University of Groningen Studies on the Role of Dopamine and Serotonin In

University of Groningen

Studies on the role of dopamine and serotonin in tumors and their microenvironment Peters, Marloes A.M.

DOI: 10.33612/diss.135597229

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CHAPTER 2

Dopamine and serotonin regulate tumor behavior by affecting angiogenesis

Marloes A.M. Peters,1 Annemiek M.E. Walenkamp,1 Ido P. Kema,2 Coby Meijer,1 Elisabeth G.E. de Vries,1 and Sjoukje F. Oosting 1

1 Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands 2 Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

Drug Resist Updat 2014; 17: 96-104.

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548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 19 ABSTRACT INTRODUCTION

The biogenic amines dopamine and serotonin are neurotransmitters and hormones, Increasingly, it is recognized that apart from tumor cells also the non-tumor cells in which are mainly produced in the central nervous system and in the gastro-intestinal the microenvironment participate in tumor behavior [1,2]. Endothelial cells are part of tract. They execute local and systemic functions such as intestinal motility and tissue this microenvironment and can create new blood vessels, which provide oxygen and 2 repair. Dopamine and serotonin are primarily stored in and transported by platelets. nutrients to the tumor, and hence promote tumor growth and metastasis [3]. Besides This review focuses on the recently recognized role of dopamine and serotonin in the cells from the microenvironment, also distant host cells are attracted to the tumor and regulation of tumor behavior by affecting angiogenesis and tumor cell proliferation. contribute to tumor development. The interaction between tumor microenvironment Preclinical studies demonstrate that dopamine inhibits tumor growth via activation of and distant host cells shows similarities with the process of wound healing, in which dopamine receptor D2 on endothelial and tumor cells. Serotonin stimulates tumor immune cells and platelets are attracted to the affected tissue and participate in repair. growth via activation of serotonin receptor 2B on endothelial cells and serotonin In tumor development, attracted host cells can have a detrimental effect; they can receptors on tumor cells. Drugs that stimulate dopamine receptor D2 or inhibit contribute to tumor growth, invasiveness and metastasis [4]. Alike during wound serotonin receptors are available and therefore clinical intervention studies for cancer healing, platelets are recruited to the tumor site and adhere to the activated vascular patients are within reach. wall [5]. This leads to release of their content consisting of pro- and anti-angiogenic factors such as vascular endothelial growth factor A (VEGF-A) [6] and dopamine and serotonin [7]. Dopamine and serotonin are produced in the central nervous system (CNS) and in the gastrointestinal tract, where they play a role in several local and systemic processes [8,9]. Moreover, dopamine and serotonin are increasingly recognized to be also involved in tumor behavior by affecting angiogenesis and tumor cell proliferation [10-12]. This review therefore aims to summarize the available knowledge on the role of dopamine and serotonin as regulators of tumor behavior with a focus on angiogenesis.

METHODS

Articles for this review were identified by searches of PubMed and Web of Knowledge using the search terms “dopamine”, “serotonin”, “5-hydroxytryptamine”, “dopamine receptor”, “serotonin receptor”, “platelets”, “angiogenesis”, “neovascularization”, “neoplasm”, and “cancer”. Relevant references of found articles were also included. We selected English publications of all years. International Clinical Trials Registry Platform (ICTRP) accepted trial registries were searched for ongoing clinical trials.

RESULTS

Dopamine Dopamine is synthesized in the CNS and the gastrointestinal tract [13-15]. It is

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548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 20 ABSTRACT INTRODUCTION

The biogenic amines dopamine and serotonin are neurotransmitters and hormones, Increasingly, it is recognized that apart from tumor cells also the non-tumor cells in which are mainly produced in the central nervous system and in the gastro-intestinal the microenvironment participate in tumor behavior [1,2]. Endothelial cells are part of tract. They execute local and systemic functions such as intestinal motility and tissue this microenvironment and can create new blood vessels, which provide oxygen and repair. Dopamine and serotonin are primarily stored in and transported by platelets. nutrients to the tumor, and hence promote tumor growth and metastasis [3]. Besides 2 This review focuses on the recently recognized role of dopamine and serotonin in the cells from the microenvironment, also distant host cells are attracted to the tumor and regulation of tumor behavior by affecting angiogenesis and tumor cell proliferation. contribute to tumor development. The interaction between tumor microenvironment Preclinical studies demonstrate that dopamine inhibits tumor growth via activation of and distant host cells shows similarities with the process of wound healing, in which dopamine receptor D2 on endothelial and tumor cells. Serotonin stimulates tumor immune cells and platelets are attracted to the affected tissue and participate in repair. growth via activation of serotonin receptor 2B on endothelial cells and serotonin In tumor development, attracted host cells can have a detrimental effect; they can receptors on tumor cells. Drugs that stimulate dopamine receptor D2 or inhibit contribute to tumor growth, invasiveness and metastasis [4]. Alike during wound serotonin receptors are available and therefore clinical intervention studies for cancer healing, platelets are recruited to the tumor site and adhere to the activated vascular patients are within reach. wall [5]. This leads to release of their content consisting of pro- and anti-angiogenic factors such as vascular endothelial growth factor A (VEGF-A) [6] and dopamine and serotonin [7]. Dopamine and serotonin are produced in the central nervous system (CNS) and in the gastrointestinal tract, where they play a role in several local and systemic processes [8,9]. Moreover, dopamine and serotonin are increasingly recognized to be also involved in tumor behavior by affecting angiogenesis and tumor cell proliferation [10-12]. This review therefore aims to summarize the available knowledge on the role of dopamine and serotonin as regulators of tumor behavior with a focus on angiogenesis.

METHODS

RESULTS

Dopamine Dopamine is synthesized in the CNS and the gastrointestinal tract [13-15]. It is

