NGF on glioblastoma cell line (U87-MG) was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. To evaluate the relationship between NGF and Notch1 signaling, Notch1 and Hes1 expression were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To confirm the effects of NGF on Notch1 signaling, Notch1 and Hes1 small interfering RNAs (siRNAs) were used. RESULTS: In immunohistochemistry, Notch1 expression was higher in glioblastoma than in normal brain tissue. MTT assay showed that NGF stimulates U87-MG cells in a dose-dependent manner. RT-PCR and Western blot analysis demonstrated that Notch1 and Hes1 expression were increased by NGF in a dose-dependent manner. After transfection with Notch1 and Hes1 siRNAs, there was no significant difference between controls and 100 nM NGF-β, which means that U87-MG cell proliferation was suppressed by Notch1 and Hes1 siRNAs. CONCLUSION: These results indicate that NGF stimulates glioblastoma cell proliferation via Notch1 signaling through Hes 1.

Brucine inhibits bone metastasis of breast cancer cells by suppressing Jagged1/Notch1 signaling pathways / 中国结合医学杂志

<p><b>OBJECTIVE</b>To examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Compared 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).</p><p><b>CONCLUSION</b>Brucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.</p>

Circulating-free DNA Mutation Associated with Response of Targeted Therapy in Human Epidermal Growth Factor Receptor 2-positive Metastatic Breast Cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The 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).</p><p><b>METHODS</b>From 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSION</b>Targeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.</p>

Caracterização da inter-relação entre as vias de sinalização Notch e TLR na paracoccidioidomicose experimental / Characterization of the inter-relationship between Notch and TLR signaling pathways in experimental paracoccidioidomycosis

A paracoccidioidomicose é uma micose sistêmica de natureza profunda que afeta preferencialmente o tecido pulmonar podendo disseminar via linfo-hematogênica para outros órgãos e tecidos, sendo causada principalmente pelo Paracoccidioides brasiliensis, fungo que apresenta dimorfismo térmico. O sistema imune inato mediado por macrófagos é extremamente importante para o controle de infecções e está envolvido na indução e regulação da resposta imune/inflamatória. Estas células são capazes de reconhecer patógenos por meio de receptores de reconhecimento de padrões (PRRs), tais como receptores Toll-like (TLR). Além desses PRRs, recentemente, demonstrou-se a importância da via de sinalização Notch no sistema imune inato e na regulação da atividade dos macrófagos. Nossos dados demonstram que a cepa Pb18 do P. brasiliensis é capaz de ativar o receptor Notch1 em macrófagos J774. A ativação desse receptor concomitante com a ativação de TLR 4 (via LPS) induz a produção de IL-6, e apresenta elevada carga fúngica e menor fagocitose, o que favorece a patogenia. Ao utilizarmos um inibidor farmacológico da γ-secretase (DAPT) para inibir a ativação do receptor Notch1 em macrófagos, é possível observar diminuição da carga fúngica, diminuição de IL-6, aumento de TNF-α e aumento da fagocitose. Entretanto, a ausência do receptor TLR 4 em macrófagos derivados de medula óssea de camundongos TLR 4-/-, na presença de DAPT, percebe-se diminuição da capacidade fagocítica desses macrófagos e também diminuição da carga fúngica, evidenciando a relação entre TLR 4 e Notch1. Em adição, realizamos um tratamento em camundongos BALB/c com DAPT previamente à infecção com Pb18. Nossos resultados evidenciaram que animais com este tratamento apresentaram diminuição da carga fúngica dos pulmões, diminuição de IL-6, ativação de macrófagos e aumento de IgG, após 45 dias de infecção, indicando um perfil de cura desses animais. O mesmo tratamento foi realizado em camundongos BALB/c NUDE, seguido da infecção com Pb18. Nestes animais, verificamos que há maior produção de citocinas pró-inflamatórias no pulmão, aumento de células CD19+ e a carga fúngica dos animais tratados manteve-se similar ao dos animais não tratados, indicando que o perfil protetor observado em animais com DAPT é dependente da resposta das células T. Juntos, esses resultados evidenciam que o Pb18 é capaz de ativar o receptor Notch1 em macrófagos e utiliza a via de sinalização Notch-TLR 4 como um possível mecanismo de escape, podendo fornecer uma nova abordagem de estudo da imunidade envolvida na paracoccidioidomicose experimental

