Angiogenesis In Chick Chorioallantoic Membrane Research Articles

HIF1α Counteracts TGFβ1-Driven TSP1 Expression in Endothelial Cells to Stimulate Angiogenesis in the Hypoxic Tumor Microenvironment.

Emerging evidence suggests that transforming growth factor β1 (TGFβ1) can inhibit angiogenesis, contradicting the coexistence of active angiogenesis and high abundance of TGFβ1 in the tumor microenvironment. Here, we investigated how tumors overcome the anti-angiogenic effect of TGFβ1. TGFβ1 treatment suppressed physiological angiogenesis in chick chorioallantoic membrane and zebrafish models but did not affect angiogenesis in mouse hepatoma xenografts. The suppressive effect of TGFβ1 on angiogenesis was recovered in mouse xenografts by a hypoxia-inducible factor 1α (HIF1α) inhibitor. In contrast, a HIF1α stabilizer abrogated angiogenesis in zebrafish, indicating that hypoxia may attenuate the anti-angiogenic role of TGFβ1. Under normoxic conditions, TGFβ1 inhibited angiogenesis by upregulating anti-angiogenic factor thrombospondin 1 (TSP1) in endothelial cells (ECs) via TGFβ type I receptor (TGFβR1)-SMAD2/3 signaling. In a hypoxic microenvironment, HIF1α induced microRNA-145 (miR145) expression; miR145 abolished the inhibitory effect of TGFβ1 on angiogenesis by binding and repressing SMAD2/3 expression and subsequently reducing TSP1 levels in ECs. Primary ECs isolated from human hepatocellular carcinoma (HCC) displayed increased miR145 and decreased SMAD3 and TSP1 compared to ECs from adjacent non-tumor livers. The reduced SMAD3 or TSP1 in ECs was associated with increased angiogenesis in HCC tissues. Collectively, this study identified that TGFβ1-TGFβR1-SMAD2/3-TSP1 signaling in ECs inhibits angiogenesis. This inhibition can be circumvented by a hypoxia-HIF1α-miR145 axis, elucidating a mechanism by which hypoxia promotes angiogenesis.

Studying the Efficacy of Copper Oxide Loaded Graphitic Carbon Nitride Nanosheets on VEGF-Induced Angiogenesis in Chick Chorioallantoic Membrane (CAM Assay) In Vivo Studies and Cytotoxicity in Human Hep3B Liver Cancer Cell Lines

Studying the Efficacy of Copper Oxide Loaded Graphitic Carbon Nitride Nanosheets on VEGF-Induced Angiogenesis in Chick Chorioallantoic Membrane (CAM Assay) In Vivo Studies and Cytotoxicity in Human Hep3B Liver Cancer Cell Lines

Moraea sisyrinchium inhibits proliferation, cell cycle, and migration of cancerous cells, and decreases angiogenesis in chick chorioallantoic membrane.

Experimental studies reported that some plants in the genus of Moraea (Iridaceae family) show anticancer potential. This study aimed to evaluate the effects of Moraea sisyrinchium on U87 glioblastoma multiforme and HepG2 liver cancer cells. The cells were incubated for 24 hr with hydroalcoholic extract of the stem, flower, and bulb of M. sisyrinchium. Then, the cell proliferation (MTT) assay, cell cycle analysis (propidium iodide staining), cell migration test (scratch), Western blotting (Bax and Bcl-2 expression), and gelatin zymography (for matrix metalloproteinases, MMPs) were performed. Oxidative stress was evaluated by determining the levels of reactive oxygen species and lipid peroxidation. Angiogenesis was evaluated on chick embryo chorioallantoic membrane. The extracts of the flower, stem, and bulb significantly decreased the proliferation of HepG2 and U87 cells. This effect was more for U87 than HepG2 and for the bulb and stem than the flower. In U87 cells, the bulb extract increased oxidative stress, cell cycle arrest, and the Bax/Bcl-2 ratio. Also, this extract suppressed the migration ability of HepG2 and U87 cells, which was associated with the inhibition of MMP2 activity. In addition, it significantly reduced the number and diameter of vessels in the chorioallantoic membrane. Liquid chromatography-mass spectrometry revealed the presence of xanthones (bellidifolin and mangiferin), flavonoids (quercetin and luteolin), isoflavones (iridin and tectorigenin), and phytosterols (e.g., stigmasterol) in the bulb. M. sisyrinchium bulb decreased the proliferation and survival of cancer cells by inducing oxidative stress. It also reduced the migration ability of the cells and inhibited angiogenesis.

