Expression Levels Of Notch1 Research Articles

Induction of apoptosis in renal cell carcinoma by cinobufotalin through inhibition of Notch1 signal activation*

Abstract Objective The aim of this study was to investigate the effect of cinobufotalin on apoptosis in renal cell carcinoma and its possible mechanism of action. Methods The expression levels of Notch1 in renal cancer cells, as well as in adjacent and normal tissues were assessed in 64 patients with renal cellcarcinoma. The 769-P cells were treated with 0, 10, 20 and 40 mg/L cinobufotalin and the proliferation activity and apoptotic rate of the cells were measured. The expression levels of Notch1, Bcl-2, and Pro-caspase 3 were detected by RT-PCR and Western-blot. Results (1) The rates of Notch1 expression in renal cancer cells, adjacent tissues, and normal tissues were 75.0%, 45.3%, and 9.4%, respectively. Notch1 expression had significant effects on tumor, node and metastasis (TNM) staging, Fuhrman grade, and tumor size in patients with renal cell carcinoma (P < 0.05); (2) The inhibition rates of cinobufotalin on 769-P cells were 0%, 6.85%, 11.37%, and 16.33% at 24 h; 0%, 13.57%, 20.14%, and 31.69% at 48 h; 0%, 19.97%, 28.53% and 51.42% at 72 h. At 24 h, the apoptotic rates were 8.2 ± 3.1%, 19.8 ± 5.6%, 33.7 ± 5.0%, and 51.5 ± 6.8%.The effect of cinobufotalin on apoptosis of 769-P cells was dose-dependent; (3) RT-PCR assay showed that protein expression levels for Notch1, Bcl-2, and Pro-caspase 3 were significantly decreased with the increase of drug concentration. Western-blot analysis also showed that Notch1, Bcl-2 and Pro-caspase 3 protein levels showed a significant downward trend with the increase of drug concentration. Conclusion Cinobufotalin inhibits the growth of renal cancer cells and induces apoptosis in renal cell carcinoma, which may be related to the inhibition of Notch1 signal activation.

Notch1 in Cancer Therapy: Possible Clinical Implications and Challenges

The Notch family consists of four highly conserved transmembrane receptors. The release of the active intracellular domain requires the enzymatic activity of γ-secretase. Notch is involved in embryonic development and in many physiologic processes of normal cells, in which it regulates growth, apoptosis, and differentiation. Notch1, a member of the Notch family, is implicated in many types of cancer, including breast cancer (especially triple-negative breast cancer), leukemias, brain tumors, and many others. Notch1 is tightly connected to many signaling pathways that are therapeutically involved in tumorigenesis. Together, they impact apoptosis, proliferation, chemosensitivity, immune response, and the population of cancer stem cells. Notch1 inhibition can be achieved through various and diverse methods, the most common of which are the γ-secretase inhibitors, which produce a pan-Notch inhibition, or the use of Notch1 short interference RNA or Notch1 monoclonal antibodies, which produce a more specific blockade. Downregulation of Notch1 can be used alone or in combination with chemotherapy, which can achieve a synergistic effect and a decrease in chemoresistance. Targeting Notch1 in cancers that harbor high expression levels of Notch1 offers an addition to therapeutic strategies recruited for managing cancer. Considering available evidence, Notch1 offers a legitimate target that might be incorporated in future strategies for combating cancer. In this review, the possible clinical applications of Notch1 inhibition and the obstacles that hinder its clinical application are discussed. SIGNIFICANCE STATEMENT: Notch1 plays an important role in different types of cancer. Numerous approaches of Notch1 inhibition possess potential benefits in the management of various clinical aspects of cancer. The application of different Notch1 inhibition modalities faces many challenges.

Blocking the Notch signal transduction pathway promotes tumor growth in osteosarcoma by affecting polarization of TAM to M2 phenotype.

