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Objective:To investigate the effects and mechanism of sciadopitysin combined with CK2 inhibitor CX-4945 on proliferation and apoptosis of glioblastoma U87 cells.Methods:Glioblastoma U87 cells were cultured in vitro, and treated with 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin respectively. U87 cells were treated with 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945. U87 cells were divided into control group (without any treatment), sciadopitysin group (100.00 μmol/L of sciadopitysin), CX-4945 group (5.00 μmol/L of CX-4945), sciadopitysin combined with CX-4945 group (100.00 μmol/L of sciadopitysin plus 5.00 μmol/L of CX-4945). MTT method was used to detect cell viability, Caspase3/7 activity assay and Annexin Ⅴ/ PI double staining were used to detect cell apoptosis, and Western blotting was used to detect the expressions of Notch1 pathway related proteins ICN1, HES1 and DLL3. Results:The cell viabilities of U87 cells treated with 0, 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin were (100.00±6.30) %, (112.02±7.63) %, (140.84±6.73) %, (113.92±7.92) %, (102.60±7.12) % and (73.16±2.74) % respectively, and there was a statistically significant difference ( F=55.21, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 0.01, 0.10, 1.00, 100.00 μmol/L of sciadopitysin treatment ( P=0.009; P<0.001; P=0.003; P<0.001). The cell viability of U87 cells was inhibited by 100.00 μmol/L of sciadopitysin, while sciadopitysin at other low concentrations manifested as enhancement or no obvious effect. The cell viabilities of U87 cells treated with 0, 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945 were (100.00±5.53) %, (108.70±10.24) %, (93.14±2.82) %, (81.46±4.92) %, (56.92±3.99) % and (31.24±2.67) % respectively, and there was a statistically significant difference ( F=135.18, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 1.25, 5.00, 10.00, 20.00 μmol/L of CX-4945 treatment ( P=0.022; P<0.001; P<0.001; P<0.001). Low concentration (1.25 μmol/L) of CX-4945 enhanced the cell viability of U87 cells, however higher concentrations (5.00, 10.00, 20.00 μmol/L) of CX-4945 shown inhibitory effect. The cell viabilities of U87 cells in the control group, sciadopitysin group, CX-4945 group and sciadopitysin combined with CX-4945 group were (100.00±5.53) %, (71.96±2.10) %, (77.66±4.12) % and (42.56±4.22) % respectively, and there was a statistically significant difference ( F=160.56, P<0.001). There were statistically significant differences between the control group and each treatment groups (all P<0.001). There were statistically significant differences between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (both P<0.001). The Caspase3/7 activities of U87 cells in the above four groups were 2.34±0.47, 4.02±0.22, 3.67±0.32 and 5.85±0.28 respectively, and there was a statistically significant difference ( F=55.80, P<0.001). The apoptosis rates of each groups were (0.40±0.10) %, (17.37±0.57) %, (3.00±0.66) % and (33.47±0.87) % respectively, and there was a statistically significant difference ( F=1 822.18, P<0.001). Further pairwise comparison showed that there were statistically significant differences in Caspase3/7 activities and apoptosis rates between the control group and each treatment groups ( P<0.001, P=0.001, P<0.001; P<0.001, P=0.001, P<0.001). There were statistically significant differences in Caspase3/7 activities and apoptosis rates between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (all P<0.001). The protein expression levels of Notch 1 pathway related proteins ICN1 (0.55±0.07 vs. 1.01±0.09), HES1 (0.66±0.08 vs. 1.00±0.06) and DLL3 (0.74±0.04 vs. 1.01±0.09) in U87 cells decreased significantly after treatment with 100.00 μmol/L of sciadopitysin ( t=5.94, P=0.004; t=5.15, P=0.007; t=4.00, P=0.016) . Conclusion:Sciadopitysin can synergize with CK2 inhibitor CX-4945 to inhibit the proliferation and promote apoptosis of glioblastoma U87 cells by inhibiting Notch1 signaling pathway.