Bioactive glasses-reinforced human amniotic membrane for tissue engineering applications

In this study, we prepared a biocomposite made of decellularized human amniotic membrane (dHAM) and mesoporous bioactive glasses (MBGs). dHAM was solubilized by using pepsin and acetic acid and then impregnated with copper- and cobalt-doped 13-93B3 MBGs (CuCo-MBGs). The prepared composition was then lyophilized to prepare biocomposite membranes. SEM displayed the homogenous presence of glass particles inside dHAM. The composites showed compatibility with fibroblast cells. dHAM + MBG/CuCo-MBG had higher migratory effects on human umbilical vein endothelial cells (HUVECs) compared to glass-free samples. The angiogenic activity of the samples was further confirmed by the chicken chorioallantoic membrane angiogenesis (CAM) assay. The incorporation of MBG and CuCo-MBG into dHAM resulted in enhancing the antibacterial activity. In conclusion, Cu/Co-doped MBGs could improve the biological functions of dHAM without any adverse significant effects in vitro. In conclusion, this composite could be considered the next generation of wound dressings after performing in vivo experiments.

Preparation and characterization of the farnesiferol C-loaded solid lipid nanoparticles decorated with folic acid-bound chitosan and evaluation of its in vitro anti-cancer and anti-angiogenic activities

Farnesiferol C (FC) has long been used as a cancer prevention agent. In the present study, FC was loaded into solid lipid nanoparticles (SLN) coated with folic acid (FA) bound chitosan (CS), FC-SLN-CS-FA, by homogenization followed by ultrasonication. Then its toxicity effect on cancer cells compared to normal cells was investigated by the MTT method. In the following, the pro-apoptotic effects of synthesized nanoparticles were evaluated by flow cytometry and acridine orange, propidium iodide, and DAPI staining, and the expression of genes involved in apoptosis was evaluated. The anti-oxidant effects of formulations have been evaluated using DPPH and ABTS assay. To investigate the anti-angiogenic effects of nanoparticles, the chicken chorioallantoic membrane (CAM) assay was used and the expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) genes was used by real-time quantitative PCR (qPCR). The formed nanoparticles with a size of 150 nm and a dispersion index of 0.30 had a surface charge of +17 mV. These nanoparticles exhibited significant growth and proliferation inhibitory effects in PANC, AGS, HT-29, MCF7, and A549 human cancer cells from the lowest concentration (15.6 µg/mL, *** p < 0.001). The flow cytometry analysis shows 10.1%, 48.8%, and 74.4% subG1 arrest at the concentrations of 6, 36, and 66 µg/mL of the FC-SLN-CS-FA, respectively. The formulation caused apoptotic cell death by 3-fold overregulating BAX at concentrations of 36 and 66 µg/mL, and 2- and 2.5-fold overexpression of caspase-9 at concentrations of 36 and 66 µg/mL respectively. The treatment with FC-SLN-CS-FA resulted in decreasing BCL2 and NF-κB gene expression. The ABTS and DPPH assays showed the anti-oxidant properties of formulation in a concentration-dependent manner. FC-SLN-CS-FA significantly inhibited angiogenesis in CAM by reducing the length and number of blood vessels. Considering the pro-apoptotic and anti-angiogenic effects of FC-SLN-CS-FA, it can be said that these nanoparticles can effectively suppress the carcinogenesis of multipotent cancer stem cells. Therefore, they have the potential to be studied as an efficient anticancer agent.

