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Cancer is the second cause of mortality worldwide. Angiogenesis is an important process involved in the growth of primary tumors and metastasis. New approaches for controlling the cancer progression and invasiveness can be addressed by limiting the angiogenesis process. An increasingly large number of natural compounds are evaluated as angiogenesis inhibitors. The chorioallantoic membrane (CAM) assay represents an in vivo attractive experimental model for cancer and angiogenesis studies as prescreening to the murine models. Since the discovery of tumor angiogenesis, the CAM has been intensively used in cancer research. The advantages of this in vivo technique are in terms of low time-consuming, costs, and a lower number of sacrificed animals. Currently, a great number of natural compounds are being investigated for their effectiveness in controlling tumor angiogenesis. Potential reducing of angiogenesis has been investigated by our group for pentacyclic triterpenes, in various formulations, and differences in their mechanism were registered. This chapter aims to give an overview on a number of phytocompounds investigated using in vitro, murine models and the chorioallantoic membrane assay as well as to emphasize the use of CAM assay in the study of natural compounds with potential effects in malignancies.
Department of Phamacognosy, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Roxana Ghiulai*
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Ioana Zinuca Pavel
Department of Phamacognosy, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Marius Mioc
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Roxana Babuta
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Mirela Voicu
Department of Phamacology, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Dorina Coricovac
Department of Toxicology, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Corina Danciu
Department of Phamacognosy, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Cristina Dehelean
Department of Toxicology, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
Codruta Soica
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania
*Address all correspondence to: roxana.ghiulai@umft.ro
1. Introduction
Angiogenesis represents the process by which new vessels are formed from preexisting vessels [1] and has important implications associated with tumor growth and metastasis [2]. Studies have shown that neovascularization is essential for tumor survival and growth, whereas in angiogenic absent conditions, tumor may display necrosis or even apoptosis [3, 4]. The angiogenic switch represents the process in which endothelial cells are led to a rapid growth state induced by stimuli secreted by the tumor microenvironment, comprising tumor and stromal cells, extracellular matrix components, immunologic cells, fibroblasts, adipocytes, muscle cells, and pericytes [5]. The switch may also involve downregulation of endogenous inhibitors of angiogenesis such as endostatin, angiostatin, or thrombospondin.
The undergoing of tumor angiogenesis represents a four-step process [6]: (i) tissue basement membrane injury; (ii) migration of endothelial cells, activated by angiogenic factors; (iii) endothelial cell proliferation and stabilization; (iv) continuous angiogenesis induced by angiogenic factors. Therefore, key elements in the angiogenesis process are the endogenous angiogenic factors. The most relevant angiogenic activators, signal mediators, and signaling effects are represented in Figure 1.
A class of proteins that is widely responsible for tumor angiogenesis is represented by growth factors, such as the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived endothelial growth factor (PDGF), tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), placental growth factor (PGF), transforming growth factor (TGF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), angiostatin, and angiogenin [7]. However, VEGF is thought to be the main proangiogenic growth factor, because it induces all four phases of angiogenesis by augmenting vascular permeability, endothelial cell proliferation, endothelial cell migration, and capillary like tube formation [8]. Angiogenic cytokines or other growth factors such as VEGF are expressed under hypoxia conditions or by various oncogenes (e.g., mutant ras, erbB-2/HER2) [9].
As shown in Figure 1, after binding the tyrosine kinase specific domain of the receptors, multiple ways of signaling are possible for the angiogenic factors. Important molecular mechanisms involve activation of RAS/RAF1/kinase through the extracellular signal (ERK-1 și-2), inducing proliferation and differentiation; RAS/p38 mitogen-activated kinase (MAPK) and JUN/kinase 1-3 N-terminal, modulating inflammation, apoptosis, and differentiation; phosfatidyl-3-inositol kinase-1 (PI3K) and AKT dependent, regulating cell survival, mammalian receptor for rapamycin (mTOR), highly involved in proliferation and cell growth. Other inductor factors of the signaling pathways of angiogenesis are found in the cytoplasm (e.g., GAB1, SHC, SRC, PI3K, and phosfolipase γ C) [10].
VEGF and its receptors, the VEGFR family, remain intensively researched for targeting angiogenesis in different tumors. At the same time, other angiogenesis suppressing-related targets are being studied for the development of anticancer therapies for tumors resistant to anti-VEGF therapy. A number of therapeutic agents are currently in use for several malignancies: monoclonal antibodies against angiogenic growth factors (e.g., antibody against VEGF, Bevacizumab), inhibitors of angiogenic factors synthesis (e.g., mTOR inhibitor Rapamycin), and inhibitors of angiogenic factor receptors (tyrosine-kinase inhibitors, e.g., imatinib and sorafenib) [11]. Unfortunately, clinical response to the new molecular advances in cancer therapy by targeting angiogenesis is unsatisfactory. Resistance and low survival rates are signaled. New therapeutic approaches with minimal side effects are desired to act by targeting the multiple factors that are activated during tumor progression.
