Proliferation Of ECV304 Cells Research Articles

Lycopene alleviates oxidative stress-induced cell injury in human vascular endothelial cells by encouraging the SIRT1/Nrf2/HO-1 pathway

ABSTRACT Background and objective: Epidemiological research have displayed that dietary intake rich in lycopene, an antioxidant, is negatively correlated with the risk of cardiovascular disease (CVD). This study aimed to investigate whether the intervention with different concentrations of lycopene could attenuate H2O2-induced oxidative stress injury in human vascular endothelial cells (VECs). Methods: The human VECs HMEC-1 and ECV-304 were incubated with a final concentration of 300 µmol/L H2O2, followed by they were incubated with lycopene at doses of 0.5, 1, or 2 µm. Subsequently, cell proliferation, cytotoxicity, cell adhesion, reactive oxygen species (ROS) contents, adhesion molecule expression, oxidative stress levels, pro-inflammatory factor production, the apoptosis protein levels, and the silent information regulator-1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway protein levels were tested by CCK-8 kit, lactate dehydrogenase (LDH) kit, immunofluorescence labeling, cell surface enzyme immunoassays (EIA), enzyme-linked immunosorbent assay (ELISA), as well as Western blot assays, respectively. Results: Under H2O2 stimulation, HMEC-1 and ECV-304 cell proliferation and the SIRT1/Nrf2/HO-1 pathway protein expression were significantly reduced, whereas cytotoxicity, apoptosis, cell adhesion molecule expression, pro-inflammatory and oxidative stress factors production were apparently encouraged, which were partially countered by lycopene intervention in a dose-dependent manner. Conclusion: Lycopene alleviates H2O2-induced oxidative damage in human VECs by reducing intracellular ROS levels, inflammatory factor production, cell adhesiveness, and apoptosis rate under oxidative stress conditions through activation of the SIRT1/Nrf2/HO-1 pathway.

Tumor-suppressor microRNA-139-5p restrains bladder cancer cell line ECV-304 properties via targeting Connexin 43.

Background:In our previous paper, we demonstrated that Connexin 43 (CX43) was highly expressed in bladder cancer (BC) tissues. But the molecular mechanism about microRNAs (miRNAs) regulation upstream of CX43 in BC has not been well elucidated and remains to be further studied. MicroRNA-139-5p (miR-139-5p) is a tumor suppressor in progression of multifarious cancers including BC. Nevertheless, the underlying mechanisms of CX43/miR-139-5p in tumorigenesis of BC are still not well illustrated. The specific objective of our study was to inquiry the effect of CX43/miR-139-5p on BC progression and its underlying mechanism.Methods:The bioinformatics analysis softwares were applied to predict the miRNAs in the upstream of CX43. First, the expression levels of miR-139-5p in BC tissues (tumor) and paracancer tissues (normal) were investigated using the data from The Cancer Genome Atlas database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of miR-139-5p in three human BC cell lines 5637, T24, ECV-304 and a human bladder epithelial immortalized cell line SV-HUC-1 (normal control). Then si-CX43, si-control, miR-139-5p mimic, and its negative control (NC) were transfected into BC cell line ECV-304. The relationship of miR-139-5p and CX43 was analyzed by dual-luciferase reporter assay. The qRT-PCR and Western blotting were used to test the mRNA and protein expression level of CX43. The proliferation of ECV-304 and T24 cells were examined by cell counting kit-8. The migration and invasion of ECV-304 cells were tested by transwell assay. To determine whether miR-139-5p would affect cell proliferation, migration and invasion by targeting CX43, we executed the rescue assay. The comparison between two groups was analyzed by Student's t test, and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test.Results:The expression of miR-139-5p was remarkably down-regulated in BC tissues (tumor vs. normal, 2.286 ± 0.017 vs. 3.211 ± 0.034, t = 11.540, P < 0.0001) and cell lines (P < 0.01 in all BC cell lines). Besides, we also indicated that over-expression of miR-139-5p reduced the proliferation of ECV-304 (P = 0.001) and T24 cells (P = 0.005). Moreover, miR-139-5p over-expression weakened the invasion (P = 0.001) and migration (P = 0.001) of ECV-304 cells. Furthermore, the relative luciferase activity of CX43-wild type construct was distinctly lessened by up-regulation of miR-139-5p (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.916 ± 0.063 vs. 0.356 ± 0.048, t = 7.085, P = 0.002), nevertheless the activity of CX43-mutant type construct was untouched (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.918 ± 0.057 vs. 0.878 ± 0.039, t = 0.577, P = 0.595). Finally, the rescue assay revealed that CX43 deletion enhanced the depressor effect of miR-139-5p on ECV-304 cell proliferation (P < 0.01), invasion (P = 0.028), and migration (P = 0.014).Conclusion:MiR-139-5p, as a tumor-suppressor, repressed cell proliferation, invasion, and migration in BC, which might be achieved by regulating CX43.