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Objective:To evaluate the role of Notch1/hairy and enhancer of split homolog1(Hes1) signaling pathway in high glucose and hypoxia-reoxygenation (H/R) injury to cardiomyocytes.Methods:H9c2 cardiomyocytes were cultured in low-glucose DMEM culture medium supplemented with 10% fetal bovine serum.The cells were divided into 6 groups ( n=12 each) using a random number table method: control group (group C), H/R group, H/R+ Jagged-1 group (group H/R+ J), high glucose group (group HG), high glucose+ H/R group (group HG+ H/R) and high glucose+ H/R+ Jagged-1 group (group HG+ H/R+ J). The cells were incubated in low-glucose culture medium for 72 h in group C. After incubated in low-glucose culture medium for 72 h, the cells were exposed to 24-h hypoxia in an incubator filled with 95% N 2-5% CO 2 at 37℃, immediately followed by 6-h reoxygenation in an incubator filled with 95% O 2-5% CO 2 at 37℃ in group H/R.In group H/R+ J, Jagged-1 (Notch1/Hes1 signaling pathway specific activator) 5μg/ml was added to low-glucose culture medium and the cells were incubated for 72h before H/R.In group HG, H9c2 cardiomyocytes were incubated in high-glucose culture medium containing 33 mmol/L glucose for 72 h. In group HG+ H/R, the cells were incubated in high-glucose medium for 72 h before H/R.In group HG+ H/R+ J, Jagged-1 5μg/ml was added to high-glucose culture medium, and the cells were incubated for 72 h before H/R.At 6 h of reoxygenation, the supernatant of the culture medium was collected for detection of the activities of superoxide dismutase (SOD) and lactic dehydrogenase (LDH), the cell viability (by CCK-8 assay) and the cell apoptosis rate (by flow cytometry) and for determination of expression of Notch1, Hes1 and c-caspase-3 (by Western blot). Results:Compared with group C, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in H/R, H/R+ J and HG groups, expression of Notch1, Hes1 and c-caspase-3 was up-regulated in H/R and H/R+ J groups, and the expression of Notch1 and Hes1 was down-regulated and c-caspase-3 expression was up-regulated in group HG ( P<0.05). Compared with group H/R, the cell survival rate and SOD activity was significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group H/R+ J, and the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in HG+ H/R and HG+ H/R+ J groups ( P<0.05), and expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG+ H/R, the cell survival rate and SOD activity were significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group HG+ H/R+ J ( P<0.05). Compared with group H/R+ J, the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R+ J ( P<0.05). Conclusion:Activation of Notch1/Hes1 signaling pathway is the endogenous protective mechanism of high glucose and H/R injury to cardiomyocytes.
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Humans , Disease-Free Survival , Endothelial Cells , Prognosis , Receptor, Notch1 , Triple Negative Breast NeoplasmsABSTRACT
Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells.Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group,Nc-shRNA group and Notch1-shRNA group.The Nc-shRNA group was a negative control RNAi lentivirus group,and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group.The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1.The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay.Apoptosis was detected by Annexin V/7-AAD double staining.Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1.Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000 ± 0.000,0.937 ± 0.025,0.490 ± 0.036 and 1.000 ± 0.000,1.077 ± 0.070,0.373± 0.038,with statistically significant differences (F =359.707,P ＜0.001;F =210.455,P ＜0.001),further paired comparison,the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P ＜ 0.05).Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results.MTT assay showed that the 24 h A values of A549 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.209 ± 0.005,0.219 ± 0.009,0.159 ±0.006,48 h A values were 0.293 ± 0.004,0.302 ± 0.004,0.205 ± 0.005,72 h A values were 0.450 ± 0.003,0.430 ± 0.012,0.348 ± 0.017,with statistically significant differences (F =79.487,P＜0.001;F =508.664,P ＜0.001;F =57.156,P ＜0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24,48,72 h (all P ＜ 0.05).The 48 h A values of SPC-A-1 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.438 ±0.022,0.412 ± 0.015,0.364 ± 0.010,72h A values were 0.540 ± 0.016,0.519 ± 0.009,0.438 ± 0.019,with statistically significant differences (F =15.667,P =0.004;F =37.299,P ＜ 0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P ＜ 0.05).The sphere sizes of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667 ± 6.506) μm,(136.667 ± 7.095) μm,(86.676 ± 7.638) μm,with statistically significant difference (F =65.940,P ＜ 0.001).The sphere sizes of the three groups in SPC-A-1 cells were (118.667 ± 6.658) μm,(128.000 ± 7.000) μm,(60.675 ± 4.509) μm,with statistically significant difference (F =105.372,P ＜0.001).Further paired comparison,the sphere size of Notch1shRNA group was significandy smaller than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).The apoptosis rates of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1cells were (0.489 ± 0.014)％,(0.633 ± 0.021)％,(1.683 ± 0.221)％ and (1.323 ± 0.194)％,(1.690 ± 0.188) ％,(3.017 ± 0.356) ％,with statistically significant differences (F =77.660,P ＜ 0.001;F=32.200,P =0.001),further paired comparison,the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).Western blotting showed that the expressions of PCNA,Bcl-2 and Hes-1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F =155.343,P ＜ 0.001;F =22.576,P =0.002;F =70.108,P＜0.001),and the expressions of PCNA,Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F =49.419,P ＜0.001;F =28.090,P =0.001;F =12.040,P =0.007).Further paired comparison,the expressions of PCNA,Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells,and the differences were statistically significant (all P ＜0.05).Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis,which may be related to the down-regulation of its downstream gene Hes-1.