BackgroundOsteosarcoma is a primary malignant tumor that seriously affects the health and life of patients. It is of great clinical significance to explore the molecular mechanism of osteosarcoma development and develop the corresponding therapeutic targets. Th1/Th2 cytokines in the normal human body are in a state of dynamic balance. When this balance is destroyed, it is related to many diseases such as a tumor, autoimmune disease, microbial infection, transplant rejection, among many others.MethodThe model of mouse tumor-associated macrophage (TAM) was induced by being co-cultured with inducer granulocyte-macrophage colony stimulating factor (GM-CSF) and osteosarcoma S180 cells. The Notch1 knockout mice were obtained by gene targeting technology. The distribution of M1- and M2-type TAMs in the tumor was visualized by immunofluorescence staining. And the western-blot testing was used to detect and quantified the protein level of Notch1 and Th1/Th2-type cytokines.ResultsIn this study, the polarization of TAMs to the M2 phenotype occurred after coculture with osteosarcoma S180 cells and secretion level Th1/Th2-type cytokines changed. Also, the expression level of Notch1 reduced significantly. Further, the critical transcription factor Notch1 of the Notch signaling pathway was knocked out in mice. The tumor volume of Notch1 knockout mice was significantly more extensive than of the control mice. The results of microstructural observation on tumor showed that M2-type TAMs infiltrated into tumor increased with increased expression of Th2-type cytokines, but M1-type TAMs reduced with reduced expression of Th1-type cytokines.ConclusionsAccording to our results, the Notch signal transduction pathway participates in tumor occurrence and growth with a negative role by maintaining Th1/Th2 balance.

Notch signaling is involved in the antiapoptotic effects of liraglutide on rat H9c2 cardiomyocytes exposed to hypoxia followed by reoxygenation.

ObjectiveLiraglutide (Lir) protects cardiomyocytes against high glucose-induced myocardial damage. This study investigated whether Notch signaling participated in the antiapoptotic effects of Lir on rat H9c2 cardiomyocytes subjected to hypoxia followed by reoxygenation (H/R).MethodsWe used H9c2 rat cardiomyocytes as a model of H/R and measured viability, apoptosis, and expression of the apoptotic genes Bax and Bcl-2 and Notch signaling genes Notch1 and Jagged1. Notch1 was depleted by siRNA to test the effect of Notch1 deficiency on the antiapoptotic effects of Lir on H/R-treated H9c2 cardiomyocytes.ResultsAfter H/R treatment, viability was significantly decreased, and the apoptosis rate was greater in the H/R group than in the control (CT). Lir at 50, 100, and 200 nM significantly increased viability and decreased apoptosis in H/R-treated H9c2 cells. Treatment with 50 nM Lir for 2 hours before H/R significantly increased the expression levels of Notch1, Jagged1, and Bcl-2 compared with the CT levels. Bax was downregulated, which indicated that Lir activated Notch signaling and inhibited apoptosis. Notch1 depletion partially abolished the antiapoptotic effect of Lir on H/R-treated H9c2 cells by altering apoptotic gene expression.ConclusionLir activated Notch signaling, which was responsible for the antiapoptotic effect of Lir on H9c2 cardiomyocytes.

Mechanism of Hematopoietic Reconstruction in Mice Treated with Danggui Buxue Decoction combined with Muscle-Derived Stem Cell Transplantation

To investigate the mechanism of hematopoietic reconstruction in mice treated with Danggui Buxue Decoction (DBD) combined with the muscle-derived stem cell transplantation (MDSCT). Female Kunming mice were randomly divided into the 6 groups: irradiation model, the bone marrow transplantation, the MDSC transplantation, the DBD 1 (4.5 g/kg), 2 (13.5 g/kg), and 3 (22.5 g/kg) + MDSC transplantation. After a week of oral administration of normal saline or different doses of DBD, The mice were exposied to 8 Gy 137Cs γ ray and were followed by bone marrow or MDSC transplantation. The expression levels of Notch1, Jagged1 and Hes1 in bone marrow, thymus and spleen were measured at 3 and 8 weeks after irradiation and transplantation. In the bone marrow, 3 weeks after above-mentioned treatment, the expression of Notch1 mRNA increased obviously and the expression of Jagged1, Hes1 mRNA decreased obviously in each intervention group, compared with the irradiation model group. 8th week after treatment, the expression of Notch1 mRNA decreased obviously in each intervention group, the Jagged1 mRNA expression decreased obviously except the bone marrow group, and Hes1 mRNA expression increased (P＜0.05) in each intervention group. 3 weeks after treatment, compared with the irradiation model group, the expression of Notch1 mRNA in the thymocytes increased only in DBD1+MDSC group, Jagged1, Hes1 mRNA was increased in the MDSC transplantation group and the DBD1、2+MDSC group. 8th week after treatment, the expression of Notch1, Jagged1 mRNA expression decreased in each intervention group, the expression of Hes1 mRNA increased obviously in the MDSC transplantation group and the DBD1、2+MDSC group (P＜0.05). In the spleen, 3 weeks after treatment, the expression of Notch1, Jagged1 mRNA in the spleen of each intervention group decreased obviously, compared with the irradiation model group. The expression of Jagged1, Hes1 mRNA in each intervention group were increased obviously 8th week after treatment (P＜0.05). MDSC transplantation after pretreatment of DBD can improve the hematopoietic reconstitution in mice with lethal dose radiation damage. Notch1、Jagged1 and Hes1 play different roles in this process, but the concrete mechanism needs to be further studied.