Extract of Marsdenia tenacissima (Roxb.) Moon [Apocynaceae] Suppresses Hepatocellular Carcinoma by Inhibiting Angiogenesis.

The extract of Marsdenia tenacissima (Roxb.) Moon [Apocynaceae] (MTE) has shown a significant anti-cancer effect on hepatocellular carcinoma (HCC), but its mechanism remains unclear. In this study, we used transcriptomics methods to investigate the underlying mechanism of MTE against HCC. Both MHCC97H and HepG2 cell lines were treated with MTE. The cell viability and migration were measured using the cell counting kit-8 assay and transwell assay. RNA-sequencing was used to identify differentially expressed genes (DEGs) between HepG2 cells treated with and without MTE. The expression levels of selected DEGs—vascular endothelial growth factor-A (VEGFA), platelet-derived growth factor receptor-β (PDGFRB), and von Willebrand factor (VWF)—were verified by RT-PCR and Western blot. The effect of conditioned medium from HCC cells with MTE treatment (CM-MTE) on blood vessels was observed by tube formation assay of HUVECs and chick chorioallantoic membrane (CAM) assay. A mouse model of HCC patient-derived tumor xenograft (PDX) was established and treated with MTE. The effect of MTE on the growth and angiogenesis of HCC-PDX was analyzed. The results demonstrated that MTE inhibited the viability and migration of HCC cells. RNA-seq showed that MTE treatment downregulated multiple genes associated with metabolism and angiogenesis. The expression levels of VEGFA, VWF, PDGFB, and PDGFRB in HCC cells were significantly suppressed by MTE. Meanwhile, MTE effectively inhibited the tube-forming capability of HUVECs and the angiogenesis of chick CAM. In vivo experiments revealed that the extract reduced tumor volume, inhibited the proliferation of HCC cells, and expanded the necrotic area of the tumor. Immunohistochemical results showed that the expression levels of CD31, PDGFB, VEGF, VWF, and PDGFRB in the HCC-PDX tumor tissues were all downregulated by MTE in a dose-dependent manner. Taken together, MTE could inhibit angiogenesis by repressing the expression of VEGF, VWF, PDGF, and PDGFRB in HCC cells, a mechanism that may enable MTE to counter HCC development.

Abstract 5968: Ethnodyne Visio™ affects EGFR signaling in breast cancer

Abstract Breast Cancer (BC), the most common form of female cancer, is one of the most extensively studied diseases. Metastasis is the process by which a cancer cell travels to another part of the body and starts to multiply. This detrimental occurrence is one of the primary forms of disease relapse in cancer patients and accounts for an estimated 90 percent of cancer deaths. Ethnodyne Visio™ is a new drug for Age-Related Macular Degeneration (AMD), the primary cause for blindness in people 65 and older. It is a mixture of phytochemicals Withaferin A, Gallic Acid, and Bacopaside I. However, the exact mechanism of action of Ethnodyne Visio™ is not known. Macular Degeneration occurs in two primary forms: wet and dry. Wet Macular Degeneration shows surprising similarities with metastatic Breast Cancer in terms of their progression and causation. As both diseases evolve, the retina (in the case of Wet AMD) and the tumor (in the case of BC) secrete Vascular Endothelial Growth Factor (VEGF) to encourage vascular development. In Wet AMD, this new and unregulated vascular growth, or angiogenesis, causes “leaky” blood vessels that allow fluid to enter the retina, ultimately causing blindness. In BC, this blood vessel growth provides a means for the tumor to obtain nutrients as well as disseminate metastatic cells. We determined if there exists any easily accessible nutraceuticals possessing effective antiangiogenic properties. We used CAMs (chick chorioallantoic membrane), grown ex-ovo, to study angiogenesis. We found that Ashwagandha (Withaferin A) blocked angiogenesis of CAMs by 67%. Taking clues from these observations, we wanted to determine possible molecular signaling mechanisms triggered by Ethnodyne Visio™ that may counter progression of BC. We hypothesized that Ethnodyne Visio™ may have anti-angiogenic properties. Using multiple online gene association platforms, we compiled a list of transcriptomic changes caused by Withaferin A, Gallic Acid, and Bacopaside I on breast tumor cells. A g:Profiler analysis of the combined transcriptomic changes revealed Epidermal Growth Factor Receptor (EGFR) signaling as a downstream target of the components of Ethnodyne Visio™. Our analysis reveals that Ethnodyne Visio™ potentially regulates VEGF signaling through the EGFR pathway. Citation Format: Rishab Rajeev Samant, Rajeev Samant. Ethnodyne Visio™ affects EGFR signaling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5968.