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548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 21 produced in the cytoplasm from the amino-acid tyrosine by tyrosine hydroxylase [14]. Serotonin is metabolized in cells of the brain, gastrointestinal tract, liver, lungs and In the blood, ~99% of dopamine is stored in platelets [7], in plasma ~1% circulates in platelets by monoamine oxidase (MAO), and thereafter excreted by the kidney as 5- its free form and the remainder as inactive dopamine sulfate [16]. hydroxyindoleacetic acid (5-HIAA) [9]. In the CNS, dopamine is involved in reward mechanisms and limb movement control. In addition, after being transported by sympathetic nerves and platelets it also Platelets 2 has peripheral effects throughout the body like regulation of vascular tone [8]. Apart Platelets store dopamine and serotonin in dense granules and are their main circulating from exhibiting its own function, it is also a precursor of epinephrine and reservoir [7,21]. Platelet activation and content release play a critical role in hemostasis, norepinephrine in the CNS and the adrenal glands [14]. Dopamine exerts its function thrombosis, and angiogenesis [5,22]. In cancer, platelets adhere to the activated wall of by binding to dopamine receptors (DRs). DRs are located on the cell membrane, and tumor vessels and release their content, consisting of dopamine and serotonin, but expressed in the brain, heart, kidneys, adrenal cortex, sympathetic nerve terminals, and also calcium, factor V, fibrinogen and VEGF-A [5,7]. VEGF-A is stored in α-granules blood vessels. There are two types of DRs: D1-like DR and D2-like DR. D1-like DR [23]. In this review, we will use VEGF if the articles cited do not specify whether consist of subtypes DRD1 and DRD5, and D2-like DR of subtypes DRD2, DRD3, VEGF-A or another isoform is studied. and DRD4. Activation of D1-like DR stimulates cellular cyclic adenosine monophosphate (cAMP) accumulation, whereas activation of D2-like DR inhibits this In vitro effects of dopamine on endothelial cells [8]. Dopamine transporters are located at the plasma membrane. They actively Dopamine predominantly affects tumor behavior via inhibition of angiogenesis. Many transport dopamine from the synaptic cleft or the blood into cells where it is stored or in vitro studies addressed the effect of dopamine on endothelial cells. catabolized. These transporters are expressed in the substantia nigra and ventral Dopamine (1 µM) inhibited VEGF-induced proliferation and migration of tegmental area of the brain, stomach, kidney, pancreatic duct [17], and platelets [18]. human umbilical vein endothelial cells (HUVEC) [24] and stimulated early apoptosis Dopamine is predominantly degraded in the liver, kidney and brain by in murine mesentery endothelial cells [11]. Furthermore, pretreatment with dopamine sulfoconjugation, oxidative deamination, and O-methylation [14]. (10 µM) abolished VEGF-induced HUVEC permeability in an in vitro permeability assay. Immunoprecipitation showed that this was induced by restabilization of Serotonin adherent junctions, tight junctions, and occludin [25]. Serotonin, also known as 5-hydroxytryptamine or 5-HT, is produced in the CNS and DRD2 agonists had a similar effect as dopamine. In vitro, the DRD2 agonists in enterochromaffin cells of the gastrointestinal tract. It is converted from one amino- bromocriptine or quinpirole (50 µM) inhibited HUVEC proliferation and migration, in acid L-tryptophan by tryptophan hydroxylase. Less than 1% serotonin circulates in its contrast to DRD1 agonist SKF39393 (50 µM). Quinpirole (10 µM) also inhibited free form in the blood. The remaining serotonin is stored in platelets, presynaptic HUVEC permeability [24,25]. The inhibitory effect of dopamine could be abrogated neurons, and enterochromaffin cells [7,9]. by the DRD2 antagonist eticlopride (50 µM), but not by the DRD1 antagonist Serotonin plays a role in many physiological processes. It modulates heart SCH23390 (50 µM). This indicates that dopamine affects HUVEC proliferation and development [19], intestinal motility, vascular tone, and platelet aggregation [9]. migration via DRD2 activation. Fluorescence activating cell sorting (FACS) analysis Serotonin receptors (5-HTR) are located on the cell membrane, and are present in the revealed that dopamine and DRD2 agonists can induce VEGF receptor type 2 CNS, heart, gastro-intestinal tract, blood vessels, and on platelets. There are 7 types of (VEGF-R2) internalization [24]. 5-HTR (5-HTR1-7), some of which are subdivided into for example 5-HTR1A, 5- Dopamine not only affects endothelial cells, but also mesenchymal stem cells HTR1B, etc. Activation of 5-HTR1 and 5-HTR5 inhibits intracellular cAMP (MSC) and endothelial progenitor cells (EPC) [26,27], which can be incorporated in accumulation, whereas activation of 5-HTR4 and 5-HTR7 stimulates this. 5-HTR2 newly developing blood vessels [3,26,27]. For these experiments, MSCs and EPCs activation induces intracellular Ca2+ release and 5-HTR3 activation stimulates Na+/K+ were obtained from the bone marrow of healthy mice and expanded in vitro. cation channels resulting in membrane depolarization. Serotonin transporters are Dopamine (1 µM) reduced VEGF-induced MSC and EPC migration [26,27]. The located at the plasma membrane and actively transport serotonin from the interstitial effect on EPCs could be abolished by the DRD2 antagonist eticlopride (10 µM). space and the blood into cells [9]. They are expressed in the brain [17], heart [20], Dopamine also reduced VEGF-A induced extracellular signal-regulated kinase (ERK) gastro-intestinal tract, adrenal gland [17], blood vessels [20], and on platelets [17]. 1/2 phosphorylation. Furthermore, the pro-matrix metalloproteinase 9 (MMP-9) level - 22 - - 23 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 22 produced in the cytoplasm from the amino-acid tyrosine by tyrosine hydroxylase [14]. Serotonin is metabolized in cells of the brain, gastrointestinal tract, liver, lungs and In the blood, ~99% of dopamine is stored in platelets [7], in plasma ~1% circulates in platelets by monoamine oxidase (MAO), and thereafter excreted by the kidney as 5- its free form and the remainder as inactive dopamine sulfate [16]. hydroxyindoleacetic acid (5-HIAA) [9]. In the CNS, dopamine is involved in reward mechanisms and limb movement control. In addition, after being transported by sympathetic nerves and platelets it also Platelets has peripheral effects throughout the body like regulation of vascular tone [8]. Apart Platelets store dopamine and serotonin in dense granules and are their main circulating 2 from exhibiting its own function, it is also a precursor of epinephrine and reservoir [7,21]. Platelet activation and content release play a critical role in hemostasis, norepinephrine in the CNS and the adrenal glands [14]. Dopamine exerts its function thrombosis, and angiogenesis [5,22]. In cancer, platelets adhere to the activated wall of by binding to dopamine receptors (DRs). DRs are located on the cell membrane, and tumor vessels and release their content, consisting of dopamine and serotonin, but expressed in the brain, heart, kidneys, adrenal cortex, sympathetic nerve terminals, and also calcium, factor V, fibrinogen and VEGF-A [5,7]. VEGF-A is stored in α-granules blood vessels. There are two types of DRs: D1-like DR and D2-like DR. D1-like DR [23]. In this review, we will use VEGF if the articles cited do not specify whether consist of subtypes DRD1 and DRD5, and D2-like DR of subtypes DRD2, DRD3, VEGF-A or another isoform is studied. and DRD4. Activation of D1-like DR stimulates cellular cyclic adenosine monophosphate (cAMP) accumulation, whereas activation of D2-like DR inhibits this In vitro effects of dopamine on endothelial cells [8]. Dopamine transporters are located at the plasma membrane. They actively Dopamine predominantly affects tumor behavior via inhibition of angiogenesis. Many transport dopamine from the synaptic cleft or the blood into cells where it is stored or in vitro studies addressed the effect of dopamine on endothelial cells. catabolized. These transporters are expressed in the substantia nigra and ventral Dopamine (1 µM) inhibited VEGF-induced proliferation and migration of tegmental area of the brain, stomach, kidney, pancreatic duct [17], and platelets [18]. human umbilical vein endothelial cells (HUVEC) [24] and stimulated early apoptosis Dopamine is predominantly degraded in the liver, kidney and brain by in murine mesentery endothelial cells [11]. Furthermore, pretreatment with dopamine sulfoconjugation, oxidative deamination, and O-methylation [14]. (10 µM) abolished VEGF-induced HUVEC permeability in an in vitro permeability assay. Immunoprecipitation showed that this was induced by restabilization of Serotonin adherent junctions, tight junctions, and occludin [25]. Serotonin, also known as 5-hydroxytryptamine or 5-HT, is produced in the CNS and DRD2 agonists had a similar effect as dopamine. In vitro, the DRD2 agonists in enterochromaffin cells of the gastrointestinal tract. It is converted from one amino- bromocriptine or quinpirole (50 µM) inhibited HUVEC proliferation and migration, in acid L-tryptophan by tryptophan hydroxylase. Less than 1% serotonin circulates in its contrast to DRD1 agonist SKF39393 (50 µM). Quinpirole (10 µM) also inhibited free form in the blood. The remaining serotonin is stored in platelets, presynaptic HUVEC permeability [24,25]. The inhibitory effect of dopamine could be abrogated neurons, and enterochromaffin cells [7,9]. by the DRD2 antagonist eticlopride (50 µM), but not by the DRD1 antagonist Serotonin plays a role in many physiological processes. It modulates heart SCH23390 (50 µM). This indicates that dopamine affects HUVEC proliferation and development [19], intestinal motility, vascular tone, and platelet aggregation [9]. migration via DRD2 activation. Fluorescence activating cell sorting (FACS) analysis Serotonin receptors (5-HTR) are located on the cell membrane, and are present in the revealed that dopamine and DRD2 agonists can induce VEGF receptor type 2 CNS, heart, gastro-intestinal tract, blood vessels, and on platelets. There are 7 types of (VEGF-R2) internalization [24]. 5-HTR (5-HTR1-7), some of which are subdivided into for example 5-HTR1A, 5- Dopamine not only affects endothelial cells, but also mesenchymal stem cells HTR1B, etc. Activation of 5-HTR1 and 5-HTR5 inhibits intracellular cAMP (MSC) and endothelial progenitor cells (EPC) [26,27], which can be incorporated in accumulation, whereas activation of 5-HTR4 and 5-HTR7 stimulates this. 5-HTR2 newly developing blood vessels [3,26,27]. For these experiments, MSCs and EPCs activation induces intracellular Ca2+ release and 5-HTR3 activation stimulates Na+/K+ were obtained from the bone marrow of healthy mice and expanded in vitro. cation channels resulting in membrane depolarization. Serotonin transporters are Dopamine (1 µM) reduced VEGF-induced MSC and EPC migration [26,27]. The located at the plasma membrane and actively transport serotonin from the interstitial effect on EPCs could be abolished by the DRD2 antagonist eticlopride (10 µM). space and the blood into cells [9]. They are expressed in the brain [17], heart [20], Dopamine also reduced VEGF-A induced extracellular signal-regulated kinase (ERK) gastro-intestinal tract, adrenal gland [17], blood vessels [20], and on platelets [17]. 1/2 phosphorylation. Furthermore, the pro-matrix metalloproteinase 9 (MMP-9) level - 22 - - 23 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 23 in the supernatant was decreased [27]. MMP-9 is involved in EPC mobilization from vascularization in these lesions [31,33]. the bone marrow and is under control of the classic mitogen-activated protein kinase Endometrial biopsies of patients with endometriosis showed less DRD2 and (MAPK) pathway [28,29] (Figure 1). more VEGF RNA and protein expression than endometrial biopsies of controls. In summary, dopamine and DRD2 agonists inhibit VEGF-induced endothelial These patients had higher VEGF-R2 RNA expression in the red (active) cell proliferation as well as MSC and EPC migration. endometriosis lesions than in white and black (inactive) lesions [31]. These findings led 2 to a clinical trial in nine endometriosis patients. During laparoscopic treatment, one- half of the red endometriosis lesions were removed, while the other lesions were marked with silk sutures. Thereafter, patients received increasing doses DRD2 agonist quinagolide (0.025-0.075 mg/day orally for 18-20 weeks). In a second laparoscopic procedure, the remaining endometriosis lesions were evaluated and removed. Quinagolide treatment reduced the size of the lesions by 69.5% and 35% of the lesions disappeared. VEGF-R2 protein expression was decreased in the remaining lesions [34]. Larger clinical trials have to be conducted to investigate whether DRD2 agonists are a relevant standard treatment for endometriosis, but to our knowledge these are currently not performed.