Combination of Quercetin, Hirudin and Cinnamaldehyde Promotes Schwann Cell Differentiation and Myelination against High Glucose by Inhibiting ERK Signaling Pathway.

To investigate the therapeutic and synergistic effects of QHC (combination of quercetin (Q), hirudin (H) and cinnamaldehyd (C)) on Schwann cell differentiation and myelination against high glucose (HG) induced injury. Primary-culture Schwann cells exposed to HG (50 mmol/L) for 72 h and Schwann cell-dorsal root ganglion (DRG) neuron cocultures exposed to HG (50 mmol/L) for 7 days were employed as in vitro model of diabetic neuropathy. The cells were randomly divided into 10 groups: control (CON, 25 mmol/L glucose), HG (50 mmol/L glucose), HG plus 10 μmol/L quercetin (Q), HG plus 0.04 IU/mL hirudin (H), HG plus 100 nmol/L cinnamaldehyd (C), HG plus 10 μmol/L quercetin and 0.04 IU/mL hirudin (QH), HG plus 10 μmol/L quercetin and 50 nmol/L cinnamaldehyd (QC), HG plus 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (HC), HG plus 10 μmol/L quercetin, 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (QHC) or 10 μmol/L U0126. Cell differentiation was evaluated by periaxin immunofluorescence staining. The protein expression levels of myelin protein zero (P0), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), extracellular signal-regulated kinase (ERK), p-ERK, p-c-Jun, c-Jun, notch intracellular domain (NICD) and the mRNA expression levels of P0, MBP, MAG, Krox-20, Notch1 and Jagged1 were detected by Western blotting and real-time quantitative PCR analysis. The secretion of ciliary neurotrophic factor (CNTF) was determined by enzyme-linked immunosorbent assay (ELISA). The number and length of the myelin segments were evaluated by MBP immunofluorescence staining. The expression and the location of p-ERK in cocultures were detected by MAG and p-ERK immunofluorescence double staining. Co-treatment with Q, C, H and their combination promoted Schwann cell differentiation, increased CNTF secretion, up-regulated the protein and mRNA expressions of myelin, and increased the number and length of the myelin segments (P<0.01 or P<0.05). In particular, the combination therapy of Q, H and C was superior to the respective monotherapy (P<0.01). Combination therapy of QHC exhibited higher inhibitory activities for ERK signaling related molecules than each monomer or the combination of the two monomers (P<0.01). QHC combination yielded synergy in promoting Schwann cell differentiation and myelination and the protective effect may involve in the inhibition of ERK signaling pathway, providing scientific evidence for better understanding of combination of Q, H and C in clinical applications.

Effects of Notch signaling components from breast cancer cells treated in culture with resveratrol

Resveratrol (Res) has an anti-tumor effect. Notch signaling components from breast cancer cells treated in culture with Resveratrol was investigated. MDA-MB-231cells were divided into control group (Res-untreated) and Res-treated groups including six concentrations 0 μM, 10 μM, 20 μM, 40 μM, 80 μM and 160 μM. Cytotoxicity test were evaluated by CCK-8. The mRNA and Protein expression levels of Notch1, Jagged1, Dll4 and Hes-5 were detected by RT-PCR and Western blot. The mRNA expression of Notch1, Jagged1, Hes-5 and Dll4 in the Res administration group decreased significantly (0.01 < p < .05), and Hes-5 and Dll4 were extremely significant (p < .01). Compared with the blank control group, the protein expression of Notch1 and Dll4 decreased significantly in each concentration Res group, but the decrease of protein expression of Jagged1 and Hes-5 was not significant. In conclusion, Res regulates mRNA and protein expression of Notch1, Dll4 of MDA-MB-231 cells via Notch pathway.