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Objective To investigate the predicting value of Notch1 levels expressed in peripheral blood mononuclear cell (PBMC) for coronary artery calcification.Methods 300 consecutive patients with coronary artery disease (CAD) who hospitalizing in the department of cardiology in Guangzhou first people's hospital from January 2016 to June 2017 were enrolled.All Patients received 320-slice multi-detector row computed tomography scanning and coronary artery calcium sore(CCS)were measured.Patients were divided into three groups:control group (CCS =0),Low CCS group (CCS ＜97.6) and high CCS group (CCS ≥97.6) according to the mean value of CCS (CCS =97.6).Notch1 expressed in PBMC,serum interlekin-6 (IL-6) and high sensitivity C reactive protein (hs-CRP)of patients were examined and compared among three groups.Results The levels of Notch1 in PBMC and serum IL-6,hs-CRP of patients in high CCS group were significant higher than the other two groups [Notch1:7.02 ± 0.86 vs 6.32 ± 0.78 vs 5.49 ± 0.71;IL-6:(133.66 ± 10.18) μg/L vs (127.49 ± 10.79) μg/L vs (111.62 ± 9.87) μg/L;hs-CRP:(3.98 ± 1.02) mg/L vs (3.11 ±0.95)mg/L vs (2.56 ±0.76)mg/L] (P ＜0.05).The Spearman correlation analysis showed that the levels of Notch1 in PBMC were positive correlated with the levels of serum IL-6 and hs-CRP in enrolled patients with coronary calcification (P ＜ 0.05).Multivariate logistic regression analysis showed that the levels of Notch1 in PBMC and serum IL-6 were two strong independent risk factors for severity of coronary calcification in patients with CAD (P ＜ 0.05).Conclusions Notch1 expression in PBMC of patients with CAD was valuable to predicate the severity of coronary calcification.That the Notch1 signal path regulating the inflammation conditions in patients may be one of the most important mechanisms in the formation and progress of coronary calcification.
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Objective: To analyze the associations of Notch 1 expression with the lymph node metastasis and distant metastasis of papillary thyroid carcinoma (PTC) by Meta analysis. Methods: Computer retrieve was conducted in PubMed (MEDLINE), Cochrane Library, EMBASE, China Journal Full-text Database (CNKI), China Biology Medicine disc (CBMdisc) to search the studies which were about the association of Notch1 signal with the lymph node and distant metastases of PTC, and published from 2010 to 2017. The literatures were screened and evaluated, then the information was extracted independently by 2 researchers according to the literature selection criteria. Meta analysis was performed using RevMan 5.3 and STATA 12.0 software. The odds ratio (OR) and 95% confidence interval (CI) were calculated. The sensitivity analysis and publication bias test were performed. Results: A total of 7 clinical case-control studies involving 743 patients with PTC were selected. Meta-analysis showed that the expression of Notch1 was significantly positively correlated with lymph node metastasis of PTC (OR = 4.68, 95% CI: 3.00-7.30), furthermore the test for overall effect showed that Z = 6.80 and P < 0.000 01. However, there was no significant correlation between Notch1 expression and the distant metastasis of PTC (OR = 1.59, 95% CI: 0.88-2.89), the test for overall effect showed that Z and P values were 1.53 and 0.1 3 respectively. Conclusion: The Notch1 signaling pathway plays a promoting role in the lymph node metastasis of PTC, which suggests that the expression of Notch1 has a certain predictive value for the clinical prognosis of PTC.