Ovarian Hyperstimulation Syndrome (OHSS) In 0.5-5% of women undergoing fertilization treatment with gonadotrophins, OHSS occurs as a complication [35]. This condition is characterized by swelling of the Fig. 1 Dopamine and serotonin are mainly produced in the CNS and enterochromaffin cells of the ovaries and increased vascular permeability resulting in ascites, and is mediated gastrointestinal tract. Platelets store and release dopamine and serotonin. Serotonin can stimulate through VEGF-induced VEGF-R2 activation [36]. tumor cell proliferation by activation of differentially expressed receptors on various tumor types When HUVECs were treated in vitro with dopamine (2 μM) before exposure and endothelial cell proliferation and migration via activation of 5-HTR1 and 5-HTR2. Dopamine inhibits VEGF-induced endothelial cell proliferation via activation of DRD2 and VEGF-induced EPC to follicular fluid of OHSS patients, phosphorylation of VEGF-R2 and tight and mobilization by diminished production of MMP-9. Hereby, dopamine prevents EPC to transform to adherent junctions was decreased. Endothelial permeability assay showed that endothelial cells at the site of action. dopamine treatment also reduced HUVEC permeability. HUVEC migration and proliferation however, were only inhibited by a higher dose dopamine (10 µM) [37]. In a rat OHSS model, treatment with cabergoline (0.1 or 0.5 mg/kg/day) Dopamine in non-malignant diseases reduced vascular hyperpermeability. The higher dose however, also disrupted luteal In non-malignant diseases, the effect of dopamine on angiogenesis has been clearly angiogenesis, which is required for follicle maturation and potential subsequent demonstrated in preclinical and clinical studies. These data support the concept that pregnancy development [38]. dopamine also plays a role in tumor behavior. Women with polycystic ovary syndrome (PCOS) have an increased risk to develop OHSS during ovulation-inducing treatment. Key symptoms of PCOS are Endometriosis hyperandrogenism and chronic anovulation [39]. These patients have a lower ovarian In endometriosis ectopic endometrial tissue is implanted outside the uterus, which is and follicular DRD2 protein expression and higher follicular microvessel density than associated with pelvic pain and infertility. Angiogenesis is essential for the healthy controls [40]. maintenance and progression of this disease [30]. A meta-analysis of 570 women at high risk of developing OHSS during In mice bearing implanted endometriosis fragments, orally administered assisted reproduction showed that cabergoline (0.5 mg/day) reduced this risk with DRD2 agonist cabergoline (0.05 and 1 mg/kg/day) decreased VEGF RNA and 12% (95% confidence interval (CI) 6.1–18.2%). This treatment decreased ascites and protein expression [31,32], VEGF-R2 protein expression [31], and impaired did not adversely affect the ~40% pregnancy rate in these women [41-43]. In a - 24 - - 25 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 24 in the supernatant was decreased [27]. MMP-9 is involved in EPC mobilization from vascularization in these lesions [31,33]. the bone marrow and is under control of the classic mitogen-activated protein kinase Endometrial biopsies of patients with endometriosis showed less DRD2 and (MAPK) pathway [28,29] (Figure 1). more VEGF RNA and protein expression than endometrial biopsies of controls. In summary, dopamine and DRD2 agonists inhibit VEGF-induced endothelial These patients had higher VEGF-R2 RNA expression in the red (active) cell proliferation as well as MSC and EPC migration. endometriosis lesions than in white and black (inactive) lesions [31]. These findings led to a clinical trial in nine endometriosis patients. During laparoscopic treatment, one- 2 half of the red endometriosis lesions were removed, while the other lesions were marked with silk sutures. Thereafter, patients received increasing doses DRD2 agonist quinagolide (0.025-0.075 mg/day orally for 18-20 weeks). In a second laparoscopic procedure, the remaining endometriosis lesions were evaluated and removed. Quinagolide treatment reduced the size of the lesions by 69.5% and 35% of the lesions disappeared. VEGF-R2 protein expression was decreased in the remaining lesions [34]. Larger clinical trials have to be conducted to investigate whether DRD2 agonists are a relevant standard treatment for endometriosis, but to our knowledge these are currently not performed.

