Cell Proliferation Of Human Umbilical Vein Endothelial Cells Research Articles

Inhibition of high glucose-induced apoptosis by uncoupling protein 2 in human umbilical vein endothelial cells

Studies have shown that an overproduction of mitochondrial reactive oxygen species (ROS) is an initiating cause in the pathogenesis of diabetic complications. However, uncoupling protein 2 (UCP2) can protect retinal vascular endothelial cells from damage by inhibiting the overproduction of mitochondrial ROS, although the protective mechanism involved is not completely clear. This study aimed to assess the effect and mechanism of UCP2 on the apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in normal glucose (NG, 5.5 mmol/l) or high glucose (HG, 30 mmol/l) medium in the presence or absence of UCP2(+/+) lentiviral transfection. Lentivirus-mediated UCP2 overexpression inhibited the apoptosis of HUVECs induced by HG. Treatment with HG resulted in the upregulation of caspase-3 and cytochrome c and the downregulation of Bcl-2 in vitro. Furthermore, compared with the NG group, the rate of apoptosis was significantly increased in the HG group. On day two post-infection, NG cells showed significantly greater HUVEC cell proliferation than HG cells. Notably, UCP2 overexpression inhibited these processes. Taken together, these results suggest that UCP2 promotes cell proliferation and inhibits HG-induced apoptosis in HUVECs via the Bcl-2 up‑ and downregulation of caspase-3 and cytochrome c in vitro. This may provide experimental evidence for the application of UCP2 as a new protective factor for diabetic complications, such as diabetic retinopathy.

LAT1 Is a Central Transporter of Essential Amino Acids in Human Umbilical Vein Endothelial Cells

Endothelial cell proliferation supporting angiogenesis requires sufficient nutrient supply because of facilitated intracellular metabolism. However, little is known about the mechanism for the promotion of nutrient incorporation in proliferating endothelial cells. Here we show that L-type amino acid transporter 1 (LAT1) is a major transporter of essential amino acids in human umbilical vein endothelial cells (HUVECs). Growing HUVECs express a certain level of LAT1. A LAT1-specific inhibitor suppressed leucine uptake, cell proliferation, and tube formation of HUVECs. Therefore, LAT1 acts to support effective uptake of amino acids, which is critical for the optimal function of HUVECs for angiogenesis.

Abstract 5664: The antiangiogenic effect of two drugs loaded nanocarriers and its potential application for cancer metronomic treatment.

Abstract Purpose: To synthesize and evaluate the antiangiogenic effect of individual and mixed paclitaxel (PTX) and rapamycin (RAP) conjugated micelles Methods: RAP or PTX were conjugated to poly(ethyleneglycol)-block-poly(aspartate-hydrazide) utilizing a pH-sensitive linker based on hydrazone prodrug strategy. The synthesized drug-polymer conjugates were characterized using NMR and gel permeation chromatography (GPC). RAP and PTX polymer conjugates, PEG-RAP and PEG-PTX respectively were assembled into individual and mixed micelles. The size of these micelles was determined using dynamic light scattering. The micelle drug content was quantified by RP-HPLC. The antiangiogenic effect of individual and mixed micelles was evaluated in in vitro Human Umbilical Vein Endothelial Cells (HUVEC) model through proliferation, tube formation and migration assays. The HUVEC cell proliferation was evaluated by the CellTiter-Blue® Cell Viability Assay, the tube formation was evaluated by measuring the total area covered by the HUVEC capiliary formation while the HUVEC cell migration was determined by xCELLigence System. Results: PEG-RAP and PEG-PTX were successfully synthesized and the molecular weight of the polymers was determined by GPC and NMR. The number of drug molecules attached to the polymer for RAP and PTX were 22 and 25 respectively. PEG-RAP and PEG-PTX were assembled into individual and mixed micelles as mono dispersed populations in the range of 40-100nm. The IC50 of PEG-RAP and PEG-PTX individual micelles were 12μM and 2nM respectively. The mixed micelles showed synergistic effect in inhibiting the HUVEC cell proliferation. Individual and mixed micelles inhibited HUVEC tube formation and migration with the mixed micelles showing a synergistic effect. Conclusions: We were successful in synthesizing new conjugates of RAP and PTX with a pH-sensitive linker. These polymers were formulated into individual and mixed micelles. Antiangiogenic effect was observed with individual and mixed micelles. The antiangiogenic effect was synergistically enhanced with the use of mixed micelles. Citation Format: Adam G. Alani, Gyan Mishra. The antiangiogenic effect of two drugs loaded nanocarriers and its potential application for cancer metronomic treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5664. doi:10.1158/1538-7445.AM2013-5664