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Objective To observe the expression of Notch1 signaling pathway in the aorta of chronic kidney disease (CKD) rats with vascular calcification and to explore the role of this signaling pathway activation in aortic calcification of CKD.Methods A total of 40 male SD rats were randomly divided into normal group (Nor group) and CKD with vascular calcification group (CKD+VC group).Rats in each group were sacrificed at 4 weeks,6 weeks and 8 weeks respectively after the success of modeling.Their 24-hour urine was reserved to test 24 hour urine protein (24 h Upro);blood sample was collected from abdominal aorta to test blood urea nitrogen (BUN),serum creatinine (Scr),Ca and P.The histopathology of renal was detected by HE staining.The aortic calcification was detected by alizarin red S staining.Immunohistochemistry (IHC) was used to test the protein expressions of alpha-smooth muscle actin (or-SMA),Runt-related transcription factor 2 (Runx2),Notchl,recombination signal-binding protein for immunoglobulin kappa J region (RBP-Jκ),Msh homeobox 2 (Msx2),Jagged1 and Notch1 intracellular domain (N1-ICD) in the aorta.Real time PCR was applied to detect the mRNA expressions of α-SMA,smooth muscle 22 alpha (SM22α),Runx2,alkaline phosphatase (ALP),Notch1,RBP-Jκ,Msx2 and Jagged1.Results Compared with those in Nor group,24 h Upro,BUN and Scr increased in the CKD+VC group at 4th,6th and 8th weeks (all P ＜ 0.05).Numerous continuous calcified nodules were detected in the vascular wall of the CKD+VC group,but none in Nor group.As compared with Nor group,the expression of α-SMA was low,while the expression of Runx2 was relatively high in the CKD+VC rats at each time point (all P ＜ 0.05).The expressions of Notchl,RBP-Jκ,Msx2,Jaggedl and NI-ICD in the Nor group were slightly appeared in the aortic wall,while in CKD+VC group these signal protein expressions increased relatively during the experimental period (all P ＜ 0.05).As compared with Nor group,the expressions of α-SMA and SM22α mRNA were low,yet the expressions of Runx2 and ALP mRNA were high in the CKD+VC rats at each time point (all P ＜ 0.05).The mRNA levels of Notch1,RBP-Jκ,Msx2 and Jagged1 in the CKD+VC group at each time point were significantly up-regulated as compared with the Nor group (all P ＜ 0.05).Conclusions There exist phenotypic changes in smooth muscle cells and activations of Notch1/RBP-Jκ/Msx2 signaling pathway in CKD rats with vascular calcification.It may be one of the important signal transduction pathways.
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Objective To investigate the role of Notch signaling pathway to maintain the stem cell-like characteristics of osteosarcoma and its underlying mechanism.Methods Lentiviral NICD1 or Numb-shRNA was transduced into MG63 osteosarcoma cells to activate Notch activity in vitro.The impact of Notch on osteosarcoma stem cells were assessed by the tumor sphere formation assay and flow cytometry analysis of cell surface markers STRO-1/CD117.The expression of stem cell related genes (Sox2,Oct4) were evaluated by Western blot and qPCR.The nude mice were randomly divided into 3 groups:the NICD1 overexpression (NICD-OE) group,the DAPT group and the control (CON) group.The tumor growth was monitored for 8 weeks and the tumor volume and weight were recorded weekly.To investigate whether Notch regulates Eph pathway,Eph pathway related protein EphB,pEphB was measured by Western blot.The impact of ephrinB 1 on NICD overexpression cell were assessed by tumor sphere formation assay.The expression of Sox2 and Oct4 was evaluated by Western blot.Results NICD1 overexpression or Numb-shRNA increased the activity of Notch pathway.The Notch-activated osteosarcoma showed enhanced in vitro tumor spheroid formation capacity,increased Stro-1/CD117double positive ratio,and upregulated expression of Sox2 and Oct4 in vitro.In animal experiments,it was found that activation of Notch pathway promoted tumor formation in vivo and Notch inhibition decreased it.The primary osteosarcoma cells were obtained from mice xenograft treated with DAPT and its tumor sphere formation capacity was significantly reduced.Finally,The Notch pathway inhibits the phosphorylation of EphB,as well as the downstream signal pathway of EphB,but there is no significant change in total EphB.The activation of Eph pathway inhibited Notch induced up-regulation of tumor sphere formation and Sox2 and Oct4 expression.Conclusion Notch signaling pathway maintains the stem cell-like characteristics of osteosarcoma probably by inhibiting the Eph pathway.
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Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR),and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2).Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study.The PDR patients were divided into non-injection group (30 patients,32 eyes) and injection group (27 patients,28 eyes).The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery.The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy.Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls.The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression ofNotch1,Dll4 and VEGFR2.In the meantime,the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted.Results The immunohistochemical staining revealed that there were positive expression ofNotch1,Dll4 and VEGFR2 in all PDR membranes,regardless of the injection of the ranibizumab.The levels ofNotch1,Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45,6.01,4.08;P=0.030,0.008,0.023).In injection group,the number of endothelial cells in the membranes reduced (17.17 ± 2.48) compared with that of the non-injection group (41.50± 5.57).There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05).RT-PCR showed that the differences of the mRNA expression ofNotch1,Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50,12.50,12.02;P＜0.05).The expression ofNotch1,Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients.In the PDR group,the expression of Notch1,Dll4 and VEGFR2 of non-injection group was higher than that of injection group.Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83),VEGFR2 and Notch1 (r=0.81),Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05).Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group,and it is positively correlated with the expression of the VEGFR2.Notchl and Dll4 play a regulatory rule in the neovascularization in PDR,the acting way may be correlated with VEGFR2.