Based on the preventive effect that healthy diets have on the epidemiology of cancer, medicinal plants, spices, fruits, and vegetables represent an interesting source of phytochemicals. Natural compounds or even plant extracts are now considered important and accessible therapeutic or chemopreventive agents in cancer. In the search of the suitable phytocompounds to test for specific effects, virtual screening methods can be successfully applied in the selection of selective compounds for specific targets [12]. To avoid lack of selectivity, computational filtering schemes can be used [13]. Extensive studies demonstrate the high potential of plant-derived chemicals in controlling tumor angiogenesis with minimal secondary effects and drug resistance, by targeting multiple key pathways in a synergistic manner.
2. Experimental models for tumor angiogenesis: focus on the CAM assay
An important issue in angiogenesis studies is the appropriate choice of the assays. To evaluate the efficacy of potential phytocompounds and to identify potential targets within the angiogenic process, several methods both in vitro and in vivo can be applied. Each of them having one or more drawbacks, ideally more techniques are to be applied. In vitro techniques are used by co-culturing endothelial cell and other tumor microenvironment factors with tumor cells in 2D or even 3D models which facilitate the identification of the involved molecular mechanisms. Despite the advances made in the direction of designing in vitro assays, the in vivo environment can be difficultly reproduced with such protocols [14]. To better assess the key aspects of tumor angiogenesis and therapeutic approaches, in vivo assays can be applied, such as the chick chorioallantoic membrane (CAM), the zebrafish, the sponge implantation, the corneal, or dorsal air sac and tumor angiogenesis models in rodents or rabbits [15]. Several drawbacks can still be cited, especially for the murine models, including high costs, complex technical and surgical abilities, and important quantities of test compounds.
2.1. Chorioallantoic membrane assay
The chorioallantoic membrane (CAM) assay represents an attractive in vivo experimental model for angiogenesis and cancer studies. The advantages of this in vivo technique in terms of costs, time, simplicity, reproducibility, and ease of the approval by the ethic committee make it a good prescreening assay to murine models in the research of biological systems and new therapeutic targets. Especially tumor angiogenesis and metastasis protocols benefit for a much shorter time for the tumor to grow and metastasize than the classical animal models.
The limitations of the model include a restricted number of reagents to work with due to low compatibility, nonspecific inflammatory reactions, keratinization of the membrane, and a vascular reaction that interferes with the visualization of vascular modifications. Technical skills may be significant to counteract these limitations [16, 17].
The chorioallantoic membrane is the vascularized respiratory extraembryonic tissue of avian species. First, this biologic system has been used for embryologic, immunological, and tumor grafting studies [18], and more recently, since the discovery of tumor angiogenesis [19], it is intensively applied in cancer research [20]. During the stages of embryo development, the immunologic, nervous, and nociceptive systems are not fully developed [21]. Several types of CAM assay protocols have been developed.
2.2. Uses in biological studies
The method can be applied for bioengineering development, morphology, biochemistry, transplant biology, cancer research, and drug development, but also in immunology, wound healing, tissue repair, or drug toxicity [22, 23]. The possibilities of imaging and evaluation have attracted many research studies. Nutritional therapeutics is an example of products approved by the U.S. Food and Drug Administration (FDA) that were preclinically evaluated in the CAM model [16].
Phytocompounds can be tested in order to evaluate their potential bioavailability, tolerability, and lack of irritation effects. For this purpose, the variations of the HET-CAM protocol can be applied, according the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommendations published in November 2006 in Appendix G of reference [24]. Our previous evaluations proved its applicability in testing different sets of compounds, i.e., surfactants and aflatoxins [25].
In the attempt of finding new means for cancer chemoprevention, the chorioallantoic membrane assay can be used to test natural compounds that could reduce or inhibit several pathways involved in malignancies, especially pro-inflammatory cytokine activation and excessive angiogenesis. Tumor microenvironment, including inflammation and angiogenesis next to the development of new therapeutic targets for these pathological conditions, is intensively researched on murine models [26]. Previously, we have evaluated mast cell involvement in the angiogenesis process implementing a mastocytoma model on the CAM assay [27], which can be further developed for the evaluation of natural compounds on mast cells as key participants in the tumor microenvironment.
2.3. General in ovo method
Ex ovo or in ovo techniques are applicable. The ex ovo protocol involves the transfer of the egg content on day 3 of incubation into a Petri dish. It facilitates the visualization of the experiment, but the unnatural milieu of development of the embryo is detrimental to the survival rate of the specimens. Therefore, we prefer the in ovo protocol and is the type of method described here.
Fertilized eggs are horizontally incubated 7 days prior to use, at 37°C, in a controlled wet atmosphere. On the third day of incubation, in order to detach the chorioallantoic membrane, a volume of 2–3 ml of albumen is aspired through a perforation at the more pointed end of the eggs. The hole is resealed and returned to the incubator. The next day, a window is cut and resealed on the superior side of the shell. The eggs are returned to incubation until the day of the experiment [28]. Generally, 5–10 eggs are used for each test sample. Samples are applied inside a sterile plastic ring on the surface of the membrane. Samples are applied in triplicate. In ovo investigation by means of a stereomicroscope is performed throughout the experiment. Photographs are recorded for further analysis (Figure 2).
Figure 2.