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 25 randomized controlled trial with lower doses of cabergoline (0.25 mg/day) a similar also diminished proliferation of non-Hodgkin's lymphoma cells. This effect was absolute risk reduction could be achieved in 100 women at high risk of developing abolished by the free radical scavenger sodium metabisulfite. The authors suggested OHSS compared with 100 women not receiving cabergoline [44]. dopamine to act via induction of oxidative stress [53]. Colon and breast cancer cell proliferation and migration were not affected by dopamine (1.2 µM) [54]. Differences Parkinson's disease in outcome might be the consequence of different tumor types and dopamine doses 2 Parkinson’s disease is characterized by central dopamine depletion due to degeneration used. of dopamine producing neurons in the substantia nigra. Major symptoms are rigidity, Various tumor cell types express DRs and dopamine transporters [11,53,55- tremor and akinesia [45]. Parkinson's disease can be treated with the dopamine 57]. Tumor cell behavior can be affected by DRD2 agonists. Clonal growth of human precursor L-3,4-dihydroxyphenylalanine (L-DOPA), which induces dyskinesia as a small cell lung cancer cells was inhibited by DRD2 agonist bromocriptine (0.1 nM) side-effect in over half of patients [46]. [57]. Pretreatment with the DRD2 agonist quinpirole (50 µM) inhibited insulin-like Rats that developed L-DOPA induced dyskinesia had increased VEGF-A growth factor 1 (IGF1)-induced proliferation of human gastric cancer cells via protein levels in the dorsolateral striatum and substantia nigra pars reticulata [47] and reduced cellular phosphorylation of IGF1 receptor and its downstream molecule Akt enhanced endothelial cell proliferation and blood vessel length in the basal ganglia [55]. [48,49]. Signs of dyskinesia and angiogenesis similar as induced by L-DOPA were seen In various animal experiments, dopamine’s role in cancer was further after treatment with the DRD1 agonist SKF38393 (1.5 mg/kg/day), but not with the investigated. In mice, bone marrow dopamine concentrations decreased 7-fold after DRD2 agonist bromocriptine (2.5 mg/kg/day). The DRD1 antagonist SCH23390 murine sarcoma transplantation [27]. In another mouse model, 6-hydroxydopamine (0.25 mg/kg/day) abolished the effect of L-DOPA, but the DRD2 antagonist injections ablated peripheral dopaminergic nerves and therefore induced dopamine eticlopride (0.01 mg/kg/day) did not. The pro-angiogenic effect of L-DOPA in depletion. These mice had larger subcutaneously (sc) injected murine melanomas and Parkinson's disease thus seems to be mediated through DRD1 [48]. sarcomas than mice with intact peripheral dopaminergic nerves. Dopamine depleted The mesencephalon of 6 Parkinson patients at autopsy contained more nuclei mice had also enhanced tumor microvessel density and permeability as well as of endothelial cells than the mesencephalon of 10 non-Parkinson patients. Since it increased VEGF-R2 phosphorylation of tumor endothelial cells [58,59]. was not reported whether these patients used L-DOPA, it remains unclear whether In contrast to dopamine depleted mice, dopamine transporter knockout mice this phenomenon is a result of L-DOPA treatment or of the disease itself [50]. have a hyperdopaminergic system resulting in high systemic dopamine levels. When lung carcinoma cells were sc implanted, these mice had smaller tumors with lower Wound healing microvessel density compared with wild-type mice [60]. Apomorphine-susceptible Wound healing partially depends on angiogenesis [51]. Since DRD2 agonists have an (APO-SUS) rats selected from an outbred population of Wistar rats have a anti-angiogenic effect, DRD2 antagonists were evaluated for pro-angiogenic and hyperreactive dopaminergic system with a higher amount of cerebral tyrosine hence wound healing capacity in mice. In mice with cutaneous wounds, treatment with hydroxylase mRNA and DRD2 protein [61]. Seven days after sc implantation, DRD2 antagonist eticlopride (10 mg/kg/day) did indeed accelerate wound healing and mammary tumors were smaller with a lower tumor microvessel density in APO-SUS increased microvessel density in the wound compared to saline treatment [52]. rats than in their apomorphine-unsusceptible (APO-UNSUS) counterparts. Eticlopride also enhanced the number of circulating MSC in the peripheral blood Furthermore, APO-SUS rats developed fewer lung metastases than APO-UNSUS rats compared to saline treatment. The number of bromodeoxyuridine (BrdU) labeled after intravenous (iv) injection of mammary breast cancer cells [62]. MSCs that were iv injected were also higher in the wound bed after eticlopride Dopamine treatment resulted in decreased tumor growth with a lower treatment [26]. microvessel density in several animal models [11,24,54,63-65]. In general, dopamine treatment consisted of intra peritoneal administration of 50 mg/kg/day, resulting in Dopamine in cancer dopamine plasma levels of 1.2 µM in mice and 2.4 µM in rats one minute after Several in vitro experiments indicated that dopamine can directly affect tumor cells. In injection. This is 5% of the lethal dose in rodents. Dopamine also reduced vascular ovarian cancer cells, dopamine (12.5-50 µM) decreased tumor cell invasion in a permeability in mice bearing human colon and breast tumors [54] and murine ovarian membrane invasion culture system and increased apoptosis [11]. Dopamine (5 µM) tumors, resulting in less accumulation of ascites in the latter model [24]. Tumor - 26 - - 27 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 26 randomized controlled trial with lower doses of cabergoline (0.25 mg/day) a similar also diminished proliferation of non-Hodgkin's lymphoma cells. This effect was absolute risk reduction could be achieved in 100 women at high risk of developing abolished by the free radical scavenger sodium metabisulfite. The authors suggested OHSS compared with 100 women not receiving cabergoline [44]. dopamine to act via induction of oxidative stress [53]. Colon and breast cancer cell proliferation and migration were not affected by dopamine (1.2 µM) [54]. Differences Parkinson's disease in outcome might be the consequence of different tumor types and dopamine doses Parkinson’s disease is characterized by central dopamine depletion due to degeneration used. 2 of dopamine producing neurons in the substantia nigra. Major symptoms are rigidity, Various tumor cell types express DRs and dopamine transporters [11,53,55- tremor and akinesia [45]. Parkinson's disease can be treated with the dopamine 57]. Tumor cell behavior can be affected by DRD2 agonists. Clonal growth of human precursor L-3,4-dihydroxyphenylalanine (L-DOPA), which induces dyskinesia as a small cell lung cancer cells was inhibited by DRD2 agonist bromocriptine (0.1 nM) side-effect in over half of patients [46]. [57]. Pretreatment with the DRD2 agonist quinpirole (50 µM) inhibited insulin-like Rats that developed L-DOPA induced dyskinesia had increased VEGF-A growth factor 1 (IGF1)-induced proliferation of human gastric cancer cells via protein levels in the dorsolateral striatum and substantia nigra pars reticulata [47] and reduced cellular phosphorylation of IGF1 receptor and its downstream molecule Akt enhanced endothelial cell proliferation and blood vessel length in the basal ganglia [55]. [48,49]. Signs of dyskinesia and angiogenesis similar as induced by L-DOPA were seen In various animal experiments, dopamine’s role in cancer was further after treatment with the DRD1 agonist SKF38393 (1.5 mg/kg/day), but not with the investigated. In mice, bone marrow dopamine concentrations decreased 7-fold after DRD2 agonist bromocriptine (2.5 mg/kg/day). The DRD1 antagonist SCH23390 murine sarcoma transplantation [27]. In another mouse model, 6-hydroxydopamine (0.25 mg/kg/day) abolished the effect of L-DOPA, but the DRD2 antagonist injections ablated peripheral dopaminergic nerves and therefore induced dopamine eticlopride (0.01 mg/kg/day) did not. The pro-angiogenic effect of L-DOPA in depletion. These mice had larger subcutaneously (sc) injected murine melanomas and Parkinson's disease thus seems to be mediated through DRD1 [48]. sarcomas than mice with intact peripheral dopaminergic nerves. Dopamine depleted The mesencephalon of 6 Parkinson patients at autopsy contained more nuclei mice had also enhanced tumor microvessel density and permeability as well as of endothelial cells than the mesencephalon of 10 non-Parkinson patients. Since it increased VEGF-R2 phosphorylation of tumor endothelial cells [58,59]. was not reported whether these patients used L-DOPA, it remains unclear whether In contrast to dopamine depleted mice, dopamine transporter knockout mice this phenomenon is a result of L-DOPA treatment or of the disease itself [50]. have a hyperdopaminergic system resulting in high systemic dopamine levels. When lung carcinoma cells were sc implanted, these mice had smaller tumors with lower Wound healing microvessel density compared with wild-type mice [60]. Apomorphine-susceptible Wound healing partially depends on angiogenesis [51]. Since DRD2 agonists have an (APO-SUS) rats selected from an outbred population of Wistar rats have a anti-angiogenic effect, DRD2 antagonists were evaluated for pro-angiogenic and hyperreactive dopaminergic system with a higher amount of cerebral tyrosine hence wound healing capacity in mice. In mice with cutaneous wounds, treatment with hydroxylase mRNA and DRD2 protein [61]. Seven days after sc implantation, DRD2 antagonist eticlopride (10 mg/kg/day) did indeed accelerate wound healing and mammary tumors were smaller with a lower tumor microvessel density in APO-SUS increased microvessel density in the wound compared to saline treatment [52]. rats than in their apomorphine-unsusceptible (APO-UNSUS) counterparts. Eticlopride also enhanced the number of circulating MSC in the peripheral blood Furthermore, APO-SUS rats developed fewer lung metastases than APO-UNSUS rats compared to saline treatment. The number of bromodeoxyuridine (BrdU) labeled after intravenous (iv) injection of mammary breast cancer cells [62]. MSCs that were iv injected were also higher in the wound bed after eticlopride Dopamine treatment resulted in decreased tumor growth with a lower treatment [26]. microvessel density in several animal models [11,24,54,63-65]. In general, dopamine treatment consisted of intra peritoneal administration of 50 mg/kg/day, resulting in Dopamine in cancer dopamine plasma levels of 1.2 µM in mice and 2.4 µM in rats one minute after Several in vitro experiments indicated that dopamine can directly affect tumor cells. In injection. This is 5% of the lethal dose in rodents. Dopamine also reduced vascular ovarian cancer cells, dopamine (12.5-50 µM) decreased tumor cell invasion in a permeability in mice bearing human colon and breast tumors [54] and murine ovarian membrane invasion culture system and increased apoptosis [11]. Dopamine (5 µM) tumors, resulting in less accumulation of ascites in the latter model [24]. Tumor - 26 - - 27 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 27 endothelial cells of dopamine-treated mice had decreased phosphorylation of VEGF- cell proliferation as revealed in experiments with siRNA targeting DRD2. In stressed R2 [63] and downstream targets, like focal adhesion kinase (FAK) and MAPK [54]. mice bearing human SKOV3ip1 or HeyA8 ovarian tumors, dopamine’s inhibitory In a very elegant study dopamine was shown to decrease EPC mobilization effect was abolished if it was co-injected with nanoparticles containing siRNA into the peripheral circulation and EPC incorporation in murine sarcoma vasculature. targeting murine DRD2 present on endothelial and other host cells. However, These mice first received a bone marrow transplantation from transgenic Tie2 mice. dopamine co-injected with nanoparticles containing siRNA targeting human DRD2 2 Four weeks later a murine sarcoma was transplanted, followed by dopamine treatment. present on tumor cells abolished dopamine’s inhibitory effect only on HeyA8 ovarian LacZ+ EPC from the transgenic mice were then identified by X-gal staining. This tumors. As HeyA8 cell viability in vitro is also affected by dopamine treatment, this staining demonstrated a lower EPC number in sarcoma vasculature of dopamine- suggests that DRD2 present on HeyA8 tumor cells is involved in tumor growth. Why treated mice than of untreated mice. MMP-9, involved in EPC mobilization [27,29], SKOV3ip1 tumor cells are not affected by dopamine treatment or siRNA targeting was also lower in the bone marrow of dopamine-treated mice compared to untreated human DRD2 remains puzzling, because this cell line also expresses DRD2 [11]. ones [27]. In a study with stressed mice bearing human ovarian cancer, it was shown From these studies, it can be concluded that dopamine inhibits tumor angiogenesis that dopamine treatment increased pericyte coverage of tumor vasculature [65]. and thereby tumor growth via activation of DRD2; in some tumors also DRD2 Increased pericyte coverage is one of the characteristics of vessel normalization mediated inhibition of tumor cell proliferation may play a role. induced by antiangiogenic therapy [66]. There is conflicting evidence regarding the influence of DRD1 activation on The effect of dopamine in combination therapy was investigated in a study in tumor angiogenesis. One study showed that DRD1 knockout mice bearing murine which mice bearing sc human breast tumors received dopamine alone, doxorubicin lung carcinoma had smaller tumors than wild-type mice. In wild-type mice, DRD1 alone, dopamine and doxorubicin, or vehicle. Dopamine, doxorubicin, and antagonist SCH23390 (0.3 mg/kg/2 days) inhibited tumor growth and microvessel combination of dopamine and doxorubicin resulted in decreased tumor volume density [60]. However, in a study in mice bearing murine ovarian tumors neither (171%, 133%, and 63% of original size respectively) compared with vehicle treatment DRD1 antagonist SCH23390 (10 mg/kg), nor DRD1 agonist SKF38390 (10 mg/kg) (413% of original size) and increased life span (with 24%, 38%, and 90% respectively) affected tumor vascularization or extent of malignant ascites [24]. Also in stressed compared with vehicle treated mice. A similar phenomenon was observed in mice mice bearing human SKOV3ip1 and HeyA8 ovarian tumors DRD1 antagonist bearing orthotopic human colon tumors treated with dopamine alone, 5-fluorouracil butaclamol (1.5 mg/kg) did not affect dopamine-induced inhibition of angiogenesis alone, dopamine and 5-fluorouracil, or vehicle [54]. In stressed mice bearing ovarian and tumor growth. Butaclamol did however inhibit the increased pericyte coverage of cancer, dopamine in combination with cisplatin enhanced cisplatin concentration in tumor vessels caused by dopamine treatment in these models. In these mice, the tumor as demonstrated by an increased tumor/kidney and tumor/liver cisplatin administration of DRD1 agonist SKF82958 (1 mg/kg) also resulted in increased ratio. This combination treatment resulted in a 6-fold decrease in tumor weight pericyte coverage of tumor vessels. Combination treatment with cisplatin and DRD1 compared to cisplatin only treated animals [65]. agonist SKF82958 resulted in two-fold increased tumor/liver and tumor/kidney The role of DRD2 was also investigated in animal experiments. DRD2 cisplatin concentration ratios and a five-fold decrease in tumor growth compared to knockout mice bearing murine sarcoma or melanoma had increased tumor size, cisplatin only treated controls [65]. These observations suggest that dopamine-induced microvessel density and permeability compared to wild-type mice [27,58]. Dopamine DRD1 activation results in vascular normalization. treatment did not affect EPC mobilization in these mice, suggesting that DRD2 is Dopamine concentrations have also been investigated in cancer patients. In necessary for dopamine to execute its function [27]. colon cancer tissue of 36 patients, the dopamine level determined by 3[H] dopamine DRD2 agonists bromocriptine and quinpirole (10 mg/kg) inhibited tumor binding assay was 3-10-fold lower than in adjacent healthy colon tissue [67]. angiogenesis in mice bearing murine ovarian cancer [24]. DRD2 antagonists Dopamine and tyrosine hydroxylase were not detectable by high performance liquid eticlopride or domperidon (10 mg/kg/day) administered prior to dopamine treatment, chromatography in gastric cancer tissue of 22 patients, whereas presence of both was on the other hand, abolished dopamine’s inhibitory effect on human gastric and demonstrated in healthy gastric tissue of 22 patients with adenomatous