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Objective@#To investigate the expression levels of S100A6, Notch1 in multiple myeloma (MM) patients and its clinical significance.@*Mathods@#The expression levels of S100A6, Notch1 in 28 MM cases and 20 healthy controls were determined by real time quantitative PCR (RQ-PCR) , and their relationships with clinical features and outcomes were analyzed. Immunohistochemical was used to analysis the levels of S100A6 and Notch1 in bone marrow biopsy samples and intramedullary metastases soft tissues. RQ-PCR and Western blot were used to test the changes of Notch1 mRNA and Notch1 protein in U266 MM cells after S100A6 silenced by siRNA.@*Results@#①The expression levels of S100A6, Notch1 in primary MM patients was 2.19±1.25, 2.98±0.64, significantly higher than those in controls (0.71±0.20, 0.58±0.39, P<0.05) and patients in platform status (0.85±0.26, 0.72±0.40, P<0.05) . 8 cases with intramedullary metastasis had significantly higher levels of S100A6 (3.36±1.23) and Notch1 (5.71±3.96) , as compared to those without extra medullary metastases. ②S100A6 expression was positive correlation with Notch1 (r=0.505, P=0.007) . ③S100A6 and Notch1 proteins were positive in plasma cells of bone marrow biopsy samples and intramedullary metastases soft tissues. ④The Notch1 mRNA and Notch1 expression decreased significantly after 48 hours treatment by S100A6 siRNA in U266 cells.@*Conclusion@#S100A6 and Notch1 were closely associated with MM progress and intramedullary metastasis. They have significant correlation and might be as two prognostic molecular markers in MM.
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Objective To explore the expressions and changes of endogenous neural stem cells (ENSCs) and Notch-1 after acute spinal cord injury in adult rats,and to explore its role in the nerve regeneration process.Methods The 30 Sprague Dawley (SD) female rats (3-6 months) (300-350 g) were divided into control groups (n =5) and experimental group (n =25) by digital random method.The control group only accepted lamina decompression,and the experimental group was the spinal cord injury group.Five rats were drawn when at 1 d,3 d,1 w,2 w,and 4 w,then behavior,histology,immunohistochemistry and immunofluorescence method were used to detect the proliferation and expression of endogenous neural stem cells and Notch-1 protein.Results Behavioral observation showed the experimental group rats were disfunction.Histological observation showed nerve fiber structure turbulence,edema and denaturation in the experimental group.Immunohistochemistry staining showed the Notch-1 expression every experimental group.Notch-1 positive cell peaked at 3 days.Immunofluorescence test showed,in the experimental group damage to segment the area surrounding the BrdU positive staining cells was significantly higher than the control group.Using three-dimensional quantitative study method detected in 1 w after spinal cord injury was the number of newborn cells mitosis,most about was about 75 times in the control group.Linear regression method was used to analyze the different time points after BrdU and Notch-1 protein expression positive area,the area of the results found that both into linear correlation.Conclusions The new born neurons after spinal cord injury in rats Notch-1 expression has a certain relevance.In addition,the expression of signal protein Notch-1,might be associated with early proliferation of ENSCs in rats.
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Objective To explore the expressions and changes of endogenous neural stem cells (ENSCs) and Notch-1 after acute spinal cord injury in adult rats,and to explore its role in the nerve regeneration process.Methods The 30 Sprague Dawley (SD) female rats (3-6 months) (300-350 g) were divided into control groups (n =5) and experimental group (n =25) by digital random method.The control group only accepted lamina decompression,and the experimental group was the spinal cord injury group.Five rats were drawn when at 1 d,3 d,1 w,2 w,and 4 w,then behavior,histology,immunohistochemistry and immunofluorescence method were used to detect the proliferation and expression of endogenous neural stem cells and Notch-1 protein.Results Behavioral observation showed the experimental group rats were disfunction.Histological observation showed nerve fiber structure turbulence,edema and denaturation in the experimental group.Immunohistochemistry staining showed the Notch-1 expression every experimental group.Notch-1 positive cell peaked at 3 days.Immunofluorescence test showed,in the experimental group damage to segment the area surrounding the BrdU positive staining cells was significantly higher than the control group.Using three-dimensional quantitative study method detected in 1 w after spinal cord injury was the number of newborn cells mitosis,most about was about 75 times in the control group.Linear regression method was used to analyze the different time points after BrdU and Notch-1 protein expression positive area,the area of the results found that both into linear correlation.Conclusions The new born neurons after spinal cord injury in rats Notch-1 expression has a certain relevance.In addition,the expression of signal protein Notch-1,might be associated with early proliferation of ENSCs in rats.