Effect of an EDA-A1 gene mutant on the proliferation and cell cycle distribution of cultured human umbilical vein endothelial cells.

Ectodysplasin (EDA) gene mutation is associated with hypohidrotic ectodermal dysplasia (HED). The aim of this study was to investigate the effect of ectodysplasin, transcript variant 1 (EDA-A1) on the proliferation and cell cycle of ECV304 human umbilical vein endothelial cells (HUVECs). Recombinant eukaryotic expression vectors containing mutant (M) and wild-type (W) EDA-A1 coding sequences, pcDNA3.1 (-)-EDA-A1-M and pcDNA3.1 (-)-EDA-A1-W, respectively, were transfected into ECV304 cells. The EDA-A1 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), and the protein was detected by western blotting. The EDA-A1 gene and protein were detected in ECV304 cells transfected with pcDNA3.1 (-)-EDA-A1-M and pcDNA3.1 (-)-EDA-A1-W, but not in ECV304 cells transfected with empty plasmid or cells that had not undergone transfection. Compared with the control group, the EDA-A1 gene mutant significantly decreased the proliferation of ECV304 cells and its inhibition rate was 45.70% (P<0.01), whereas the wild-type EDA-A1 gene did not cause such growth inhibition (P>0.05). A significant increase of the fraction of cells in the G0/G1 phase of the cell cycle was observed in the ECV304 cells of the mutant group compared with wild type group, with an increase in the S phase population and a concomitant reduction in the G2/M phase population (P<0.05). These results indicate that compared with the wild-type gene, transfection with a mutant EDA-A1 gene inhibited the proliferation and cell cycle of cultured HUVECs.

Construction and assessment of recombinant plasmid pRNAT-U6.1/CFB siRNA and its inhibitory effect on proliferation of human umbilical vein endothelial cells

