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Neovascularization occurs by two distinct mechanisms: angiogenesis, in which new blood vessels sprout from the pre-existing blood vessels, and vasculogenesis, in which new blood vessels are derived from the circulating bone marrow-derived endothelial progenitor cells (EPCs) [1–5]. This process of new blood vessel formation is essential not only in normal physiological situations (e.g., the menstrual cycle, implantation, embryogenesis, and wound-healing), but also in the growth and metastasis of malignant tumors [1–5].

In the normal physiological milieu, angiogenesis is tightly regulated by intricately balanced endogenous pro- and anti-angiogenic molecules [6–8]. However, in malignancy, this fine tuning of the balance between pro- and anti-angiogenic molecules is disabled by either the overexpression of pro-angiogenic molecules or the down-regulation of anti-angiogenic molecules, resulting in the activation of the angiogenic switch [6–8]. The overexpression of pro-angiogenic growth factors, such as vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), fibroblast growth factor (FGF), interleukin-8 (IL-8), placenta growth factor (PlGF), transforming growth factor β (TGF-β), and platelet derived growth factor (PDGF) tilt the tumor microenvironment in favor of angiogenesis and allow the transition of an avascular dormant tumor into a growing vascular tumor mass [1–3]. Because targeting growth factor-induced angiogenesis has shown clinical promise, designing therapies to target tumor neovessels is of interest owing to the decreased toxicity of the approach, the minimal drug resistance, and the ability to increase the efficacies of anti-cancer drugs and radiation therapy [1–10].

Catecholamines are a group of neurotransmitters that includes dopamine (DA), norepinephrine (NE), and epinephrine (E) [11]. In addition to their conventional roles in the brain, these molecules also have important functions in the periphery [11]. Recent discoveries of a regulatory role for different catecholamines in tumor angiogenesis are of current interest from a clinical viewpoint for the development of anti-angiogenic drugs to treat cancer patients [12–14]. These newly identified roles for catecholamines also enable us to understand the biology of catecholamines in peripheral systems [13]. The available information regarding the roles of catecholamines in the regulation of tumor angiogenesis is discussed in this chapter.

NE and E are Endogenous Promoters of Tumor Angiogenesis

NE and E act on their target cells through α (α1 and α2) and β (β1 and β2) adrenoceptors [11]. Evidence indicates that exposure to chronic stress promotes tumor growth [12, 15] through the stress mediators NE and E [16, 17], and the up-regulation of tumor angiogenesis is suggested to be the underlying mechanism [13, 18]. In a model of orthotopically xenografted human ovarian tumors in nude mice, a tumor growth-promoting effect was observed in animals following exposure to chronic stress or treatment with the β-adrenergic agonist isoproterenol, and this effect was abrogated by the β-adrenergic antagonist propranolol [19]. Interestingly, this increase in tumor growth was associated with the up-regulation of VEGF in tumor tissues, which led to the induction of tumor angiogenesis [19]. However, inhibition of the VEGF pathway suppressed the tumor growth stimulatory effect of the β-adrenergic agonist [19]. In vitro studies also demonstrated the NE-mediated secretion of VEGF by ovarian carcinoma cells [20]. In addition, there are reports that indicate that NE, by acting on the adrenoceptors present in the tumor-associated macrophages (TAM), induces angiogenesis by stimulating the production of matrix metalloproteinase 9 (MMP-9) [21].

In addition to the NE-mediated increase in the expression of the pro-angiogenic cytokine VEGF, studies have also indicated that in different tumor cells bearing the β-adrenoceptor (such as melanoma, ovarian, and nasopharyngeal cancer cells), NE induces a significant increase in the synthesis and release of other pro-angiogenic factors, including IL-6, IL-8, MMP-2, and MMP-9 [21–24]. Interestingly, nicotine has been shown to increase xenografted human colon tumor growth in nude mice. This increased tumor growth is associated with elevated plasma E levels and tumor angiogenesis. However, blocking the β-adrenoceptors with specific antagonists significantly abrogated the nicotine-induced tumor growth through the down-regulation of tumor angiogenesis [25]. In addition, NE has also been shown to stimulate VEGF mRNA synthesis in endothelial cells through the cAMP-PKA pathway and to promote proliferation of these cells by activating ERK [26].

Molecular Mechanisms of NE- and E-induced Tumor Angiogenesis

By acting through β2-adrenoceptors, NE has been shown to promote angiogenesis in the orthotopically grown ovarian cancers HEY-8 and SKOV3ipI [19]. The underlying molecular mechanisms of this phenomenon were determined to be increased VEGF synthesis and the overexpression of matrix metalloproteinases, such as MMP-2 and MMP-9 [19]. Further investigations demonstrated that this VEGF-induced overexpression of matrix metalloproteinases is mediated through the cAMP-PKA signaling pathway following stimulation of β2-adrenoceptors by NE, indicating a novel signaling pathway, such as NE-β2-adrenoceptors-cAMP-PKA-VEGF [19]. NE treatment has also shown similar results in the human nasopharyngeal cell line HONE 1 [23]. In several human multiple myeloma cell lines (NCI-H-929, MM-M1, and FLAM-76) NE treatment has also shown similar results by acting through β1 and β2 adrenoceptors present in these cells [27]. However, a recent study has demonstrated that in vitro treatment of human prostate (PC3), breast (MDA-MB-231), and liver (HCC SK-Hep1) cancer cells with NE or isoproterenol stimulated the expression of HIF-1α and synthesis of VEGF in a dose-dependent manner [28]. This increased VEGF synthesis was decreased when the tumor cells were transfected with HIF-1α siRNA [28]. This observation was further strengthened when HIF-1α was up- or down-regulated in these tumor cells following pretreatment with the adenylate cyclase activator forskolin or the protein kinase A (PKA) inhibitor H-89, respectively [28]. Finally, pretreatment of tumor cells with a β-adrenergic blocker propranolol completely abolished the expression of VEGF and HIF-1α protein amount in these cells [28]. Therefore, in brief, NE induces VEGF expression in cancer cells through NE-β-adrenoceptor-PKA-HIF-1α-VEGF signaling pathway (Fig. 7.1).

Fig. 7.1
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NE stimulates VEGF synthesis in the tumor cells. NE by activating β-adrenergic receptors activates cAMP-PKA axis and stimulates VEGF synthesis by up-regulating the transcription factor Hypoxia Inducible Factor-1α (HIF-1α) in tumor cells

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This catecholamine neurotransmitter also stimulates the synthesis and release of another pro-angiogenic factor, IL-6, in the human ovarian tumor cell lines SKOV3ip1, HEY-A8 and EG in vitro [24]. By acting through β-adrenoceptors in these tumor cells, NE significantly increased both IL-6 mRNA synthesis and promoter activity [24]. Additional results have demonstrated an abrogation of this NE-mediated effect on IL-6 synthesis following treatment with β-adrenoceptor antagonists, confirming the NE-mediated regulation of IL-6 gene transcription through the activation of β-adrenoceptors [24]. NE-mediated β-adrenoceptor activation was also shown to increase Src kinase phosphorylation, which subsequently increased IL-6 mRNA synthesis through the up-regulation of the IL-6 promoter activity [24]. This suggestion of a NE-β-adrenoceptor-Src kinase-IL-6 pathway for increased tumor angiogenesis was further strengthened by immunohistochemical analysis of human ovarian cancer tissues, in which a significant correlation between the overexpression of Src kinase and the degree of tumor neovascularization was observed [24]. Src activation was also instrumental in increasing the synthesis of other pro-angiogenic molecules, such as VEGF and IL-8 [24]. However, another alternate signaling pathway was recently identified in which NE and E stimulated MMPs in human ovarian tumor cell lines independent of the β1, β2-adrenoceptors-PKA pathway. This pathway involves STAT-3, a transcription factor known to initiate several signaling pathways in cancer cells [29].

DA as an Endogenous Inhibitor of Tumor Angiogenesis

In addition to acting as a precursor molecule in the biosynthetic pathway of NE and E, DA also acts as an important neurotransmitter in both the brain and the peripheral organs [30]. In the brain, DA regulates several major functions, including cognition, motor activities, and the reward effect in the form of pleasure [30, 31]. In peripheral systems, DA regulates cardiac and renal functions. In addition, recent evidence has indicated that DA influences other diverse functions, such as blood pressure, insulin synthesis in beta cells of the pancreas, and the functions of immune effector cells [32–34]. Recently, another role of this neurotransmitter in the peripheral system has been demonstrated: DA functions as an endogenous inhibitor of angiogenesis by acting through its D2 class of receptors present in the endothelial cells and EPCs [35–41].

A significant increase in B-16 melanoma growth has been found in D2 DA receptor (−/−) mice [37]. Another study has revealed significantly decreased growth of mammary carcinoma in hyperdopaminergic Wistar rats (APO-SUS), and increased growth of the same tumors was observed in hypodopaminergic rats (APO-UNSUS) [36]. This increased or decreased tumor growth in hypo- or hyperdopamineregic rats is closely associated with increased or decreased angiogenesis in tumors [36]. Furthermore, in human gastric cancer patients, a significant reduction of DA in malignant stomach tissues compared to the surrounding normal tissues has been observed, and exogenous administration of DA or a D2 DA receptor agonist significantly inhibited stomach tumor growth [38]. The mechanism of this phenomenon has been attributed to the inhibition of angiogenesis in the tumor tissues [38].

Molecular Mechanisms of DA-induced Inhibition of Tumor Angiogenesis

VEGF is the predominant cytokine that regulates angiogenesis by mediating proliferation, migration, and tube formation in endothelial cells from pre-existing vessels [3, 10]. VEGF also plays a pivotal role in the migration and subsequent mobilization of EPCs from the bone marrow into the neovessels of tumors by acting through VEGFR-2 receptors present on these cells [41]. In vivo studies have demonstrated that DA treatment significantly inhibits tumor angiogenesis [35–41]. Tumor endothelial cells isolated from human breast (MCF-7) and colon (HT29) tumor-bearing mice displayed suppression of VEGFR-2 phosphorylation with subsequent inhibition of its downstream signaling cascades (e.g., MAPK and focal adhesion kinase (FAK)), which regulate the proliferation and migration of endothelial cells; this regulation is essential for tumor neovessel formation (Fig. 7.2) [40]. Recent studies also reveal important contributions of bone marrow-derived EPCs in tumor angiogenesis [4, 5, 42]. Indeed, additional reports indicate that in the bone marrow niche, DA is synthesized in stromal cells [43] and is depleted in tumor-bearing mice [41], thus indicating a role of DA in the regulation of the mobilization of these precursor cells from bone marrow into circulation [41]. Importantly, the administration of exogenous DA, which inhibited tumor angiogenesis, also inhibited the mobilization of these cells from bone marrow [41]. This inhibitory effect of DA is abrogated when the animals are treated with a D2 DA receptor antagonist [41]. The inhibitory effect on the mobilization of EPCs from the bone marrow and hence on tumor angiogenesis is due to the D2 DA receptor-mediated down-regulation of MMP-9 synthesis by these bone marrow progenitor cells through the inhibition of the ERK1/ERK2 pathway (Fig. 7.3) [41]. These observations were further supported in D2 DA receptor (−/−) mice: increased numbers of circulating EPCs were evident in tumor-bearing mice compared to wild type controls, and the D2 DA receptor antagonist treatment failed to elicit any effect in the animals [41].

Fig. 7.2
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Dopamine inhibits tumor endothelial cell proliferation and migration. Dopamine by activating its D2 receptors inhibits VEGFR-2 phosphorylation and downstream signaling molecules like mitogen-activated protein kinase (MAPK) and Focal adhesion kinase (FAK)

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Fig. 7.3
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Dopamine inhibits mobilization of bone marrow-derived endothelial progenitor cells for tumor neovessel formation. Activation of D2 dopamine receptors in endothelial progenitor cells (EPCs), dopamine inhibits migration of these cells from bone marrow to the circulation and subsequently to neovessels of the tumors by inhibiting synthesis of matrix metalloproteinase-9 (MMP-9) in these progenitor cells

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These studies have clearly demonstrated that DA acts as an endogenous inhibitor of angiogenesis, and hence of tumor growth, by targeting the VEGF-induced proliferation and migration of endothelial cells and EPCs through several newly uncovered mechanisms [35–41].

Discussion

Together, current findings show that catecholamine neurotransmitters act as regulators of tumor angiogenesis and, hence, regulate the growth of malignant tumors [13]. The available evidence suggests that DA acts through its D2 class of receptors to inhibit tumor angiogenesis by targeting the proliferation and migration of tumor endothelial cells as well as the mobilization of EPCs [35–41]. In contrast, NE and E act through β-adrenoceptors to stimulate angiogenesis by stimulating the synthesis of pro-angiogenic cytokines (e.g., VEGF, IL-8, and IL-6) and MMPs (e.g., MMP-2 and MMP-9) in tumor cells through different signaling pathways [13]. Briefly, in the D2 DA receptor-mediated down-regulation of angiogenesis, the target cells are endothelial cells and EPCs, whereas in the NE-mediated up-regulation of tumor angiogenesis, the target cells are principally tumor cells [13]. Therefore, based on these opposing effects of stimulation and inhibition of tumor angiogenesis by the catecholamine neurotransmitters (DA, NE, and E), it is suggested that catecholamines function as an angiogenic switch in the tumor microenvironment (Fig. 7.4). The expression profile of D2 DA receptors or β-adrenoceptors in any organ may be altered in response to the onset of malignancy in that organ. These alterations may tilt the microenvironment of the tumor in favor of angiogenesis, thereby transforming an avascular, dormant tumor mass into a vascular, rapidly growing tumor. Epidemiological evidence has also indicated that the use of NE antagonists reduces the risk of cancer incidence [44]. Therefore, it will be prudent to undertake further detailed investigations to dissect the specific roles of catecholamines in relation to their function as an angiogenic switch in tumor growth. These studies will enable clinicians to develop DA or NE/E receptor agonists or antagonists as anti-angiogenic drugs.

Fig. 7.4
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Model of an angiogenic switch in tumor: Diagram of catecholamine-mediated operation of an angiogenic switch in tumor microenvironment. Dopamine inhibits angiogenesis, whereas norepinephrine and epinephrine up-regulate angiogenesis in tumor tissues

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