MiR-29a modulates the angiogenic properties of human endothelial cells

Although extensive investigation has been made on miR-29a in relation to malignancies, only a little information has been provided about the angiogenic property of this miRNA so far. Herein, we sought to investigate the role of miR-29a in regulating cell cycle and angiogenic phenotype of endothelial cells. The results showed that miR-29a is highly expressed and upregulated by hypoxia-mimicking reagents in human umbilical vein endothelial cells (HUVEC). Consistent with this preliminary finding, introduction of exogenous agomiR-29a, or Antagomir-29a altered cell cycle progression and promoted, or repressed the proliferation and tube formation of HUVEC, respectively. Furthermore, by using luciferase reporter assay, the expression of HBP1, a suppressor transcription factor was directly regulated by miR-29a through 3′-UTR. Increased or decreased HBP1 protein level was associated with the inhibition or overexpression of miR-29a, respectively. We conclude that miR-29a has a significant role in regulating cell cycle, proliferation and angiogenic properties of HUVEC, and this function is likely mediated through HBP1 protein at the post-transcriptional level. As a novel molecular target, miR-29a may have a potential value for the treatment of angiogenesis-associated diseases such as cardiovascular diseases and cancers.

Effects of dried longan seed (Euphoria longana Lam.) extract on VEGF secretion and expression in colon cancer cells and angiogenesis in human umbilical vein endothelial cells

Angiogenesis is a critical event in cancer metastasis, via delivery of needed oxygen and nutrients to tumor cells. Anti-angiogenesis is one strategy for controlling cancer progression. We herein report anti-angiogenesis activity of dried longan seeds using colon adenocarcinoma cells (SW480 cells) and human umbilical vein endothelial cells (HUVECs). Sephadex LH-20 column chromatography was used for separate three dried longan seed fractions. We firstly evaluated vascular endothelial cell growth factor (VEGF) secretion, expression and colony formation of SW480 cells, using enzyme-linked immunosorbent assay (ELISA), Western blot analysis and soft agar assays. Meanwhile cell proliferation, gelatinase activity and tube formation of HUVECs were determined via proliferation assay, gelatin zymography and in vitro tube formation assay, respectively. The results suggest that dried longan seed fractions could be potential angiogenic inhibitors not only interruption of VEGF secretion and expression in SW480 cells but also abrogation of cell proliferation, the activity of gelatinase and tube formation of HUVECs.

Melatonin modulates aromatase activity and expression in endothelial cells

Melatonin is known to suppress the development of endocrine-responsive breast cancers by interacting with the estrogen signaling pathways. Paracrine interactions between malignant epithelial cells and proximal stromal cells are responsible for local estrogen biosynthesis. In human breast cancer cells and peritumoral adipose tissue, melatonin downregulates aromatase, which transforms androgens into estrogens. The presence of aromatase on endothelial cells indicates that endothelial cells may contribute to tumor growth by producing estrogens. Since human umbilical vein endothelial cells (HUVECs) express both aromatase and melatonin receptors, the aim of the present study was to evaluate the ability of melatonin to regulate the activity and expression of aromatase on endothelial cells, thus, modulating local estrogen biosynthesis. In the present study, we demonstrated that melatonin inhibits the growth of HUVECs and reduces the local biosynthesis of estrogens through the downregulation of aromatase. These results are supported by three lines of evidence. Firstly, 1 mM of melatonin counteracted the testosterone-induced cell proliferation of HUVECs, which is dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. Secondly, we found that 1 mM of melatonin reduced the aromatase activity of HUVECs. Finally, by real‑time RT-PCR, we demonstrated that melatonin significantly downregulated the expression of aromatase as well as its endothelial-specific aromatase promoter region I.7. We conclude that melatonin inhibits aromatase activity and expression in HUVECs by regulating gene expression of specific aromatase promoter regions, thereby reducing the local production of estrogens.

The tetraspanin CD151-ARSA mutant inhibits angiogenesis via the YRSL sequence.

Previous studies have shown that the tetraspanin CD151 is essential for pathological or physiological angiogenesis. However, the cellular signaling mechanism and the role of the CD151 YRSL sorting motif in in vitro vasculogenesis remains unknown. In this study, the results showed that both CD151 and CD151-ARSA gene delivery were capable of increasing the expression of CD151 at the protein level in human umbilical vein endothelial cells (HUVECs). Moreover, there was no significant difference in CD151 protein expression between the CD151 group and the CD151-ARSA group. Overexpression of CD151 promoted HUVEC cell proliferation, migration and capillary network formation in vitro. However, in the CD151-ARSA group, the abilities of cell proliferation, migration and capillary network formation were all decreased, compared with the CD151 group. Furthermore, the activation of PI3K, Akt and ERK signaling pathways was attenuated in the CD151-ARSA mutant group compared with the CD151 group. This study suggests that the YRSL motif of CD151 plays a key role in CD151-induced angiogenesis. Our observations provide insights into a new mechanism of CD151 regulating angiogenesis via vesicle trafficking.

Synergic antitumor effect of SKLB1002 and local hyperthermia in 4T1 and CT26

A de novo VEGFR2-inhibited compound SKLB1002 which is independently developed in our laboratory has been described for antiangiogenesis and displays a potent antitumor activity in vivo and in vitro. In the present investigation, we aim to prove that combination therapy of SKLB1002 with hyperthermia plays a synergy as an antitumor agent in solid tumor. In this study, we analyzed their synergetic inhibitory action on human umbilical vein endothelial cells (HUVEC), murine mammary cancer 4T1, murine colon carcinoma CT26 in vitro. Multiply-table tournament was performed to detect cell proliferation in vitro. 4T1 implantation and CT26 implantation in BALB/c mice were used to examine the activity of combination therapy of SKLB1002 with hyperthermia in vivo. Vascular density was determined by CD31 immunohistochemistry. TUNEL was used to measure apoptosis in tumor tissue. Metastasis assay was investigated via measurement of pulmonary metastasis nodules under the microscope. Potential toxicity of combination therapy was observed by histologic analysis of main organs stained with H&E. In vitro, the combination therapy significantly inhibited cell proliferation of HUVEC, 4T1 and CT26. In vivo, 4T1 and CT26 model experiments showed that combination therapy remarkably inhibited tumor growth and prolonged life span. When compared with controls, combination therapy reached 61% inhibition index of tumor growth against CT26 and 51% against 4T1. Moreover, it reduced angiogenesis and increased tumor apoptosis and necrosis. It was further found that combination therapy could efficiently prevent tumor from metastasizing to lung. Importantly, it had no toxicity to main organs including heart, liver, spleen, lung and kidney. Combination treatment has been proved to be a novel and strong strategy in clinical antitumor therapy. Our findings suggest that the combination therapy of SKLB1002 with hyperthermia has a synergistic antiangiogenesis, anticancer and promotion of apoptosis efficacy compared with controls. These findings could pave a new way in clinical tumor therapy.

Barbigerone, an isoflavone, inhibits tumor angiogenesis and human non-small-cell lung cancer xenografts growth through VEGFR2 signaling pathways

We previously reported that barbigerone (BA), an isoflavone isolated from Suberect Spatholobus, exhibited inhibitory effects on proliferation of many cancer cell lines in vitro. The objective of this study was to explore whether BA could effectively suppress tumor angiogenesis and tumor growth. Zebrafish model and Matrigel assay were performed to access the anti-angiogenesis effects of BA. A549 and SPC-A1 tumor xenografts in mice models were used to examine the antitumor activity of BA. The anti-angiogenic effects and underlying mechanisms were also investigated using human umbilical vein endothelial cells (HUVECs) and A549 cells. In zebrafish model, 2.5 μmol/L of BA significantly inhibited angiogenesis. Intravenous administration of BA effectively inhibited the tumor growth of A549 and SPC-A1 xenograft models in mice. The anti-angiogenic effect was indicated by CD31 immunohistochemical staining, Matrigel plug assay, and mouse aortic ring assay. BA could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, and capillary-like tuber formation of HUVECs in a dose-dependent manner, suggesting that BA inhibited tumorigenesis by targeting angiogenesis. Western blots revealed that BA directly inhibited the phosphorylation of VEGFR2, followed by inhibiting the activations of its downstream protein kinases, including ERK, p38, FAK, AKT, and expression of iNOS, but had no effect on COX2. Additionally, BA could also down-regulate VEGF secretion in A549 cancer cells, which may correlate with the suppression of ERK, AKT activation, indicating that BA inhibits tumor angiogenesis and tumor growth through VEGFR2 signaling pathways. These findings suggest that BA may be a novel candidate in inhibiting tumor angiogenesis and NSCLC tumor growth.

Anti-inflammatory and anti-angiogenic effects of flavonoids isolated from Lycium barbarum Linnaeus on human umbilical vein endothelial cells

Anti-inflammatory and anti-angiogenic effects of flavonoids isolated from Lycium barbarum fruits, a traditional Chinese medicine, on human umbilical vein endothelial cells (HUVECs) were investigated. Initially, flavonoids were extracted with 80% ethanol and separated using a Cosmosil 140 C18-OPN column, with the acidic fraction eluted with deionized water being composed of chlorogenic acid, caffeoyl quinic acid, caffeic acid and p-coumaric acid and the neutral fraction eluted with methanol composed of quercetin-diglycoside, rutin and kaempferol-O-rutinoside. Flavonoid extract was effective in inhibiting expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) induced by TNF-α in HUVECs. The RT-PCR analysis indicated that ICAM-1 mRNA induced by TNF-α was inhibited by flavonoid extract. The flavonoid extract attenuated TNF-α-induced IκB phosphorylation as well as NF-κB, p65 and p50 translocation from cytosol to nucleus, through inhibition on TNF-α- and H(2)O(2)-induced intracellular reactive oxygen species (ROS) production. For the anti-angiogenic study, the flavonoid extract inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation and migration in HUVECs, as well as angiogenesis. However, the flavonoid extract did not inhibit VEGF signaling. Surprisingly, HUVECs adhesion to the extracellular matrix was compromised and adhesion-induced signaling was retarded by the flavonoid extract.

Melatonin prevents human pancreatic carcinoma cell PANC‐1‐induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression

Melatonin is an important natural oncostatic agent, and our previous studies have found its inhibitory action on tumor angiogenesis, but the mechanism remains unclear. It is well known that vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis and has become an important target for antitumor therapy. Pancreatic cancer is a representative of the most highly vascularized and angiogenic solid tumors, which responds poorly to chemotherapy and radiation. Thus, seeking new treatment strategies targeting which have anti-angiogenic capability is urgent in clinical practice. In this study, a co-culture system between human umbilical vein endothelial cells (HUVECs) and pancreatic carcinoma cells (PANC-1) was used to investigate the direct effect of melatonin on the tumor angiogenesis and its possible action on VEGF expression. We found HUVECs exhibited an increased cell proliferation and cell migration when co-cultured with PANC-1 cells, but the process was prevented when melatonin added to the incubation medium. Melatonin at concentrations of 1 μm and 1 mm inhibited the cell proliferation and migration of HUVECs and also decreased both the VEGF protein secreted to the cultured medium and the protein produced by the PANC-1 cells. In addition, the VEGF mRNA expression was also down-regulated by melatonin. Taken together, our present study shows that melatonin at pharmacological concentrations inhibited the elevated cell proliferation and cell migration of HUVECs stimulated by co-culturing them with PANC-1 cells; this was associated with a suppression of VEGF expression in PANC-1 cells.

Dihydroartemisinin inhibits angiogenesis in pancreatic cancer by targeting the NF-κB pathway

Dihydroartemisinin (DHA) has recently shown antitumor activity in human pancreatic cancer cells. However, its effect on antiangiogenic activity in pancreatic cancer is unknown, and the mechanism is unclear. This study was aimed to investigate whether DHA would inhibit angiogenesis in human pancreatic cancer. Cell viability and proliferation, tube formation of human umbilical vein endothelial cells (HUVECs), nuclear factor (NF)-κB DNA-binding activity, expressions of vascular endothelial growth factor (VEGF), interleukin (IL)-8, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9 were examined in vitro. The effect of DHA on antiangiogenic activity in pancreatic cancer was also assessed using BxPC-3 xenografts subcutaneously established in BALB/c nude mice. DHA inhibited cell proliferation and tube formation of HUVECs in a time- and dose-dependent manner and also reduced cell viability in pancreatic cancer cells. DHA significantly inhibited NF-κB DNA-binding activity, so as to tremendously decrease the expression of NF-κB-targeted proangiogenic gene products: VEGF, IL-8, COX-2, and MMP-9 in vitro. In vivo studies, DHA remarkably reduced tumor volume, decreased microvessel density, and down-regulated the expression of NF-κB-related proangiogenic gene products. Inhibition of NF-κB activation is one of the mechanisms that DHA inhibits angiogenesis in human pancreatic cancer. We also suggest that DHA could be developed as a novel agent against pancreatic cancer.

Metronomic docetaxel chemotherapy inhibits angiogenesis and tumor growth in a gastric cancer model

Low-dose metronomic (LDM) chemotherapy represents a new strategy to treat solid tumors by stronger antiangiogenic activity and less side effects. The aim of the study is to rationally develop a docetaxel metronomic regimen in preclinical settings of gastric cancer. In vitro cell proliferation, apoptosis, and thrombospondin-1/vascular endothelial growth factor (TSP-1/VEGF) expression analyses were performed on human umbilical vein endothelial cells (HUVECs) and gastric cancer (BGC-823) cells exposed for 144h to metronomic concentrations of docetaxel. BGC-823 human gastric cancer xenograft model was used, and tumor growth and side effects were closely monitored. Quantitative real-time PCR was used to determine TSP-1/VEGF mRNA levels in tumor samples. Expression of VEGF and CD31 was observed by immunohistochemistry. Our results indicated that LDM docetaxel preferentially inhibited HUVEC cell proliferation and induced HUVEC apoptosis. Docetaxel significantly increased TSP-1 expression and secretion in HUVEC cells whereas the expression and secretion of VEGF significantly decreased in BGC-823 cells. LDM docetaxel significantly inhibited BGC-823 tumor growth in the absence of toxicity, which was accompanied by decreases in microvessel density (MVD) and VEGF and increases in TSP-1 gene expression in tumor tissues. In vitro results show the antiangiogenic properties of LDM docetaxel. In vivo, LDM docetaxel treatment is effective against gastric tumor and microvessel growth without toxic effect on nude mice.

Formononetin accelerates wound repair by the regulation of early growth response factor-1 transcription factor through the phosphorylation of the ERK and p38 MAPK pathways

Formononetin, a phytoestrogen from the root of Astragalus membranaceus, is used as a blood enhancer and to improve blood microcirculation in complementary and alternative medicine. The present study investigated the influence of formononetin on the expression of early growth response factor-1 (Egr-1) and growth factors contributing to wound healing. Formononetin significantly increased growth factors such as transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) in human umbilical vein endothelial cells (HUVECs). Formononetin also increased the expression of Egr-1 transcription factor by 3.2- and 10.5-fold, compared with recombinant VEGF 125 in HUVECs. The formononetin-mediated 12%–43% increase induced endothelial cell proliferation and recovered the migration of wounded HUVECs. In an ex vivo angiogenesis assay, formononetin produced a larger capillary sprouting area than produced using recombinant VEGF 125. Cell proliferation and migration of HUVECs were also greater in the presence of formonectin than VEGF 125. Western blot analysis of scratch-wounded confluent HUVECs showed that formononetin induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slightly inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The formononetin-mediated sustained activation of Egr-1 was suppressed by the ERK inhibitor PD98059 and the p38 inhibitor SB203580. PD98059 inhibited the formononetin-induced endothelial proliferation and repair in scratch-wounded HUVECs, SB203580 increased the cell proliferation and wound healing. Formononetin accelerate wound closure rate as early as day 3 after surgery and consistently observed until day 10 after in wound animal model. These data suggest that formononetin promotes endothelial repair and wound healing in a process involving the over-expression of Egr-1 transcription factor through the regulation of the ERK1/2 and p38 MAPK pathways.

Anti-angiogenic/proliferative behavior of a “4-aryl-4H-chromene” on blood vessel’s endothelial cells: A possible evidence on dual “anti-tumor” activity

Development of therapies aimed at inhibiting the angiogenesis, in combination with classical anti-cancer therapies, is among the most intensively studied approaches to the treatment of cancer. As a continuation of the efforts to discover and develop “4-aryl-4H-chromenes” as novel anti-cancer agents, in this study, we have been synthesizing (and examining) these compounds that appear to inhibit metastasis and, in particular, angiogenesis by using successful approaches such as three-dimensional capillary tube formation as well as matrix metalloproteinase (MMP) gelatinase assay in the endothelial cell-based experimental system. Anti-angiogenic and anti-proliferative effects of chromene compound 1 were especially checked on three-dimensional culture of human umbilical vein endothelial cells (HUVECs) in collagen matrix and HUVECs proliferation assay, respectively. Compound 1 was identified to be a highly potent anti-angiogenic agent at well-tolerated concentrations. In mechanistic studies, baseline MMP activities were inhibited in the presence of compound 1, in a dose-dependent manner. Based on our data, there is this possibility that chromene compounds (especially 1) are useful in treatment of some cancers because of their ability to both induce cancer cell apoptosis and reduce the stimulatory factors involved in HUVEC cell proliferation and angiogenesis.

Antiangiogenic and antitumor activity of LP-261, a novel oral tubulin binding agent, alone and in combination with bevacizumab.

LP-261 is a novel tubulin targeting anticancer agent that binds at the colchicine site on tubulin, inducing G2/M arrest. Screening in the NCI60 cancer cell lines resulted in a mean GI50 of approximately 100 nM. Here, we report the results of testing in multiple mouse xenograft models and angiogenesis assays, along with bioavailability studies. To determine the antiangiogenic activity of LP-261, both in vitro and ex vivo experiments were performed. Human umbilical vein endothelial cells (HUVECs) were incubated with LP-261 at 50 nM to 10 μM. LP-261 was also tested in a rat aortic ring assay, from 20 nM to 10 μM. Multiple mouse xenograft studies were performed to assess in vivo antitumor activity. LP-261 was tested as a single agent in colon adenocarcinoma (SW620) and prostate cancer (LNCaP and PC3) xenografts, evaluating several different dosing schedules. LP-261 was also used in combination with bevacizumab in the SW620 xenograft model. LP-261 also exhibited high oral bioavailability and apparent lack of efflux by intestinal transporters such as ABCB1. LP-261 is a very potent inhibitor of angiogenesis, preventing microvessel outgrowth in the rat aortic ring assay and HUVEC cell proliferation at nanomolar concentrations. Complete inhibition of tumor growth was achieved in the PC3 xenograft model and shown to be schedule dependent. Excellent inhibition of tumor growth in the SW620 model was observed, comparable with paclitaxel. Combining oral, low dose LP-261 with bevacizumab led to significantly improved tumor inhibition. Oral LP-261 is very effective at inhibiting tumor growth in multiple mouse xenograft models and is well tolerated.

Abstract 3571: The Thioredoxin Reductase Inhibitor Chaetocin has Potent Antineoplastic Effects in Solid Tumors

Abstract Background: We had previously reported that the natural product chaetocin has potent and selective in vitro, ex vivo and in vivo anti-myeloma activity attributable to the imposition of cellular oxidative stress (ROS) in part mediated via competitive inhibition of the redox enzyme thioredoxin reductase. Having also observed chaetocin-induced cytotoxicity in solid tumor cell lines, we now extend prior work to characterize the effects of chaetocin in solid tumor cell lines and in human umbilical vein endothelial cells (HUVECs). Methods: The effects of chaetocin in solid tumor cell lines were assessed using colony forming assays, trypan blue exclusion assays, apoptosis and autophagy assays, electron microscopy, transcriptional profiling, and the National Cancer Institute 60 cell line screen. Results: Chaetocin demonstrated potent anti-cancer activity in all assessed solid tumor cell lines with IC50 values between 2-10 nM (24 h exposures, colony forming assays). While apoptosis was induced in a cell line-dependant fashion, it was not required for chaetocin-induced cytotoxicity, as ZVAD-fmk prevented apoptosis but not cell death. Markers of autophagy were not altered by chaetocin treatment. Interestingly, results form the NCI 60 cell line screen showed that hematological cell lines were generally more resistant to chaetocin than solid tumor lines despite our prior report indicating the activity of chaetocin in myeloma. Transcriptional profiling results were consistent with those anticipated from an agent producing cell death via imposition of cellular ROS, with heme oxidase-1 prominently induced along with other transcripts in pathways related to inflammatory response and cell death. Results from OxyBlot protein oxidation kit analyses (Millipore, Billerica, MA) confirmed a generalized increase in the carbonyl modification of proteins, a hallmark of cellular oxidative damage, in response to chaetocin treatment. Experiments using Rho0 mitochondrial inactive cells indicated that cellular ROS is induced by chaetocin independent of respiratory functional mitochondria. Chaetocin was also shown to block the interleukin-, fibroblast growth factor- or EGM2 media-induced proliferation of HUVEC cells at low-nanomolar concentrations. Conclusions: Chaetocin has wide-ranging antineoplastic activity across not only hematological, but also solid tumor, cell lines and displays evidence of antiangiogentic activity in HUVEC proliferation assays. Overall, chaetocin appears to be an attractive agent for further development as a candidate anti-cancer therapeutic in a variety of neoplasms. Supported in part by CA125750. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3571.

Moscatilin, a bibenzyl derivative from the India orchid Dendrobrium loddigesii, suppresses tumor angiogenesis and growth in vitro and in vivo

Attacking angiogenesis is considered an effective strategy for controls the expansion and metastasis of tumors and other related-diseases. The aim of this study was to assess the effects of moscatilin, a bibenzyl derivative, on VEGF and bFGF-induced angiogenesis in cultured human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Moscatilin significantly inhibited growth of lung cancer cell line A549 (NSCLC) and suppressed growth factor-induced neovascularization. In addition, VEGF- and bFGF-induced cell proliferation, migration, and tube formation of HUVECs was markedly inhibited by moscatilin. Western blotting analysis of cell signaling molecules indicated that moscatilin inhibited ERK1/2, Akt, and eNOS signaling pathways in HUVECs. These results suggest that inhibition of angiogenesis by moscatilin may be a major mechanism in cancer therapy.

Influence of Delta-like ligand 4/Notch signal transduction pathway upon the biological behavior of human umbilical vein endothelial cells

To study the influence of inhibiting Delta-like ligand 4 (Dll4)/Notch signal transduction pathway upon the biological behavior of human umbilical vein endothelial cells (HUVEC). Used rAAV vectors expressing an active small interfering RNA (siRNA) (vector 6) targeting the Dll4 (rAAV-Dll4-shRNA) to infect HUVEC. And an empty plasmid (rAAV-EGFP) was infected into the same cell line as control group. The stable transfection and expression of Dll4 mRNA in HUVEC were determined by semi-quantitative RT-PCR. The protein expression of Dll4 was examined by Western blotting. Distribution of cell cycle was assessed by flow cytometry. The cell growth was analyzed by MTT assay. HUVEC were separated by type I collagen and cultured in a three-dimensional culture system for tubule like structure (TLS) formation. Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed the expression of Dll4 mRNA (0.636 +/- 0.082, 0.972 +/- 0.022 vs 0.948 +/- 0.046) and protein (0.632 +/- 0.052, 2.016 +/- 0.048 vs 1.946 +/- 0.066) were down-regulated in the stable cell (P = 0.024, 0.033). The rAAV vectors expressing an active small interfering RNA (siRNA) targeting the Dll4 effectively stimulated HUVEC cell growth and proliferation while empty plasmid had no such specific effect. The proliferation index of experimental group was (39.9 +/- 2.2)% versus untreated group (25.7 +/- 4.5)% (P = 0.036). TLS formation was significantly induced by rAAV vector. And the average length of TLS were more than those of control group (12.5 +/- 0.5, 8.7 +/- 7.7, 8.5 +/- 3.0, P = 0.028). The inhibiting Dll4/Notch signal transduction pathway stimulates the proliferation of HUVEC and facilitates the angiogenesis. Interference with Dll4/Notch signaling may be particularly desirable in tumors with highly induced Dll4/Notch pathway.

Proliferating and quiescent human umbilical vein endothelial cells (HUVECs): a potential in vitro model to evaluate contrast agents for molecular imaging of angiogenesis

The design of highly specific contrast agents for molecular imaging of angiogenesis requires the availability of adequate in vitro models. In this context, we investigated the applicability of a potential in vitro model based on human umbilical vein endothelial cells (HUVECs) mimicking physiological and angiogenic vasculature. HUVECs in supplemented medium were used to mimic proliferating neovasculature (stimulated HUVECs), whereas quiescent non-proliferating endothelium was modeled by alteration of medium supplements (unstimulated HUVECs). The features of both culture subsets were compared with features of angiogenic and physiological vessels in vivo described in the literature using different techniques. Testing of the cell model was performed by specific labeling of CD105 and VEGFR2 with fluorophores and consecutive imaging using a planar near-infrared fluorescence (NIRF) imager. Light microscopy revealed tubular alignment of unstimulated HUVECs, which was absent in stimulated HUVECs. Proliferation assay confirmed a high level of proliferation in stimulated HUVECs but almost no cell proliferation in unstimulated HUVECs. Flow cytometry revealed an up-regulation of CD105, but not of VEGFR2 on stimulated HUVECs. CD105 and VEGFR2 gene expression was detectable both in proliferating and in non-proliferating cells. NIRF-imaging revealed highest fluorescence signal for CD105 in proliferating endothelial cells. No relevant fluorescence signal could be observed for VEGFR2. The established cell model exhibits features of physiological and angiogenic vasculature. NIRF-imaging using the proposed model was feasible. We conclude that the presented cell model might be useful in future angiogenesis applications, like evaluating new fluorophores and other contrast media.