Background Choroidal neovascularization (CNV) is one of the causes of blindness in multiple eye diseases. Researches showed that complement system participates in the pathogenesis of CNV. Objective This study was to construct the recombinant of complement factor B-small interference RNA (CFB-siRNA) expression vector and to observe its inhibitory effect on human umbilical vein endothelial cells (ECV-304). Methods CFB gene primers were designed based on human CFB gene, and an expression vector of CFB-siRNA was constructed by inserting CFB-siRNA into pRNAT-U6. 1/Neo plasmid. Recombinant plasmids were confirmed by the digestion analysis of restriction endonuclease, and all inserted sequences were verified by DNA sequencing. The recombinant pRNAT-U6. 1/CFB-siRNA plasmid and the blank plasmid were transfected into ECV-304 cells in the CFB-siRNA group and blank plasmid group by electroblot, respectively, and non-transfected cells served as the normal control group. The cells were observed under the fluorescence microscope 48 hours after transfection, and the transfective efficiency was calculated. The relative expression of CFB mRNA in the cells of different groups was detected by semi-quantitative reverse transcription PCR (RT-PCR). MTT was employed to calculated the growth inhibitory rates of the cells 24, 48 and 72 hours after transfection. The percentages of the cells in different cell cycles were detected by flow cytometry. Results The sequence of the target vector was identical to the designed sequence. The green fluorescence protein (GFP) was seen in both the CFB-siRNA group and the blank plasmid group. The relative expression levels of CFB mRNA were 0. 07±0. 04, 0. 14±0. 02 and 0. 14±0. 03 in the CFB-siRNA group, the blank plasmid group and the normal control group, respectively, a significant difference was obtained among the three groups (F=233. 05, P=0. 00); the expression level of CFB mRNA in the CFB-siRNA group was significantly declined in comparison with the blank plasmid group and the normal control group (both at P<0. 05). The growth inhibitory rates of the cells were (23. 45±0. 01)%, (33. 48±0. 02)% and (45. 49±0. 01)% at 24, 48 and 72 hours after transfection, respectively, a significant difference was obtained among the three groups (Fgroup=212. 99, P=0. 00); the growth inhibitory rates in CFB-siRNA group were significantly higher than that in the blank plasmid group and normal control group (all at P<0. 05). The percentages of G1 phase cells were (44. 4±0. 5)%, (25. 8±0. 4)% and (27. 9±0. 6)% in the CFB-siRNA group, the blank plasmid group and the normal control group respectively, a significant difference was obtained among the three groups (F=58. 98, P=0. 00). The percentages of G1 phase and G2 phase cells in the CFB-siRNA group were significantly higher than those in the blank plasmid group and the normal control group (all at P<0. 05). Conclusions Recombinant pRNAT-U6. 1/CFB siRNA inhibits the proliferation of ECV-304 cells effectively by arresting the cells in G1 intermediate phase of the growth cycle. Key words: Choroidal neovascularization; Small interfering RNA; Complement factor B; Umbilical vein endothelial cell; Human; ECV-304

Facile immobilization of heparin on bioabsorbable iron via mussel adhesive protein (MAPs)

Motivated by adhesive proteins in mussels, strategies using dopamine to modified surface have become particularly attractive. In the present work, we developed a novel and convenient method to modify the biodegradable Fe plates with heparin. Iron was first treated by a facile one-step pH-induced polymerization of dopamine, and then a high density heparin was successfully grafted onto the surface via coupling with polydopamine (PDA) active layer. Heparin immobilization contributed much longer blood clotting coagulation time than the pure Fe sample, and hence reduced the risk of thrombosis. Cell viability tests suggested that the heparin modified Fe plates were more favorable to the proliferation of ECV304 cells. In summary, the heparin modified Fe plates with good anti-thrombus properties and inhibiting the proliferation of VSMC cells provide great prospects for biodegradable iron.

Endoscopic deployment of multiple plastic stents for biliary stricture after living donor liver transplantation

Objective To construct a recombinant adenovirus vector carrying hSulf-1 gene and to investigate its effect on the proliferation and migration of human umbilical vein endothelial cells.Methods The hSulf-1 protein was inserted into adenovirus genome to generate recombinant adenovirus Ad5-hSulf1,and then the virus was used to infect ECV-304 cell line.The expression of hSulf-1 protein was detected by Western blotting analysis,the cell viability was examined by MTT assay,and the cell migration ability was evaluated by wound-healing assay in ECV-304 cells.Results The recombinant adenovirus Ad5-hSulf1 was successfully constructed.Western blotting analysis showed that over-expression of hSulf1 down-regulated the phosphorylation levels of Akt and ERK in ECV-304 cells.MTT assay showed that over-expression of hSulf1 inhibited the proliferation of ECV-304 cells,with the cell survival rate decreased to(68.49±0.05)% at MOI of 50 pfu/ml and(67.78±0.06)% at MOI of 100 pfu/ml(P0.05).Wound-healing assay showed that over-expression of hSulf1 significantly inhibited the migration of ECV-304 cells compared with the control group(P0.01).Conclusion The recombinant adenovirus Ad5-hSulf1-mediated hSulf-1 over-expression can markedly inhibit the proliferation and migration of ECV-304 cells,laying a foundation for hSulf-1 related gene therapy of tumor and angiogenesis-associated diseases.

Hyperoside Downregulates the Receptor for Advanced Glycation End Products (RAGE) and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK) Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs) in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK) activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes.

