Antiangiogenic Effects of Coumarins against Cancer: From Chemistry to Medicine

Angiogenesis, the process of formation and recruitment of new blood vessels from pre-existing vessels, plays an important role in the development of cancer. Therefore, the use of antiangiogenic agents is one of the most critical strategies for the treatment of cancer. In addition, the complexity of cancer pathogenicity raises the need for multi-targeting agents. Coumarins are multi-targeting natural agents belonging to the class of benzopyrones. Coumarins have several biological and pharmacological effects, including antimicrobial, antioxidant, anti-inflammation, anticoagulant, anxiolytic, analgesic, and anticancer properties. Several reports have shown that the anticancer effect of coumarins and their derivatives are mediated through targeting angiogenesis by modulating the functions of vascular endothelial growth factor as well as vascular endothelial growth factor receptor 2, which are involved in cancer pathogenesis. In the present review, we focus on the antiangiogenic effects of coumarins and related structure-activity relationships with particular emphasis on cancer.


Introduction
Angiogenesis (also known as neovascularization), the growth of blood vessels from the existing vasculature, has been shown to play a critical role in the development of various diseases, including rheumatoid arthritis, diabetic retinopathy, asthma, endometriosis, psoriasis, obesity, and cancer [1][2][3]. Inflammation, tissue ischemia, and hypoxia which cause the release of the angiogenesis factors, such as vascular endothelial growth factor (VEGF), cytokines, cell adhesion molecules, and nitric oxide (NO), are among the most important triggers of angiogenesis [4]. In 1971, Folkman reported that tumor Molecules 2019, 24, 4278; doi:10.3390/molecules24234278 www.mdpi.com/journal/molecules metastasis occurs as a consequence of angiogenesis [5]. This was the starting point for the design and use of bevacizumab, thalidomide, sunitinib, and axitinib as antiangiogenic drugs in the treatment of a variety of cancers [6][7][8]. Considering the crucial role of angiogenesis in the progression of cancer, investigating novel and potential antiangiogenic compounds is of great importance to combat cancer. Several naturally occurring compounds, including vinblastine, vincristine, paclitaxel, were reported as antiangiogenic and anticancer agents. Besides, other natural compounds with antiangiogenic activities, such as resveratrol, artemisinin, boswellic acid, and cannabidiol, have shown enormous potential for cancer prevention and therapy [9][10][11][12][13]. For instance, endocannabinoid 2-arachidonoyl-glycerol showed a promising anticancer effect in several cell lines [14]. Overall, cancer remains a clinical challenge, despite advancements in its treatment. This raises the need to investigate novel multi-target agents to attenuate multiple signaling pathways involved in tumor progression. Growing evidence has introduced coumarins as potential multi-targeting agents with various pharmacological effects and medicinal uses [15]. Coumarins, with their 2H-1-benzopyran-2-one structure, are natural compounds that exist in various plant families, including Apiaceae, Asteraceae, Fabaceae, Rutaceae, Moraceae, Oleaceae, and Thymelaeaceae [16]. Apiaceae is the greatest family of plants containing coumarin compounds [16]. Also, due to the antioxidant [17], anti-inflammatory [18], anxiolytic [19], analgesic [20], neuroprotective [21], cardioprotective [22], antidiabetic [23], and anticancer [24] activities of coumarins [25], researchers have studied the synthesis of various coumarin derivatives, in addition to their purification from natural sources [26]. Both synthetic and natural coumarins have shown noticeable anticancer effects in vitro and in vivo through various mechanisms [27], including the inhibition of angiogenesis [28,29]. From a mechanistic point of view, some coumarins have shown promising antiangiogenic effects through the interaction with and repression of signaling mediators involved in angiogenesis [30,31].
In this review, we focus on the cellular signaling pathways of angiogenesis and recent pharmacological antiangiogenic agents, emphasizing natural and synthetic coumarins with antiangiogenic effects as well as their pharmacological mechanisms and structure-activity relationship in cancer.
On the other hand, angiogenesis could be suppressed by inhibiting proteins, which are classified into either direct or indirect angiogenesis inhibitors. The first class of inhibitors directly suppress ECs in the growing vasculature, while the second class indirectly suppress either tumor cells or other tumor-associated stromal cells [35,38]. This direct inhibitory effect could also be mediated by integrin receptors through several intracellular signaling pathways [39]. Angiostatin, endostatin, arrestin, canstatin, and tumstatin are released by the proteolysis of distinct endothelial cell-matrix molecules and prevent vascular ECs from proliferating and migrating in response to angiogenesis inducers [40]. Interferons, retinoic acid, IL-1, IL-12, tissue inhibitor of metalloproteinases, and multimerin 2 are other angiogenesis inhibitors [41,42].
As previously mentioned, angiogenesis is controlled by a balance between activators and inhibitors of angiogenesis. Hypoxia, as a critical determinant, causes an imbalance between activators and inhibitors by inducing the upregulation of HIF-1α, which elevates the expression of pro-angiogenesis agents as well as suppresses the expression of angiogenesis inhibitors [43]. Therefore, all the mediators in these pathways could be therapeutic targets to inhibit angiogenesis.