Chorioallantoic membrane assay—in ovo practical approach: incubation of the eggs (a–c); albumen removal, shell opening, and resealing (d–f); visualization of the CAM, sample application, and sample application inside a plastic ring (g–i) [30].
Starting with day 11 of incubation, samples can be considered active on excessive angiogenesis. The rapid growth of the vessels occurs during days 7–11; therefore, applying substances during this interval can be evaluated in terms of antiangiogenic effects. Morphometric evaluation of the angiogenic reaction can be conducted using a 0–5 arbitrary scale, the mean values expressing the vascular density around the site of application [20]. Finally, specimens are sacrificed and membranes are submitted to histological and immunohistological evaluation. On slides with immunohistochemical marked vessels, the mean microvascular density can be determined using the hotspot method, and counting the blood vessels, to calculate an antiangiogenic index, with the aid of the formula: AAI = 1 – NoBVtest/NoBVcontrol, AAI = antiangiogenic index, BV = blood vessels [29].
2.4. Tumor angiogenesis model on CAM
Tumor cells are used on the CAM in order to obtain tumors, to study their microenvironment and the effects that phytochemicals might have. Tumor grafts can be used as well. Usually, cultured cancer cells are inoculated on the surface of the CAM, on day 10 of incubation, after being trypsinized and resuspended in culture medium to final concentrations in the range of 105–106 ml−1. Cells can be applied directly on the CAM using a plastic ring for localizing the cells or using Matrigel impregnated with cells. Further, test compound solutions diluted with minimal DMSO (dimethyl sulfoxide) concentration in phosphate buffer can be applied on the same spot as the cancer cell samples. In ovo stereomicroscopic follow-up is performed daily to register the changes in the vascular response around the tumor developing area that will be used for the morphometric analysis. On the final day of the experiment, after sacrificing the embryos, tumor masses are measured; the chorioallantoic membrane, the formed tumors, and some organs suspected to have metastasis are harvested and histologically processed.
In order to observe morphologic changes in the chorioallantoic membrane, hematoxylin eosin staining is analyzed. Different panels of immunohistochemical markers can be further applied: tumor cell markers and specific antibodies for different key proteins involved in the tumor microenvironment (e.g., endothelial cell marker-factor VIII, smooth muscle actin (SMA) marker, vascular endothelial growth factors, and its receptors, mast cells marker—Tryptase, the proliferation marker—Ki67). Results can reveal molecular modifications and serve to vascular density quantification.
Our experience is related to testing phytocompounds and plant extracts for the effect on angiogenesis. Using the angiogenesis method in the rapid stage of CAM development, we found that pentacyclic triterpenes, betulinic (BA) acid, and betulin (Bet) in various formulations with cyclodextrin and in nanoemulsion are potential antiangiogenic compounds, acting differently, both through direct and indirect mechanisms [31, 32]. Immunohistochemical staining for smooth muscle actin (SMA) on the specimens treated with betulin in nanoemulsion, next to blank and control samples, are shown in Figure 3. The low expression of the marker in the betulin-treated specimen indicates a minimal implication of pericytes in the angiogenesis process [32]. On the contrary, we found that betulinic acid determined rapid maturation of the vessels and high levels of SMA [31]. We also evaluated triterpenes and other types of natural compounds in melanoma models on CAM, which confirms the inhibitory effect on tumor angiogenesis (data not published).
Figure 3.
Light microscopic evaluation of CAM sections from ID 11 smooth muscle actin marker: (a) blank specimen, ×40, (b) control specimen treated with nanoemulsion, ×40, (c) specimen treated with betulin in nanoemulsion, ×40 [32].
Most studies that use the CAM assay are evaluated through stereomicroscopy that allows a series of quantitative measurements, and by histologic an immunohistological interpretation. Advances in the evaluation techniques include fluorescence microscopy, confocal microscopy, microCT scanning, and imaging, in situ hybridization (ISH), quantitative PCR (qPCR) determination of specific targets [16, 33].
3. Phytocompounds targeting cancer angiogenesis: in vitro, on the chorioallantoic membrane assay, in animal model
Chemicals derived from plant sources as well as various types of extracts have been already investigated for their effects on angiogenesis and on cancer. Currently, based on the failure of the approved therapeutics and also by crediting the traditional medicine philosophy that pathologies are imbalances that have to be rebalanced, the idea of multiple targeting through synergetic phytocompounds mixtures is gaining more attention. Extensive research is being dedicated to the understanding of their mechanism and their efficacy using in vitro and in vivo methods. The most in depth evidence comes from the results on cell cultures. In vivo methods also offer other accurate data on their effects. The chorioallantoic membrane assay is being used by more and more researchers for the evaluation of plant-derived chemicals or extracts. Correlations can be made using the results obtained for in vitro, animal and CAM assays, which will improve the knowledge and the future analysis to perform for the active compounds. We reviewed here some of the most investigated phytocompounds concerning the results obtained on all the three experimental models (Table 1).
Table 1.
Common phytocompounds with in vitro and in vivo antiangiogenic activity.