Abstract P303: Panaxnotogensengsaponins Promotes Angiogenesis and Enhances the Buildup of Effective Coronary Collateral Circulation in Ischemic Myocardium

Background: Ischemic heart disease (IHD) is a condition that the blood vessels are narrowed or blocked due to the deposition of cholesterol plaques on the blood vessel walls. This affects the supply of blood to the heart and reduces the supply of oxygen and nutrients to the heart musculature, which has been regarded as the largest threat to the public health worldwide. PNS is a purified natural product extract that has been proved effective and widely used clinically in preventing and treating IHD in China. However, the detailed mechanisms of action of PNS remain to be addressed. The present study aims to explore the effect of PNS on modulating angiogenesis in vitro and in vivo to better understand its mechanism of action in treating IHD. Methods and Results: In this study, myocardial ischemia rat model and endothelial cell derived from human capillary blood vessel (ECV304) were utilized. Our data revealed that PNS significantly increased capillary blood vessel density in isoproterenol-induced rat ischemic myocardium model. It also decreased the pathological damage and ameliorated the ultrastructure of myocardial cells in rat ischemic myocardium as assessed by light microscopy, transmission electron microscopy and immunohistochemistry. At cellular level, PNS significantly decreased the number of apoptotic cells in rat ischemic myocardium model. Mechanistic study utilizing microarray and real-time PCR were further performed to examine the gene expression profile. The data revealed that PNS significantly upregulated the mRNA expression of a panel of angiogenic growth factors and their receptors such as VEGF, VEGFR2 and FGFR1. In vitro study revealed that PNS did not exhibit significant effect on the proliferation of ECV304 cells under normoxia culture condition; however, it facilitated the proliferation of ECV304 cells under hypoxia condition, suggesting the selective effect of PNS on angiogenesis under ischemic/hypoxic conditions. Conclusions: Our study demonstrated for the first time that PNS exerts its therapeutic effect on IHD through the mechanisms that in part involve regulating the expression of important angiogenic growth factor, which coordinately enhance the build-up of effective coronary collateral circulation in ischemic myocardium.

Gene expression of ECV304 cells after treated with endostatin detected by microarray analysis

Objective To observe the altered gene expression of ECV304 cell after treated with endostatin (ES) using cDNA microarray, and to investigate its molecular mechanism. Methods BiostarH40S gene expression DNA microarrays were used to profile changes in gene expression of ECV304 cells after treated with ES for 36 hours. To prepare the probes, mRNA from both control and treated cells were isolated and purified, and then reversely transcribed to cDNA with the incorporation of fluorescent-labeled dUTP: Cy3 and Cy5 respectively. The probes were hybridized with expressed cDNA microarray, the fluorescent signals of Cy3 and Cy5 were scanned and analyzed by a computer system. Results ES inhibited the proliferation of ECV304 cells. The result of electrophoresis indicated that the extracted total RNA had high quality. Approximately 5. 96% of all human genes examined on 4096 gene microarrays showed altered expression, with 225 down-regulated genes and 19 up-regulated genes. Conclusion The expression of some genes of ECV304 cells decrease after treated with ES, which may be related to the mechanism of inhibition of neovascularization. Key words: Gene expression profiling/utilization; Microchip analytical procedures/method; Endostatins/pharmacology; Gene expression

Construction of NF-κB-targeting RNAi adenovirus vector and the effect of NF-κB pathway on proliferation and apoptosis of vascular endothelial cells

To construct a recombinant adenovirus vector expressing a RNAi for the Nuclear Factor kappa B (NF-κB)/p65 gene and use it to explore the role of the NF-κB pathway on the regulation of proliferation and apoptosis of vascular endothelial cells. A recombinant adenovirus containing a RNAi cassette targeting the p65 gene was constructed, and its silencing effect on p65 was detected by Western blot analysis in ECV304 cells. Expression of the p65 protein in ECV304 cells was efficiently down-regulated by the RNAi adenovirus for more than 6 days. ECV304 cells proliferation and apoptosis were measured using the MTT assay and flow cytometry, respectively. Blocking the NF-κB pathway with the RNAi adenovirus substantially decreased the proliferation of ECV304 cells, but only slightly affected cell apoptosis. We used a NF-κB/p65-targeting RNAi adenovirus to demonstrate the role of the NF-κB pathway in the regulation of ECV304 cell proliferation. This adenovirus may serve as an important tool to study the NF-κB pathway.

Hepatocyte growth factor protects endothelial cells against gamma ray irradiation-induced damage

To investigate the effect of HGF on proliferation, apoptosis and migratory ability of human vascular endothelial cells against gamma ray irradiation. ECV304 cells derived from adult human umbilical vein endothelial cells (HUVEC) were irradiated with a single gamma ray dose of 20 Gy. Immunocytochemistry and Western blot analysis were used to detect c-Met protein expression and HGF/c-Met signal pathway. In the HGF-treated groups, ECV304 cells were incubated with HGF (20 or 40 ng/mL) 3 h prior to irradiation. At 48 h post-irradiation, the proliferation of ECV304 cells was measured by MTT assay, the apoptosis was assessed by flow cytometry, and the migratory ability of ECV304 cells was measured by transwell chamber assay. c-Met protein is expressed in ECV304 cells and can be activated by HGF. Gamma ray irradiation inhibits proliferation and migration of ECV304 cells in a dose-dependent manner. HGF significantly promoted the proliferation of ECV304 cells, and flow cytometry revealed that HGF can inhibit apoptosis of ECV304 cells. Transwell chamber assay also showed that HGF increases migration activity of endothelial cells. HGF may afford protection to vascular endothelial cells against gamma ray irradiation-induced damage.

Prokaryotic expression, purification and functional characterization of human FHL3

Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42 degrees C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.

Inhibition of Proliferation of ECV304 Cells by a Disintegrin from Chinese Green Tree Viper

A novel disintegrin, stejnin, was purified from the Trimeresurus stejnegeri venom by gel filtration and reverse phase high performance liquid chromatography. The molecular weight of stejnin was determined to be 7428 Da by MALD-TOF MS analysis. The cDNA encoding the precursor of stejnin was cloned from the venom gland. From the deduced amino acid sequence, stejnin is composed of 71 amino acid residues contains the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Stejnin strongly inhibited ADP- and ristomycin-induced human platelet aggregation with IC50 of 45 and 50 nM, respectively. Stejnin also possessed potent inhibited cell proliferation of ECV304 cells.

Inhibitory effect of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma.

To investigate the effect of adeno-associated virus-mediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC). HCC cell line Hep3B was infected with recombinant adeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2-hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined by ELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk. The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P<0.01). Intratumoral injection of rAAV2-hEndo (2 x 10(10) v.g.) led to a sustained serum endostatin level of approximately (86.71+/-5.19) ng/mL. The tumor volume and microvessel density were less in rAAV2-hEndo group than in control groups (P<0.01). Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.

Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation

To study the changes in gene expression in endothelial cells stimulated by lipopolysaccharide (LPS) we performed subtraction hybridization on control human umbilical vein endothelial cells (HUVEC) versus HUVEC stimulated by LPS. A novel cDNA, named endothelial-overexpressed lipopolysaccharide-associated factor 1 ( EOLA1), was cloned from our differentially expressed EST database of HUVEC cDNA library (GenBank Accession No. AY074889). Computational analysis showed that EOLA1 is 1404 bp long, encoding a 158 aa, 17.8 kDa protein, mapped to chromosome Xq27.4 with 5 exons, expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. Stable transfection of EOLA1 stimulates ECV304 cell proliferation. Our data suggest that the physical interaction of EOLA1 and MT2A may have an important role of cell protection in inflammation reaction.

Suppression of Tacrolimus on the expression of MMP-9 in rhHGF-stimulated ECV304 cell line

To investigate the stimulating effect of recombinant human hepatocyte growth factor (rhHGF) on the expression of matrix metalloproteinase-9 (MMP-9) in ECV304 cells and the suppression effect of FK506. The growth curve of ECV304 cells was drawn and the valid concentration of rhHGF and FK506 were determined. The expression levels of MMP-9 were analyzed by flow cytometry (FCM). (1) The optimal growth concentration of ECV304 cell was 1.0E+4/ml and logarithm growth time was 3-5 days. The valid concentration of rhHGF was 8 ng/ml and IC50 was 8.27 ng/ml. The valid concentration of FK506 was 50 ng/ml and IC50 was 51.63 ng/ml. (2) The expression level of MMP-9 was 39.74% in controls. Cell livability was 1.388169 +/-0.102033 (P<0.01) when 8 ng/ml rhHGF was added to culture medium, and the expression level of MMP-9 was 40.32%. Cell livability was 0.398764 +/-0.092476 (P<0.01) when 50 ng/ml FK506 was added to culture medium, and the expression level of MMP-9 was 14.61%. Cell livability was 0.767203 +/-0.02639 (P<0.01) when 8 ng/ml rhHGF was added 15 minutes after 50 ng/ml FK506 was added to culture medium, and the expression level of MMP-9 was 35.08%. rhHGF can stimulate the proliferation of ECV304 cells and increase the protein expression level of MMP-9. FK506 can suppress the proliferation of ECV304 cells and decrease the expression level of MMP-9. FK506 can inhibit the proliferation of ECV304 cells induced by rhHGF and decrease MMP-9 protein level.

Inhibition of Tumor Metastasis by Sodium Caffeate and Its Effect on Angiogenesis

Objective: Sodium caffeate (SC), the sodium salt of caffeic acid, was synthesized in our laboratory. We studied the antimetastatic effect induced by SC and its inhibition of tumor angiogenesis using various in vitro and in vivo metastasis assays. Methods: The in vivo inhibitory effect of SC on metastasis and angiogenesis was examined in the Lewis lung carcinoma pulmonary metastasis model and chicken chorioallantoic membrane (CAM) model, respectively. MTT assay and flow cytometry were used to measure the inhibition by SC of the proliferation of transformed human umbilical vein endothelial cells (ECV304) and the apoptosis induced by SC in ECV304 cells, respectively. A cell attachment assay was used to evaluate inhibition by SC of the adhesion activity of human high metastatic giant cell carcinoma of the lung (PG) cells. A cell invasion assay was used to evaluate the effect of SC on the ability of PG cells to cross tissue barriers. Inhibition by SC of gelatinase secretion in PG cells was determined by zymography. Results: In vivo results showed that SC (1 g/kg i.p. for 14 days) inhibited pulmonary metastasis at a rate of 55%. There were no differences in animal weights among the groups. The angiogenesis of CAM was inhibited by SC (200 µg/egg) at a rate of 70%. In vitro studies showed that SC inhibited the proliferation of ECV304 cells by inducing apoptosis. SC also reduced the adhesion and invasion ability of PG cells and inhibited the secretion of MMP-2 and MMP-9 in PG cells. Conclusion: SC might be a potential antimetastatic agent with an antiangiogenetic effect.

Cloning and characterization of Adinbitor, a novel disintegrin from the snake venom of Agkistrodon halys brevicaudus stejneger.

Adinbitor was cloned from Agkistrodon halys brevicaudus stejneger and characterized as a novel disintegrin. In this study, total RNA was extracted from venom gland and used in RT-PCR to generate a cDNA which is 219 bp long. The sequence encoded a polypeptide composed of 73 amino acids, including 12 cysteines, an RGD motif, and the signature motif of disintergrin. Recombinant Adinbitor (rAdinbitor) was expressed in E. coli and purified by using the His. Bind affinity chromatography. The IC(50) for inhibiting human platelet aggregation and bFGF-induced proliferation of ECV304 cells was 6 microM and 0.89 microM respectively. Furthermore, Adinbitor significantly inhibited angiogenesis both in vivo and in vitro. Taken together, these results suggested that Adinbitor had typical functions of disintegrins.

Resveratrol suppresses the angiogenesis and tumor growth of gliomas in rats.