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PURPOSE: The Notch signaling pathway is widely expressed in normal, reactive, and neoplastic tissues; however, its role in thyroid tissues has not been fully elucidated. Therefore, this study was conducted to characterize the expression of the Notch signaling pathway in papillary thyroid cancer (PTC) cells and anaplastic thyroid cancer (ATC) cells. MATERIALS AND METHODS: Expression of activated Notch1 in ATC and PTC paraffin-embedded tissues was determined by immunohistochemistry. The small interfering RNA techniquewas employed to knock down Notch1 expression in ATC and PTC cell lines. RESULTS: The expression of activated Notch1 was higher in ATC cases than in PTC cases. Inhibition of Notch1 significantly reduced proliferation and migration of ATC cells, but not PTC cells. In addition, inhibition of Notch1 in ATC cells significantly reduced the expression of key markers of epithelial-mesenchymal transition and cancer stem cells. Conversely, changes in the expression of these proteins were not observed in PTC cells. CONCLUSION: The results of this study suggest that Notch1 expression plays different roles in tumor progression in ATC and PTC cells. We also found that Notch1 expression was significantly related to the highly invasive or proliferative activity of ATC cells.
Subject(s)
Cell Line , Epithelial-Mesenchymal Transition , Immunohistochemistry , Neoplastic Stem Cells , RNA, Small Interfering , Thyroid Carcinoma, Anaplastic , Thyroid Gland , Thyroid NeoplasmsABSTRACT
Objective To explore the relationship between resveratrol and Notch 1 signalling pathway in podocytes.Methods Interference RNA (RNAi) and doxycycline (Dox) were used to inhibit the Sirtuin (SIRT) 1 expression in the wild-type and inducible SIRT1 shRNA (CAGGS) podocytes respectively.Recombinant mouse delta-like ligand 4 (DLL4) was used to activate Notch1 signalling.The message RNA of SIRT1,Notch1 downstream gene Hes1 and Hey2,as well as the key enzymes of Notch1 signalling pathway were detected by using real-time PCR.Western blotting was used to detect intracellular domain of Notch 1 (ICD1),SIRT1,and metalloprotease (ADAM) 10 and components of γ-secretase complex protein expression.Results In WT murine podoytes,resveratrol up-regulated ICD1 protein production,as well as the mRNA of Hes1 and Hey2 in a dose-dependent manner.Treatment with resveratrol resulted Nicastrin mRNA and protein increase in podocytes (P ＜0.05),as well as inhibit ADAM10 expression (P ＜ 0.05),but all these changes were prevented after the use of SIRT1 RNAi(P ＜ 0.05).DLL4 up-regulated the expression of mRNA of Hes1 and Hey2,as well as ICD1 protein production in a dose-dependent manner.Treatment with doxycycline resulted decrease of SIRT1 gene and protein expression in CAGGS podocytes after 24 h and 48 h respectively(P ＜ 0.05),which weakend the role of DLL4 significantly(P ＜ 0.05).Conclusion Resveratrol induces Nicastrin expression,as well as activation of Notch1 signalling pathway in a SIRT1-dependent manner.
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Objective To investigate the effects of Notch1 siRNA on VEGF and angiogenesis of myeloma cell line RPMI-8226 in vitro and in vivo.Methods In vitro,Notch siRNA was transfected into RPMI-8226 cells,and then cell supernatant VEGF secretion was detected using ELISA method.Expression levels of Notch1 and VEGF proteins were assayed by Western blotting.RPMI-8226 cells were subcutaneously transplanted in NOD/SCID mice,and then the tumor mice were divided into three groups randomly:NS group (Notch1 siRNA-transfected group),CS group (Control siRNA-transfected group) and UN group (Untransfected group),and the changes of tumor volume were observed.Immunohistochemical staining was used to detect the changes in expression levels of Notch1,VEGF and CD34.Results Notch1 and VEGF proteins expressions of RPMI-8226 cells were significantly decreased by Notch1 siRNA.At 48 h and 72 h,VEGF secretion level in NS group was significantly different with CS group [(120 ± 25) ng/L ∶ (175 ± 15) ng/L,t =3.27,P ＜ 0.05;(145 ± 24)ng/L ∶ (295 ± 17)ng/L,t =8.83,P＜0.01].At 13 d,17 d and 21 d,tumor volume in NS group was significantly reduced,that was significantly different with CS group [(1 548 ± 218) mm3 ∶ (1 820 ± 64) mm3,t =2.68,P ＜0.05;(1 200 ±75)mm3 ∶ (2 180 ±84)mm3,t =19.46,P＜0.01;(1 150 ±88)mm3 ∶ (2 250 ± 145)mm3,t =14.50,P ＜0.01].The expression levels of Notch1 and VEGF protein were decreased by Notch1 siRNA.The expression levels of Notchl and VEGF in NS group were different with CS group [(16.33 ±2.52)％∶ (75.33 ±2.52)％,t=28.71,P＜0.01;(5.00±1.00)％∶29.67±2.08 ％,t=18.50,P ＜ 0.01].Notch1 siRNA reduced the number of transplanted tumor neovascularization in NS group.Microvascular density in NS group was significantly less than that in CS group [(14.67 ± 2.52) ∶ (30.00 ± 5.00),t =4.74,P ＜ 0.01].Conclusion In vitro,Notch siRNA reduces human myeloma cell RPMI-8226 cell supernatant VEGF secretion.In vivo,Notch siRNA can reduce tumor volume and the number of new blood vessels in transplanted-multiple myeloma mice.Thus,Notchl is an effective molecular target for anti-angiogenesis in myeloma.