Implementation of clinical trials is vital for the validation and future use of the active phytocompounds as additional therapies to the oncologic protocols or as chemopreventive strategies. These types of experiments are difficult to implement and therefore not many trials are finalized for the evaluation of antiangiogenic effect in cancer. Two of the above-listed phytochemicals (Table 1) benefit from large investigations among which some are clinical trials, but the modulation of the angiogenic process does not appear as a distinct evaluation, cancer effects being the first ones to be described.
Most of the controlled clinical trials of curcumin supplementation in cancer patients aimed to determine its feasibility, tolerability, safety, and to provide early evidence of efficacy [79]. For patients with advanced colorectal cancer, oral doses up to 3.6 g/day for 4 months were well tolerated, although the systemic bioavailability of oral curcumin was low [80]. For this dose, trace levels of curcumin metabolites were measured in liver tissue, but curcumin itself was not detected [81]. These findings suggested that oral curcumin is effective as a therapeutic agent in cancers of the gastrointestinal tract. Other trials found that combining curcumin with anticancer drugs like gemcitabine in pancreatic cancer [82], docetaxel in breast cancer [83], and imatinib in chronic myeloid leukemia may confer additional benefits to conventional drugs against different types of cancer.
Green tea made from Camellia sinensis L. leaves, originated in China, is one of the most extensively consumed beverages and achieved significant attention due to health benefits against cancer. Representative compounds are polyphenols and catechins with therapeutic potential against cancer [84]. Recent clinical trials proved that green tea extract and epigallocatechin gallate (EGCG) can be active in several forms of cancer. There is an increasing trend to employ green tea extract and EGCG as conservative management for patients diagnosed with less advanced prostate cancer. Combinations of chemopreventive agents should be carefully investigated because mechanisms of action may be additive or synergistic [85]. Several clinical examinations reported different molecular mechanisms regarding green tea beneficial effects against oral cancer chemoprevention [86–88]. Lung cancer induction may also be inhibited by tea polyphenols. Some studies suggest that individuals who never drank green tea have an elevated lung cancer risk compared to those who drank green tea at least one cup per day, and the effect is more pronounced in smokers [88]. Hepatocellular carcinoma (HCC) usually develops in a cirrhotic liver due to hepatitis virus infection. Green tea catechins (GTCs) may possess potent anticancer and chemopreventive properties for a number of different malignancies, including liver cancer. Antioxidant and anti-inflammatory activities are key mechanisms through which GTCs prevent the development of neoplasms, and they also exert cancer chemopreventive effects by modulating several signaling transduction and metabolic pathways where angiogenesis is exacerbated. Several interventional trials in humans have shown that GTCs may ameliorate metabolic abnormalities and prevent the development of precancerous lesions [89].
Currently, a great number of natural compounds are being investigated for their potential effectiveness in controlling tumor angiogenesis and therefore the reduction of tumor growth and metastasis. Observing the high number of molecular pathways that are deregulated in tumor angiogenesis and that many phytocompounds are active on several key factors, it is recommendable that more in vivo studies should investigate mixture of compounds for broader targeting, having eventually lower secondary effects and resistance. The optimal experimental technique is an important factor in order to get a useful output. More types of assays are always a good choice, including in vivo assays. The chorioallantoic membrane protocol is a good candidate for one type of “golden standardized method” in tumor angiogenesis, being a versatile, rapid, easy, and cheap method to apply in the research of phytocompounds. A great number of plant-derived chemicals, alone or in combination, are studied using this method, but standardization, next to applying new analysis techniques will outcome useful data that will be easier translated to clinical trials.
This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS—UEFISCDI, project number PN-II-RU-TE-2014-4-2842 to S.A., R.G., I.Z.P. and D.C. Special thanks to the Histology and Angiogenesis Department, University of Medicine and Pharmacy Victor Babes Timisoara, for the technical support and help in setting up the CAM assay.