Analysis of changes in the expression of Notch1 and HES1 and the prognosis of osteosarcoma patients following surgery.

The present study aimed to analyze the changes in the expression of Notch1 and hairy and enhancer of split-1 (HES1) and the prognosis of patients with osteosarcoma following surgery. Samples from 62 patients with osteosarcoma treated at Shandong Cancer hospital from April, 2011 to June, 2013 were collected as the research group, and those from 52 healthy individuals undergoing physical examination were collected as the control group. The expression levels of Notch1 and HES1 in the serum of patients with osteosarcoma were measured by ELISA before and after surgery. Pearson's correlation analysis was used to analyze the correlation between Notch1 expression and HES1 expression in the osteosarcoma patients. According to the expression levels of Notch1 and HES1, the patients were divided into the high expression group and the low expression group, and the 5-year survival rate of the patients was observed. The expression levels of Notch1 and HES1 in the osteosarcoma patients before surgery were higher than those after surgery (P<0.05). The sensitivity, specificity and AUC of Notch1 for osteosarcoma were 93.55%, 58.06% and 0.732 respectively, and those of HES1 were 82.26%, 61.29% and 0.766, respectively. The expression level of Notch1 positively correlated with the expression level of HES1 in the osteosarcoma patients (r=0.795, P<0.001). According to the expression levels of Notch1 and HES1, the patients were divided into the high and low expression groups. The survival rate of the low expression group was significantly higher than that of the high expression groups (P=0.045). Finally, multiple factors were analyzed by logistic regression, and it was found that tumor location, chemotherapy response, tumor size, Notch1 and HES1 were independent risk factors for prognosis. Notch1 and HES1 exhibited a low expression in patients following surgery. ROC curve analysis revealed that the two indicators had good diagnostic efficacy and were expected to become markers for diagnosis and prognosis of osteosarcoma.

MicroRNA‑204‑5p inhibits the osteogenic differentiation of ankylosing spondylitis fibroblasts by regulating the Notch2 signaling pathway.

Ankylosing spondylitis (AS) is a chronic inflammatory systemic disease and is difficult to detect in the early stages. The present study aimed to investigate the role of microRNA (miR)-204-5p in osteogenic differentiation of AS fibroblasts. Bone morphogenetic protein 2 (BMP-2) was used to induce osteogenic differentiation. Cells were divided into the following groups: AS group, AS + BMP-2 group, AS + BMP-2 + miR-negative control group, AS + BMP-2 + miR-204-5p mimics group and AS + BMP-2 + miR-204-5p mimics + pcDNA-Notch2 group. The expression levels of miR-204-5p, Notch2, runt-related transcription factor 2 (RUNX2) and osteocalcin were detected via reverse transcription-quantitative PCR analysis. The binding site between Notch2 and miR-204-5p was predicted using TargetScan software and verified via the dual-luciferase reporter assay. Alkaline phosphatase (ALP) activity was assessed via the ALP assay, while the mineralized nodules area was determined via the Alizarin Red S staining assay. The results demonstrated that Notch2 is a target gene of miR-204-5p. Furthermore, treatment with BMP-2 significantly decreased miR-204-5p expression, and significantly increased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Conversely, transfection with miR-204-5p mimics significantly increased miR-204-5p expression, and significantly decreased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Notably, transfection with pcDNA-Notch2 significantly reversed the inhibitory effects induced by miR-204-5p mimics on the osteogenic differentiation of ligament fibroblasts (all P<0.05). Furthermore, miR-204-5p inhibited the osteogenic differentiation of ligament fibroblasts in patients with AS by targeting Notch2. Thus, miR-204-5p may negatively regulate Notch2 expression and may be a potential therapeutic target for AS. Collectively, the results of the present study provide a theoretical basis for the effective treatment of patients with AS.

Downregulation of LncRNA-MEG3 promotes HTR8/SVneo cells apoptosis and attenuates its migration by repressing Notch1 signal in preeclampsia.