Paracoccidioidomycosis is a systemic mycosis of deep nature that primarily affects the lung and can spread via lymphatic and hematogenous to other organs and tissues. It is mainly caused by Paracoccidioides brasiliensis fungus which exhibit thermal dimorphism. The innate immune system mediated by macrophages is extremely important for the control of infection and is involved in the induction and regulation of immune/inflammatory response. These cells are able to recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLR). Beyond these PRRs, the importance of Notch signaling has recently been demonstrated in the innate immune system and the regulation of macrophage activity. Our data demonstrate that the Pb18 strain of P. brasiliensis is able to activate the Notch1 receptor in J774 macrophages. Activation of this receptor with also activation of TLR 4 (via LPS) induces IL-6 production, induces phagocytosis and decreases fungal burden, which favors the pathogenesis. By using a γ-secretase pharmacological inhibitor (DAPT) for inhibiting the activation of Notch1 receptor on macrophages, it is possible to observe decreased fungal burden, less production of IL-6, and increased TNF-α and phagocytosis. However, due to the absence of TLR 4 receptor in bone marrow derived macrophages from TLR 4-/- mice, these macrophages showed decreased phagocytic ability and also reduced fungal burden in the presence of DAPT, showing a relationship between TLR 4 and Notch1. In addition, we made a treatment with DAPT in BALB/c mice prior to infection with Pb18. And our results showed that DAPT-treated animals exhibited a decrease of fungal burden in the lungs, and a decrease of IL-6. Furthermore, we observed an increase of IgG after 45 days of infection, indicating probably a healing of these animals. Same treatment was made in BALB/c NUDE mice, followed by infection with Pb18. In these animals, we observed an increased production of proinflammatory cytokines in the lung and increased CD19+ cells, but fungal burden was similar in both group (treated and untreated), which indicates that treatment with DAPT is dependent on T cell response. Taken together, these results showed that Pb18 is able to activate the Notch 1 receptor on macrophages and uses the Notch-TLR 4 signaling pathway as a possible escape mechanism, and may provide a new immunity study approach in experimental paracoccidioidomycosis

Up-regulation of Notch1 inhibits proliferation and differentiation of osteoclast in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed to explore the effect of the up-regulation of Notch1 on osteoclastogenesis induced to osteoclasts by receptor activator for nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factors (MCSF) in vitro.</p><p><b>METHODS</b>The bone marrow stem cells (BMSCs) of Rosa(-notch1) mice were cultured and induced to osteoclasts by RANKL and MCSF. The BMSCs were transfected with the Ad-Cre-green fluorescent protein (GFP) virus or Ad-GFP virus. Total RNA from cells was extracted, and the gene expression levels of Notch1, Notch2, Notch3, Notch4, Deltal, Delta3, Delta4, Jagged1, Hes1, and tartrate resistant acid phosphatase (TRAP) were detected at the defined stage by reverse transcription-polymerase chain reaction (RT-PCR). Osteoclast formation was analyzed by TRAP assay.</p><p><b>RESULTS</b>The number of TRAP-positive multinuclear cells of the experimental group significantly decreased compared with that of the control group. The mRNA expression levels of Notch1, Notch3, Jagged1, Delta3, and Hesl of the experimental group were significantly higher than those of the control group, whereas the TRAP mRNA expression of the experimental group was significantly lower than that of the control group (P<0.05).</p><p><b>CONCLUSION</b>Up-regulation of Notch1 inhibit osteoclastogenesis of BMSCs induced by RANKL and MCSF in vitro.</p>