stomach ovarian tumor growth in mice and rats [11,63,65]. Dopamine-induced pericyte polyps [63]. coverage was not affected by DRD2 antagonist eticlopride (10 mg/kg) [65]. One clinical intervention study was performed, aiming to achieve systemic There are however also data indicating that DRD2 activation can inhibit tumor dopamine levels which could inhibit tumor cell growth. Four patients with metastatic - 28 - - 29 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 28 endothelial cells of dopamine-treated mice had decreased phosphorylation of VEGF- cell proliferation as revealed in experiments with siRNA targeting DRD2. In stressed R2 [63] and downstream targets, like focal adhesion kinase (FAK) and MAPK [54]. mice bearing human SKOV3ip1 or HeyA8 ovarian tumors, dopamine’s inhibitory In a very elegant study dopamine was shown to decrease EPC mobilization effect was abolished if it was co-injected with nanoparticles containing siRNA into the peripheral circulation and EPC incorporation in murine sarcoma vasculature. targeting murine DRD2 present on endothelial and other host cells. However, These mice first received a bone marrow transplantation from transgenic Tie2 mice. dopamine co-injected with nanoparticles containing siRNA targeting human DRD2 Four weeks later a murine sarcoma was transplanted, followed by dopamine treatment. present on tumor cells abolished dopamine’s inhibitory effect only on HeyA8 ovarian 2 LacZ+ EPC from the transgenic mice were then identified by X-gal staining. This tumors. As HeyA8 cell viability in vitro is also affected by dopamine treatment, this staining demonstrated a lower EPC number in sarcoma vasculature of dopamine- suggests that DRD2 present on HeyA8 tumor cells is involved in tumor growth. Why treated mice than of untreated mice. MMP-9, involved in EPC mobilization [27,29], SKOV3ip1 tumor cells are not affected by dopamine treatment or siRNA targeting was also lower in the bone marrow of dopamine-treated mice compared to untreated human DRD2 remains puzzling, because this cell line also expresses DRD2 [11]. ones [27]. In a study with stressed mice bearing human ovarian cancer, it was shown From these studies, it can be concluded that dopamine inhibits tumor angiogenesis that dopamine treatment increased pericyte coverage of tumor vasculature [65]. and thereby tumor growth via activation of DRD2; in some tumors also DRD2 Increased pericyte coverage is one of the characteristics of vessel normalization mediated inhibition of tumor cell proliferation may play a role. induced by antiangiogenic therapy [66]. There is conflicting evidence regarding the influence of DRD1 activation on The effect of dopamine in combination therapy was investigated in a study in tumor angiogenesis. One study showed that DRD1 knockout mice bearing murine which mice bearing sc human breast tumors received dopamine alone, doxorubicin lung carcinoma had smaller tumors than wild-type mice. In wild-type mice, DRD1 alone, dopamine and doxorubicin, or vehicle. Dopamine, doxorubicin, and antagonist SCH23390 (0.3 mg/kg/2 days) inhibited tumor growth and microvessel combination of dopamine and doxorubicin resulted in decreased tumor volume density [60]. However, in a study in mice bearing murine ovarian tumors neither (171%, 133%, and 63% of original size respectively) compared with vehicle treatment DRD1 antagonist SCH23390 (10 mg/kg), nor DRD1 agonist SKF38390 (10 mg/kg) (413% of original size) and increased life span (with 24%, 38%, and 90% respectively) affected tumor vascularization or extent of malignant ascites [24]. Also in stressed compared with vehicle treated mice. A similar phenomenon was observed in mice mice bearing human SKOV3ip1 and HeyA8 ovarian tumors DRD1 antagonist bearing orthotopic human colon tumors treated with dopamine alone, 5-fluorouracil butaclamol (1.5 mg/kg) did not affect dopamine-induced inhibition of angiogenesis alone, dopamine and 5-fluorouracil, or vehicle [54]. In stressed mice bearing ovarian and tumor growth. Butaclamol did however inhibit the increased pericyte coverage of cancer, dopamine in combination with cisplatin enhanced cisplatin concentration in tumor vessels caused by dopamine treatment in these models. In these mice, the tumor as demonstrated by an increased tumor/kidney and tumor/liver cisplatin administration of DRD1 agonist SKF82958 (1 mg/kg) also resulted in increased ratio. This combination treatment resulted in a 6-fold decrease in tumor weight pericyte coverage of tumor vessels. Combination treatment with cisplatin and DRD1 compared to cisplatin only treated animals [65]. agonist SKF82958 resulted in two-fold increased tumor/liver and tumor/kidney The role of DRD2 was also investigated in animal experiments. DRD2 cisplatin concentration ratios and a five-fold decrease in tumor growth compared to knockout mice bearing murine sarcoma or melanoma had increased tumor size, cisplatin only treated controls [65]. These observations suggest that dopamine-induced microvessel density and permeability compared to wild-type mice [27,58]. Dopamine DRD1 activation results in vascular normalization. treatment did not affect EPC mobilization in these mice, suggesting that DRD2 is Dopamine concentrations have also been investigated in cancer patients. In necessary for dopamine to execute its function [27]. colon cancer tissue of 36 patients, the dopamine level determined by 3[H] dopamine DRD2 agonists bromocriptine and quinpirole (10 mg/kg) inhibited tumor binding assay was 3-10-fold lower than in adjacent healthy colon tissue [67]. angiogenesis in mice bearing murine ovarian cancer [24]. DRD2 antagonists Dopamine and tyrosine hydroxylase were not detectable by high performance liquid eticlopride or domperidon (10 mg/kg/day) administered prior to dopamine treatment, chromatography in gastric cancer tissue of 22 patients, whereas presence of both was on the other hand, abolished dopamine’s inhibitory effect on human gastric and demonstrated in healthy gastric tissue of 22 patients with adenomatous stomach ovarian tumor growth in mice and rats [11,63,65]. Dopamine-induced pericyte polyps [63]. coverage was not affected by DRD2 antagonist eticlopride (10 mg/kg) [65]. One clinical intervention study was performed, aiming to achieve systemic There are however also data indicating that DRD2 activation can inhibit tumor dopamine levels which could inhibit tumor cell growth. Four patients with metastatic - 28 - - 29 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 29 melanoma received dopamine infusion at a maximum dose of 20 μg/kg/min for 48- increased by serotonin (10 µM) via reduction of adherens and tight junction protein 120 hours resulting in plasma dopamine levels between 1-10 µM. The study was expression [79]. prematurely terminated due to severe cardiovascular side-effects after the Serotonin exerts its effect on endothelial cells through multiple receptors as administration of only one treatment cycle [68]. An ex vivo proliferation assay of shown in experiments with various 5-HTR agonists and antagonists. Serotonin- biopsies prior to and immediately after this treatment cycle [69] showed a 10-fold induced migration of HAEC was inhibited by 5-HTR1 antagonist GR55562 (0.1 µM), 2 decrease from 1.0-3.0% to 0.1-0.2% radioactive thymidine-labeled tumor cells [68]. but not inhibited by a low dose 5-HTR2 antagonist ketanserin (0.1 µM) [76]. Higher DRD2 was expressed in gastric cancer tissue of 65 patients, but was lower in dose of ketanserin (18 µM) did inhibit migration in HUVEC [77]. Also serotonin- tumors than in benign polyps or normal gastric tissue of 83 controls [70]. induced proliferation of HUVEC and dog aortic endothelial cells could be abolished Furthermore, the allele frequency of germ line functional single-nucleotide by ketanserin (18 µM) and 5-HTR2B antagonist LY281067 (0.02 µM) (see Figure 1) polymorphisms (SNP) of the DRD2 gene in cancer patients was investigated in three [77,80]. 5-HTR3 antagonist 3-tropanylindole-3-carboxylate methiodide (7 µM) studies. -141C/del DRD2 SNP allele frequency was higher in 370 colorectal cancer inhibited serotonin-induced HUVEC proliferation and migration [77], while for 5- patients than in 327 controls (odds ratio (OR) = 2.28) [71]. In a colon polyp HTR4 opposing results were obtained. 5-HTR4 agonist mosapride citrate (10 µM) prevention trial with over 2,000 participants who underwent polypectomy, this DRD2 induced arrest of HUVEC in the G0/G1 phase and inhibited its proliferation, SNP and others were evaluated. The -141C/del allele frequency was higher in 673 migration and tube-like formation [81]. However, in another study 5-HTR4 antagonist patients with recurrence of colorectal adenoma (OR = 1.3), and the TaqIA allele RS39604 (10-30 µM) inhibited HUVEC proliferation, migration and tube-like frequency was higher in 109 patients with recurrence of advanced colorectal adenoma formation [82]. (OR = 2.40) [72]. Another study showed the -141C/del allele to be more frequently Serotonin activates several intracellular second messenger pathways. In the present in 335 lung carcinoma patients than in 413 controls (all former or current four endothelial cell lines HUVEC, HAEC, human microvascular endothelial cells, and smokers) (OR = 2.19) [73]. Since these studies only investigated the DRD2 SNP allele human pulmonary artery endothelial cells, the signaling kinases ERK, P70S6K, Src, frequency and not mRNA expression, the functional effect of having these SNPs PI3K, Akt, mTOR, and p38 MAPK were activated upon serotonin stimulation (1 µM). remains unclear. Serotonin induces Akt phosphorylation and upregulation of orphan nuclear In summary, dopamine and tyrosine hydroxylase are present in lower transcription factor TR3/Nur77 in HAEC respectively HUVEC [75,77,80,83,84]. concentrations in tumor tissue than in benign tissue. Increasing dopamine levels by Western blot analysis revealed that blocking serotonin with 5-HTR1B dopamine treatment seems to inhibit tumor cell proliferation in melanoma patients, antagonist SB316641 (100 nM) inhibited P70S6K, ERK, and the but cannot be used due to toxicity. DRD2 is present in gastric cancer tissue and is an Src/PI3K/Akt/mTOR pathway [84]. attractive target for therapy with DRD2 agonists. 5-HTR2B antagonist SB2047415 (10 µM) inhibited ERK1/2 and endothelial Surprisingly, there are no data available regarding the relation of dopamine nitric oxide synthase (eNOS) phosphorylation in HUVEC [85]. Another 5-HTR2B with angiogenesis in paraganglioma and pheochromocytoma, which are antagonist, SB206553 (100 nM), and the 5-HTR7 antagonist SB269970 (100 nM) neuroendocrine tumors known for their dopamine production [74]. inhibited serotonin-induced activation of p38 MAPK [84]. As 5-HTR1B and 5-HTR2B antagonists inhibit ERK phosphorylation and In vitro effects of serotonin on endothelial cells ERK inhibition abolished serotonin-induced HAEC migration [75], these data suggest The effect of serotonin on endothelial cells was investigated in several in vitro studies. that serotonin stimulated ERK-induced endothelial cell migration via activation of 5- Serotonin (0.1 µM and 1 µM) enhanced migration, tubule length and proliferation of HTR1B and 5-HTR2B. HUVEC and human aortic endothelial cells (HAEC) [75-77]. In a culture of human Besides endothelial cells, serotonin also affects aortic smooth muscle cells. In umbilical cord CD34+ cells together with serotonin (0.2 µM) and thrombopoietin, vitro, serotonin (1 μM) increased proliferation of bovine aortic smooth muscle cells stem cell factor and FL-3 ligand (TSF), an increased number of endothelial stem cells [86]. Migration of rat aortic smooth muscle cells was enhanced after addition of and EPCs was demonstrated with FACS analysis compared with CD34+ cells cultured serotonin (minimum of 1 nM) to smooth muscle cell derived migration factor (25 with TSF alone. This indicates that serotonin stimulated CD34+ cells to expand the μm). Pretreatment with 5-HTR2 antagonist MCI-9042 (0.1 µM) abolished serotonin- endothelial stem cell and EPC population [78]. Lastly, HUVEC permeability was induced (1 μM) migration [87]. - 30 - - 31 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 30 melanoma received dopamine infusion at a maximum dose of 20 μg/kg/min for 48- increased by serotonin (10 µM) via reduction of adherens and tight junction protein 120 hours resulting in plasma dopamine levels between 1-10 µM. The study was expression [79]. prematurely terminated due to severe cardiovascular side-effects after the Serotonin exerts its effect on endothelial cells through multiple receptors as administration of only one treatment cycle [68]. An ex vivo proliferation assay of shown in experiments with various 5-HTR agonists and antagonists. Serotonin- biopsies prior to and immediately after this treatment cycle [69] showed a 10-fold induced migration of HAEC was inhibited by 5-HTR1 antagonist GR55562 (0.1 µM), decrease from 1.0-3.0% to 0.1-0.2% radioactive thymidine-labeled tumor cells [68]. but not inhibited by a low dose 5-HTR2 antagonist ketanserin (0.1 µM) [76]. Higher 2 DRD2 was expressed in gastric cancer tissue of 65 patients, but was lower in dose of ketanserin (18 µM) did inhibit migration in HUVEC [77]. Also serotonin- tumors than in benign polyps or normal gastric tissue of 83 controls [70]. induced proliferation of HUVEC and dog aortic endothelial cells could be abolished Furthermore, the allele frequency of germ line functional single-nucleotide by ketanserin (18 µM) and 5-HTR2B antagonist LY281067 (0.02 µM) (see Figure 1) polymorphisms (SNP) of the DRD2 gene in cancer patients was investigated in three [77,80]. 5-HTR3 antagonist 3-tropanylindole-3-carboxylate methiodide (7 µM) studies. -141C/del DRD2 SNP allele frequency was higher in 370 colorectal cancer inhibited serotonin-induced HUVEC proliferation and migration [77], while for 5- patients than in 327 controls (odds ratio (OR) = 2.28) [71]. In a colon polyp HTR4 opposing results were obtained. 5-HTR4 agonist mosapride citrate (10 µM) prevention trial with over 2,000 participants who underwent polypectomy, this DRD2 induced arrest of HUVEC in the G0/G1 phase and inhibited its proliferation, SNP and others were evaluated. The -141C/del allele frequency was higher in 673 migration and tube-like formation [81]. However, in another study 5-HTR4 antagonist patients with recurrence of colorectal adenoma (OR = 1.3), and the TaqIA allele RS39604 (10-30 µM) inhibited HUVEC proliferation, migration and tube-like frequency was higher in 109 patients with recurrence of advanced colorectal adenoma formation [82]. (OR = 2.40) [72]. Another study showed the -141C/del allele to be more frequently Serotonin activates several intracellular second messenger pathways. In the present in 335 lung carcinoma patients than in 413 controls (all former or current four endothelial cell lines HUVEC, HAEC, human microvascular endothelial cells, and smokers) (OR = 2.19) [73]. Since these studies only investigated the DRD2 SNP allele human pulmonary artery endothelial cells, the signaling kinases ERK, P70S6K, Src, frequency and not mRNA expression, the functional effect of having these SNPs PI3K, Akt, mTOR, and p38 MAPK were activated upon serotonin stimulation (1 µM). remains unclear. Serotonin induces Akt phosphorylation and upregulation of orphan nuclear In summary, dopamine and tyrosine hydroxylase are present in lower transcription factor TR3/Nur77 in HAEC respectively HUVEC [75,77,80,83,84]. concentrations in tumor tissue than in benign tissue. Increasing dopamine levels by Western blot analysis revealed that blocking serotonin with 5-HTR1B dopamine treatment seems to inhibit tumor cell proliferation in melanoma patients, antagonist SB316641 (100 nM) inhibited P70S6K, ERK, and the but cannot be used due to toxicity. DRD2 is present in gastric cancer tissue and is an Src/PI3K/Akt/mTOR pathway [84]. attractive target for therapy with DRD2 agonists. 