• Laboraroty Animal Research
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• v.34 no.4
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• pp.248-256
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• 2018

O-2-$^{18}F$-fluoroethyl-l-tyrosine ($[^{18}F]FET$) has been widely used for glioblastomas (GBM) in clinical practice, although evaluation of its applicability in non-clinical research is still lacking. The objective of this study was to examine the value of $[^{18}F]FET$ for treatment evaluation and prognosis prediction of anti-angiogenic drug in an orthotopic mouse model of GBM. Human U87MG cells were implanted into nude mice and then bevacizumab, a representative anti-angiogenic drug, was administered. We monitored the effect of anti-angiogenic agents using multiple imaging modalities, including bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET/CT). Among these imaging methods analyzed, only $[^{18}F]FET$ uptake showed a statistically significant decrease in the treatment group compared to the control group (P=0.02 and P=0.03 at 5 and 20 mg/kg, respectively). This indicates that $[^{18}F]FET$ PET is a sensitive method to monitor the response of GBM bearing mice to anti-angiogenic drug. Moreover, $[^{18}F]FET$ uptake was confirmed to be a significant parameter for predicting the prognosis of anti-angiogenic drug (P=0.041 and P=0.007, on Days 7 and 12, respectively, on Pearson's correlation; P=0.048 and P=0.030, on Days 7 and 12, respectively, on Cox regression analysis). However, results of BLI or MRI were not significantly associated with survival time. In conclusion, this study suggests that $[^{18}F]FET$ PET imaging is a pertinent imaging modality for sensitive monitoring and accurate prediction of treatment response to anti-angiogenic agents in an orthotopic model of GBM.

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.656-662
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• 2001

Angiogenesis is known as a crucial process in the growth and spreading of tumor cells. Accordingly, the effective inhibition of this process would appear to be a promising way to cure angiogenesis-related diseases, including cancer. This study demonstrates that acalycixenolide E (AX-E) from the marine organism Acalycigorgia inermis exhibits a potent anti-angiogenic activity both in vitro and in vivo. AX-E inhibits the bFGF-induced proliferation of HUVECs in a dose dependent manner, along with the bFGF-induced migration, invasion, and tube formation of HUVECs. Moreover, AX-E potently inhibits the in vivo neovascularization of the chorioallantoic membranes (CAMs) of growing chick embryos. interestingly, AX-E suppresses the expression of metalloproteases 2 and 9, yet shows no effect on their activities. The novel chemical structure and potent anti-angiogenic activity of AX-E will be of great value in elucidating the molecular mechanism of angiogenesis as well as in the development of a novel anti-angiogenic drug.

• Biomolecules & Therapeutics
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• v.29 no.5
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• pp.506-518
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• 2021

The imprinted tumour suppressor NOEY2 is downregulated in various cancer types, including ovarian cancers. Recent data suggest that NOEY2 plays an essential role in regulating the cell cycle, angiogenesis and autophagy in tumorigenesis. However, its detailed molecular function and mechanisms in ovarian tumours remain unclear. In this report, we initially demonstrated the inhibitory effect of NOEY2 on tumour growth by utilising a xenograft tumour model. NOEY2 attenuated the cell growth approximately fourfold and significantly reduced tumour vascularity. NOEY2 inhibited the phosphorylation of the signalling components downstream of phosphatidylinositol-3'-kinase (PI3K), including phosphoinositide-dependent protein kinase 1 (PDK-1), tuberous sclerosis complex 2 (TSC-2) and p70 ribosomal protein S6 kinase (p70S6K), during ovarian tumour progression via direct binding to vascular endothelial growth factor receptor-2 (VEGFR-2). Particularly, the N-terminal domain of NOEY2 (NOEY2-N) had a potent anti-angiogenic activity and dramatically downregulated VEGF and hypoxia-inducible factor-1α (HIF-1α), key regulators of angiogenesis. Since no X-ray or nuclear magnetic resonance structures is available for NOEY2, we constructed the three-dimensional structure of this protein via molecular modelling methods, such as homology modelling and molecular dynamic simulations. Thereby, Lys15 and Arg16 appeared as key residues in the N-terminal domain. We also found that NOEY2-N acts as a potent inhibitor of tumorigenesis and angiogenesis. These findings provide convincing evidence that NOEY2-N regulates endothelial cell function and angiogenesis by interrupting the VEGFR-2/PDK-1/GSK-3β signal transduction and thus strongly suggest that NOEY2-N might serve as a novel anti-tumour and anti-angiogenic agent against many diseases, including ovarian cancer.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.117-125
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• 2019

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.6
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• pp.705-712
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• 2018

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). In vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a $2{\times}2$ stitched area from the $4{\times}$ object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, $IC_{50}$ values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

• BMB Reports
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• v.47 no.4
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• pp.227-232
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• 2014

Histone deacetylase-3 (HDAC3) is involved in cellular proliferation, apoptosis and transcriptional repression. However, the role of HDAC3 in angiogenesis remains unknown. HDAC3 negatively regulated the expression of angiogenic factors, such as VEGF and plasminogen activator inhibitor-1 (PAI-1). HDAC3 showed binding to promoter sequences of PAI-1. HDAC3 activity was necessary for the expression regulation of PAI-1 by HDAC3. VEGF decreased the expression of HDAC3, and the down-regulation of HDAC3 enhanced endothelial cell tube formation. HDAC3 negatively regulated tumor-induced angiogenic potential. We show the novel role of HDAC3 as a negative regulator of angiogenesis.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.6
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• pp.1602-1607
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• 2004

3-O-D-galactopyranosylglyceride (GPG; fatty acids R1, R2 = myristic acid 11.62%, palmitic acid 61.90% and oleic acid 26.48%) was isolated from Chrysanthemum coronarium L that has been used for treating renal and cardiovascular diseases as one of vegetables or medicinal drug. However, little was known about the anti-angiogenic activity of GPG. Thus, anti-angiogenic effect of GPG was evaluated in human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. GPG effectively inhibited bFGF-induced migration and invasion of HUVECs in a concentration-dependent manner, whereas it did not inhibit bFGF-induced proliferation and capillary-like tube formation of HUVECs. To examine the mechanism of anti-angiogenic activity of GPG, gelatin zymography was carried out. GPG downregulated the expression of matrix metalloproteinase-2 in a concentration-dependent manner. Furthermore, GPG significantly disrupted bFGF-induced neovascularization on the chick chorioallantoic membrane assay in vivo. These results suggest that 3-O--D-galactopyranosylglyceride may inhibit neovascularization by inhibiting angiogenic activity of endothelial cells via regulation of matrix metalloproteinase-2 (MMP-2).

• Molecules and Cells
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• v.39 no.3
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• pp.229-241
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• 2016

We have previously reported the role of miR-326-HDAC3 loop in anti-cancer drug-resistance. CAGE, a cancer/testis antigen, regulates the response to anti-cancer drug-resistance by forming a negative feedback loop with miR-200b. Studies investigating the relationship between CAGE and HDAC3 revealed that HDAC3 negatively regulated the expression of CAGE. ChIP assays demonstrated the binding of HDAC3 to the promoter sequences of CAGE. However, CAGE did not affect the expression of HDAC3. We also found that EGFR signaling regulated the expressions of HDAC3 and CAGE. Anti-cancer drug-resistant cancer cell lines show an increased expression of $pEGFR^{Y845}$. HDAC3 was found to negatively regulate the expression of $pEGFR^{Y845}$. CAGE showed an interaction and co-localization with EGFR. It was seen that miR-326, a negative regulator of HDAC3, regulated the expression of CAGE, $pEGFR^{Y845}$, and the interaction between CAGE and EGFR. miR-326 inhibitor induced the binding of HDAC3 to the promoter sequences in anti-cancer drug-resistant $Malme3M^R$ cells, decreasing the tumorigenic potential of $Malme3M^R$ cells in a manner associated with its effect on the expression of HDAC3, CAGE and $pEGFR^{Y845}$. The down-regulation of HDAC3 enhanced the tumorigenic, angiogenic and invasion potential of the anti-cancer drug-sensitive Malme3M cells in CAGE-dependent manner. Studies revealed that $PKC{\delta}$ was responsible for the increased expression of $pEGFR^{Y845}$ and CAGE in $Malme3M^R$ cells. CAGE showed an interaction with $PKC{\delta}$ in $Malme3M^R$ cells. Our results show that HDAC3-CAGE axis can be employed as a target for overcoming resistance to EGFR inhibitors.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7651-7656
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• 2013

Background: Breast cancer is the most common malignancy among women in both developed and developing countries. The burden is increasing in low-income and middle-income countries (LMCs) and threatens the public health of such societies. Introduction of expensive monoclonal antibodies to cancer treatment regimens poses a real challenge in the health systems of LMCs. Despite controversy of cost-effectiveness of bevacizumab in breast cancer, some studies indicate gain of patients from this drug. The present study aimed to propose a priority setting model for administration of anti-angiogenic agents in breast cancer via assessment of tumor angiogenesis by the microvessel density (MVD) method and associations with clinicopathological characteristics (including simultaneous mutations of TP53 and HER-2 genes). Materials and Methods: Age, axillary lymph nodes status, tumor size, stage and grade, estrogen and progesterone receptors status, HER-2/neu status (by immunohistochemistry and FISH test), TP53 mutation, Ki-67 (for proliferation assay) and CD34 (for angiogenesis assay) were assessed in 111 breast cancer patients. The molecular subtype of each tumor was also determined and correlations of simultaneous mutations of HER-2 and p53 genes with angiogenesis and other clinicopathological characteristics were evaluated. Results: There were significant associations between simultaneous mutations of HER-2 and p53 genes and all other parameters except tumor size. The degree of angiogenesis in the ERBB2 subtype was greater than the others. Younger patients showed a higher angiogenesis rate rather those older than 50 years. Conclusions: Our results demonstrated that patients with simultaneous mutations of HER-2 and p53 genes, those with ERBB2 molecular subtype and also younger women (often triple negative) seem more eligible for obtaining anti-angiogenic agents. These results suggest a model for priority setting of patients with breast cancer for treatment with anti-angiogenic drugs in LMCs.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.6
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• pp.1608-1612
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• 2004

This study was conducted to investigate angiogenic inhibitory effect of Zingiberis Rhizoma methanol extract using ECV-304 cells and HT1080 fibrosarcoma cells. The viability of ECV-304 was 30% at 50㎍/㎖ of Zingiberis extract and that of HT1080 was 30% at 100㎍/㎖. Using the BrdU incorporation assay, Zingiberis inhibited the DNA synthesis of ECV-304 and HT1080 by 70% and 50% at 200㎍/㎖. In tube formation assay, at 10㎍/㎖ of Zingiberis, tube network began to degrade and at higher doses, it was completely destroyed. Zymography demonstrated that Zingiberis extract decreased MMP-9 at 10㎍/㎖ and higher doses remarkably inhibited the expression of MMP-9. These data indicate that Zingiberis Rhizoma has angiogenic inhibitory effects and shows the possibility of future anti-metastatic drug.