A successful pregnancy crucially depends on well-regulated extravillous trophoblast migration and invasion. Maternally expressed gene 3 (MEG3) is a long noncoding RNA that plays an important role in regulating trophoblast cells cell function. As previously reported, the expression of MEG3 was reduced in preeclampsia, and downregulation of MEG3 could suppress trophoblast cells migration and promote its apoptosis. However, the downstream regulatory mechanism of MEG3 remains unknown. As reported, MEG3 could inhibit cell proliferation in endometrial carcinoma by regulating Notch signaling. Our previous studies have demonstrated that Notch1 is downregulated in preeclampsia and that inhibiting the expression of Notch1 could promote trophoblast cell apoptosis. Therefore, this study was designed to investigate the role of MEG3 and its the relationship with Notch1 in trophoblasts. In this study, the mRNA expression levels of both MEG3 and Notch1 were decreased in preeclampsia placenta (n = 15) compared to the normal samples (n = 15). Exogenous upregulation and downregulation of MEG3 in HTR8/SVneo cells were performed to investigate the role of MEG3 in cell biological behavior and its effects on Notch1 expression. The results showed that MEG3 enhancement promoted trophoblast cell migration and invasion and inhibited cell apoptosis. Downregulation of MEG3 elicited the opposite results. Associated factors, such as matrix metalloproteinases 2 (MMP2), BAX, and Bcl-2, were examined at the mRNA and protein levels. Our study demonstrated that MEG3 could regulate Notch1 expression to modulate trophoblast cell migration, invasion, and apoptosis, which may represent the molecular mechanism of poor placentation during preeclampsia.

Cytotoxicity study of the interleukin-12-expressing recombinant Newcastle disease virus strain, rAF-IL12, towards CT26 colon cancer cells in vitro and in vivo

BackgroundOncolytic viruses have emerged as an alternative therapeutic modality for cancer as they can replicate specifically in tumour cells and induce toxic effects leading to apoptosis. Despite the great potentials and promising results shown in multiple studies, it appears that their efficacy is still moderate and deemed as not sufficient in clinical studies. In addressing this issue, genetic/molecular engineering approach has paved its way to improve the therapeutic efficacy as observed in the case of herpes simplex virus (HSV) expressing granulocyte–macrophage colony-stimulating factor (GM-CSF). This study aimed to explore the cytotoxicity effects of recombinant NDV strain AF2240-i expressing interleukin-12 (rAF-IL12) against CT26 colon cancer cells.MethodsThe cytotoxicity effect of rAF-IL12 against CT26 colon cancer cell line was determined by MTT assay. Based on the IC50 value from the anti-proliferative assay, further downward assays such as Annexin V FITC and cell cycle progression were carried out and measured by flow cytometry. Then, the in vivo study was conducted where the rAF-IL12 viral injections were given at the intra-tumoral site of the CT26 tumour-burden mice. At the end of the experiment, serum biochemical, T cell immunophenotyping, serum cytokine, histopathology of tumour and organ section, TUNEL assay, and Nanostring gene expression analysis were performed.ResultsThe rAF-IL12 induced apoptosis of CT26 colon cancer cells in vitro as revealed in the Annexin V FITC analysis and also arrested the cancer cells progression at G1 phase of the cell cycle analysis. On the other hand, the rAF-IL12 significantly (p < 0.05) inhibited the growth of CT26 tumour in Balb/c mice and had regulated the immune system by increasing the level of CD4 + , CD8 + , IL-2, IL-12, and IFN-γ. Furthermore, the expression level of apoptosis-related genes (bax and p53) was up-regulated as a result of the rAF-IL12 treatment. Additionally, the rAF-IL12 had also down-regulated the expression level of KRAS, BRAF, MAPK1, Notch1, CCL2, and VEGF oncogenes. Besides, rAF-IL12 intra-tumoral delivery was considered safe and not hazardous to the host as evidenced in pathophysiology of the normal tissues and organs of the mice as well as from the serum biochemistry profile of liver and kidney.ConclusionsThese results indicated that rAF-IL12 had better anti-tumoral and cytotoxicity effects compared to its parental wild-type, AF2240-i in combatting the CT26 colon cancer model.

Membrane protein CAR promotes hematopoietic regeneration upon stress.

Adult hematopoietic stem cells (HSC) are quiescent most of the time, and how HSC switch from quiescence to proliferation following hematopoietic stress is unclear. Here we demonstrate that upon stress the coxsackievirus and adenovirus receptor CAR (also known as CXADR) is upregulated in HSC and critical for HSC entry into the cell cycle. Wild-type HSC were detected with more rapid repopulation ability than the CAR knockout counterparts. After fluorouracil treatment, CAR knockout HSC had lower levels of Notch1 expression and elevated protein level of Numb, a Notch antagonist. The Notch signaling inhibitor DAPT, dominant negative form of MAML (a transcriptional coactivator of Notch), or dominant negative mutant of LNX2 (an E3 ligase that acts on Numb and binds to CAR), all were capable of abrogating the function of CAR in HSC. We conclude that CAR activates Notch1 signaling by downregulating Numb protein expression to facilitate entry of quiescent HSC into the cell cycle during regeneration.

LncRNA ROR promotes proliferation of endometrial cancer cells via regulating Notch1 pathway.

The aim of this study was to investigate the effects of long non-coding ribonucleic acid regulator of reprogramming (lncRNA ROR) on the proliferation and apoptosis of endometrial cancer (EC) cells, and to explore its possible underlying mechanism. The expression levels of lncRNA ROR and Notch1 in EC tissues were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The changes in Notch1 protein were detected via Western blotting. Subsequently, the regulatory mechanism of lncRNA ROR on Notch1 was analyzed using Luciferase reporter gene assay. Moreover, the changes in cell proliferation and apoptosis were determined through cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. Both lncRNA ROR and Notch1 were highly expressed in EC tissues (p<0.05). After overexpression of lncRNA ROR, HEC-1A cells had significantly enhanced proliferation (p<0.05) and weakened apoptosis (p<0.05). Meanwhile, the mRNA and protein levels of Notch1 rose remarkably compared with those in control group (p<0.05). Luciferase reporter gene assay revealed that lncRNA ROR could bind to the Notch1 regulatory factor miR-34a and inhibit its activity. LncRNA ROR regulates the proliferation and apoptosis of EC cells via promoting the expression of Notch1 protein.

Rubimaillin suppresses proliferation, migration and invasion of prostate cancer cells via the Notch-1/MMP signaling pathway

This study was aimed to explore the effect of Rubimaillin on the survival, migration, and invasion of prostate cancer cell lines DU145 and PC3, and its mechanism. CCK-8 method was used to detect the effects of different concentrations of rubs (0, 5, 10, 20, 40 and 80 μM) on the activity of DU145 cells and PC3 cells. Transwell cell lab test was used to detect the migration and invasion of cells. Western blot was used to detect Notch-1, MMP-2, MMP-9 and Hes-1 protein levels. The CCK-8 assay showed that Rub inhibited the activity of Du145 and PC3 cells in a concentration dependent manner. When the concentration reached 40 μM, the inhibition reached the maximum. After Rub intervention, the migration and invasion ability of Du145 and PC3 cells decreased significantly, while the expression levels of Notch-1, MMP-2, MMP-9 and Hes-1 protein decreased significantly. Rub can inhibit the growth, migration and invasion of prostate cancer cell line DU145 and PC3. The mechanism may be related to the inhibition of Notch-1, MMP-2, MMP-9 and Hes-1 by Rub.

Rehabilitation training improves nerve injuries by affecting Notch1 and SYN.

ObjectiveThe aim of this study was to investigate the effects of rehabilitation training on Notch1 and synaptophysin (SYN) levels in brain tissues of rats with chronic cerebral ischemia.MethodsEighty-one male Sprague-Dawley rats were divided into nine groups: three Sham groups, three Model groups, and three training groups. There were nine rats in each group. At different time points, the apoptosis cell rate was analyzed by the TUNEL assay, and the expression levels of Notch1 and SYN in brain tissues were analyzed by immunohistochemical staining and RT-qPCR assay.ResultsThe apoptosis cell rate of training groups was significantly higher on day 28 (P < 0.05). The protein and mRNA levels of both Noth1 and SYN in training groups were significantly higher on day 28 (P < 0.05).ConclusionRehabilitation training could improve nerve cell apoptosis by increasing the expression of both Notch1 and SYN.

Mechanism of Notch1‑saRNA‑1480 reversing androgen sensitivity in human metastatic castration‑resistant prostate cancer.