Recent Advancement in Pharmacological Antiangiogenic Agents
Several angiogenesis inhibitors have been found since Folkman first presented the concept of introducing angiogenesis inhibitors as anticancer drugs [5]. RNA interference (RNAi) therapy, chimeric antigen receptor T cell therapy, gene therapy, and pharmacological agents are auspicious antiangiogenic interventions [44]. According to the United States Food and Drug Administration (FDA), approved antiangiogenic agents are classified into two major groups, namely, monoclonal antibodies (mAbs) and small molecules [45].
Angiopoietins (Ang1-4) bind to the Tie2 receptor. While Ang1 helps the vessels stabilize, Ang2 is secreted by ECs in response to proangiogenic factors, including hypoxia, cytokines, and inflammation [64]. Ang/Tie2-targeted therapy is challenging, since it could be either antitumor or protumor, depending on the context [65].
The rearranged during transfection (RET) protein binds receptor tyrosine kinases (RTKs) associated with normal development, maintenance, and maturation of cells and tissues [66]. However, its mutation is related to the growth and progression of tumors [66,67]. Therefore, RET inhibition could be of great importance in combating cancer.
Multi-targeting antiangiogenic drugs are shown in Figure 1. These drugs exert anticancer effects through simultaneously modulating several signaling pathways involved in angiogenesis.

Biological and Pharmacological Effects of Coumarins
Coumarins have several biological and pharmacological effects. For example, coumarins isolated from the bark of M. glabra showed antioxidant properties [73]. In this line, antimicrobial effects of coumarins from the fruits of H. mantegazzianum Sommier & Levier as well as Peucedanum
On the other hand, hepatotoxicity, nausea, and diarrhea were reported as the side effects of coumarin derivatives [88,89].

Coumarins as Anticancer Agents
As the second leading cause of death worldwide, cancer is one of the most critical diseases that threaten public health and imposes a high cost on countries' health systems each year. Because of the resistance of cancer cells to conventional drugs used in chemotherapy as well as the side effects of these drugs, it is necessary to find new anticancer agents. Undoubtedly, medicinal plants are one of the richest sources of biologically active compounds and potential novel anticancer drugs. Coumarins are natural compounds with low to moderate side effects, which have been introduced by researchers as promising anticancer compounds [29,[90][91][92]. Several coumarins also inhibit cytochrome P450, thereby affecting the blood concentration of various anticancer drugs. In this line, bergamottin inhibits cytochrome P450 and reduces the effects of various carcinogenic agents [93].
The anticancer and cytotoxic activities of synthetic and natural coumarins with different functional groups on their basic structure ( Figure 2) have been reported by several investigators. These studies showed the anticancer activity of coumarins against breast cancer [89], colon cancer [94], lung cancer [24], ovarian cancer [95], hepatocellular carcinoma [96], bladder carcinoma [97], leukemia [98], and other types of cancer in vitro and in vivo, via different mechanisms, including free-radical scavenging, antioxidant activity [99], induction of cell cycle arrest [100], interaction with various signaling pathways with important role in cell differentiation and proliferation [101], telomerase and carbonic anhydrase inhibition [102,103], and antiangiogenic activity [104]. For example, Taniguchi and co-workers [105] isolated eight coumarins from the leaves of Rhizophora mucronata and reported the anticancer effects of methoxyinophyllum P, calocoumarin B, and calophyllolide against HeLa cells (cervical cancer), with IC 50 equal to 3.8, 29.9, and 36.4 µM, respectively, and HL-60 cells (promyelocytic leukemia cells) with IC 50 of 12.9, 2.6, and 2.2 µM, respectively. Also, three hemiterpene ether coumarins, isolated from Artemisia armeniaca Lam, showed cytotoxic effects in HL-60 and K562 cells (chronic myelogenous leukemia cells). In their study, armenin showed the highest cytotoxic effect through cycle arrest, with IC 50 equal to 22.5 and 71.1 µM for K562 and HL-60 cells, respectively [106].
On the other hand, several studies have been carried out on the synthesis of coumarins to produce coumarin derivatives with improved anticancer effects. Among them, synthetic scopoletin derivatives have shown promising antitumor activities. In their study, out of 20 synthesized derivatives of scopoletin, 5 compounds showed the greatest effects (IC 50 < 2 µM) in MCF-7 and MDA-MB 231 cells (human breast adenocarcinoma cell line) as well as in HT29 cells (human colorectal adenocarcinoma cell line). The relationship between the increase in Log P value and the increase in cytotoxic activity was established in this investigation. Cell cycle arrest was also suggested as the anticancer mechanism of these compounds [108].
Considering the above investigations on the anticancer effects of coumarin derivatives, these compounds could be developed as anticancer drugs.
Pan and co-workers [119] showed the antiangiogenic effects of scopoletin, isolated from Erycibe obtusifolia Benth stem, in vitro and in vivo. In their study, scopoletin markedly reduced the number of blood vessel branch points after a 48-h treatment with the dose of 100 nmol/egg in the chick chorioallantoic membrane (CAM) model. Additionally, the inhibitory effects of scopoletin on migration, proliferation, and tube formation of human umbilical vein endothelial cells (HUVECs) induced by VEGF (10 ng/mL) were observed. These investigators showed that the proliferation of HUVECs was significantly inhibited by 100 µM scopoletin after 72 h, and tube formation and migration of HUVECs were inhibited following treatment with 100 µM scopoletin by 52.4% and 38.1%, respectively [119].
Decursin and decursinol angelate, isolated from Angelica gigas root, showed substantial antiangiogenic effects in vitro and in vivo [120]. These natural coumarins significantly decreased the development of blood vessels in transgenic zebrafish embryos at 20 µM concentration as well as in the CAM model at 6 µM/egg. In their study, the inhibition of VEGFR2 (one of the most important receptors of VEGF) and other angiogenesis signaling pathway related to VGEF, such as phosphorylated extracellular signal-regulated kinase (p-ERK) and MAPK as well as phosphorylated-c-Jun N-terminal kinase (JNK) in ECs were observed [116].