5-HTR2B antagonist SB2047415 (10 µM) inhibited ERK1/2 and endothelial Surprisingly, there are no data available regarding the relation of dopamine nitric oxide synthase (eNOS) phosphorylation in HUVEC [85]. Another 5-HTR2B with angiogenesis in paraganglioma and pheochromocytoma, which are antagonist, SB206553 (100 nM), and the 5-HTR7 antagonist SB269970 (100 nM) neuroendocrine tumors known for their dopamine production [74]. inhibited serotonin-induced activation of p38 MAPK [84]. As 5-HTR1B and 5-HTR2B antagonists inhibit ERK phosphorylation and In vitro effects of serotonin on endothelial cells ERK inhibition abolished serotonin-induced HAEC migration [75], these data suggest The effect of serotonin on endothelial cells was investigated in several in vitro studies. that serotonin stimulated ERK-induced endothelial cell migration via activation of 5- Serotonin (0.1 µM and 1 µM) enhanced migration, tubule length and proliferation of HTR1B and 5-HTR2B. HUVEC and human aortic endothelial cells (HAEC) [75-77]. In a culture of human Besides endothelial cells, serotonin also affects aortic smooth muscle cells. In umbilical cord CD34+ cells together with serotonin (0.2 µM) and thrombopoietin, vitro, serotonin (1 μM) increased proliferation of bovine aortic smooth muscle cells stem cell factor and FL-3 ligand (TSF), an increased number of endothelial stem cells [86]. Migration of rat aortic smooth muscle cells was enhanced after addition of and EPCs was demonstrated with FACS analysis compared with CD34+ cells cultured serotonin (minimum of 1 nM) to smooth muscle cell derived migration factor (25 with TSF alone. This indicates that serotonin stimulated CD34+ cells to expand the μm). Pretreatment with 5-HTR2 antagonist MCI-9042 (0.1 µM) abolished serotonin- endothelial stem cell and EPC population [78]. Lastly, HUVEC permeability was induced (1 μM) migration [87]. - 30 - - 31 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 31 In summary, serotonin in vitro stimulates proliferation of endothelial cells via cell growth. It was suggested that blocking one 5-HTR leads to increased susceptibility activation of 5-HTR1, 5-HTR2, and 5-HTR3 and aortic smooth muscle cells via 5- of serotonin-induced activation of the other [102]. HTR2. The role of 5-HTR4 is less clear as opposing results have been found. In animal experiments the effect of serotonin depletion and repletion on tumor behavior was studied. Hepatocellular carcinoma xenografts were not able to Serotonin in inflammation grow in tryptophan hydroxylase deficient (and thus serotonin depleted) mice [99]. 2 Angiogenesis is not only a hallmark of cancer, but also of inflammation [88]. Murine colon or lung carcinomas in tryptophan hydroxylase deficient mice were 3-fold Preclinical and clinical evidence suggests that serotonin can affect inflammation. In an respectively 1.5-fold smaller than in wild-type mice. Microvessel density in colon air-pouch model in rats, inflammation can be induced by sc injection of sterile air and cancers was also decreased in tryptophan hydroxylase deficient mice. Colon and lung subsequently 1 mL 1% carrageenan solution. Treatment with 5-HTR3 antagonist carcinoma growth could be restored when 5-hydroxytryptophan (50 mg/kg twice granisetron (50-200 μM) decreased angiogenesis and inflammation, as shown by daily) was sc injected 2 days before tumor inoculation. Tryptophan hydroxylase reduced hemoglobin levels and leukocytes in the granulation tissue [89]. Rheumatoid deficient mice had tumor VEGF and VEGF-R2 concentrations similar to wild-type arthritis and osteoarthritis are examples of chronic inflammation. In a double-blind mice, but higher MMP-12 and angiostatin concentrations [12]. MMP-12 cleaves study in 36 patients with rheumatoid arthritis or osteoarthritis, a single intra-articular plasminogen into angiostatin, which is an endogenous angiogenesis inhibitor [103]. injection with 5-HTR3 antagonist tropisetron (10 mg) reduced symptoms with similar Therefore, it was suggested that serotonin affects the angiostatin-pathway instead of efficacy as methylprednisolone (40 mg). Since angiogenic markers were not evaluated the VEGF-pathway [12]. Absence of the serotonin transporter induces lower plasma after tropisetron treatment, it cannot be confirmed that tropisetron induced symptom and tumor serotonin levels. This resulted in smaller tumors in serotonin transporter reduction via inhibition of angiogenesis [90]. deficient mice bearing murine lung carcinoma or melanoma compared to wild-type mice. However, tumor microvessel density was not affected. To obtain more insight in Serotonin in cancer the cause of decreased tumor growth, eNOS concentrations were determined and In vitro, serotonin stimulated tumor cell proliferation and prevented cell death in found to be reduced in the tumors of serotonin transporter deficient mice. As eNOS several tumor cell lines (Figure 1) [91-100]. In contrast, human melanoma cell can cause vasodilatation, the authors suggested decreased blood flow to be the cause proliferation was inhibited by serotonin (500 µM) [101]. In a human of the smaller tumors in serotonin transporter deficient mice [81]. The human cholangiocarcinoma cell line, tryptophan hydroxylase mRNA was 2.5-50-fold higher cholangiocarcinoma growth reduction in mice after treatment with the tryptophan and MAO-A mRNA was ~2-fold lower compared to non-malignant cholangiocytes. hydroxylase inhibitor CPA (150 mg/kg trice a week) for ~2 months further supported This resulted in increased serotonin production by these tumor cells [91]. In the the evidence that absence of serotonin affects tumor behavior [91]. human hepatocellular carcinoma cell line Huh7, serotonin stimulated proliferation in In mouse studies, research regarding the role of 5-HTR in tumor behavior serum deprived medium via upregulation and phosphorylation of FOXO3a. This only focused on the role of 5-HTR2B. Treatment with 5-HTR2B antagonist effect did not occur in two other human hepatocellular carcinoma cell lines HepG2 en SB204741 (20 mg/kg) decreased tumor growth and microvessel density in mice Hep3b [95]. bearing murine lung cancer or melanoma [85]. Tryptophan hydroxylase inhibitors block the conversion of tryptophan to In patients immunohistochemistry studies have been performed. Stainings of serotonin. In vitro experiments showed reduced proliferation of neuroendocrine the neuroendocrine tumor cell markers chromogranin A and serotonin were used to tumor cells after treatment with tryptophan hydroxylase inhibitor 7-HTP (1 nM). identify neuroendocrine foci in prostate cancer. The presence of serotonin-positive Cholangiocarcinoma cell proliferation could be blocked by tryptophan hydroxylase cells was associated with a higher microvessel density [104] and VEGF expression inhibitor p-chlorophenylalanine (CPA) (1 mM) [91,94]. [105]. 5-HTR was present in several tumor types. 5-HTR1A and 5-HTR1B expression In vitro experiments showed various 5-HTRs to be present on several tumor was increased in hepatocellular carcinoma tissue of 109 patients compared to cell types. Tumor cell growth could be inhibited by specific antagonists targeting the surrounding healthy liver tissue, whereas 5-HTR2B and 5-HTR7 expression was 5HTR expressed on the tumor cell (Supplementary Table 1). In small cell lung cancer similar in both tissue types. 5-HTR1A, 5-HTR1B and 5-HTR2B expression were cells, both 5-HTR1A and 5-HTR1D had to be targeted by antagonists (500 nM associated with a higher proliferation index in tumors of 176 hepatocellular cancer spiperone respectively GR127935) to achieve maximal inhibition of serotonin-induced patients. In addition, expression of 5-HTR1B was correlated with tumor size in these - 32 - - 33 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 32 In summary, serotonin in vitro stimulates proliferation of endothelial cells via cell growth. It was suggested that blocking one 5-HTR leads to increased susceptibility activation of 5-HTR1, 5-HTR2, and 5-HTR3 and aortic smooth muscle cells via 5- of serotonin-induced activation of the other [102]. HTR2. The role of 5-HTR4 is less clear as opposing results have been found. In animal experiments the effect of serotonin depletion and repletion on tumor behavior was studied. Hepatocellular carcinoma xenografts were not able to Serotonin in inflammation grow in tryptophan hydroxylase deficient (and thus serotonin depleted) mice [99]. Angiogenesis is not only a hallmark of cancer, but also of inflammation [88]. Murine colon or lung carcinomas in tryptophan hydroxylase deficient mice were 3-fold 2 Preclinical and clinical evidence suggests that serotonin can affect inflammation. In an respectively 1.5-fold smaller than in wild-type mice. Microvessel density in colon air-pouch model in rats, inflammation can be induced by sc injection of sterile air and cancers was also decreased in tryptophan hydroxylase deficient mice. Colon and lung subsequently 1 mL 1% carrageenan solution. Treatment with 5-HTR3 antagonist carcinoma growth could be restored when 5-hydroxytryptophan (50 mg/kg twice granisetron (50-200 μM) decreased angiogenesis and inflammation, as shown by daily) was sc injected 2 days before tumor inoculation. Tryptophan hydroxylase reduced hemoglobin levels and leukocytes in the granulation tissue [89]. Rheumatoid deficient mice had tumor VEGF and VEGF-R2 concentrations similar to wild-type arthritis and osteoarthritis are examples of chronic inflammation. In a double-blind mice, but higher MMP-12 and angiostatin concentrations [12]. MMP-12 cleaves study in 36 patients with rheumatoid arthritis or osteoarthritis, a single intra-articular plasminogen into angiostatin, which is an endogenous angiogenesis inhibitor [103]. injection with 5-HTR3 antagonist tropisetron (10 mg) reduced symptoms with similar Therefore, it was suggested that serotonin affects the angiostatin-pathway instead of efficacy as methylprednisolone (40 mg). Since angiogenic markers were not evaluated the VEGF-pathway [12]. Absence of the serotonin transporter induces lower plasma after tropisetron treatment, it cannot be confirmed that tropisetron induced symptom and tumor serotonin levels. This resulted in smaller tumors in serotonin transporter reduction via inhibition of angiogenesis [90]. deficient mice bearing murine lung carcinoma or melanoma compared to wild-type mice. However, tumor microvessel density was not affected. To obtain more insight in Serotonin in cancer the cause of decreased tumor growth, eNOS concentrations were determined and In vitro, serotonin stimulated tumor cell proliferation and prevented cell death in found to be reduced in the tumors of serotonin transporter deficient mice. As eNOS several tumor cell lines (Figure 1) [91-100]. In contrast, human melanoma cell can cause vasodilatation, the authors suggested decreased blood flow to be the cause proliferation was inhibited by serotonin (500 µM) [101]. In a human of the smaller tumors in serotonin transporter deficient mice [81]. The human cholangiocarcinoma cell line, tryptophan hydroxylase mRNA was 2.5-50-fold higher cholangiocarcinoma growth reduction in mice after treatment with the tryptophan and MAO-A mRNA was ~2-fold lower compared to non-malignant cholangiocytes. hydroxylase inhibitor CPA (150 mg/kg trice a week) for ~2 months further supported This resulted in increased serotonin production by these tumor cells [91]. In the the evidence that absence of serotonin affects tumor behavior [91]. human hepatocellular carcinoma cell line Huh7, serotonin stimulated proliferation in In mouse studies, research regarding the role of 5-HTR in tumor behavior serum deprived medium via upregulation and phosphorylation of FOXO3a. This only focused on the role of 5-HTR2B. Treatment with 5-HTR2B antagonist effect did not occur in two other human hepatocellular carcinoma cell lines HepG2 en SB204741 (20 mg/kg) decreased tumor growth and microvessel density in mice Hep3b [95]. bearing murine lung cancer or melanoma [85]. Tryptophan hydroxylase inhibitors block the conversion of tryptophan to In patients immunohistochemistry studies have been performed. Stainings of serotonin. In vitro experiments showed reduced proliferation of neuroendocrine the neuroendocrine tumor cell markers chromogranin A and serotonin were used to tumor cells after treatment with tryptophan hydroxylase inhibitor 7-HTP (1 nM). identify neuroendocrine foci in prostate cancer. The presence of serotonin-positive Cholangiocarcinoma cell proliferation could be blocked by tryptophan hydroxylase cells was associated with a higher microvessel density [104] and VEGF expression inhibitor p-chlorophenylalanine (CPA) (1 mM) [91,94]. [105]. 5-HTR was present in several tumor types. 5-HTR1A and 5-HTR1B expression In vitro experiments showed various 5-HTRs to be present on several tumor was increased in hepatocellular carcinoma tissue of 109 patients compared to cell types. Tumor cell growth could be inhibited by specific antagonists targeting the surrounding healthy liver tissue, whereas 5-HTR2B and 5-HTR7 expression was 5HTR expressed on the tumor cell (Supplementary Table 1). In small cell lung cancer similar in both tissue types. 5-HTR1A, 5-HTR1B and 5-HTR2B expression were cells, both 5-HTR1A and 5-HTR1D had to be targeted by antagonists (500 nM associated with a higher proliferation index in tumors of 176 hepatocellular cancer spiperone respectively GR127935) to achieve maximal inhibition of serotonin-induced patients. In addition, expression of 5-HTR1B was correlated with tumor size in these - 32 - - 33 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 33 patients [106]. However, in prostate cancer tissue of 25 patients, 5-HTR2B expression REFERENCES was similar among all Gleason grades, whereas 5-HTR4 was only expressed in Gleason grade 3-4 prostate cancer tissue [93]. In breast cancer tissue of 102 patients, 1. Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the 5-HTR1A, 5-HTR1B, 5-HTR2B and 5-HTR4 were present. In these tumors, receptor tumor microenvironment. Cancer Cell 2012; 21: 309-322. expression did not correlate with tumor grade [107]. In 159 bone metastases of 2. Hanahan D, Weinberg RA. The hallmarks of cancer: the next generation. Cell 2011; 144: 2 carcinoma and sarcoma patients reverse phase protein microarray analysis showed that 646-674. expression of serotonin in combination with tumor necrosis factor receptor 1 was 3. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249- associated with a poor survival [108]. Similar to dopamine, there are no data relating 257. serotonin production to angiogenesis in carcinoid tumors [74]. 4. Schäfer M, Werner S. Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol 2008; 9: 628-638. 5. Pinedo HM, Verheul HM, D’Amato RJ, et al. Involvement of platelets in tumour CONCLUSIONS AND PERSPECTIVES angiogenesis? Lancet 1998; 352: 1775-1777. 6. Holmsen H, Weiss HJ. Secretable storage pools in platelets. Annu Rev Med 1979; 30: 119- Based on the available literature it can be concluded that dopamine inhibits tumor 134. growth, whereas serotonin stimulates tumor growth. In vitro and animal studies 7. Da Prada M, Picotti GB. Content and subcellular localization of catecholamines and 5- showed that dopamine inhibits endothelial cell proliferation and tumor growth via hydroxytryptamine in human and animal blood platelets: monoamine distribution between activation of DRD2. Treatment with dopamine is not feasible because of severe platelets and plasma. Br J Pharmacol 1979; 65: 653-662. 8. Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine cardiovascular toxicity. Therefore clinical intervention studies with DRD2 agonists are receptors. Pharmacol Rev 2011; 63: 182-217. attractive, especially as these agents are already being used in the clinic for other 9. Mohammad-Zadeh LF, Moses L, Gwaltney-Brant SM. Serotonin: a review. J Vet Pharmacol indications such as Parkinson’s disease and hyperprolactinemia [8]. To our knowledge, Ther 2008; 31: 187-199. no clinical trials with DRD2 agonists in cancer patients are currently performed. 10. Chakroborty D, Sarkar C, Basu B, et al. Catecholamines regulate tumor angiogenesis. Serotonin’s role in tumor behavior has been studied less extensively and data Cancer Res 2009; 69: 3727-3730. are predominantly derived from in vitro experiments. These experiments showed 11. Moreno-Smith M, Lu C, Shahzad MM, et al. Dopamine blocks stress-mediated ovarian serotonin to stimulate endothelial cells via activation of 5-HTR1 and 5-HTR2. Tumor carcinoma growth. Clin Cancer Res 2011; 17: 3649-3659. cell proliferation could be inhibited by selected 5-HTR antagonists, depending on 12. Nocito A, Dahm F, Jochum W, et al. Serotonin regulates macrophage-mediated tumor cell type. Tryptophan hydroxylase inhibitors are currently used in clinical trials angiogenesis in a mouse model of colon cancer allografts. Cancer Res 2008; 68: 5152-5158. in irritable bowel syndrome patients [109] and for serotonin-producing carcinoid 13. Eisenhofer G, Aneman A, Friberg P, et al. Substantial production of dopamine in the tumor patients to evaluate the effect on the carcinoid syndrome. However further human gastrointestinal tract. J Clin Endocrinol Metab 1997; 82: 3864-3871. 14. Kopin IJ. Catecholamine metabolism: basic aspects and clinical significance. Pharmacol Rev animal experiments to reveal serotonin’s mechanism of action are warranted. 1985; 37: 333-364.

15. Mezey E, Eisenhofer G, Harta G, et al. A novel nonneuronal catecholaminergic system: Acknowledgements exocrine pancreas synthesizes and releases dopamine. Proc Natl Acad Sci U S A 1996; 93: We would like to thank E.M.E. van Straten for assistance in graphical design. 10377-10382. 16. Eisenhofer G, Coughtrie MW, Goldstein DS. Dopamine sulphate: an enigma resolved. Clin Exp Pharmacol Physiol Suppl 1999; 26: S41-53. 17. Torres GE, Gainetdinov RR, Caron MG. Plasma membrane monoamine transporters: structure, regulation and function. Nat Rev Neurosci 2003; 4: 13-25. 18. Frankhauser P, Grimmer Y, Bugert P, et al. Characterization of the neuronal dopamine transporter DAT in human blood platelets. Neurosci Lett 2006; 399: 197-201. 19. Nebigil CG, Choi DS, Dierich A, et al. Serotonin 2B receptor is required for heart development. Proc Natl Acad Sci U S A 2000; 97: 9508-9513. - 34 - - 35 -

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 34 patients [106]. However, in prostate cancer tissue of 25 patients, 5-HTR2B expression REFERENCES was similar among all Gleason grades, whereas 5-HTR4 was only expressed in Gleason grade 3-4 prostate cancer tissue [93]. In breast cancer tissue of 102 patients, 1. Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the 5-HTR1A, 5-HTR1B, 5-HTR2B and 5-HTR4 were present. In these tumors, receptor tumor microenvironment. Cancer Cell 2012; 21: 309-322. expression did not correlate with tumor grade [107]. In 159 bone metastases of 2. Hanahan D, Weinberg RA. The hallmarks of cancer: the next generation. Cell 2011; 144: carcinoma and sarcoma patients reverse phase protein microarray analysis showed that 646-674. 2 expression of serotonin in combination with tumor necrosis factor receptor 1 was 3. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249- associated with a poor survival [108]. Similar to dopamine, there are no data relating 257. serotonin production to angiogenesis in carcinoid tumors [74]. 4. Schäfer M, Werner S. Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol 2008; 9: 628-638. 5. Pinedo HM, Verheul HM, D’Amato RJ, et al. Involvement of platelets in tumour CONCLUSIONS AND PERSPECTIVES angiogenesis? Lancet 1998; 352: 1775-1777. 6. Holmsen H, Weiss HJ. Secretable storage pools in platelets. Annu Rev Med 1979; 30: 119- Based on the available literature it can be concluded that dopamine inhibits tumor 134. growth, whereas serotonin stimulates tumor growth. In vitro and animal studies 7. Da Prada M, Picotti GB. Content and subcellular localization of catecholamines and 5- showed that dopamine inhibits endothelial cell proliferation and tumor growth via hydroxytryptamine in human and animal blood platelets: monoamine distribution between activation of DRD2. Treatment with dopamine is not feasible because of severe platelets and plasma. Br J Pharmacol 1979; 65: 653-662. 8. Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of dopamine cardiovascular toxicity. Therefore clinical intervention studies with DRD2 agonists are receptors. Pharmacol Rev 2011; 63: 182-217. attractive, especially as these agents are already being used in the clinic for other 9. Mohammad-Zadeh LF, Moses L, Gwaltney-Brant SM. Serotonin: a review. J Vet Pharmacol indications such as Parkinson’s disease and hyperprolactinemia [8]. To our knowledge, Ther 2008; 31: 187-199. no clinical trials with DRD2 agonists in cancer patients are currently performed. 10. Chakroborty D, Sarkar C, Basu B, et al. Catecholamines regulate tumor angiogenesis. Serotonin’s role in tumor behavior has been studied less extensively and data Cancer Res 2009; 69: 3727-3730. are predominantly derived from in vitro experiments. These experiments showed 11. Moreno-Smith M, Lu C, Shahzad MM, et al. Dopamine blocks stress-mediated ovarian serotonin to stimulate endothelial cells via activation of 5-HTR1 and 5-HTR2. Tumor carcinoma growth. Clin Cancer Res 2011; 17: 3649-3659. cell proliferation could be inhibited by selected 5-HTR antagonists, depending on 12. Nocito A, Dahm F, Jochum W, et al. Serotonin regulates macrophage-mediated tumor cell type. Tryptophan hydroxylase inhibitors are currently used in clinical trials angiogenesis in a mouse model of colon cancer allografts. Cancer Res 2008; 68: 5152-5158. in irritable bowel syndrome patients [109] and for serotonin-producing carcinoid 13. Eisenhofer G, Aneman A, Friberg P, et al. Substantial production of dopamine in the tumor patients to evaluate the effect on the carcinoid syndrome. However further human gastrointestinal tract. J Clin Endocrinol Metab 1997; 82: 3864-3871. 14. Kopin IJ. Catecholamine metabolism: basic aspects and clinical significance. Pharmacol Rev animal experiments to reveal serotonin’s mechanism of action are warranted. 1985; 37: 333-364.