We wanted to investigate the antitumor effects and effect on angiogenesis of resveratrol in rat RT-2 gliomas. RT-2 glioma cells were treated with resveratrol, and then cytotoxicity was assayed, apoptosis was measured by flow-activated cell sorter flow cytometry, and expression of vascular endothelial growth factor was measured by reverse transcription-PCR. Tumor size, animal survival time, and survival rate were followed in resveratrol-treated rats with s.c. or intracerebral gliomas. Furthermore, in vitro proliferation was assayed to explore the effect of resveratrol on the proliferation of ECV304 human umbilical vein endothelial cells. Expression of CD31 in resveratrol-treated gliomas was followed immunohistochemically to study the effect of resveratrol on the glioma-induced angiogenesis. Resveratrol was demonstrated to exert cytotoxic effects and induce glioma cell apoptosis in a concentration- and time-dependent manner (P < 0.05). Resveratrol (40 mg/kg/day) exerted significant antitumor effects on s.c. tumors, including slower tumor growth rate, longer animal survival time, and higher animal survival rate (P < 0.05). In contrast, resveratrol affected intracerebral tumors at only an increased dose (100 mg/kg/day), prolonging animal survival (P < 0.05) without affecting survival rate. The expression of vascular endothelial growth factor in the glioma cells and the proliferation of ECV304 cells were inhibited by resveratrol in a concentration-dependent manner. Immunohistochemical analyses showed that the s.c. gliomas from resveratrol-treated rats had fewer microvessel densities than did control rats (P < 0.01). Resveratrol caused significant glioma cell cytotoxicity and apoptosis, exerted antitumor effects on the s.c. and intracerebral gliomas, and inhibited angiogenesis in s.c. gliomas. Thus, resveratrol might be considered a possible treatment strategy for gliomas.

Effects of endostatin-vascular endothelial growth inhibitor chimeric recombinant adenoviruses on antiangiogenesis.

To investigate the inhibitory effects of endostatin-vascular endothelial growth inhibitor (VEGI151) recombinant adenoviruses on neovascularization. We used recombinant adenoviruses to treat human vascular endothelial cell line ECV304, human hepatocellular carcinoma cell line HepG2, and murine fibroblast cell line L929, in order to study the chimeric gene expression in these cell lines. Chick choriallantic membrane (CAM) model, rabbit inflammatory corneal neovascularization (CNV) model, and liver cancer-bearing nude mice model were employed to investigate the negative biological effect of fusion molecules on neovascularization in vivo. Western blot showed that the molecular weight of fusion protein was about 41 kD after infection of ECV304, HepG2 and L929 cells with supernatant of AdhENDO-VEGI151. The fusion protein showed a specific inhibitory effect on the proliferation of ECV304 cells, but no inhibitory effect on the growth of HepG2 and L929 cells (F=13112.13, P=0.0001). In the chick choriallantic membrane (CAM) assay, the expressed fusion protein significantly inhibited neovascularization. Rabbit inflammatory corneal neovascularization (CNV) induced by intrastromal sutures resulted in a uniform neovascular response. In this model, direct subconjunctival injection of AdhENDO-VEGI151 expressed the fusion protein in vivo and suppressed the development of CNV. Topical application of AdhENDO-VEGI151 led to a significant suppression of CNV (F=1413.11, P=0.0001), as compared with the control group of AdLacZ. Immunohistochemical staining showed the fusion protein dominantly expressed in corneal epithelium. Compared with the control group of AdLacZ (4075.9+/-1849.9 mm(3)), the average tumor size of group AdhENDO-VEGI151 reduced in size (487.7+/-241.2 mm(3)) (F=14.80, P=0.0085), with an inhibition rate of 88.03%. Immunohistochemical staining showed the adenoviruses carried the fusion gene expressed on liver cancer cell membrane. MVD decreased more significantly in treated mice (30.75+/-3.31%) than in AdLacZ control (50.25+/-8.65%) (F=17.72, P=0.0056) with an inhibition rate of 39%. Fusion protein expressed by recombinant adenoviruses has a significant inhibitory effect on neovascularization.