OBJECTIVE: To determine the effect of EphA2 protein on the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9) proteins in HCT116 cells. METHODS: High expression of EphA2 protein in HCT116 cells was confirmed by Western blot. HCT116 cells were transfected with EphA2 antisense oligonucleotide. The expression of the transfection efficiency was analyzed by Western blot. VEGF proteins in the cell supernatants were detected by enzyme linked immunosorbent assay(ELISA), and the expressions of MMP9 in cell supernatants were examined by gelatin zymography. RESULTS: EphA2 antisense oligonucleotide suppressed the expression of VEGF and MMP9 proteins in HCT116 cells. CONCLUSION EphA2 could decrease the invasion and metastasis of HCT116 cells by suppressing the expression of VEGF and MMP9.
HCT116 Cells ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Oligonucleotides, Antisense ; Receptor, EphA2 ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

BACKGROUND: Eph receptors and ephrin ligands have several functions including angiogenesis, cell migration, axon guidance, fluid homeostasis, oncogenesis, inflammation and injury repair. The EphA2 receptor potentially mediates the regulation of vascular permeability and inflammation in response to lung injury. METHODS: Mice were divided into 3 experimental groups to study the role of EphA2 signaling in the lipopolysaccharide (LPS)-induced lung injury model i.e., IgG+phosphate-buffered saline (PBS) group (IgG instillation before PBS exposure), IgG+LPS group (IgG instillation before LPS exposure) and EphA2 monoclonal antibody (mAb)+LPS group (EphA2 mAb pretreatment before LPS exposure). RESULTS: EphA2 and ephrinA1 were upregulated in LPS-induced lung injury. The lung injury score of the EphA2 mAb+LPS group was lower than that of the IgG+LPS group (4.30+/-2.93 vs. 11.45+/-1.20, respectively; p=0.004). Cell counts (EphA2 mAb+LPS: 11.33x10(4)+/-8.84x10(4) vs. IgG+LPS: 208.0x10(4)+/-122.6x10(4); p=0.018) and total protein concentrations (EphA2 mAb+LPS: 0.52+/-0.41 mg/mL vs. IgG+LPS: 1.38+/-1.08 mg/mL; p=0.192) were decreased in EphA2 mAb+LPS group, as compared to the IgG+LPS group. In addition, EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase 110gamma, phospho-Akt, nuclear factor kappaB, and proinflammatory cytokines. CONCLUSION: This results of the study indicated a role for EphA2-ephrinA1 signaling in the pathogenesis of LPS-induced lung injury. Furthermore, EphA2 antagonism inhibits the phosphoinositide 3-kinase-Akt pathway and attenuates inflammation.
Animals ; Axons ; Capillary Permeability ; Carcinogenesis ; Cell Count ; Cell Movement ; Cytokines ; Homeostasis ; Inflammation ; Ligands ; Lipopolysaccharides ; Lung Injury* ; Methods ; Mice ; Receptor, EphA1 ; Receptor, EphA2* ; Receptors, Eph Family

OBJECTIVE: To investigate the expression and clinical significance of EphA2 and E cadherin proteins in papillary thyroid carcinoma tissues, and to explore the relationship between them. METHOD: Using immunohistochemical SP/PV method, we detected the expression of EphA2 and E cadherin in tumors of 43 papillary thyroid carcinomas, 11 thyroid adenoma and 10 normal thyroid tissues, then studied their relationships with clinic pathological factors. RESULT: The total positive rates of EphA2 and E cadherin expression were 58. 14% and 32. 56% in papillary thyroid carcinoma tissues, 18. 18% and 81. 81% in thyroid adenoma.tissues and they were 10. 00% and 100. 00% in normal thyroid tissues respectively. The positive expression of EphA2 in carcinoma tissues was higher than in the thyroid adenoma tissues and normal thyroid tissues (P<0. 05) and the positive expression of E cadherin in carcinoma tissues was lower than that in the thyroid adenoma tissues and normal thyroid tissues (P<0. 05). The positive expression of EphA2 and E cadherin was associated with lymph node metastasis and histological grade (P<0. 05), but it was not associated with all the clinic-pathological factors including age, sex and the tumor size (P>0. 05). In papillary thyroid carcinoma tissues, the expression of EphA2 was negatively correlated with the expression of E cadherin protein (r= -0. 416, P<0. 01). CONCLUSION EphA2 and E cadherin may be involved in carcinogenesis and development of papillary thyroid carcinoma.
Adenoma ; metabolism ; pathology ; Antigens, CD ; Cadherins ; metabolism ; Carcinoma ; metabolism ; pathology ; Carcinoma, Papillary ; Humans ; Lymphatic Metastasis ; Receptor, EphA2 ; metabolism ; Thyroid Cancer, Papillary ; Thyroid Gland ; metabolism ; Thyroid Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the effects of EphA2 on the angiogenesis and cervical lymph node metastasis of squamous cell carcinoma of the head and neck (SCCHN) in vivo.

METHODSEphA2 short hairpin (shRNA) lentiviral particles were used to knockdown the expression of EphA2 in SCCHN cell line M2 with high lymph nodes metastasis rate. Stable clones, obtained by puromycin screening, were assayed by RT-PCR and Western blot to validate the gene silencing efficiency and were used to establish SCCHN metastatic xenograft mouse model. Hematoxylin-eosin staining was applied to identify cervical lymph node metastasis of SCCHN in xenografted tumors. Immunohistochemistry was used to observe microvessel density. Western blot was used to investigate the protein expressions of EphA2 and vascular endothelial, growth factor (VEGF).

RESULTSEphA2 shRNA lentiviral particles efficiently decreased the mRNA and protein expressions of EphA2 in SCCHN cell line M2, which were further successfully utilized to establish SCCHN metastatic xenograft mouse model. Compared with xenografted tumors in control group, xenografted tumors in M2EphA2RNAi(+) group decreased significantly tumor volume [(430.7 ± 190.0) mm(3) (x(-) ± s) vs (1179.0 ± 289.4) mm(3)] and weight [(0.26 ± 0.10) g vs (0.54 ± 0.12) g] (both P < 0.05). More importantly, bilateral cervical lymph node metastasis rate in M2EphA2RNAi(+) was also greatly declined (Mann-Whitney U = 10.0, P < 0.05). Decreased protein expressions of EphA2 and VEGF and microvessel density were observed in M2EphA2RNAi(+) group (t = 26.751, P < 0.01).

CONCLUSIONSKnockdown of EphA2 expression led to the inhibition of tumor growth and metastasis in SCCHN nude mouse model. More importantly, SCCHN angiogenesis was also impeded, which might be associated with the decreased expression of VEGF.

Animals ; Carcinoma, Squamous Cell ; pathology ; Cell Line, Tumor ; Gene Silencing ; Head and Neck Neoplasms ; pathology ; Humans ; Lymphatic Metastasis ; Mice ; Mice, Nude ; Neovascularization, Pathologic ; Prognosis ; RNA, Small Interfering ; Receptor, EphA2 ; genetics

OBJECTIVETo investigate the expression of EphA2 and EphrinA1 and its relationship with angiogenesis in renal cell carcinoma and its relevance to clinicopathologic features.

METHODSThe expression of the EphA2 and EphrinA1 was detected by immunohistochemistry (IHC) in the tissues samples from 68 renal cell carcinomas and 24 normal kidneys, and quantitatively analyzed. The microvessel density (MVD) was determined by CD34 immunostaining of microvascular endothelial cells. Statistical analysis was performed using the software SPSS (version 13.0).

RESULTSThe expression of EphA2, EphrinA1 and MND in the cancerous tissues were significantly higher (P<0.01) than that in the normal ones. Significantly increased expression of EphA2, EphrinA1 and MVD (P<0.01) was detected in cancer tissues with higher grade differentiation, more advanced stage and more lymph node metastasis, respectively (P<0.05 for each group). Expression of the EphA2 and EphrinA1 protein was shown to be positively associated with the MVD assessed by Spearman's correlation and factor analysis (r=0.555, r=0.485, P<0.01). The MVD was also significantly correlated with the diameter of the tumor (P<0.01).

CONCLUSIONEphA2 and EphrinA1 are highly expressed in renal cell carcinoma, and positively correlated with histological differentiation, clinical stage and angiogenesis in the cancer.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Renal Cell ; metabolism ; pathology ; Ephrin-A1 ; metabolism ; Female ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Microvessels ; pathology ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic ; metabolism ; pathology ; Receptor, EphA2 ; metabolism ; Tumor Burden ; Young Adult

OBJECTIVETo investigate the role of EPHA2 in regulating apoptosis, proliferation and vasculogenic mimicry of osteosarcoma cells, by gene silencing through RNA interference.

METHODSEPHA2-siRNA plasmids were achieved by gene cloning. The plasmids were transfected into human osteosarcoma cells (MG63). The expression level of EPHA2 protein was measured by Western blotting. The proliferation, apoptosis and vasculogenic mimicry features of osteosarcoma MG63cells were assessed by light microscopy, MTIP assay, flow cytometry, annexin V-FITC/PI and HE staining, respectively.

RESULTSThe EPHA2-siRNA plasmid was confirmed by DNA sequencing. After treatment with Sequence-specific siRNA targeted EPHA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher and earlier apoptosis and less osteosarcoma-generated vasculogenic mimicry.

CONCLUSIONEPHA2 gene may be involoved in apoptosis and proliferation of osteosarcoma cells, and may be necessary for vasculogenic mimicry. Down-regulation of EPHA2 expression by sequence-specific siRNA may be considered as a new option in the treatment of EPHA2 over-expressing cancer including osteosarcoma in future.

Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Neovascularization, Pathologic ; pathology ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, EphA2 ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the regulatory effect of erythropoietin-producing hepatocellular receptor (EphA2) on the expression of VEGF protein, a pro-angiogenic factor, via p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in squamous cell carcinoma of the head and neck(SCCHN) in vitro.

METHODSSCCHN Tu686 cells were transfected with EphA2 overexpression vector pEGFP-N1-EphA2. Western blot was used to detect the expression of p38 MAPK and enzyme-linked immunosorbent assay (ELISA) was applied to assay of VEGF. SB203580 as a inhibitor of p38 MAPK signaling pathway was used.

RESULTSThe expression of VEGF protein was significantly up-regulated in Tu686 cells transfected with EphA2 overexpression vector (535.31 ± 45.71) pg/ml, when compared with Tu686 cells transfected with empty vector (400.99 ± 33.50) pg/ml and Tu686 cells with no transfection (385.30 ± 33.50) pg/ml (F = 17.091, P < 0.01). The expression of phosphorylated p38 MAPK was obviously increased in Tu686 cells with EphA2 overexpression. SB203580 inhibited the expressions of VEGF and phosphorylated p38 MAPK proteins in Tu686 cells with EphA2 overexpression.

CONCLUSIONEphA2 can regulate the expression of VEGF protein and stimulate p38 MAPK signaling pathway.

Carcinoma, Squamous Cell ; metabolism ; Cell Line, Tumor ; Enzyme Inhibitors ; pharmacology ; Head and Neck Neoplasms ; metabolism ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; Pyridines ; pharmacology ; Receptor, EphA2 ; physiology ; Vascular Endothelial Growth Factor A ; metabolism ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

ObjectiveTo investigate the effect of Qilan Capsules (QLC) on the expressions of the related proteins HIF-1α, VEGF-α, EphA2 and MMP-1 in the formation of vasculogenic mimicry (VM) in prostate cancer.

METHODSProstate cancer PC-3 cells were cultured, transfected with siRNA, and divided into eight groups, blank control, HIF-1α siRNA, VEGF-α siRNA, EphA2 siRNA, QLC intervention, QLC + HIF-1α siRNA, QLC + VEGF-α siRNA, and QLC + EphA2 siRNA. The expressions of the HIF-1α, VEGF-α and EphA2 proteins in the pathway of VEGF were determined by Western blot.

RESULTSCompared with the blank control group, the expression of HIF-1α was evidently decreased in the HIF-lα siRNA and QLC + HIF-lα siRNA groups (0.624 7 ± 0.042 8 vs 0.032 8 ± 0.002 5 and 0.036 8 ± 0.018 1, P < 0.05), so were that of VEGF-α in the VEGF-α siRNA and QLC + VEGF-α siRNA groups (0.068 9 ± 0.005 1 vs 0.016 9 ± 0.000 7 and 0.010 9 ± 0.000 8, P < 0.05), that of EphA2 in the EphA2 siRNA and QLC + EphA2 siRNA groups though with no statistically significant difference (0.1684 ± 0.0126 vs 0.134 5 ± 0.028 6 and 0.165 4 ± 0.039 8, P > 0.05), and that of MMP-1 in the HIF-lα siRNA, VEGF-α siRNA and EphA2 siRNA groups (1.696 1 ± 0.152 7 vs 0.435 9 ± 0.036 9, 0.198 7 ± 0.009 0 and 0.0218 ± 0.000 7, P < 0.05).