References
1.Ribatti D, Djonov V. Intussusceptive microvascular growth in tumors. Cancer Letters. 2012;316(2):126-131
2.Folkman J. Tumor angiogenesis: Therapeutic implications. The New England Journal of Medicine. 1971;285(21):1182-1186
3.Holmgren L, O’Reilly MS, Folkman J. Dormancy of micrometastases: Balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nature Medicine. 1995;1:149-153
4.Parangi S, O’Reilly M, Christofori G, Holmgren L, Grosfeld J, Folkman J, Hanahan D. Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(5):2002-2007
6.Denekamp J. Angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. The British Journal of Radiology. 1993;66(783):181-196
7.Nishida N, Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vascular Health and Risk Management. 2006;2(3):213-219
8.Prager GW, Poettler M, Unseld M, Zielinski CC. Angiogenesis in cancer: Anti-VEGF escape mechanisms. Translational Lung Cancer Research. 2012;1(1):14-25
9.Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature. 2005;438(7070):967-974
10.Oklu R, Walker TG, Wicky S, Hesketh R. Angiogenesis and current antiangiogenic strategies for the treatment of cancer. Journal of Vascular Interventional Radiology. 2010;21(12):1791-805; quiz 1806
11.Wang Z, Dabrosin C, Yin X, Fuster MM, Arreola A, Rathmell WK, Generali D, Nagaraju GP, El-Rayes B, Ribatti D, Chen YC, Honoki K, Fujii H, Georgakilas AG, Nowsheen S, Amedei A, Niccolai E, Amin A, Ashraf SS, Helferich B, Yang X, Guha G, Bhakta D, Ciriolo MR, Aquilano K, Chen S, Halicka D, Mohammed SI, Azmi AS, Bilsland A, Keith WN, Jensen LD. Broad targeting of angiogenesis for cancer prevention and therapy. Seminars in Cancer Biology. 2015;35:S224–S243
12.Bora A, Avram S, Ciucanu I, Raica M, Avram S. Predictive models for fast and effective profiling of kinase inhibitors. Journal of Chemical Information and Modeling. 2016;56(5):895-905
13.Avram SI, Pacureanu LM, Bora A, Crisan L, Avram S, Kurunczi L. ColBioS-FlavRC: A collection of bioselective flavonoids and related compounds filtered from high-throughput screening outcomes. Journal of Chemical Information and Modeling. 2014;54(8):2360-2370
14.Roudsari LC, West JL. Studying the influence of angiogenesis in in vitro cancer model systems. Advanced Drug Delivery Reviews. 2016;97:250-259
15.Staton CA, Reed MWR, Brown NJ. A critical analysis of current in vitro and in vivo angiogenesis assays. International Journal of Experimental Pathology. 2009;90(3):195-221
16.Dupertuis YM, Delie F, Cohen M, Pichard C. In ovo method for evaluating the effect of nutritional therapies on tumor development, growth and vascularization. Clinical Nutrition Experimental. 2015;2:9-17
17.Nowak-Sliwinska P, Segura T, Iruela-Arispe ML. The chicken chorioallantoic membrane model in biology, medicine and bioengineering. Angiogenesis. 2016;17(4):779-804
18.Harris RJ. Multiplication of Rous No. 1 sarcoma agent in the chorioallantoic membrane of the embryonated egg. British Journal of Cancer. 1954;8(4):731-736
19.Folkman J, Cotran R. Relation of vascular proliferation to tumor growth. International Review of Experimental Pathology. 1976;16:207-248
20.Ribatti D. The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis. Springer Netherlands; 2010
21.Friend JV, Crevel RW, Williams TC, Parish WE. Immaturity of the inflammatory response of the chick chorioallantoic membrane. Toxicology In Vitro. 1990;4(4-5):324-326
22.Rashidi H, Sottile V. The chick embryo: Hatching a model for contemporary biomedical research. Bioessays. 2009;31(4):459-465
23.Vargas A, Zeisser-Labouèbe M, Lange N, Gurny R, Delie F. The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems. Advanced Drug Delivery Reviews. 2007;59(11):1162-1176
24.Scheel J, Kleber M, Kreutz J, Lehringer E, Mehling A, Reisinger K, Steiling W. Eye irritation potential: Usefulness of the HET-CAM under the globally harmonized system of classification and labeling of chemicals (GHS). Regulatory Toxicology and Pharmacology. 2011;59(3):471-492
25.Ardelean S, Feflea S, Ionescu D, Năstase V, Dehelean CA. Toxicologic screening of some surfactants using modern in vivo bioassays. Revista Medico-Chirurgicala a Societatii De Medici Si Naturalisti Din Iasi Nat. din Iaşi. 2011;115(1):251-258
26.Lokman NA, Elder ASF, Ricciardelli C, Oehler MK. Chick chorioallantoic membrane (CAM) assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis. International Journal of Molecular Sciences. 2012;13(8):9959-9970
27.(Feflea) Avram S, Cimpean AM, Raica M. Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos. Brazilian Journal of Medical and Biological Research. 2013;46(1):52-57.
28.Ribatti D. The chick embryo chorioallantoic membrane in the study of tumor angiogenesis. Romanian Journal of Morphology and Embryology. 2008;49(2):131-135
29.Demir R, Peros G, Hohenberger W. Definition of the ‘Drug-Angiogenic-Activity-Index’ that allows the quantification of the positive and negative angiogenic active drugs: A study based on the chorioallantoic membrane model. Pathology and Oncology Research. 2011;17(2):309-313
30.Feflea S. Stimulators and Inhibitors Of Angiogenesis in Experimental Model. (Doctoral Dissertation). University of Medicine and Pharmacy Victor Babes Timisoara; Timisoara;2013
31.Dehelean CA, Feflea S, Ganta S, Amiji M. Anti-angiogenic effects of betulinic acid administered in nanoemulsion formulation using chorioallantoic membrane assay. Journal of Biomedical Nanotechnology. 2011;7(2):317-324
32.Dehelean CA, Feflea S, Gheorgheosu D, Ganta S, Cimpean AM, Muntean D, Amiji MM. Anti-angiogenic and anti-cancer evaluation of betulin nanoemulsion in chicken chorioallantoic membrane and skin carcinoma in Balb/c mice. Journal of Biomedical Nanotechnology. 2013;9(4):577-589
33.Xue X, Xiaoying Z, Huixin M, Zhang J, Huang G, Zhang Z, Li P. Chick chorioallantoic membrane assay: A 3D animal model for study of human nasopharyngeal carcinoma. PLoS One. 2015;10(6):e0130935
34.Kunnumakkara AB, Guha S, Krishnan S, Diagaradjane P, Gelovani J, Aggarwal BB. Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis, and inhibition of nuclear factor-kappa B-regulated gene products. Cancer Research. 2007;67(8):3853-3861
35.Gururaj AE, Belakavadi M, Venkatesh DA, Marmé D, Salimath BP. Molecular mechanisms of anti-angiogenic effect of curcumin. Biochemical and Biophysical Research Communications. 2002;297(4):934-942
36.Ranjan AP, Mukerjee A, Helson L, Gupta R, Vishwanatha JK. Efficacy of liposomal curcumin in a human pancreatic tumor xenograft model: Inhibition of tumor growth and angiogenesis. Anticancer Research. 2013;33(9):3603-3609.
37.Shirakami Y, Shimizu M, Adachi S, Sakai H, Nakagawa T, Yasuda Y, Tsurumi H, Hara Y, Moriwaki H. (-)-Epigallocatechin gallate suppresses the growth of human hepatocellular carcinoma cells by inhibiting activation of the vascular endothelial growth factor-vascular endothelial growth factor receptor axis. Cancer Science. 2009;100(10):1957-1962
38.Siddiqui IA, Adhami VM, Bharali DJ, Hafeez BB, Asim M, Khwaja SI, Ahmad N, Cui H, Mousa SA, Mukhtar H. Introducing nanochemoprevention as a novel approach for cancer control: Proof of principle with green tea polyphenol epigallocatechin-3-gallate. Cancer Research. 2009;69(5):1712-1716
39.Wu H, Xin Y, Xu C, Xiao Y. Capecitabine combined with (-)-epigallocatechin-3-gallate inhibits angiogenesis and tumor growth in nude mice with gastric cancer xenografts. Experimental and Therapeutic Medicine. 2012;3(4):650-654
40.Martínez-Poveda B, Quesada AR, Medina MÁ. Hyperforin, a bio-active compound of St. John’s Wort, is a new inhibitor of angiogenesis targeting several key steps of the process. International Journal of Cancer. 2005;117(5):775-780
41.Rothley M, Schmid A, Thiele W, Schacht V, Plaumann D, Gartner M, Yektaoglu A, Bruyère F, Noël A, Giannis A, Sleeman JP. Hyperforin and aristoforin inhibit lymphatic endothelial cell proliferation in vitro and suppress tumor-induced lymphangiogenesis in vivo. International Journal of Cancer. 2009;125(1):34-42
42.Trapp V, Basmina P, Papazian V, Lyndsay W, Fruehauf JP. Anti-angiogenic effects of resveratrol mediated by decreased VEGF and increased TSP1 expression in melanoma-endothelial cell co-culture. Angiogenesis. 2010;13:305-315
43.Wang H, Zhou H, Zou Y, Liu Q, Guo C, Gao G, Shao C, Gong Y. Resveratrol modulates angiogenesis through the GSK3β/β-catenin/TCF-dependent pathway in human endothelial cells. Biochemical Pharmacology. 2010;80(9):1386-1395
44.Kimura Y, Okuda H. Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice. The Journal of Nutrition. 2001;131(6):1844-1849
45.López-Jiménez A, García-Caballero M, Medina MÁ, Quesada AR. Anti-angiogenic properties of carnosol and carnosic acid, two major dietary compounds from rosemary. European Journal of Nutrition. 2013;52(1):85-95
46.Rajasekaran D, Manoharan S, Silvan S, Vasudevan K, Baskaran N, Palanimuthu D. Proapoptotic, anti-cell proliferative, anti-inflammatory and anti-angiogenic potential of carnosic acid during 7,12 dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. African Journal of Traditional, Complementary and Alternative Medicine. 2012;10(1):102-112
47.Chakraborty S, Adhikary A, Mazumdar M, Mukherjee S, Bhattacharjee P, Guha D, Choudhuri T, Chattopadhyay S, Sa G, Sen A, Das T. Capsaicin-induced activation of p53-SMAR1 auto-regulatory loop down-regulates VEGF in non-small cell lung cancer to restrain angiogenesis. PLoS One. 2014;9(6):e99743
48.Min J-K. Capsaicin inhibits in vitro and in vivo angiogenesis. Cancer Research. 2004;64(2):644-651
49.Mahmoud AM, Yang W, Bosland MC. Soy isoflavones and prostate cancer: A review of molecular mechanisms. The Journal of Steroid Biochemistry and Molecular Biology. 2014;140:116-132
50.Krenn L, Paper DH. Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolium pratense L.). Phytomedicine. 2009;16(12):1083-1088
51.Fotsis T, Pepper M, Adlercreutz H, Fleischmann G, Hase T, Montesano R, Schweigerer L. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proceedings of the National Academy of Sciences of the United States of America. 1993;90(7):2690-2694
52.Gu Y, Zhu C-F, Iwamoto H, Chen J-S. Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis. World Journal of Gastroenterology. 2005;11(41):6512-6517
53.Pratheeshkumar P, Budhraja A, Son Y-O, Wang X, Zhang Z, Ding S, Wang L, Hitron A, Lee J-C, Xu M, Chen G, Luo J, Shi X. Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR-2 regulated AKT/mTOR/P70S6K signaling pathways. PLoS One. 2012;7(10): e47516. https://DOI.org/10.1371/journal.pone.0047516
54.Kong L, Wu K, Lin H. Inhibitory effects of quercetin on angiogenesis of experimental mammary carcinoma. Chinese Journal of Clinical Oncology. 2005;2(3):631-636
55.Lou C, Zhang F, Yang M, Zhao J, Zeng W, Fang X, Zhang Y, Zhang C, Liang W. Naringenin decreases invasiveness and metastasis by inhibiting TGF-β-induced epithelial to mesenchymal transition in pancreatic cancer cells. PLoS One. 2012;7(12):e50956
56.Anand K, Sarkar A, Kumar A, Ambasta RK, Kumar P. Combinatorial antitumor effect of naringenin and curcumin elicit angioinhibitory activities in vivo. Nutrition and Cancer. 2012;64(5):714-724
57.Fang J, Zhou Q, Liu LZ, Xia C, Hu X, Shi X, Jiang BH. Apigenin inhibits tumor angiogenesis through decreasing HIF-1α and VEGF expression. Carcinogenesis. 2007;28(4):858-864
58.Germanò MP, Certo G, D’Angelo V, Sanogo R, Malafronte N, De Tommasi N, Rapisarda A. Anti-angiogenic activity of Entada africana root. Natural Product Research. 2015;29(16):1551-1556
59.Sun Z-J, Chen G, Zhang W, Hu X, Huang C-F, Wang Y-F, Jia J, Zhao Y-F. Mammalian target of rapamycin pathway promotes tumor-induced angiogenesis in adenoid cystic carcinoma: Its suppression by isoliquiritigenin through dual activation of c-Jun NH2-terminal kinase and inhibition of extracellular signal-regulated kinase. Journal of Pharmacology and Experimental Therapeutics. 2010;334(2):500-512
60.Jhanji V, Liu H, Law K, Lee VY-W, Huang S-F, Pang C-P, Yam GH-F. Isoliquiritigenin from licorice root suppressed neovascularisation in experimental ocular angiogenesis models. British Journal of Ophthalmology. 2011;95(9):1309-1315
61.Raina K, Agarwal C, Agarwal R. Effect of silibinin in human colorectal cancer cells: Targeting the activation of NF-κB signaling. Molecular Carcinogenesis. 2013;52(3):195-206
62.Yang S-H, Lin J-K, Huang C-J, Chen W-S, Li S-Y, Chiu J-H. Silibinin inhibits angiogenesis via Flt-1, but not KDR, receptor up-regulation. Journal of Surgical Research. 2005;128(1):140-146
63.Tyagi A, Singh RP, Ramasamy K, Raina K, Redente EF, Dwyer-Nield LD, Radcliffe RA, Malkinson AM, Agarwal R. Growth inhibition and regression of lung tumors by silibinin: Modulation of angiogenesis by Macrophage-Associated cytokines and nuclear Factor- B and signal transducers and activators of transcription 3. Cancer Prevention Research. 2009;2(1):74-83
64.Marimpietri D, Brignole C, Nico B, Pastorino F, Pezzolo A, Piccardi F, Cilli M, Di Paolo D, Pagnan G, Longo L, Perri P, Ribatti D, Ponzoni M. Combined therapeutic effects of vinblastine and rapamycin on human neuroblastoma growth, apoptosis, and angiogenesis. Clinical Cancer Research. 2007;13(13):3977-3988
65.Park K-J, Yu MO, Park D-H, Park J-Y, Chung Y-G, Kang S-H. Role of vincristine in the inhibition of angiogenesis in glioblastoma. Neurology Research. 2016; 38(10):871-9. doi: 10.1080/01616412.2016.1211231
66.Michaelis M, Hinsch N, Michaelis UR, Rothweiler F, Simon T, ilhelm Doerr HW, Cinatl J, Cinatl J. Chemotherapy-associated angiogenesis in neuroblastoma tumors. The American Journal of Pathology. 2012;180(4):1370-1377
67.Schirner M, Hoffmann J, Menrad A, Schneider MR. Antiangiogenic chemotherapeutic agents: Characterization in comparison to their tumor growth inhibition in human renal cell carcinoma models. Clinical Cancer Research. 1998;4(5):1331-1336.