Possible Mechanism of Human Recombinant Leptin-Induced VEGF A Synthesis via PI3K/Akt/mTOR/S6 Kinase Signaling Pathway while Inducing Angiogenesis: An Analysis Using Chicken Chorioallantoic Membrane Model

Introduction: The present study aimed to realize human recombinant leptin ’s ability to synthesize VEGF A while inducing neovascularization through PI3K/Akt/mTOR/S6 kinase involved signaling pathway. Methods: To examine the PI3K/Akt/mTOR/S6 kinase pathway involvement in leptin-induced VEGF A synthesis, the chick chorioallantoic membrane (CAM) was incubated with human recombinant leptin and specific inhibitors of the proposed signaling molecules (rapamycin and wortmannin). We analyzed the role of specified signaling molecules in human recombinant leptin-induced physiological angiogenesis via VEGF A synthesis in detail with the support of various methodologies. Results: Human recombinant leptin’s ability to synthesize VEGF A is diminished significantly in the presence of inhibitors. This observation supported the role of PI3K/Akt/mTOR/S6 kinase signaling molecules in human recombinant leptin-mediated VEGF A synthesis while inducing angiogenesis in CAM. Conclusion: Synthesis of VEGF A, followed by the growth of new blood vessels, by human recombinant leptin via the activation of the PI3K/Akt/mTOR/S6 kinase signaling pathway reflects mechanistic therapeutic application of human recombinant leptin. The data also signify the role of mTOR and S6 kinase molecules in angiogenesis under a physiological environment.

C118P, a novel microtubule inhibitor with anti-angiogenic and vascular disrupting activities, exerts anti-tumor effects against hepatocellular carcinoma

Hepatocellular carcinoma (HCC), a hypervascular solid tumor, is the most leading cause of cancer mortality worldwide. Microtubule binding agents targeting tumor vasculature have been investigated and employed clinically. C118P is a newly synthesized analog of CA4 with improved water solubility and extended half-life. The current studies investigated the pharmacological effects of C118P and its active metabolite C118. Here, we first confirmed by in vitro assays that C118 exerts microtubule depolymerization activity and by molecular docking revealed that it fits to the colchicine binding site of tubulin. In addition, we found that C118P and C118 altered microtubule dynamics and cytoskeleton in human umbilical vein endothelial cells. Accordingly, we observed that C118P and C118 inhibited angiogenesis and disrupted established vascular networks using tube formation assays and chick chorioallantoic membrane angiogenesis assays. In addition, our data showed that C118P and C118 exhibited board anti-proliferative effect on various cancer cells, including HCC cell lines, in MTT assays or Sulforhodamine B assays. Moreover, we found that C118P induced G2/M phase cell cycle arrest and apoptosis in HCC cell lines BEL7402 and SMMC7721 using flow cytometry analysis and immunoblotting assays. Finally, we confirmed that C118P suppressed HCC growth via targeting tumor vasculature and inducing apoptosis in the SMMC7721 xenograft mouse model. In conclusion, our studies revealed that C118P, as a potent microtubule destabilizing agent, exerts its multiple pharmacological effects against HCC by inducing cell cycle arrest and apoptosis, as well as targeting tumor vasculature. Thus, C118P might be a promising drug candidate for liver cancer treatment.