The aim of the present study was to explore the mechanism by which Notch1-small activating (sa)RNA restored androgen sensitivity in human metastatic castration-resistant prostate cancer (CRPC). After transfection of Notch1-saRNA-1480 in PC3 cells, the expression of Notch1 and androgen receptor (AR) was investigated by reverse transcription quantitative PCR (RT-qPCR) and western blotting. Furthermore, the protein expression level of vascular endothelial growth factor (VEGF) was measured. Then, flow cytometry was used to analyze the cell cycle and apoptosis after transfection. Moreover, the migration and invasion ability of PC3 cells were assessed by transwell assays. Then, angio-genesis experiments were conducted to analyze the abilities of PC3 cells to form blood vessels. Furthermore, in vivo experiments detected the antitumor activity of Notch1-saRNA-1480. The mRNA and protein expression levels of Notch1 were significantly increased after transfection, while the expression levels of AR and VEGF were decreased. After transfection, the cell cycle was arrested at the G0/G1 checkpoint. Notch1-saRNA-1480 significantly increased the proportion of apoptotic cells after transfection. In addition, transwell assay results showed that PC3 cell migration and invasion were inhibited. The total vessel length was significantly decreased based on angiogenesis experiments, which indicated that PC3 cell angiogenesis was inhibited. In vivo experiments showed that Notch1-saRNA-1480 could inhibit tumor growth and volume. The protein expression of Notch1, AR, VEGF receptor 2 (VEGFR2) and VEGF in tumor tissues was consistent with in vitro levels. Notch1-saRNA-1480 could significantly inhibit the proliferation of PC3 cells in vitro and the growth of tumors in vivo, which is associated with the inhibition of the AR and VEGF pathways.

MiR-210 suppresses neuronal apoptosis in rats with cerebral infarction through regulating VEGF-notch signaling pathway.

The aim of this study was to explore the effect of micro ribonucleic acid (miR)-210 on neuronal apoptosis in rats with cerebral infarction (CI) by regulating the vascular endothelial growth factor (VEGF)-Notch signaling pathway. A total of 30 clean healthy male Sprague-Dawley rats weighing 200-300 g were selected and randomly divided into Sham group (n=10), CI model group (CIM group, n=10), and CIM + miR-210 Mimic group (n=10). The protein expression levels of VEGF, Notch1, cleaved-Caspase3 (c-Caspase3), B-cell lymphoma-2 (Bcl-2), and tubulin were detected via Western blotting. The messenger RNA (mRNA) levels of VEGF and Notch1 were detected via quantitative Polymerase Chain Reaction (qPCR). Meanwhile, the expression levels of VEGF and Notch1 in tissues were determined using immunohistochemistry. Furthermore, the apoptosis of tissues was determined via Annexin V-FITC, propidium iodide (PI) double labeling, and flow cytometry. The levels of VEGF and Notch1 increased significantly in the CIM group when compared with those in the Sham group (p<0.01). However, their expressions decreased remarkably in CIM + miR-210 Mimic group when compared with CIM group (p<0.05). The mRNA expressions of VEGF and Notch1 were evidently upregulated in the CIM group when compared with the Sham group (p<0.01), whereas they were remarkably downregulated in the CIM + miR-210 Mimic group than CIM group (p<0.05). Immunohistochemistry results indicated that the expression levels of VEGF and Notch1 in tissues were consistent with Western blotting results. Besides, the protein expressions of c-Caspase3 and Bcl-2 were remarkably higher in the CIM group than Sham group (p<0.01). However, they were significantly lower in the CIM + miR-210 Mimic group than those in the CIM group (p<0.05). In addition, flow cytometry results demonstrated that the apoptosis level increased significantly in CIM group when compared with the Sham group (p<0.05), while it was remarkably inhibited in the CIM + miR-210 Mimic group (p<0.05). MiR-210 can reduce the protein expressions of VEGF and Notch1, inhibit the VEGF-Notch signaling pathway, decrease the expression of pro-apoptotic factor c-Caspase3 and increase the expression of anti-apoptotic factor Bcl-2, thereby suppressing cerebral neuronal apoptosis and preventing CI-induced neuronal apoptosis.

Effect of atorvastatin on pulmonary hypertension rats through regulating notch signaling pathway.

