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Objective: Delta-like ligand 4 (DLL4) is the main ligand of Notch signaling pathway in regulating tumor angiogenesis. Obesity is a major risk factor for colorectal cancer, and its mechanism of promoting tumorigenesis needs further investigation. In the present study, we aimed to investigate the relationship of DLL4 expression with tumor progression and prognosis, as well as obesity in colorectal cancer. Methods: For this study we collected 265 samples of colorectal cancer tissues and paracancerous tissues resected at The First Affiliated Hospital of Xi'an Jiaotong University between January 2012 and December 2013. Immunohistochemistry was used to detect the expression of DLL4. We analyzed the relationship of DLL4 expression with clinicopathological characteristics, prognosis and obesity. The survival data was analyzed by Kaplan-Meier and Cox proportional hazard regression model. Results: The positive expression rate of DLL4 in colorectal cancer tissues was significantly higher than that in adjacent tissues (77.4% vs. 17.7%, P<0.05). The patients with high expression of DLL4 had a lower degree of differentiation, but a greater depth of invasion, a higher lymph node metastasis rate and higher TNM stage (P<0.05). The disease-free survival and overall survival were shorter in DLL4 positive expression group than in the negative group (P<0.001). Cox multiple regression analysis showed that DLL4 expression was an independent prognostic factor for colorectal cancer. The positive expression rate of DLL4 in overweight and obese patients was significantly higher than that in normal weight patients. BMI was positively correlated with the expression level of DLL4 (rs=0.578, P<0.05). Conclusion: The expression of DLL4 is upregulated in colorectal cancer tissues. Increased expression of DLL4 is associated with poor prognosis of patients, suggesting that DLL4-Notch pathway may be involved in the carcinogenesis and progression of obesity-induced colorectal cancer.
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OBJECTIVE： To investigate the effects of Buyang huanwu decoction on endothelial-mesenchymal transformation （EndMT） of lung tissue in idiopathic pulmonary fibrosis （IPF） model rats， and to explore its potential mechanism. METHODS： Male SD rats were randomly divided into normal group， model gorup， dexamethasone group [0.405 mg/（kg·d）]， Buyang huanwu decoction low-dose， medium-dose and high-dose groups [6.435， 12.87， 25.74 g/（kg·d）， by raw material]， with 8 rats in each group. Except for normal group， other groups were given endotracheal injection of bleomycin to induce IPF model. On the second day after modeling， normal group and model group were given water intrgastrically [10 mL/（kg·d）]； administration groups were given relevant medicine intragastrically， once a day， for consecutive 28 days. 24 h after last medication， the expression of endothelial cell markers [platelet endothelial cell adhesion molecule 1， vascular endothelial cell cadherin] and interstitial cell markers [α-smooth muscle actin， fibroblast specific protein 1] were detected by immunohistochemistry method. The expression of Notch4 and DLL4 in lung tissue of rats were detected by Western blotting assay. RESULTS： Compared with normal group， the expression of endothelial cell markers were decreased significantly in lung tissue of model group， while the expressipon of interstitial cell markers， Notch4 and DLL4 were increased significantly （P＜0.01）. Compared with model group， the expression of endothelial cell markers in lung tissue of rats were increased significantly in administration groups， while Buyang huanwu decoction low-dose group was significantly lower than dexamethasone group； the expression of interstitial cell markers， Notch4 and DLL4 were decreased significantly， while Buyang huanwu decoction low-dose group was significantly higher than dexamethasone group （P＜0.05 or P＜0.01）. CONCLUSIONS： Buyang huanwu decoction can relieve IPF of model rats by intervening in EndMT， the mechanism of which may be associated with inhibiting DLL4/Notch4 singaling pathway.