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Objective To investigate the effect of Doxycycline on gastric cancer cell line BGC-823 proliferation and Notch1, Matrix metalloproteinase-9(MMP-9)expression in order to shine a light on new anticancer drugs. Methods Final concentration of 0, 5, 10, 20, 40 mg/L doxycycline were added into human gastric cancer cell line BGC-823. Cell pro-liferation was detected by MTT;Cell cycle distribution was assessed by flow cytometry;Notch1, MMP-9 protein expression was revealed by Immunoblot. Results Doxycycline can inhibit proliferation of human gastric cancer cells line BGC-823, and its effect is dose and time-dependent(P＜0.01). Doxycycline alters distribution of gastric cancer cell line BGC-823. With increasing drug concentration, the proportion of cells in S phase dropped(P＜0.01). Notch1 expression rose and MMP-9 expression decreased(P＜0.01). Conclusion Doxycyclinecan inhibited gastric cancer cell line BGC-823 prolif-eration and up-regulating Notch1 might be one mechanisms.
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Objective To study the expression of Notch 1,Jagged1 and Notch intracellular domain (NICD) in epithelial ovarian carcinoma tissues and analyze the clinical significance.To explore the activity of γ-secretase in epithelial ovarian carcinoma cell line SKOV3 and the effect of N-[N-(3,5-dil uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT),a γ-secretase inhibitor on the activity of γ-secretase in SKOV3.Methods Immunohistochemistry staining method was performed in 43 patients with epithelial ovarian carcinoma and 11 patients with benign epithelial ovarian tumor to detect the expression of Notch1,Jagged1 and NICD.The differences of expressionof Notch1,Jagged1 and NICD between malignant and benign ovarian tumors was compared and alsoanalyzed the correlation with clinicopathological parameters of ovarian carcinoma.Human serous ovarian cancer cell line SKOV3 and immortalized nontumorigenic ovarian epithelial cell line T29 were incubated in vitro.The activities of γ-secretase in SKOV3 and T29 with dimethyl sulfoxide (DMSO) and DAPT were detected respectively by Gal4VP16/UAS and dual luciferase reporter assay system.Results (1) The immunohistochemical composite scores (ICS) of Notch1 in epithelial ovarian carcinoma (6.7±2.2) were not significantly different with those in benign epithelial ovarian tumor (5.4± 2.7,P=0.153),while the ICS of Jagged 1 and NICD in epithelial ovarian carcinoma (5.3± 2.4,5.3± 2.3) were higher than those in benign epithelial ovarian tumor (1.6± 1.4,3.1± 1.7; all P＜0.01).The expression of Notch 1,Jagged 1 and N ICD had no correlation with patients' aged,history of carcinoma,ascites,the level of serum CA125,maximum length of ovarian tumor,Federation International of Gynecology and Obstetrics (FIGO) stage,grade and pathology subtypes (all P＞0.05).The hazard ratio between the high expression of Notch1,Jagged1,or NICD and the moderate to low expression of Notch1,Jagged1,or NICD,and Jagged1 were 0.771,1.648 and 1.316,respectively (all P＞0.05).The 5-year survival rate and median survival time between the high expression of Notch,Jagged 1 or NICD in subgroup and moderate to low expression in subgroup were of no difference (all P＞0.05).The activity of γ-secretase in SKOV3 was significantly higher than that in T29 [(12.2± 1.4)％,P=0.019].(2)After DAPT treated,the relative activity of γ-secretase in SKOV3 (50 μmol/L) was declined from (100.0±5.3)％ to (6.6±0.8)％ (P=0.001).Conclusions Jagged1 and NICD in Notch1 pathway may play a key role in the occurrence of ovarian carcinoma.The activity of γ-secretase in epithelial ovarian carcinoma was higher than that in ovarian epithelial cell which suggest that DAPT,γ-secretase inhibitor,may become the target of ovarian carcinoma treatment.
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Objective To investigate the expression of Notch1,Delta-like ligand 4 (DLL4),hairy and enhancer of split-1 (HES-1),microvessel density (MVD),lymphatic vessel density (MLD) in gastric carcinoma,so as to discuss their roles in the development of gastric carcinoma.Methods Gastric carcinoma,paracancer tissues which was apart from the edge of cancer tissue ＞ 60 mm obtained during operation and normal gastric mucosa obtained during gastroscopy were used as controls.All specimens were made tissue microarray.The expressions of Notchl,DLL4,HES-1 were detected by immunohistochemistry.Immunohistochenical double taining was used to detect MVD and MLD.The relationships between Notch1,DLL4,HES-1 expression and angiogenesis,lymphangiogenesis and their significances were analyzed.Results The positive rate of Notch1 in gastric carcinoma was 48.30％,significantly higher than that of paracancerous (25.00％,x2 =6.38,P ＜ 0.05) and control group (16.67％,x2 =10.18,P ＜0.05).The differences of the positive rate of DLL4 in gastric carcinoma (55.94％),paracancerous (45.70％) and control group (56.67％) were not significant (x2 =1.18,P ＞0.05 ; x2 =0.005,P ＞ 0.05).The differences of the positive rate of HES-1 in gastric carcinoma (36.64％),paracancerous (34.40％) and control group (33.33％) were not significant (x2 =0.05,P ＞ 0.05 ;,x2 =0.11,P ＞ 0.05).The mean of MVD in gastric carcinoma group was 28.84 ± 14.17,which was significantly higher than that in paracancerous group (17.02 ±8.54,t =4.03,P＜0.05) and control group (16.69 ±7.21,t =5.01,P＜0.05).The mean of MLD in gastric carcinoma group was 8.55 ±4.98,which was significantly higher than that in paracancerous group (4.05 ± 2.48,t =9.30,P ＜ 0.05) and control group (3.99 ± 1.56,t =10.32,P ＜ 0.05).The expression of DLL4 was correlated with MVD (t =2.77,P ＜ 0.05),but wasn't correlated with MLD (t =1.89,P ＞0.05).There were no correlations between the expression of Notch1,HES-1 and tissues MVD,MLD (P ＞0.05).Conclusion Notch1 plays important roles in the development of gastric carcinoma.There are many angiogenesis and lymphangiogenesis in gastric carcinoma.The expression of DLL4 in gastric carcinoma has certain effect in the formation of microvessel.
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Objective To explore the effects of homocysteine (Hcy) on neural stem cell (NSC) proliferation and the mRNA expression level of Notch1 and Hes1 related Notch signaling pathway from neonatal rats in vitro. Methods NSCs from neonatal rats were cultured by serum-free culture method in vitro. Cells were divided into four groups: control group (Hcy-C), low dose Hcy (Hcy-L) group, middle dose Hcy (Hcy-M) group and high dose Hcy (Hcy-H) group. NSCs were iden- tified by immunofluorescent staining using the antibodies against Nestin, β-tubulin Ⅲ and GFAP. The proliferation ability of NSCs was detected by MTT. The mRNA expressions of Notch1 and Hes1 were detected by Real-time PCR. Results In the serum free suspension medium, neurospheres that consisted of a great number of nestin-positive cells were found. β-tu- bulin Ⅲ positive neurons and GFAP positive astrocytes were detected by immunofluorescence staining on the 6 th day of cell induction. MTT assay showed that the cell viability was significantly lower in three Hcy treatment groups than those of con- trol group (P ＜ 0.05). And the effect of concentration-dependent was observed. The results of RT-PCR showed that mRNA expression of Hes1 was significantly lower in three Hcy treatment groups than that in control group (P ＜ 0.05). The mRNA ex- pression of Notch1 was significantly lower in Hcy-H group than that of other three groups (P ＜ 0.05). The mRNA expression of Notch1 was significantly lower in Hcy-M group than that of Hcy-L group and control group (P ＜ 0.05). Conclusion Hcy could inhibit the proliferation of NSCs by down-regulating mRNA expression levels of Notch1 and Hes1 genes related to Notch signal pathway.
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Recently numerous studies have demonstrated that Notch signaling pathway plays a critical role in regulating cell proliferation and differentiation in hematopoietic microenvironment,which is associated with MM occurrence and the drug resistance.The latest researches show that there are close relations between hypoxia and Notch signaling pathway in tumor occurrence and progression.Exploring the interactions of microenvironment of hypoxia,HIF-1α and Notch signaling pathway will provide theoretical basis for MM targeted therapy.