The effect of RGD-targeted and non-targeted liposomal Galbanic acid on the therapeutic efficacy of pegylated liposomal doxorubicin: From liposomal preparation to in-vivo studies

The anti-cancer therapeutic application of Galbanic acid (Gba) as a strong antiangiogenic sesquiterpene coumarin has been limited due to its low water solubility. This issue necessitates developing new liposomal formulations for the efficient delivery of Gba in vivo. In this study, various liposomal formulations were prepared by a thin-film hydration method, and Gba was incorporated into the liposomal bilayers, which consequently increased its release profile compared to formulations in our previous study prepared by remote loading methods. The most stable formulation with desired properties was selected and decorated with RGD peptide (cyclo [Arg-Gly-Asp-D-Tyr-Cys]) to target tumor vasculature actively. The fluorescently-labeled model liposomes showed that the targeting could improve the receptor-mediated endocytosis of the liposomes higher than those prepared in our previous study in vitro in human umbilical vein endothelial cells (HUVECs), which was confirmed by chicken chorioallantoic membrane angiogenesis (CAM) model in vivo. Although not significant, it also could increase the accumulation of liposomes in colon tumors. In BALB/c mice bearing colon cancer, not only non-targeted Gba liposomes but also even RGD-targeted ones combinatorial therapy with pegylated liposomal doxorubicin could improve the anti-tumor efficacy as compared to their monotherapy. These outcomes have strong consequences for cancer therapy.

Effect of dual PPAR-α/γ agonist saroglitazar on diabetic retinopathy and oxygen-induced retinopathy

Diabetic retinopathy is a serious complication of diabetes, marked by retinal vascular damage, inflammation, and angiogenesis. This study's objective was to assess the potential benefits of saroglitazar, a peroxisome proliferator-activated receptor-alpha/gamma (PPAR-α/γ) agonist in diabetic retinopathy. Diabetic retinopathy was induced by streptozotocin in Sprague Dawley rats. The effect of saroglitazar was also assessed in the oxygen-induced retinopathy model in newborn rats and VEGF-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay.Treatment of saroglitazar (1 and 4 mg/kg, oral) for 12 weeks significantly ameliorated retinal vascular leakage and leukostasis in the diabetic rats. Saroglitazar decreased oxidative stress, VEGF receptor signalling, NF-κBp65, and ICAM-1 in the retina of diabetic rats. The beneficial effects of saroglitazar (1 and 4 mg/kg, oral) were also observed on the neovascularization in oxygen-induced retinopathy in newborn rats. Saroglitazar also reduced VEGF-induced angiogenesis in CAM assay.This study reveals that saroglitazar has the potential to prevent the progression of retinopathy in diabetic patients.

Inhibition of neovascularisation in human endothelial cells using anti NRP-1 nanobody fused to truncated form of diphtheria toxin as a novel immunotoxin

Neuropilin-1 (NRP-1) regulates a range of physiological and pathological processes, including angiogenesis. Targeting of NRP1 is considered a significant approach in cancer therapy. In the present study, a novel antiNRP1 immunotoxin (αNRP1 IT) was developed by genetic fusion of a single domain (VHH) anti-NRP-1 antibody fragment to a truncated diphtheria toxin. The αNRP1 IT was expressed into bacterial cells as an inclusion body (IB). Expression of αNRP1 IT was confirmed by SDS-PAGE and western blotting. Recombinant αNRP1 IT was purified using nickel affinity chromatography. Toxicity and antiangiogenesis effect of αNRP1 IT was investigated both in vitro and in vivo. Results showed that αNRP1 IT significantly reduced the viability of human umbilical vein endothelial cell line (HUVEC) (p < .05). The αNRP1 IT significantly inhibited tube formation of HUVEC cells (p < .001). Furthermore, αNRP1 IT inhibited angiogenesis in Chick Chorioallantoic Membrane (CAM) Assay. These data suggest the potential of αNRP1 IT as a novel therapeutic in targeted cancer therapy.

A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth

AbstractThe goal of our study was to raise monoclonal antibodies (mAbs) against endothelial cell—surface proteins specific for tumor vasculature. Here, we describe the generation and intensive characterization of mAb AA98, including its functional properties and its antigen identification. In our study, an enhanced mAb AA98 immunoreactivity was observed on stimulated human umbilical vein endothelial cells (HUVECs). In addition, mAb AA98 showed remarkably restricted immunoreactivity against intratumoral neovasculature compared with blood vessels of normal tissues. We identified the AA98 antigen as human CD146, an adhesion molecule belonging to the immunoglobulin superfamily. Data from in vitro experiments imply structural and signaling functions for endothelial CD146; however, the role of CD146 in vivo is largely unknown. Here, we show that mAb AA98 displays antiangiogenic properties in vitro and in vivo. Proliferation and migration of HUVECs were inhibited by mAb AA98 as was angiogenesis in chicken chorioallantoic membrane (CAM) assays and tumor growth in 3 xenografted human tumor models in mice. Our data provide new insights into the function of CD146 on endothelial cells, validate CD146 as a novel target for antiangiogenic agents, and demonstrate that mAb AA98 has potential as a diagnostic and therapeutic agent in vascular and cancer biology. (Blood. 2003;102:184-191)

Synergistic interactions between resveratrol and doxorubicin inhibit angiogenesis both in vitro and in vivo.

Resveratrol is a polyphenolic compound which is found in many nutrients including grapes, peanuts, raspberries, and apples. Anti-proliferative, anti-angiogenic and apoptotic effects of resveratrol have been shown on various cancer cells. Doxorubicin is considered as one of the most effective anticancer agents and reveals its antitumor activity by induction of apoptosis and inhibition of angiogenesis. Our study reports for the first time the potent ability of resveratrol in combination with doxorubicin to inhibit angiogenesis in vitro and in vivo. The cytotoxic effect of resveratrol (1.56-100 μM), doxorubicin (0.01-0.92 µM) and their combination were analyzed in the human umbilical vein endothelial cells (HUVECs) by ATP assay. In vitro angiogenesis was evaluated using tube formation assay in HUVECs. In vivo anti-angiogenic activity was assessed in a chick chorioallantoic membrane (CAM) model using fertilized chicken eggs. All test groups were compared to thalidomide as a positive control, three concentrations of resveratrol (10-5-2.5 µg/pellet) and a 2 µg/pellet concentration of doxorubicin was examined. All data were evaluated statistically. Resveratrol and doxorubicin alone displayed inhibitory effects on angiogenesis and cell viability at higher doses. However, the combination of resveratrol and doxorubicin exhibited a significant dose-dependent inhibition of CAM angiogenesis in vivo as well as proliferation and tube formation in HUVECs compared to the positive control (±)-thalidomide. Our results suggest that resveratrol in combination with doxorubicin is a novel strategy in the prevention and treatment of angiogenesis.

STUDY OF ANGIOGENIC POTENTIAL OF MORINGA OLEIFERA LAM. ON CHICKEN CHORIOALLANTOIC MEMBRANE (CAM)

Angiogenesis plays an important role in many physiological and pathological conditions. Currently, the drugs possessing pro-angiogenic property as compared to anti-angiogenic property are limited. The primary objective of study was to find the angiogenic potential of hot methanolic extract of leaves of Moringa Oleifera Lam. The hot methanolic extract of moringa leaves was prepared using soxhlet extraction. Phytochemical analysis was done by thin layer chromatography, ultra violet and infrared spectrophotometer. The egg candling instrument was prepared indigenously to observe the angiogenesis in Chick Chorioallantoic Membrane (CAM) model of hen’s egg. The eggs obtained from Aarey Poultry, Goregaon (Mumbai) were observed for their fertility and Embryonic Development Day (EDD). The growth of embryo was observed every day till 7th day of EDD. The test sample was loaded on 8th day and the angiogenic effect was observed and photographed every day till 11th day. The PlerminTM ointment (ββ PDGF) (Platelet Derived Growth Factor) was used as positive control. Quantification of blood vessels was done with growth index [(No of BLVS11th day – no of blood vessels 8th day)/ No of BLVS11th day], Abgel© sponge assay and compared with PDGF.

In vitro combination therapy of pathologic angiogenesis using anti-vascular endothelial growth factor and anti-neuropilin-1 nanobodies.

Objective(s):Emergence of resistant tumor cells to the current therapeutics is the main hindrance in cancer treatment. Combination therapy, which mixes two or more drugs, is a way to overcome resistant problems of cancer cells to current treatments. Nanobodies are promising tools in cancer therapy due to their high affinity as well as high penetration to tumor sites. Materials and Methods:Here, the inhibitory effect of mixtures of two nanobodies (anti-vascular endothelial growth factor (VEGF) and anti-neuropilin-1 (NRP-1) nanobodies) on tube formation of human endothelial cells in vitro and ex vivo were analyzed.Results:Results showed that combination of two drugs significantly inhibited proliferation and tube formation of human endothelial cells. In addition, mixtures of two nanobodies inhibited angiogenesis in chick chorioallantoic membrane (CAM) assay efficiently compared with each individual nanobody.Conclusion:Results highlight the efficacy of combination therapy of cancer compared with mono-therapy and promises development of novel anti-cancer therapeutics based on nanobodies targeting two or more targets of tumor cells.

Synthesis and biological evaluation of N-Alkylamide derivatives as anti-tumor agents

N-Alkylamides (NAAs), derived from Anacyclus pyrethrum (L.) DC, have potential anti-tumor effects. To explore the molecular mechanism and chemo-preventive capacity of NAAs, we synthesized an NAA (H-10) and evaluated whether it could inhibit the proliferation of B16, HepG2, HeLa, and HCT116 cancer cells in 2D culture. To evaluate the antiproliferative activity of H-10 in 2D and 3D culture of BD, HepG2, HeLa, and HCT116 cells, multicellular tumor spheroids were constructed to more accurately reflect the cell tumor environment. To visualize nuclear changes related to apoptosis, Hoechst 33258 staining and propidium iodide-Annexin V double staining were performed. Compound H-10 strongly inhibited the growth of all tested cell lines. Hoechst 33258 staining and propidium iodide-Annexin V double staining revealed that H-10 did not cause morphological alterations in the nuclei and moderately induced late apoptosis only when treated at 180 μM. The strongest inhibitory effect was observed in HCT116 cells. Flow cytometry analysis indicated that treatment of HCT116 cells with compound H-10 resulted in robust cell growth arrest in G2 phase in 2D and 3D cell culture; in 3D-cultured HCT116 cells, growth arrest occurred in G1 phase. Treatment with compound H-10 also significantly suppressed angiogenesis of chick chorioallantoic membrane in vivo . Treatment with compound H-10 strongly affected clonogenic survival (in the long-term) and migration of HCT116 cells. Therefore, H-10, a compound of NAAs may be useful for treating cancer because of its anti-neoplastic effect and easy synthesis.

The Effects of (Otostegia Persica) Hydroalcoholic Extract on VEGF Receptor Gene Expression Changes in the Angiogenesis in Chick Chorioalantoic Membrane

Introduction: Angiogenesis is the formation of new vessels from early vessels that has effected in pathologic condition such as growing tumor, metastases, arthritis rheumatoid, diabetes and physiologic condition such as developmental and growing of organ regeneration, wound healing, and reproduction. Otostegia presica is a member of Laminacea family, has antioxidant and anti-apoptosis properties. The aim of this study was to investigate the effects of hidroalkoholic extract of Otostegia presica on VEGFR expression and angiogenesis in chick chorioallantoic membrane. Methods: In this research, 60 fertilized Ross eggs were used and randomly were divided into five groups, included: control, sham-exposed, and three experimental groups treated with hidroalkoholic extract of Otostegia presica doses 100,200,400 mg/kg. On 2nd day of incubation, a window was opened for eggs under sterile condition. On 8th day, a gelatin sponge with 1×4×4 diameter was placed on chorioalantoic membrane (CAM) and extract fractions with dose of 100,200,400 mg/kg were added. On 12th day, length and weight of embryos were measured and CAM photos were captured. Then number and length of vessels in special area on CAM were measured with Image J. software. Sampling was performed for assessment of VEGFR expression. Data were analyzed by t-test and ANOVA and p&lt;0.05 was considered significant. Results: Comparison between mean of length and weight of embryos and number and length of vessels in controls and sham-exposed did not show significant difference (P&gt;0.05). Mean of the number of vessels showed significant increase in all experimental groups (P&lt;0.05). VEGFR expression has increased in experimental groups rather than sham-exposed group. Conclusion: The results showed that hidroalkoholic extract of Otostegia presica has have angiogenic effects and increased VEGFR expression in all experimental groups.