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Forty patients with Hashimoto thyroiditis ( HT) and 20 healthy subjects with matched age-and sex-features ( NC) were selected. The patients with HT were further divided into normal thyroid function ( HT-A) and hypothyroidism ( HT-B) groups. Real-time PCR was performed to evaluate the expressions of Notch1, Dll4, and retinoid-related orphan receptor ( ROR )-γt mRNA. Flow-cytometry was used to detect the percentage of Th17 cells. Thyroid function, thyroid peroxidase antibody ( TPOAb) , and thyroglobulin antibody ( TgAb) were detected by electrochemiluminescence immunoassaies. The results showed that the Notch1, Dll4, ROR-γt mRNA levels and Th17 cell percentage were significantly increased in HT group compared with NC group (all P<0.01), especially in HT-B group. In HT patients, Notch1 and Dll4 mRNA expression levels were positively correlated with Th17 cell percentage and its transcription factor ROR-γt ( all P<0.01) . Besides, there were significantly positive correlations of Notch1 and Dll4 mRNA expressions with TPOAb and TgAb titers (P<0.05 or P<0.01). These results suggest that Notch1-Dll4 signaling pathway might be involved in the pathogenesis of thyroid-specific autoimmune damage by regulating Th17 cells in HT patients.
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Objective To explore the role of Notch signaling pathway in the matrix synthesis of osteoarthritis cartilage cell and its possi -ble mechanism.Methods Selected the femoral condylar cartilage of 8 patients who were admitted into our hospital and treated with knee joint replacement as the observation group,while the normal femoral condyle cartilage of one patient with above-knee amputation was selected as the control group.The specimens were given histological examination or cell isolation culture and detection.The cultured cells were divided into 4 groups,namely the normal cartilage cells,OA cartilage cells,OA cartilage cells with γ-secretase inhibitor DAPT,OA cartilage cells with recombinant human proteins Delta4.Then immunohistochemistry and Western blot were used for detecting the expression of Notch -1,Notch-3, Jagged-1,Jagged-2 and HES5 in chondrocytes in vitro.Results The expression of Notch signaling pathway and the phenotype of cartilage cells changed in the osteoarthritis.The expression of Notch-1,Jagged-1,Jagged-2 and HES5 were activated.γ-secretase inhibitor DAPT (20 μmol/L)could inhibit the expression of Notch-1,HES5,Jagged-1,and Notch-3,while it had no obvious effect on the expreesion of Jag-ged-2.Recombinant human proteins Delta 4(100 ng/μL)could promote the expression of Notch-1,Jagged-2,and HES5,and it had no obvi-ous effect on the expreesion of Jagged-1 and Notch-3.Conclusion The expression of Notch signaling pathway of cartilage cells changed in the osteoarthritis.DLL4 can activate the Notch signaling pathway and DAPT can inhibit the Notch signaling pathway.
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AIM:To investigate the effects of DL-3-n-butylphthalidle (NBP) on angiogenesis of human um-bilical vein endothelial cells ( HUVECs) and the role of vascular endothelial growth factor ( VEGF)/VEGF receptor 2 (VEGFR2)-Notch1/Delta-like ligand 4 (Dll4) signaling pathway in this process. METHODS:The serum-free medium and anoxic tank were used to simulate the conditions of hypoxia and ischemia ( H/I). HUVECs were divided into control group, H/I group, H/I+NBPhigh group and H/I+NBPlow group. The HUVECs in control group were conventionally cul-tured, and those in H/I group were cultured under H/I intervention. The HUVECs in H/I+NBPhigh group were treated with NBP at 20 μmol/L under H/I intervention. The HUVECs in H/I+NBPlow group were treated with NBP at 5 μmol/L under H/I intervention. The cell viability of each group was measured by CCK-8 assay. The migration ability of the HUVECs in each group was detected by cell scratch test. The vessel formation ability of the HUVECs was examined by in vitro angiogenesis assay. The expression of VEGFR2, Notch1 and Dll4 at mRNA and protein levels was determined by qPCR and Western blot, and the expression of VEGF was determined by qPCR and ELISA. RESULTS:NBP increased the viability of HUVECs, and promoted the migration ability and the formation of blood vessels in vitro under H/I interven-tion. These effects of NBP at high dose were more significant than those at low dose. NBP increased the expression of VEGF, VEGFR2, Notch1 and Dll4 at mRNA and protein levels (P<0.05). CONCLUSION:NBP promotes HUVECs to form blood vessels under H/I intervention. The mechanism may be related to the activation of VEGF/VEGFR2-Notch1/Dll4 signaling pathway.
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Objective To determine the expression of Notch pathway receptors (Notch1 and Notch4) and ligands (Jagged1 and Dll4) in cutaneous malignant melanoma (CMM) tissues,and to preliminarily explore the role of the Notch signaling pathway in the pathogenesis of CMM.Methods Immunohistochemical study was performed to determine the expression pattern and intensity of Notch1,Notch4,Jagged1 and Dll4 in 40 paraffin-embedded CMM specimens and 15 paraffin-embedded pigmented nevus specimens.Statistical analysis was carried out by chi-square test and Spearman rank correlation analysis with the SPSS 21.0 software.Results Notchl was detected in 31 (77.5％) of 40 CMM specimens,as well as in 3 of 15 pigmented nevus specimens,and the positive rates significantly differed between the two groups (x2 =15.281,P ＜ 0.001).However,no significant difference in the expression intensity of Notch1 was observed between 18 in situ melanoma tissues and 22 invasive melanoma tissues (x2 =0.631,P =0.427).In addition,the positive rates of Notch4,Jagged1 and Dll4 were also significantly higher in the CMM group than those in the pigmented nevus group (all P ＜ 0.05),and the expression intensity of Notch4,Jagged1 and Dll4 significantly differed between in situ and invasive melanoma tissues (all P ＜ 0.05).In CMM tissues,the expression of Notch1 was positively correlated with that of Jagged1 (rs =0.350,P =0.027) and Dll4 (rs =0.562,P ＜ 0.001),while the expression of Jaggedl was negatively correlated with that of Dl14 (rs =-0.734,P ＜ 0.001).Conclusion Abnormality of the Notch signaling pathway may be involved in the pathogenesis of melanoma,but further researches are still needed to elucidate the detailed mechanism.
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AIM: To observe the inhibitory effect of recombinant human endostatin (rhES) on plaque angio-genesis, and to explore the regulatory mechanism of Dll4 /Notch pathway in the anti-angiogenic effect of rhES.METH-ODS: Male Wistar rats were randomized into 3 groups: normal control group (N group), atherosclerotic model group (AS group), and rhES treated group (AS +rhES group).The rats in N group were fed a normal diet, while the remaining 2 groups were established to atherosclerotic rat model via high-cholesterol diet, intraperitoneal injection of vitamin D3 and aor-tic balloon injury.The rats in AS +rhES group received intraperitoneal injection of rhES.The blood total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), interleukin-1 (IL-1) and troponin I (TnI) were measured.The atherosclerotic abdominal aortas were taken for pathological observation.Immu-nohistochemical staining was used to measure the density of neovessels in the plaques, which were marked by CD31.The protein levels of Dll4 and Notch1 in the aortas were analyzed by Western blot.RESULTS: The levels of blood TC, TG, LDL-C, CRP and IL-1 in AS group and AS +rhES group were much higher than those in N group (P <0.05), and no sta-tistical difference between AS group and AS +rhES group was observed.The expression of CD31 in AS group was the high-est among all groups.Compared with AS group, the density of neovessels in the plaques of AS +rhES group decreased sig-nificantly (P <0.05).The protein expression of Dll4 and Notch1 in AS group was lower than that in N group (P <0.05). Compared with AS group, the protein expression of Dll4 and Notch1 increased significantly (P <0.05).CONCLUSION:rhES has the ability to inhibit plaque angiogenesis in rats.The activation of Dll4 /Notch pathway may be the mechanism of rhES in inhibiting plaque angiogenesis.