Synthetic Coumarins with Antiangiogenic Effects
Angiogenesis is a critical process in the development and progression of cancer, as demonstrated by various pre-clinical and clinical evidence. Among natural-based entities, coumarins present little cytotoxicity, while demonstrating more powerful antiangiogenic effects than conventional cytotoxic drugs [131,132]. Semi-synthesized and synthesized products from natural coumarins, used as lead compounds, led to the discovery of interesting antiangiogenic and non-cytotoxic molecules. Coumarins with interesting antiangiogenic and non-cytotoxic properties almost entirely mimic the behavior of the physiological ligands of the main therapeutic targets [132]. Recently, several sulfonyl derivatives of coumarins have been studied as cytotoxic and antiangiogenic agents against HepG2 hepatocellular carcinoma cells in vitro. All synthesized coumarins showed no cytotoxic effect but exhibited a high antimigration activity through the inhibition of MMP-2. CD105 was over-expressed in all cases and, therefore, was not involved in the antimigration activity [132]. In other cases, no statistically significant difference in gene expression of CD44 was found. A synthesized coumarin, 2-oxo-2H-chromene-6-sulfonyl derivative, was found to be the most promising antiangiogenic agent, since it was able to inhibit the migratory activity mediated by MMP-2 and down-regulate CD105; however, it did not show any effect on CD44.
On the other hand, the antiangiogenic capacity of several sulfonyl derivatives of coumarins was evaluated using molecular docking studies. In these studies, it was also observed that the compounds showed better docking scores with respect to the I52 ligand, with the nitro derivatives being the best, due to the ability of the nitro group to better coordinate the Zn ++ ion within the binding site. However, the in vitro antiangiogenic activity of sulfonyl derivatives of coumarins was not statistically significant. Only the 2-oxo-2H-chromene-6-sulfonyl derivative with N-acetylpyrazolone substitution at the 6-position showed a promising antiangiogenic activity, exhibiting better binding interactions with the active site and a docking score comparable to that of the inhibitor I52 (−16.22 vs. 18.18 kJmol-1, respectively) [132].
NF-κB is another protein factor, which plays a pivotal role in gene expression and, therefore, is involved in proliferation, angiogenesis, and metastasis, as well as in drug resistance in cancer. In light of this, the development of angiogenesis inhibitors is of significant importance in the treatment of many cancers. Recently, the effects of 26 new synthetic coumarins were tested against hepatocellular carcinoma cells [133]. The investigators identified (7-carbethoxyamino-2-oxo-2H-chromen-4-yl)methylpyrrolidine-1-carbodithioate as the most promising one, because it was cytotoxic in a time-and concentration-dependent manner and it was able to hinder the binding of NF-κB to DNA, therefore inhibiting the expression of several genes, such as cyclin D1, Bcl-2, survivin, MMP12, and c-Myc. Furthermore, it was able to reduce cell migration and invasion induced by CXCL12, a cytokine that plays a pivotal role in angiogenesis by recruiting endothelial progenitor cells from the bone marrow [133].
Analysis of data present in the existing literature shows that it is possible to identify, through structure-activity analysis, coumarin derivatives more suitable for a given type of tumor. The coumarin derivatives that possess an N-aryl carboxamide, a phenyl substitution at the C-3 position, and 1, 2, 3-triazolyl, trihydroxystilbene, and amino substitutions at the C-4 position were the most effective in targeting lung cancer [24].
Preliminary in vitro results revealed that some coumarin-tethered isoxazolines exhibited significant antiproliferative effect against a human melanoma cancer cell line (UACC 903). Only one derivative with a 3,4-dimethoxy substitution did not show any cytotoxicity against a normal fibroblast cell line (FF2441) in the same concentration range. These results were corroborated in the Ehrlich ascites carcinoma animal model, highlighting significantly lowered cell viability, body weight, ascites volume as well as a down-regulation of angiogenesis and tumor growth [134].
Another interesting coumarin derivative investigated is (E)-2-(4-methoxybenzyloxy)-3-prenyl-4-methoxy-N-hydroxycinamide (BMX), a semisynthetic derivative of osthole and a coumarin found in several plant species, such as Cnidium monnieri L, Angelica archangelica L, and Angelica pubescens Maxim. BMX was found to inhibit VEGF-induced proliferation, migration, and endothelial tube formation in HUVECs. These activities were also corroborated by ex vivo and in vivo studies decreasing VEGF-induced microvessel sprouting from aortic rings and HCT116 colorectal cancer cells. Moreover, BMX inhibited HCT116 cell proliferation and the growth of xenografts of HCT116 cells in vivo [136].

Conclusions
Considering the crucial role of angiogenesis in cancer development, antiangiogenic agents have significant potential to fight cancer. Thus, investigating novel drugs to attenuate or prevent angiogenesis-associated complications in cancer is of great importance. The several clinical limitations and side effects related to the administration of current antiangiogenic agents for cancer treatment raise the need to find alternative treatments. Natural and synthetic coumarins have shown a variety of pharmacological properties. They have demonstrated prominent anticancer effects by targeting multiple signaling pathways involved in several types of cancer.
Recently, studies have focused on the antiangiogenic effects of coumarins according to their structure-activity relationships. The present review reports the currently available literature data on the signaling and regulatory pathways of angiogenesis, as well as on antiangiogenic and anticancer mechanisms of natural and synthetic coumarins, critically analyzing and highlighting their use as possible therapeutic strategies. These studies are essential to identify novel and effective anticancer agents with fewer side effects than conventional drugs. It is also critical to identify potential synergies that may allow reducing the side effects of cytotoxic medicines and increasing the quality of life of patients. Additional studies should focus on additional in vitro and in vivo experiments followed by well-controlled clinical trials to reveal the exact signaling pathways involved in cancer angiogenesis as well as the precise pharmacological mechanisms of coumarins. In addition, there is a need to investigate and adjust novel antiangiogenic coumarin lead compounds to develop more potent and efficient anticancer drugs with lower toxicity. In addition, an appropriate drug delivery system should be introduced to overcome the existing pharmacokinetic challenges of coumarin administration. Such research will unveil the potential of coumarins in the prevention, attenuation, and treatment of angiogenesis in cancer. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.