68.Dehelean CA, Feflea S, Molnár J, Zupko I, Soica C. Betulin as an antitumor agent tested in vitro on A431, hela and MCF7, and as an angiogenic inhibitor in vivo in the CAM assay. Natural Product Communications. 2012;7(8):981-985
69.Chintharlapalli S, Papineni S, Ramaiah SK, Safe S. Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Research. 2007;67(6):2816-2823
70.Chen Q-J, Zhang M-Z, Wang L-X. Gensenoside Rg3 inhibits hypoxia-induced VEGF expression in human cancer cells. Cellular Physiology and Biochemistry. 2010;26(6):849-858
71.Xiu Yu JL, Xu H, Hu M, Luan X, Wang K, Fu Y, Zhang D. Ginsenoside Rg3 bile Salt-Phosphatidylcholine-Based mixed micelles: Design, characterization, and evaluation. Chemical and Pharmaceutical Bulletin. 2015;63(5):361-368
72.Kim J-W, Jung S-Y, Kwon Y-H, Lee J-H, Lee YM, Lee B-Y, Kwon S-M. Ginsenoside Rg3 attenuates tumor angiogenesis via inhibiting bioactivities of endothelial progenitor cells. Cancer Biology & Therapy. 2012;13(7):504-515
73.Cathcart M-C, Useckaite Z, Drakeford C, Semik V, Lysaght J, Gately K, O’Byrne KJ, Pidgeon GP. Anti-cancer effects of baicalein in non-small cell lung cancer in-vitro and in-vivo. BMC Cancer. 2016;16(1):707
74.Liu J-J, Huang T-S, Cheng W-F, Lu F-J. Baicalein and baicalin are potent inhibitors of angiogenesis: Inhibition of endothelial cell proliferation, migration and differentiation. International Journal of Cancer. 2003;106(4):559-565
75.Zhang K, Lu J, Mori T, Smith-Powell L, Synold TW, Chen S, Wen W. Baicalin increases VEGF expression and angiogenesis by activating the ERR /PGC-1 pathway. Cardiovascular Research. 2011;89(2):426-435
76.Xin W, Tian S, Song J, He G, Mu X, Qin X. Research progress on pharmacological actions and mechanism of baicalein and baicalin. Current Opinion In Complementary and Alternative Medicine. 2014;1(2):e00010
77.Vanden Berghe W, Sabbe L, Kaileh M, Haegeman G, Heyninck K. Molecular insight in the multifunctional activities of Withaferin A. Biochemical Pharmacology. 2012;84(10):1282-1291
78.Mathur R, Gupta SK, Singh N, Mathur S, Kochupillai V, Velpandian T. Evaluation of the effect of Withania somnifera root extracts on cell cycle and angiogenesis. Journal of Ethnopharmacology. 2006;105(3):336-341
79.Fanaei H, Khayat S, Kasaeian A, Javadimehr M. Effect of curcumin on serum brain-derived neurotrophic factor levels in women with premenstrual syndrome: A randomized, double-blind, placebo-controlled trial. Neuropeptides. 2016;56:25-31
80.Mall M, Kunzelmann K. Correction of the CF defect by curcumin: Hypes and disappointments. BioEssays. 2005;27(1):9-13
81.Garcea G, Jones DJL, Singh R, Dennison AR, Farmer PB, Sharma RA, Steward WP, Gescher AJ, Berry DP. Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration. British Journal of Cancer. 2004;90(5):1011-1015
82.Epelbaum R, Schaffer M, Vizel B, Badmaev V, Bar-Sela G. Curcumin and gemcitabine in patients with advanced pancreatic cancer. Nutrition and Cancer. 2010;62(8):1137-1141
83.Bayet-Robert M, Kwiatkowski F, Leheurteur M, Gachon F, Planchat E, Abrial C, Mouret-Reynier M-A, Durando X, Barthomeuf C, Chollet P. Phase I dose escalation trial of docetaxel plus curcumin in patients with advanced and metastatic breast cancer. Cancer Biology & Therapy. 2010;9(1):8-14
84.Chen L, Zhang HY. Cancer preventive mechanisms of the green tea polyphenol (-)-epigallocatechin-3-gallate. Molecules. 2007;12(5):946-957
85.Davalli P, Rizzi F, Caporali A, Pellacani D, Davoli S, Bettuzzi S, Brausi M, D’Arca D. Anticancer activity of green tea polyphenols in prostate gland. Oxidative Medicine and Cellular Longevity. 2012; 2012. DOI:10.1155/2012/984219
86.Soulieres D, Senzer NN, Vokes EE, Hidalgo M, Agarwala SS, Siu LL. Multicenter phase II study of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with recurrent or metastatic squamous cell cancer of the head and neck. Journal of Clinical Oncology. 2004;22
87.Lee U-L, Choi S-W. The chemopreventive properties and therapeutic modulation of green tea polyphenols in oral squamous cell carcinoma. ISRN Oncology. 2011;2011:1-7
88.Yang X, Thomas DP, Zhang X, Culver BW, Alexander BM, Murdoch WJ, Rao MN, Tulis DA, Ren J, Sreejayan N. Curcumin inhibits platelet-derived growth factor-stimulated vascular smooth muscle cell function and injury-induced neointima formation. Arteriosclerosis Thrombosis and Vascular Biology. 2006;26
89.Shimizu M, Shirakami Y, Sakai H, Kubota M, Kochi T, Ideta T, Miyazaki T, Moriwaki H. Chemopreventive potential of green tea catechins in hepatocellular carcinoma. International Journal of Molecular Sciences. 2015;16(3):6124-6139
Written By
Stefana Avram, Roxana Ghiulai, Ioana Zinuca Pavel, Marius Mioc,
Roxana Babuta, Mirela Voicu, Dorina Coricovac, Corina Danciu,
Cristina Dehelean and Codruta Soica
Submitted: 11 October 2016Reviewed: 13 March 2017Published: 05 July 2017
Open access peer-reviewed chapter
