IMP-1 inhibits renal cell carcinoma 786-O cell growth by targeting EphrinB2 signaling pathway

Abstract EphrinB2 plays a critical role in tumor growth. In this study, we studied the antitumor activity of imperatorin derivative IMP-1 in renal cell carcinoma (RCC) by regulating EphrinB2 pathway.. Results showed that IMP-1 inhibited the proliferation of 786-O cells in a dose- and time-dependent manner. More importantly, knockdown and transfection of EphrinB2 altered the inhibitory effect of IMP-1 on the activity of 786-O cells. IMP-1 arrested 786-O cell cycle at G0/G1 phase by decreasing the expression of cyclin D1 and cyclin E. Moreover, IMP-1 regulated Bcl-2 family proteins' expression, thus inducing apoptosis of 786-O cells. IMP-1 down-regulated the expression of EphrinB2, Syntenin1 and PICK1. Then, IMP-1 decreased the phosphorylation of Erk1/2 and AKT. In all, IMP-1 could regulate the EphrinB2 pathway in order to inhibit 786-O cell growth by arresting the cell cycle at G0/G1 phase and inducing cell apoptosis. Thus, IMP-1 may present as a potential strategy for RCC treatment.

EPhrinB2-modified mesenchymal stem cells help repair brain injury in a rat model of cerebral palsy / 中华物理医学与康复杂志

Objective To investigate any protective effect of transplanting EPhrinB2-modified bone marrow mesenchymal stem cells ( BMSCs) with a rat model of cerebral palsy. Methods BMSCs were isolated and cultured, then further modified by lentivirus-mediated transfection of the EPhrinB2 gene. Ninety-six Sprague-Dawley rats were randomly divided into a sham group, a solvent control group ( PBS group) , an empty lentivirus group ( EGFP group) and an EPhrinB2 recombinant lentivirus group ( EPhrinB2 group) , each of 24. A model of cerebral palsy was estab-lished in the rats of the PBS, EGFP and EPhrinB2 groups using hypoxic-ischemic encephalopathy. Seven days after the operation, the lateral ventricles of the PBS, EGFP and EPhrinB2 group mice were injected with phosphate-buff-ered saline solution, BMSCs or EPhrinB2-modified BMSCs respectively. EPhrinB2 protein expression in the hippo-campus was detected using immunohistochemistry 28 days after the operation. The neuron density in the CA1 region of the hippocampus was observed using hematoxylin and eosin staining, and any apoptosis of hippocampal neurons was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling. The expression of nestin and CD31 in the hippocampus was observed using immunofluorescence assays. Morris water maze testing was also conducted to e-valuate changes in learning and memory ability. Results Compared with the other 3 groups, a significant increase in the expression of protein EPhrinB2 was observed in the hippocampuses of the EPhrinB2 group rats. The pathologi-cal changes in the hippocampus among the EPhrinB2 group were significantly less severe than those in the PBS and EGFP groups. The rate of apoptosis in the hippocampuses of the EPhrinB2 group was significantly lower than that of the other groups. Immunofluorescence showed that nestin- and CD31-positive cells were significantly more numerous in the EPhrinB2 group than in the others. In the water maze the average latency of the EPhrinB2 group was signifi-cantly shorter than those of the other groups. Conclusion Lentiviral-mediated EPhrinb2 transfection of BMSCs into the hippocampus can promote EPhrinB2 gene expression, promote angiogenesis and neuron differentiation, inhibit ap-optosis and accelerate the repair of injured nerves.

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 EphrinB2 Signal in Cerebral Cortex in Ischemic Stroke Rats / 中国康复理论与实践

@#Objective To observe the expression of phosphorylated EphrinB2 in brain after focal cerebral ischemia/reperfusion in rats.Methods 24 male Sprague-Dawley rats were randomly divided into sham group (n=12) and model group (n=12). The model group was modeled as middle cerebral artery occlusion and reperfusion with nylon monofilament suture, and then was assessed with Longa's score. The expression of phosphorylated EphrinB2 in cerebral cortex was detected with immunohistochemistry and Western blotting. Results The expression of phosphorylated EphrinB2 and the number of positive cells were significantly higher in the sham group than in the control group (P<0.05). It existed in the vascular endothelium in cerebral cortex. Conclusion EphrinB2 signaling pathway is activated in ischemic stroke.

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 .

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.

Relationship of expressions of EphrinB2 and angiogenesis in human colorectal carcinoma / 肿瘤研究与临床

Objective To investigate the expression of EphrinB2 and microvessel density(MVD) in colorectal carcinoma and to analyze the relationship between the expression of EphrinB2 and the biological characteristics of colorectal carcinoma. Methods EphrinB2 and MVD of 63 colorectal carcinoma diagnosed by pathology were examined by immunohistochemistry, and 25 specimens from normal colorectal tissue were examined as controls. Results The expression of EphrinB2 protein was 82.0 % (51/63) in colorectal carcinoma and 32.0 % (8/25) in the normal colorectal tissues, the difference was significant (P <0.05). Meanwhile, MVD was significantly higher in EphrinB2 positive group (39.39±8.97) than that in negative group (33.00 ±10.19) (P<0.05). The expression of EphrinB2 protein had significant positive correlation with differentiation grade, infiltration, lymph node metastases and clinical stage (P <0.05), and had no relation to age, sex, tumor size and location(P >0.05). Conclusion The EphrinB2 protein expression increase may be involved in development of colorectal carcinoma and play important roles in the tumorigenesis and progression of colorectal carcinoma.