To study the effect of atorvastatin on pulmonary hypertension (PAH) rats through regulating the Notch signaling pathway. The rat model of PAH was established via hypoxic feeding and the Control group (n=10), PAH model group (Model group, n=10) and atorvastatin treatment group (ATO group, n=10) were set up. The right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) in each group were determined, the wet/dry weight (W/D) ratio of lung tissues was determined, and the tumor necrosis factor-α (TNF-α), myeloperoxidase (MPO) and interleukin-6 (IL-6) were detected via enzyme-linked immunosorbent assay (ELISA). Moreover, the pathological changes in lung tissues of rats were detected via hematoxylin-eosin (HE) staining and the apoptosis level was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Finally, the Notch signaling pathway and apoptosis level in tissues were detected via quantitative Polymerase Chain Reaction (qPCR), and the protein expression level of Notch pathway in lung tissues was determined through Western blotting. Both RVSP and RVHI in Model group were significantly higher than those in Control group and ATO group (p<0.05). In Model group, the levels of inflammatory factors MPO, IL-6, and TNF-α were significantly increased, and the W/D ratio was also significantly increased compared with those in Control group (p<0.05). The results of HE staining showed that the lung tissue injury in Model group was severe (p<0.05). According to the TUNEL staining results, the number of apoptotic cells in lung tissues was markedly larger in Model group than that in ATO group (p<0.05), and the expression levels of Caspase-3 and IL-6 in Model group were remarkably higher than those in ATO group (p<0.05), while the expression level of B-cell lymphoma-2 (Bcl-2) in Model group was remarkably lower than that in ATO group (p<0.05). Besides, the gene and protein expression levels of Notch1 in ATO group were evidently lower than those in Model group (p<0.05), indicating that atorvastatin can effectively suppress the expression of Notch. Atorvastatin can inhibit PAH in rats by suppressing the Notch pathway.

Low-intensity pulsed ultrasound regulates proliferation and differentiation of neural stem cells through notch signaling pathway

Low-intensity pulsed ultrasound (LIPUS) is widely used to regulate stem cell proliferation and differentiation. However, the effect of LIPUS stimulation on neural stem cells (NSCs) is not well documented. In this study, we have identified the optimal parameters, and investigated the cellular mechanisms of LIPUS to regulate the proliferation and differentiation of NSCs in vitro. NSCs were obtained and identified by nestin immunostaining. The proliferation of NSCs were measured by using Cell Counting Kit-8 (CCK-8). The expressions of nutritional factors (NTFs) were detected with immunoassay (ELISA). NSCs differentiation were detected by immunofluorescence and immunoblotting analysis. The expression level of proteins involved in the Notch signaling pathway was also measured by immunoblotting assay. Our results showed the intensity of 69.3 mW/cm2 (1 MHz, 8 V) was applicable for LIPUS stimulation. ELISA analysis demonstrated that LIPUS treatment promoted the expression of nutritional factors of NSCs in vitro. Immunofluorescence and immunoblotting analyses suggested that the LIPUS not only reduced the astrocyte differentiation, but also stimulated the differentiation to neurons. Additionally, LIPUS stimulation significantly upregulated expression level of Notch1 and Hes1. Results from our study suggest that LIPUS triggers NSCs proliferation and differentiation by modulating the Notch signaling pathway. This study implies LIPUS as a potential and promising therapeutic platform for the optimization of stem cells and enable noninvasive neuromodulation for central nervous system diseases.

Effects of Low Molecular Weight Fucoidan on the Proliferation and Apoptosis of Dysplastic Oral Keratinocyte and Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors with high incidence, rapid progress, and high mortality. There are still some defects in the treatments of OSCC, which seriously affect the quality of patients’ life. Therefore, it is urgent to find a safer and more effective treatment for OSCC. Low molecular weight fucoidan (LMWF) has various biological activities, such as antitumor, anti-inflammatory, and antithrombotic, and has no obvious side effects, but the effect of LMWF on OSCC has not been reported. In this study, the effects of LMWF on dysplastic oral keratinocyte (DOK) and OSCC cells SCC-9 and SCC-25 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, TUNEL, colony formation, and wound healing assay, and the expression levels of Notch-1 and Jagged-1 treated with LMWF were detected by RT-qPCR. The results showed that LMWF could inhibit the proliferation and migration of DOK and SCC-9, and promote apoptosis. Low molecular weight fucoidan upregulated the expression of Notch-1 and Jagged-1 in DOK and SCC-9 cells. It indicated that LMWF might promote the apoptosis of DOK and SCC-9 by upregulating the Notch signal pathway, thereby inhibiting cell proliferation and playing an antitumor role. It provided theoretical basis to develop LMWF as a novel therapeutic drug for oral cancer.