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Objective To investigate the expression and significance of DLL4 in endometriosis.Methods 83 cases due to ovarian cyst surgery from December 2010 to September 2013,were selected in the Second Affiliated Hospital of Zhengzhou University,and were randomly divided into two groups in accordance with the random number table.Group A:48 cases were induded after staining from 83 patients who were tahen a part of cyst wall;Group B:of laparoscopic ovarian cyst surgery in 83 patients,37 cases of simultaneously hysteroscopy due to fertility requirements,exclude endometrial lesions,were taken reign uterine endometrium.The American fertility association revised standard (r-AFS)in 1985 was used to divide the clinical stage:Group A:21 cases of Ⅰ ~Ⅱ stage,27 cases of Ⅲ ~Ⅳ;Group B:16 cases of Ⅰ ~Ⅱ stage,21 cases of Ⅲ ~Ⅳ stage;Group C:30 cases hysteroscopic uterine septum resection with electricity due to lower mediastinal uterus were selected at the same period.The expression levels of DLL4 protein in Group A,B were detected by S-P method,and compared with the control group (Group C),then analyze its relationship with EMs stage. Results In Group A,B and C,the expression of DLL4 protein were (144.99 ±10.12 ),(1 15.42 ±10.39 ),(90.08 ±7.29 ),and there were significantly diffiences among three groups (F=314.220,P<0.05);The expression of DLL4 protein atⅠ~Ⅱstage were significantly lower than that inⅢ~Ⅳ stage (P<0.05).Conclusion DLL4 protein may play an important role in the pathogenisis of endometriosis.
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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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Purpose To assess the expression of DLL4 in non-small cell lung cancer ( NSCLC) patients and to determine its associa-tion with clinicopathological parameters and prognosis. Methods DLL4 expression was evaluated in NSCLC tissues and adjacent non-cancerous normal lung tissues from 89 patients undergoing surgical treatment by immunohistochemistry. Results DLL4 had high ex-pression in 52 of 89 cases of NSCLC (58. 4%), which was significantly higher than that in adjacent non-cancerous lung tissues (P<0. 05). Moreover, DLL4 overexpression was significantly correlated with TNM stage (P=0. 010 78). Kaplan-Meier survival analysis showed that the overall survival times in patients expressing DLL4 in NSCLC were shorter. Conclusion High level of DLL4 expression is significantly correlated with NSCLC progression and unfavorable prognosis. Thus, DLL4 expression may be used as a clinical param-eter for predictive prognostication of NSCLC patients.
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Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.
Subject(s)
Azacitidine/metabolism , Cell Differentiation/drug effects , GATA4 Transcription Factor/metabolism , Homeodomain Proteins/metabolism , Humans , Mesenchymal Stem Cells/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Receptor, Notch1/metabolism , Receptors, Notch/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Umbilical Cord/cytologyABSTRACT
Purpose To investigate the effect of anti-HER-2 engineered antibody chA21 on the angiogenesis in nude mice xenografts of human ovarian cancer SKOV3 cells that overexpresses HER-2.Methods An animal model with SKOV3 cells involved in nude mice was established and the mice were randomized into control group and chA21 group;chA21 was administrated via caudal vein for 5 weeks, meanwhile the same volume of chA21 buffer was injected in control group.The mice were terminated after 5 weeks and the xenografts dislodged,VEGF and DLL4 expression in two groups were quantitatively analyzed by immunohistochemistry on tissue microarray sections combined with a micro-image analyzing system, and microvessel density (MVD) values were compared between the two groups by immunohistochemical staining of CD31.Results The expression of VEGF, DLL4 and MVD values in chA21 group were lower than those in control group (P<0.05).Conclusion chA21 inhibits the angiogenesis of transplantations of human ovarian cancer SKOV3 cells effectively.
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Angiogenesis is regulated by complex interactions of multiple activators and inhibitors.Vascular endothelial growth factor(VEGF)and Notch signaling pathway are involved in such process.Recent studies have demonstrated that Notch signaling pathway plays a key role in the embryonic development and tumour angiogenesis,and identified the role of Dll4-Notch signaling during vascular development and the mechanism of the vascular defects which result from reduction of Notch signaling,thus Dll4-Notch became regarded as novel and important drug targets for disrupting tumour angiogenesis.This review emphasizes on introduction of the constitution of Notch signaling pathway,the role of Dll4-Notch in angiogenesis and the significance of Dll4-Notch for tumour therapy.
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Objective To investigate the expression of Notch1 and its ligand DLL4 in human gastric carcinoma tissues and its correlation with tumor angiogenic metastasis.Methods Immunohistochemical SP method was used to detect the expression of Notch1,DLL4 and VEGF in 45 gastric carcinoma tissues and paired adjacent normal gastric mucosa,and the relationship between them and clinico-pathological parameters were analyzed.Results The positive expression rate of Notch1,DLL4 and VEGF in gastric carcinoma were higher than that in normal gastric mucosa(P