Liposomal formulation of Galbanic acid improved therapeutic efficacy of pegylated liposomal Doxorubicin in mouse colon carcinoma

Galbanic acid (Gba), a sesquiterpene coumarin, with strong antiangiogenic activity could serve as an excellent anti-cancer agent. However, Gba is a poor water-solube which hampered its clinical application. In this study, a pegylated liposomal Gba (PLGba) with HSPC/Cholesterol/mPEG2000-DSPE (56.2, 38.3, 5.3% molar ratio) was developed by the thin film hydration plus extrusion and calcium acetate gradient remote loading method, to address the issue of poor Gba solubility. Moreover, an integrin-targeting ligand (RGD peptide, cyclo[Arg-Gly-Asp-D-Tyr-Cys]) was post-inserted into liposomes in order to increase Gba cell delivery. Using fluorescently-labeled model liposomes, it was found that the targeting could improve the integrin-mediated cellular uptake of the liposomes in vitro in human umbilical vein endothelial cells (HUVECs), and in vivo as evidenced by chicken chorioallantoic membrane angiogenesis (CAM) model. It also could enrich the liposome accumulation in C26 tumor. Interestingly, co-treatment with PLGba and pegylated liposomal doxorubicin (PLD, also known as Doxil®) had a synergistic and antagonistic antiproliferative effect on the C26 tumor cell line and the normal HUVEC, respectively. In C26 tumor bearing BALB/c mice, the PLGba and PLD combinatorial therapy improved the antitumor efficacy of the treatment as compared to those of single agents. This results have clear implications for cancer therapy.

Abstract 188: The oncometabolite d-2-hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway

Abstract Mutation of isocitrate dehydrogenase (IDH) 1 (R132H) and IDH2 (R172K) and induction of hypoxia in various solid tumors results in changes to metabolic profiles, including production of the d- or l-forms of 2-hydroxyglutarate (2HG) from α-ketoglutarate in aerobic metabolism in the TCA cycle. However, it is unclear whether the oncometabolite d-2HG increases angiogenesis in endothelial cells. Therefore, in this study, we analyzed the levels of various metabolites, including d-2HG, under hypoxic conditions and in IDH2R172K mutant breast cancer cells by mass spectrometry. We then further evaluated the effects of this metabolite on angiogenesis in breast cancer cells. Our results showed that treatment with d-2HG increased the levels of secreted vascular endothelial growth factor (VEGF) in cancer cells and enhanced endothelial cell proliferation in a concentration-dependent manner. Wound healing and transwell migration were significantly increased by d-2HG to a level similar to that induced by VEGF. Tube formation was significantly stimulated by d-2HG, and chick chorioallantoic membrane angiogenesis was also enhanced by d-2HG. d-2HG activated VEGF receptor (VEGFR) 2 and VEGFR2 downstream signaling, extracellular signal-regulated kinase 1/2, focal adhesion kinase, AKT, and matrix metalloproteinase (MMP) 2. Taken together, our results suggested that d-2HG induced angiogenic activity via VEGFR2 signaling and increased MMP2 activity. Citation Format: You Mie Lee, Jiyoon Seok, Soo-Hyun Yoon, Sun-Hee Lee, Jong Hwa Jung. The oncometabolite d-2-hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 188.