CONCLUSIONSQilan Capsules can suppress VM formation in prostate cancer by inhibiting the expressions of HIF-1α, VEGF-α and MMP-1, which plays a role in the clinical treatment of prostate cancer by checking the growth and development of the blood supply system in the tumor tissue.

Capsules ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Matrix Metalloproteinase 1 ; metabolism ; Molecular Mimicry ; Prostatic Neoplasms ; metabolism ; RNA, Small Interfering ; metabolism ; Receptor, EphA2 ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVE: To observe the expression of EphA2, and investigate its correlation with the development, progression, invasion, metastasis, angiogenesis and prognosis in nasopharyngeal carcinoma(NPC). METHOD: Immunohistochemical staining was used to determine the expression level of EphA2 protein in 61 cases NPC and 20 cases chronic nasopharyngitis samples. The clinically pathological data and results of follow-up were collected. Microvessel density (MVD) was also measured by immunohistochemical staining method with CD34 in NPC. RESULT: The positive rate of EphA2 protein staining in NPC was 60.66% (37/61), while that in nasopharyngitis samples was 10.0% (2/20). The positive rates of EphA2 protein in NPC were 27.27% (3/11) in stage I, 56.25% (9/16) in stage II, 68.19% (15/22) in stage III, and 83.33% (10/12) in stage IV. The positive expressions of EphA2 in T1 + T2 and T3 + T4 with neck lymph node and distant metastasis were 58.33% (7/12) and 88.89% (16/18) respectively, while those in T1 +T2 and T3 + T4 without metastasis were 31.25% (5/16) and 50.00% (6/12) respectively. The cumulative survival of patients in the EphA2 positive group at 5 years was only 0.324 (12/37), while 0.500 (12/24) in the EphA2 negative group. The positive expression of EphA2 protein was correlated with the clinical stage, the neck lymph node metastasis and distant metastasis, and prognosis of NPC, respectively (P < 0.05). MVD in EphA2 protein positive group (45.32 +/- 4.91) was significantly higher than that in EphA2 protein negative group (28.69 +/- 3.99, P < 0.01). CONCLUSION EphA2 may play an important role in the development and progression of NPC. It is closely associated with the invasion, metastasis, angiogenesis and prognosis of NPC.
Adolescent ; Adult ; Aged ; Carcinoma ; Female ; Humans ; Lymphatic Metastasis ; Male ; Microvessels ; Middle Aged ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; blood supply ; metabolism ; pathology ; Neoplasm Staging ; Neovascularization, Pathologic ; pathology ; Prognosis ; Receptor, EphA2 ; metabolism ; Young Adult

OBJECTIVETo explore novel cancer gene therapy by retrovirus-mediated RNAi technique to suppress the endogenous EphA2 oncogene expression in colon adenocarcinoma HCT-8 cells.

METHODSSequence information of EphA2 mRNA was selected and two complementary oligonucleotides with hairpin loop were designed. Retrovirus-mediated RNAi expression vector (pSIREN-EphA2) was then constructed and transfected into the HCT-8 cells. Inhibition of EphA2 protein expression was quantitatively determined by Western blot and immunohistochemistry assay (SP method).

RESULTSThe construction of pSIREN-EphA2 vector was successful and confirmed by restriction enzyme analysis and DNA sequencing. The post-transfection level of EphA2 protein expressions was greatly reduced in HCT-8 cells transfected with pSIREN-EphA2, as compared with those of untransfected cells and the vector control (P < 0.001).

CONCLUSIONSEphA2 protein expression in HCT-8 cell line can be suppressed using recombinant retrovirus-mediated RNAi technique. This approach may provide a novel gene therapy against colonic adenocarcinoma.

Adenocarcinoma ; metabolism ; pathology ; Cell Line, Tumor ; Colonic Neoplasms ; metabolism ; pathology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, EphA2 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection