OBJECTIVES: Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer. But the problem of drug resistance severely limits their clinical application. This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil (5-FU) and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition. METHODS: Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment, and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group. The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression level of hypoxia inducible factor-1α (HIF-1α), multidrug resistance (MDR1) gene, P-glycoprotein (P-gp), and vascular endothelial growth factor (VEGF) which were related to 5-FU drug-resistance. We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU. RESULTS: Compared with the Normoxic+5-FU group, the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced (P<0.05), and the expression of apoptosis promoter protein caspase 8 was also decreased. Compared with the the Normoxic+5-FU group, HIF-1α mRNA expression in the Hypoxia+5-FU group was significantly increased (P<0.05), and the mRNA and protein expression levels of MDR1, P-gp and VEGF were also significantly increased (all P<0.05). The increased expression of MDR1, P-gp and VEGF had the same trend with the expression of HIF-1α. CONCLUSIONS Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy. Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.
Humans ; Fluorouracil/therapeutic use* ; Vascular Endothelial Growth Factor A/metabolism* ; Stomach Neoplasms/drug therapy* ; Drug Resistance, Multiple ; Vascular Endothelial Growth Factors/metabolism* ; Hypoxia ; ATP Binding Cassette Transporter, Subfamily B/genetics* ; ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics* ; Cell Line, Tumor ; Cell Hypoxia ; RNA, Messenger/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Tumor Microenvironment

Angiogenesis is a crucial process in the development of inflammatory diseases, including cancer, psoriasis and rheumatoid arthritis. Recently, several alkaloids from Picrasma quassioides had been screened for angiogenic activity in the zebrafish model, and the results indicated that 1-methoxycarbony-β-carboline (MCC) could effectively inhibit blood vessel formation. In this study, we further confirmed that MCC can inhibit, in a concentration-dependent manner, the viability, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as the regenerative vascular outgrowth of zebrafish caudal fin in vivo. In the zebrafish xenograft assay, MCC inhibited the growth of tumor masses and the metastatic transplanted DU145 tumor cells. The proteome profile array of the MCC-treated HUVECs showed that MCC could down-regulate several angiogenesis-related self-secreted proteins, including ANG, EGF, bFGF, GRO, IGF-1, PLG and MMP-1. In addition, the expression of two key membrane receptor proteins in angiogenesis, TIE-2 and uPAR, were also down-regulated after MCC treatment. Taken together, these results shed light on the potential therapeutic application of MCC as a potent natural angiogenesis inhibitor via multiple molecular targets.
Angiogenesis Inhibitors ; chemistry ; pharmacology ; Animals ; Carbolines ; chemistry ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Epidermal Growth Factor ; genetics ; metabolism ; Fibroblast Growth Factors ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Neovascularization, Physiologic ; drug effects ; Picrasma ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Receptor, TIE-2 ; genetics ; metabolism ; Zebrafish ; embryology

OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

OBJECTIVETo explore the effect of epidermal growth factor-like domain 7(EGFL7) on the migration and angiogenesis of endothelial cells.

METHODSEGFL7 overexpression vectors were constructed and transfected into human microvascular endothelial cells. The expression levels of EGFL7-mRNA and EGFL7 protein were examined by real-time RT-PCR and Western blot. Cell migration was analyzed by the wound healing. The capability of cell to form capillary-like tubes in vitro was evaluated on matrigel assay. Protein expression of p-AKT, AKT, p-ERK and ERK in endothelial cells was detected by Western blot upon transfection with EGFL7 overexpression vectors and vehicle control for 0, 10, 30 and 60 min.

RESULTSMigration and angiogenesis of endothelial cells were notably enhanced by EGFL7 overexpression. ERK pathway was strongly activated by EGFL7, whereas AKT remained constant in endothelial cells. Inhibition of ERK impaired EGFL7 induced ERK activation and endothelial cell migration and angiogenesis.

CONCLUSIONEGFL7 effectively promotes migration and angiogenesis through ERK signaling pathway in endothelial cells.

Blotting, Western ; Cell Movement ; Endothelial Cells ; physiology ; Endothelial Growth Factors ; genetics ; physiology ; Humans ; MAP Kinase Signaling System ; physiology ; Neovascularization, Physiologic ; RNA, Messenger ; metabolism ; Signal Transduction

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in several pathological processes of cardiovascular diseases. In this study, the effects of XCT790, a potent and selective inverse agonist of estrogen-related receptor alpha (ERRalpha), on rat VSMCs proliferation and related signal pathways were investigated. The proliferative activity of VSMCs was determined by CCK-8 assay. The mRNA levels of ERRalpha, PGC-1alpha, OPN and MCAD were assayed by RT-PCR. The protein levels of ERRalpha, ERK2 and p-ERK1/2 were evaluated by Western blotting. ELISA was used to assess the protein expression of VEGF. The results showed that XCT790 (5-20 micromol x L(-1)) inhibited rat VSMCs proliferation, and the expression of ERRalpha and its target genes, as well as p-ERK1/2, were also inhibited. XCT790 inhibited VSMCs proliferation in a dose-dependent manner at the dose range from 5 to 20 micromol x L(-1) and in a time-dependent manner at the dose range from 10 to 20 micromol x L(-1). These findings demonstrate that XCT790 inhibits rat VSMCs proliferation by down-regulating the gene level of ERRalpha and thus inhibiting the ERK signal pathway, suggesting that ERRalpha may be a novel potential target for therapeutic approaches to inhibit VSMCs proliferation, which plays an important role in several cardiovascular diseases.
Animals ; Cadherins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytoskeletal Proteins ; genetics ; metabolism ; Dose-Response Relationship, Drug ; GTPase-Activating Proteins ; genetics ; metabolism ; MAP Kinase Signaling System ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Nitriles ; administration & dosage ; pharmacology ; Nuclear Proteins ; genetics ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Phosphorylation ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; genetics ; metabolism ; Thiazoles ; administration & dosage ; pharmacology ; Transcription Factors ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

BACKGROUNDBreast cancer is one of the most common malignant female diseases worldwide. It is a significant threat to every woman's health. Vascular endothelial growth inhibitor (VEGI) is known to be abundant in endothelial cells. According to previous literature, overexpression of VEGI has been shown to inhibit tumor neovascularisation and progression in cellular and animal models, but there has been limited research on the significance of VEGI in the breast cancer.

METHODSIn our study, cell lines MDA-MB-231 were first constructed in which VEGI mediated by lentivirus over-expressed. The effects of VEGI over-expression on MDA-MB-231 cells were investigated both in vitro and in vivo. The expression of VEGI in the MDA-MB-231 cells after infection of lentivirus was analyzed using real-time PCR and Western blotting. The effect of the biological characteristics of MDA-MB-231 cells was assessed by growth, invasion, adhesion, and migration assay with subcutaneous tumor-bearing nude mice models. Then the growth curves of the subcutaneous tumors were studied. Expressions of VEGI, CD31 and CD34 in the tumors were analyzed by immunohistochemistry and apoptosis was detected by flow cytometry and immunohistochemistry.

RESULTSInfection of MDA-MB-231 cells within the lentivirus resulted in approximately a 1 000-fold increase in the expression of VEGI. As can be seen in the invasion, adhesion and migration assay, the over-expression of VEGI can inhibit the ability of MDA-MB-231 cells during migration, adhesion and invasion. The volume of the subcutaneous tumor in the over-expression group was distinctly and significantly less than that of the control groups. Immunohistochemistry analysis of the tumor biopsies clearly showed the expression of VEGI in the over-expression group increased while CD31 and CD34 decreased significantly. In vitro and in vivo, the early apoptosis rate and the apoptosis index were increased within the VEGI over-expression group as compared with the control group.

CONCLUSIONSTaken together, recombinant lentivirus that were successfully constructed, demonstrated up-regulated VEGI gene expression in breast cancer cells. Lentivirus-mediated over-expression of VEGI weakened the ability of the breast cancer cell migration, adhesion and invasion. Over-expression of VEGI diminished the tumorigenic capacity of breast cancer cells in vivo. Up-regulation of VEGI gene expression however inhibited breast cancer MDA-MB-231 cell in the early apoptosis.

Apoptosis ; genetics ; physiology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Vascular Endothelial Growth Factors ; genetics ; metabolism

OBJECTIVE: To investigate the association and mechanism of EGFL7 expression level with the invasion and metastasis of laryngeal squamous cell carcinoma. METHOD: RT-PCR and Western blotting were used respectively to detect the level of EGFL7 mRNA and protein form 33 fresh laryngeal carcinoma tissues and matched para-neoplastic non-tumor tissues. The immunohistochemistry technique was performed to determine microvessel density (MVD) in 33 tumor tissues. The association of EGFL7 expression and MVD with clinicopathological characteristics was analyzed. RESULT: EGFL7 mRNA and protein expression were both significantly higher in the tumor tissues than in the paraneoplastic non-tumor tissues (P < 0.01). Furthermore, the expression of EGFL7 mRNA was highly correlated with the expression of EGFL7 protein (r = 0.786, P < 0.01). EGFL7 expression and MVD were highly correlated with clinical stage, tumor size and the presence of lymph node metastasis (P < 0.05), but was not correlated with the patients gender, age, tumor sit and tumor site differentiation (P > 0.05). CONCLUSION EGFL7 may have a close correlation with the development of laryngeal carcinoma via its impact on tubulogenesis and vessel shape. EGFL7 might serve as a tumor marker for assessing the progression of laryngeal carcinoma and a guide of clinical therapeutic decisions.
Adult ; Aged ; Carcinoma, Squamous Cell ; blood supply ; metabolism ; pathology ; Endothelial Growth Factors ; genetics ; metabolism ; Female ; Humans ; Laryngeal Neoplasms ; blood supply ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging

BACKGROUND/AIMS: Renal hypoxia is involved in the pathogenesis of diabetic nephropathy. Pentoxifyllin (PTX), a nonselective phosphodiesterase inhibitor, is used to attenuate peripheral vascular diseases. To determine whether PTX can improve renal hypoxia, we investigated its effect in the streptozocin (STZ)-induced diabetic kidney. METHODS: PTX (40 mg/kg, PO) was administered to STZ-induced diabetic rats for 8 weeks. To determine tissue hypoxia, we examined hypoxic inducible factor-1alpha (HIF-1alpha), heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), and glucose transporter-1 (GLUT-1) levels. We also tested the effect of PTX on HIF-1alpha in renal tubule cells. RESULTS: PTX reduced the increased protein creatinine ratio in diabetic rats at 8 weeks. HIF-1alpha, VEGF, and GLUT-1 mRNA expression increased significantly, and the expression of HO-1 also tended to increase in diabetic rats. PTX significantly decreased mRNA expression of HIF-1alpha and VEGF at 4 and 8 weeks, and decreased HO-1 and GLUT-1 at 4 weeks. The expression of HIF-1alpha protein was significantly increased at 4 and 8 weeks in tubules in the diabetic rat kidney. PTX tended to decrease HIF-1alpha protein expression at 8 weeks. To examine whether PTX had a direct effect on renal tubules, normal rat kidney cells were stimulated with CoCl2 (100 microM), which enhanced HIF-1alpha mRNA and protein levels under low glucose conditions (5.5 mM). Their expressions were similar even after high glucose (30 mM) treatment. PTX had no effect on HIF-1alpha expression. CONCLUSIONS: PTX attenuates tubular hypoxia in the diabetic kidney.
Animals ; Anoxia/*drug therapy/enzymology/etiology/genetics ; Cell Line ; Cobalt/pharmacology ; Diabetes Mellitus, Experimental/*complications ; Diabetic Nephropathies/*drug therapy/enzymology/etiology/genetics ; Disease Models, Animal ; Gene Expression Regulation/drug effects ; Glucose/metabolism ; Glucose Transporter Type 1/genetics ; Heme Oxygenase (Decyclizing)/genetics/metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Kidney Tubules/*drug effects/enzymology ; Male ; Pentoxifylline/*pharmacology ; Phosphodiesterase Inhibitors/*pharmacology ; RNA, Messenger/metabolism ; Rats ; Rats, Sprague-Dawley ; Streptozocin ; Time Factors ; Vascular Endothelial Growth Factor A/genetics

Previous studies suggested that polymorphisms of proinflammatory cytokine genes are important host genetic factors in Helicobacter pylori infection. The present study evaluated whether IL-8-251 polymorphism affected H. pylori eradication rate and to investigate the effect of H. pylori eradication on angiogenesis and the inflammatory process according to the IL-8-251 polymorphism. A total of 250 H. pylori-positive patients treated by endoscopic resection of the gastric neoplasm were classified into 3 groups (134 H. pylori-eradicated group, 19 H. pylori-eradication failure group, and 97 H. pylori-infected group). H. pylori status, histology, and angiogenic factor levels were evaluated at baseline, 6 months, and 18 months. H. pylori eradication rate was 92.9% in AA genotype, 85.7% in AT genotype and 88.4% in TT genotype (P value = 0.731). Elevated IL-8 and matrix metalloproteinase-9 concentrations in H. pylori-infected gastric mucosa were reversible by successful eradication of H. pylori, independent of the IL-8-251 polymorphism. It is suggested that elevated IL-8 and MMP-9 concentrations in H. pylori-infected gastric mucosa are altered significantly after successful eradication and these conditions continue for 18 months. However, IL-8-251 polymorphism does not affect H. pylori eradication rate and the sequential changes of related angiogenic factors after H. pylori eradication in Koreans.
Aged ; Alleles ; Angiopoietin-1/analysis ; Anti-Bacterial Agents/therapeutic use ; Anti-Inflammatory Agents, Non-Steroidal/therapeutic use ; Asian Continental Ancestry Group/*genetics ; Female ; Gastric Mucosa/metabolism/pathology ; Genotype ; Helicobacter Infections/*drug therapy ; *Helicobacter pylori ; Humans ; Interleukin-8/analysis/*genetics ; Male ; Matrix Metalloproteinase 9/analysis ; Middle Aged ; *Polymorphism, Single Nucleotide ; Proton Pump Inhibitors/therapeutic use ; Republic of Korea ; Retrospective Studies ; Stomach Neoplasms/pathology/surgery ; Time Factors ; Vascular Endothelial Growth Factor A/analysis

OBJECTIVETo investigate the significance of Tiam1 in invasion and metastasis of breast carcinoma and its mechanisms.

METHODSImmunohistochemistry was used to detect Tiam1 expression in tumor tissue of 126 breast carcinomas. Tiam1 was silenced by siRNA in breast carcinoma cell line MDA-MB-435, then the expressions of phosphor-ERK 1, ERK 2 and VEGF were detected, and electrophoretic mobility shift assay (EMSA) was used to examine the transcription activiy of AP-1.

RESULTSThere was a significant relationship between Tiam1 expression and lymph node metastasis (P < 0.05). Furthermore, after silencing of Tiam1, the expressions of phosphor-ERK 1, ERK 2 and VEGF were decreased, and the transcription activity of AP-1 was down-regulated in the MDA-MB-435 cells.

CONCLUSIONTiam1 is closely related with invasion and metastasis of breast carcinoma, and the cascade Tiam1 through ERK, AP-1 and VEGF pathways may play an important role in enhancing angiogenesis, therefore, to promote invasion and metastasis of breast carcinoma.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Guanine Nucleotide Exchange Factors ; genetics ; metabolism ; Humans ; Lymphatic Metastasis ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neoplasm Invasiveness ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Signal Transduction ; T-Lymphoma Invasion and Metastasis-inducing Protein 1 ; Transcription Factor AP-1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism