Resistance to bevacizumab in ovarian cancer SKOV3 xenograft due to EphB4 overexpression

Aim of Study: Bevacizumab (BV) is broadly used to treat a number of cancers; however, BV resistance mechanisms and strategies to overcome this resistance are yet to be determined. Materials and Methods: In this study, we used ovarian xenograft model to evaluate the underlying resistance mechanisms of BV in ovarian cancer treatment. Results: Our results showed that EphB4 was overexpressed in BV-resistant xenograft models instead of other common receptor tyrosine kinases. In addition, when coadministrated with EphB4 blocker NVP-BHG712, the antitumor effect of BV was significantly enhanced in the resistant model, further confirmed the role of EphB4 in BV-resistant ovarian cancer. These results indicate that NVP-BHG712 reverses EphB4 overexpression-mediated resistance to BV. Conclusion: These findings represent a guide for the design of future medication strategy and may be useful in guiding the use of BV in combination with NVP-BHG712 in patients with resistance or intolerance ovarian cancer.

Effects of angiogenic factors on lung development in rats with congenital esophageal atresia / 国际儿科学杂志

Objective To investigate the gene expression of Efnb2 and Ephb4 in the lung tissue of congenital esophageal atresia and tracheoesophageal fistula(EA-TEF) rat,and to explore the effect on lung development in EA rat.Methods Twenty-four female Wistar rats were randomly divided into experimental group(EATEF group)and control group(CON group)with 12 rats in each group.Adriamycin(ADR)-treated rats were established by intrapedtoneal injection of adriamycin at 1.75 mg / kg on day E7-9.The rats were sacrificed by caesarean on day 15 (E15),day 18 (El8) and day 21 (E21).The expression of Ephb4 and Efnb2 in fetal lung was detected by Q-PCR and immunohistochemistry.Results (1) It showed significantly higher mRNA expression levels of Efnb2 at the canalicular stage,which decreased and returned to pseudoglandular level at the sacular stage.The trend of Ephb4 mRNA and Efnb2 mRNA in the two groups was basically the same.The expression of Efnb2 mRNA in EA-TEF group(50.65 ± 12.65) was significantly higher than that in control group(23.63 ± 11.31) at the canalicular stage (P ＜ 0.05),but there was no statistical significance between the two groups at both pseudoglandular stage and sacular stage;The expression of Ephb4 mRNA in EA-TEF group (4.32 ± 2.88) was significantly higher than that in control group (1.01 ± 0.19) at the pseudoglandular stage (P ＜ 0.05),but there was no statistical significance between the two groups at both canalicular stage and sacular stage.(2)Efnb2 and Ephb4 proteins were expressed on the surface of vascular epithelium,alveolar and bronchial epithelium at different developmental stages,and there was no statistical significance in the distribution pattern.The expression of Efnb2 (162.70 ± 10.04) and Ephb4 (152.20 ± 12.32) in EA-TEF group was enhanced at the canalicular stage,while returned to normal level at the sacular stage.Conclusion The angiogenesis factor Efnb2 and its receptor Ephb4 can regulate the branching development of fetal lung tissue,which maybe a compensatory mechanism for pulmonary dysplasia in adriamycin-induced EA-TEF model.

Advance of molecular imaging probes targeted for EphB4 receptor / 国际肿瘤学杂志

The erythropoietin-producing hepatocellular receptor (Eph)B4 receptor is closely associa-ted with tumor growth and angiogenesis,which is over-expressed in a wide variety of tumors.Molecular probes targeted for EphB4 receptor can improve the accuracy and specificity of tumor diagnosis.A lot of molecular probes targeted for EphB4 receptor have been designed,which are expected to provide new means for the early diagnosis and therapeutics of tumors.

Discovery of noveI EphB4 inhibitors based on pharmacophore modeIing and virtuaI screening techniques / 中国药科大学学报

The objective of this paper is to discover new potent inhibitors against EphB4 for cancer therapy via computer-aided drug design strategies including building 3D-QSAR models,virtual screening and molecular doc-king means.The first step is to generate pharmacophore models based on Catalyst/HypoGen algorithm.The best model,Hypo1,has the highest Correl value (0.96),the lowest RMS value (0.89),the closest total cost (101.26) to fixed cost (89.20),and the best Δcost (89.14).Subsequently,Hypo1 was subjected to test set validation and Fischer′s randomization verification and then was used as a 3D query to screen database.In order to further nar-row the number of hits,drug-likeness screening and molecular docking techniques were applied.Finally,23 novel molecules with diverse scaffolds were selected as possible candidates against EphB4 for further studies based on predicted activity analysis,docking scores,and binding modes analysis methods.

Effect of EphB4/EphrinB2 reverse signal on angiogenesis induced by Xuefu Zhuyu Capsule () containing serum in human microvascular endothelial cell 1 / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of Xuefu Zhuyu Capsule ()-containing serum (XFZY-CS) on EphB4/ephrinB2 and its reverse signal in human microvascular endothelial cell-1 (HMEC-1).</p><p><b>METHODS</b>XFZY-CS and the blank control serum were collected. HMEC-1 cells were randomly assigned to 6 groups including the concentration 1.25%, 2.5%, and 5% XFZY-CS groups and their blank serum control ones. The angiogenesis effect of XFZY-CS was tested with an in vitro tube formation assay and the best condition of pro-angiogenesis was determined. The effect of XFZY-CS on EphB4/ephrinB2 and the reverse signal were determined by Western blot and real-time quantitative polymerase chain reaction, respectively; we also confifirmed the results through activating and inhibiting the reverse signal by EphB4/fc and pyrophosphatase/ phosphodiesterase2 (PP2).</p><p><b>RESULTS</b>XFZY-CS promoted angiogenesis at the concentration of 2.5% corresponding serum after being cultured for 48 h, while inhibited angiogenesis at the concentration of 5% after culturing for 48 and 72 h. Under the 2.5% serum concentration, XFZY up-regulated the expression of EphB4-mRNA at 12 h (P<0.05), and down-regulates its expression at 24 h (P<0.01). Protein expression of EphB4 was apparently up-regulated at 12 h and down-regulated at 24 h. The phosphorylation of ephrinB2 increased at 9 h (P<0.05). In addition, 2.5% XFZY-CS played a similar role as the reverse signaling activator EphB4/Fc ranging from 0.5 to 5 μg/mL (P>0.05). XFZY-CS also reduced the inhibitive effect of PP2 in limited periods.</p><p><b>CONCLUSIONS</b>EphB4/ephrinB2 was the upstream signal in the process of angiogenesis and its reverse signaling was responsible for XFZY's effect on promoting angiogenesis.</p>

Expression of EphB4 in hepatocellular carcinoma tissues and its clinical significance / 重庆医学

Objective To study the expression of Ephrin‐B4 receptor (EphB4) in hepatocellular carcinoma (HCC) and its clinical significance ,and to analyze the effect of EphB4 on the proliferation of HCC cells .Methods The expression level of EphB4 in HCC tissues and matched paracancerous liver tissues of 60 cases of HCC patients was assessed by reverse transcriptase‐polymer‐ase chain reaction (RT‐PCR) and immunohistochemistry .The correlation between the expression of EphB4 in HCC tissues and clin‐ical pathologic parameters was analyzed by chi‐square test .Univariate survival analysis was carried out by Kaplan‐Meier Log‐rank test .The effect of EphB4 on the viability of HCC cells was furtherly analyzed by MTS .Results The results of RT‐PCR showed that the mRNA level of EphB4 in HCC tissues (1 .39 ± 0 .80) was significantly higher than in matched paracancerous liver tissues (0 .56 ± 0 .33) ,the difference was statistically significant (P< 0 .01) .Data from immunohistochemistry showed that the positive rates of EphB4 protein in HCC tissues and matched paracancerous liver tissues were 81 .7% and 23 .3% ,respectively .Moreover ,the expression of EphB4 in HCC tissues was relevant to AFP level ,tumor size and TNM stage(P<0 .05) .The three‐year survival rate of HCC patients with positive expression of EphB4 protein was 22 .5% ,and that of HCC patients with negative expression of EphB4 protein was 54 .5% ,the difference was statistically significant (P<0 .05) .Overexpression of EphB4 significantly enhanced the ability of hepatocellular carcinoma cell proliferation (P<0 .05) .Conclusion EphB4 expression was significantly up‐regulated in HCC ,which was associated with HCC progression and prognosis ,and EphB4 could promote the proliferation of HCC cells ,which could be used as a marker of HCC progression .

Compressive force regulates ephrinB2 and EphB4 in osteoblasts and osteoclasts contributing to alveolar bone resorption during experimental tooth movement / 대한치과교정학회지

OBJECTIVE: To investigate the involvement of ephrinB2 in periodontal tissue remodeling in compression areas during orthodontic tooth movement and the effects of compressive force on EphB4 and ephrinB2 expression in osteoblasts and osteoclasts. METHODS: A rat model of experimental tooth movement was established to examine the histological changes and the localization of ephrinB2 in compressed periodontal tissues during experimental tooth movement. RAW264.7 cells and ST2 cells, used as precursor cells of osteoclasts and osteoblasts, respectively, were subjected to compressive force in vitro. The gene expression of EphB4 and ephrinB2, as well as bone-associated factors including Runx2, Sp7, NFATc1, and calcitonin receptor, were examined by quantitative real-time polymerase chain reaction (PCR). RESULTS: Histological examination of the compression areas of alveolar bone from experimental rats showed that osteoclastogenic activities were promoted while osteogenic activities were inhibited. Immunohistochemistry revealed that ephrinB2 was strongly expressed in osteoclasts in these areas. Quantitative real-time PCR showed that mRNA levels of NFATc1, calcitonin receptor, and ephrinB2 were increased significantly in compressed RAW264.7 cells, and the expression of ephrinB2, EphB4, Sp7, and Runx2 was decreased significantly in compressed ST2 cells. CONCLUSIONS: Our results indicate that compressive force can regulate EphB4 and ephrinB2 expression in osteoblasts and osteoclasts, which might contribute to alveolar bone resorption in compression areas during orthodontic tooth movement.

Expression of EphB4 and VEGF in esophageal cancer tissues and their relationship with microvessel density and the curative effect under thoracoscope / 重庆医学

Objective To explore the expression of EphB4 and VEGF in esophageal cancer tissues and their relationship with microvessel density (MVD ) ,and analysis the curative effect of postoperative esophageal cancer radical under thoracoscope . Methods Theexpression of EphB4 and VEGF was detected by immunohistochemistry in tumor specimens from 76 cases of esopha-geal squamous cell carcinoma and paratumor normal specimens ,used CD34 as marker to count MVD .According to the situation of expression of EphB4 and VEGF ,we analysis their relationship with lymph node metastasis rate ,recurrence and 5-year survival rate . Results The positive expression rate of EphB4 and VEGF in cancerous tissue (57 .89% and 61 .84% ) ,were significantly higher than that in tissue adjacent to carcinoma(0 and 7 .89% )(P<0 .05) .The positive expression rate ofEphB4 and VEGF in high MVD values of patients (67 .44% and 76 .19% ) ,were significantly higher than thatin low MVD values of patients (45 .45% and 44 .11% )(P<0 .05) .The positive expression rate ofEphB4 and VEGF in the patientswith lymph node metastasis group and associ-ated with recurrence ,were significantly higher than that of group without lymph node metastasis and group without recurrence (P<0 .05) .The positive expression rate of EphB4 and VEGF in patients of greater than or equal to 5 years of survival rate(45 .00% and 45 .45% ) ,were significantly lower than in patientsof Less than 5 years of survival rate (80 .36% and 85 .19% )(P<0 .05) .Conclu-sion EphB4 and VEGF are highly expressed in esophageal cancer tissue ,which may be closely associated withmicrovessel density , and lymph node metastasis ,recurrence and 5 years survival rate ;the curative effect of positive expression rate of EphB 4 and VEGF is poor .

Decreasing effect of RNA interference vector targeting the EphB4 gene on the expression of EphrinB2 in human colon cancer cells / 医学研究生学报

Objective Studies show that the role of EphB 4 in the development and progression of cancer is correlated to its ligand EphrinB2.The present study was to observe the effect of the changes in EphB4 on the expression of EphrinB2 by constructing and identifying microRNA ( miRNA) interference vectors targeting the EphB4 gene in colon cancer cells . Mte hods According to the EphB4 gene sequence , 3 pairs of oligo DNA sequences of miRNA were designed .The single strand of oligo DNA was annealed to form double-strand DNA, and then connected with the plasmid pcDNA 6.2-GW/EmGFP-miRNA.The expression vector pcDNA6.2-GW/EmGFP-miR-EphB4 was linked to pDONR221 and pLenti6/V5-DEST to construct the lentiviral expression vector pLenti 6/V5-DEST-EphB4, which was cotransfected with packaging mix (pLP1, pLP2 and pLP/VSVG) into 293FT cells by lipofectamine 2000 transfec-tion to produce lentivirus , and the lentivirus titer was measured by infection of HEK 293 cells.The stable cell lines were selected and cultured.The expression levels of EphB4 and EphrinB2 were examined by qPCR. Results Three miRNA interference vectors SR-1, SR-2, and SR-3 targeting the EphB4 gene were successfully constructed , with SR-3 exhibiting the most significant interference efficien-cy.The constructed lentiviral vector pLenti 6/V5-DEST-EphB4 was successfully packaged in 293FT cells.The virus titer was 7 ×108 Caco-2 cells. Conclusion The exogenous EphB4 expression could be significantly inhibited by treatment with specific miRNA in co-lon cancer cells .The correlation of EphB4 and EphrinB2 may be effected by many factors and need further studies .

Construction of a lentiviral expression vector carrying EphB4 and its expression in colon cancer cells / 医学研究生学报

Objective Studies show that the abnormal expression of EphB4 plays an important role in the development and progression of colon cancer .The present study aims to provide some experimental evidence for the gene therapy of colon cancer by con -structing a lentiviral expression vector carrying the homo EphB4 gene and further establishing colon cancer cell lines with stable overex-pression of EphB4. Methods A series of oligonucleotides (oligo) encoding the homo EphB4 gene were ligated together by PCR and then cloned into a lentiviral expression vector pLenti 6.3-MCS-IRES2-EGFP.After confirmed by sequencing , the vector pLenti6.3-EphB4-IRES2-EGFP and its helper vectors were mixed and co-transfected into 293 T cells to obtain recombinant virus containing the EphB4 gene.The lentiviral titer was detected and the resulting recombinant lentiviruses carrying EphB4 or control viruses only carrying green fluorescence protein (GFP) were used to infect the human colon cancer cell lines .The expression of GFP was determined under the inverted fluorescence microscope and the level of EphB 4 mRNA in the infected cells detected by qPCR . Results The lentiviral expression vector pLenti6.3-EphB4-IRES2-EGFP carrying correct homo EphB4 gene sequence was successfully constructed .The titer of the recombinant EphB4 lentiviral supernatant Lenti6.3-EphB4 was 1 ×108 TU/mL.The expression of GFP was observed in the trans-duced cells under the fluorescence microscope , and that of EphB4 mRNA in the transfected SW480 and Coca-2 cells was significantly up-regulated as compared with the control and blank groups . Conclusion The homo EphB4 gene was successfully amplified and cloned.A lentiviral expression vector was successfully constructed , and so were colon cancer cell lines stably overexpressing EphB 4, which may shed light on the lentivirus-mediated genetic therapy for colon cancer .

Inhibitory Effects of Suramin in Combination with PG-Rg3 on the Growth of Lung Adencarcinoma in Mice / 天津医药

Objective To observe the effect of suramin combinated with PG-Rg3 on xenograft growth of lung adeno-carcinoma in mice, and the related mechanism thereof. Methods Forty C57BL/6J mice bearing Lewis cells were random-ized into five groups:control group, cisplatin (DDP) group, suramin group, PG-Rg3 group and combination group. Appropri-ate interventions were given in five groups of mice. Mice were sacrificed at day 24 after tumor inoculation. The subcutaneous tumors were stripped for histological examination. The tumor inhibitory rate was measured. The expressions of erythropoietin-producing hepatoma amplified sequences (Eph) B4 protein, Bcl-2 and tumors microvessel density (MVD) were determined by immunohistochemistry method with image analyze system. The apoptosis of tumor cells was measured by biotinyated dUTP nick and labeling (TUNEL) method. Results There were significantly lower values in subcutaneous tumor volume and weight in drug-treated groups than those in control group (P<0.05). The inhibitory rates were 39.20%, 49.11%, 54.86%and 62.49%in cisplatin group, suramin group, PG-Rg3 group and combined group (P<0.05). The values of EphB4, MVD and Bcl-2 grey values were significantly decreased, the apoptotic index was significantly increased, in suramin group, PG-Rg3 group and combined group than those of control group and DDP group (P<0.05). The values of EphB4, MVD and Bcl-2 grey values were significantly increased, the apoptotic index was significantly decreased, in suramin group and PG-Rg 3 group than those of combined group (P<0.05). Conclusion Suramin combinated with PG-Rg3 can produce a synergetic inhibitory activity against tumor growth of lung adenocarcinoma, which may be associated with the effect of suppressing the expression of EphB4 and angiogenesis, and the promotion of tumor cell apoptosis.

Knockdown of EphB4 gene by small interference RNA inhibits proliferation of glioma cells in vitro / 中国神经精神疾病杂志

Objective To explore the role of EphB4 in proliferation of glioma cells. Methods The mRNA and protein expressions of EphB4 were detected using RT-PCR, immunochemistry, and Western-blot, respectively. EphB4 siRNA was synthesized and transfected into U251 cells using Lipofectamine 2000. Glioma cell proliferation, apoptosis, and invasion were determined by MTT assay, TUNEL and transwell experiment, respectively. Results The expression (P<0.05) and proliferation of EphB4 were obviously decreased in U251 transfected with EphB4 siRNA and the proliferation was further decreased with the increased concetrations of siRNA. Compared with U251 group and siRNASCR group, EphB4 siRNA at different concentrations (25, 50 or 100 nmol/L) significantly reduced the invasion ability of cells and increased the number of apoptotic cells (P<0.05). Conclusions EphB4 plays an important role in the regulation of glioma cell proliferation, apoptosis and invasion.

The effect of RNA interference-mediated down-regulation of EphB4 on the growth of malignant glioma cell line U251 / 肿瘤

Objective: To determine the interfering effects of EphB4-targeted short interfering RNA (siRNA) on EphB4 mRNA expression and its effect on the growth of glioma U251 cell line. Method: EphB4-targeted siRNA was designed and synthesized, and then was transfected into U251 cells. The inhibition of EphB4 mRNA expression was detected by RT-PCR. The effect of EphB4-targeted siRNA on cell growth rate was measured by CCK-8 method. Cell apoptosis was tested by flow cytometry (FCM). Wound healing test was used to observe the migration ability of cells. The invasiveness of tumor cells was evaluated by counting the number of cells passing the Transwell membrane. Results: EphB4 mRNA transcription level was decreased by 75.0% after transfection of malignant glioma U251 cells with 100 nmol/L siRNA-EphB4. The inhibition of cell proliferation was in a dose-dependent manner. FCM analysis showed that cells were arrested at sub-G1 phase at different degrees and the migration capacity decreased after transfection with 100 nmol/L siRNA-EphB4 compared with the negative control. The number of cells permeating the matrigel membrane significantly were decreased in the siRNA-EphB4 transfection group compared with the control group. Conclusion: siRNA-EphB4 markedly targetes and knocks down EphB4 gene transcription. Down-regulation of EphB4 affects cell proliferation and induces apoptosis of cells. Transfection of siRNA-EphB4 into U251 cells inhibits the migration and invasion abilities of cells at various degrees. It indicates that silencing EphB4 expression might become a noval approach in the treatment of glioma.

Expression of EphB4 in non-small cell lung cancer and clinical significance / 吉林大学学报(医学版)

Objective To investigate the expression patterns of EphB4 in non-small cell lung cancer(NSCLC) and evaluate their roles in tumor initiation and development.Methods The expressions of EphB4 were detected with immunohistochemical method in 34 cases of NSCLC and 16 cases of adjacent normal lung tissues.Results The positive expression rate of EphB4 in NSCLC was 41.2%(14/34).The expression of EphB4 in 16 cases of adjacent normal lung tissue were all negative,there was significant difference between them(P0.05).Conclusion EphB4 may play a key role in carcinogenesis and progression of NSCLC.

Expression and clinical significance of EphB4 in breast infiltrating ductal carcinoma:report of 65 cases / 第三军医大学学报

Objective To study the expression and clinical significance of EphB4 in breast infiltrating ductal carcinoma.MethodsSP immunohistochemical technique was used to examine the expression of EphB4 in 65 cases of breast infiltrating ductal cancer and 12 matched adjacent tissue samples.ResultsThe level of EphB4 in breast infiltrating ductal cancer was significantly higher than that of adjacent normal tissues(P0.05).ConclusionEphB4 plays a crucial role in the occurrence and development of breast cancer.

Expressions of EphB4 and HIF-1? in human gastric carcinoma and their biological implication / 解放军医学杂志

Objective To investigate the expressions of EphB4 receptor and hypoxia inducible factor 1? (HIF-1?) protein in human primary gastric carcinoma, and to study the biological implication of their expression. Methods The expressions of EphB4 receptor and HIF-1? protein were detected in 74 specimens of human primary gastric carcinoma (gastric carcinoma group) and in 13 normal gastric mucosa tissues (control group) by immunohistochemistry. Correlations between the expression of these proteins, and clinical and pathological features were respectively analyzed. Result High expressions of EphB4 receptor and HIF-1? protein were found to be 62.2% (46/74) and 52.7% (39/74) respectively in gastric carcinoma group, while the expressions of EphB4 receptor and HIF-1? protein in control group were 15.38% (2/13) and 0% (0/13), respectively. The differences were significant between two grrups (P