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 35 20. Ni W, Watts SW. 5-hydroxytryptamine in the cardiovascular system: focus on the serotonin factor and interleukin-8 in ovarian hyperstimulation syndrome: dopamine targets their transporter (SERT). Clin Exp Pharmacol Physiol 2006; 33: 575-583. common pathways. Hum Reprod 2010; 25: 757-767. 21. Da Prada M, Pletscher A. Differential uptake of biogenic amines by isolated 5- 38. Gomez R, Gonzalez-Izquierdo M, Zimmermann RC, et al. Low-dose dopamine agonist hydroxytryptamine organelles of blood platelets. Life Sci 1969; 8: 65-72. administration blocks vascular endothelial growth factor (VEGF)-mediated vascular 22. Marcus AJ, Safier LB. Thromboregulation: multicellular modulation of platelet reactivity hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a 2 in hemostasis and thrombosis. FASEB J 1993; 7: 516-522. rat ovarian hyperstimulation model. Endocrinology 2006; 147: 5400-5411. 23. Italiano JE Jr, Richardson JL, Patel-Hett S, et al. Angiogenesis is regulated by a novel 39. Franks S. Polycystic ovary syndrome. N Engl J Med 1995; 333: 853-861. mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha 40. Gómez R, Ferror H, Delgado-Rosas F, et al. Evidences for the existence of a low granules and differentially released. Blood 2008; 111: 1227-1233. dopaminergic tone in polycystic ovarian syndrome: implications for OHSS development 24. Basu S, Nagy JA, Pal S, et al. The neurotransmitter dopamine inhibits angiogenesis and treatment. J Clin Endocrinol Metab 2011; 96: 2484-2492. induced by vascular permeability factor/vascular endothelial growth factor. Nat Med 2001; 41. Alvarez C, Martí-Bonmatí L, Novella-Maestre E, et al. Dopamine agonist cabergoline 7: 569-574. reduces hemoconcentration and ascites in hyperstimulated women undergoing assisted 25. Bhattacharya R, Sinha S, Yang SP, et al. The neurotransmitter dopamine modulates reproduction. J Clin Endocrinol Metab 2007; 92: 2931-2937. vascular permeability in the endothelium. J Mol Signal 2008; 3: 14. 42. Papaleo E, Doldi N, De Santis L, et al. Cabergoline influences ovarian stimulation in 26. Shome S, Dasgupta PS, Basu S. Dopamine regulates mobilization of mesenchymal stem hyperprolactinaemic patients with polycystic ovary syndrome. Hum Reprod 2001; 16: 2263- cells during wound angiogenesis. PLoS One 2012; 7: e31682. 2266. 27. Chakroborty D, Chowdhury UR, Sarkar C, et al. Dopamine regulates endothelial 43. Youssef M, Van Wely M, Hassan MA, et al. Can dopamine agonists reduce the incidence progenitor cell mobilization from mouse bone marrow in tumor vascularization. J Clin and severity of OHSS in IVF/ICSI treatment cycles? A systematic review and meta- Invest 2008; 118: 1380-1389. analysis. Hum Reprod 2010; 16: 459-466. 28. Chakraborti S, Mandal M, Das S, et al. Regulation of matrix metalloproteinases: an 44. Shaltout A, Shohyab A, Youssef MA. Can dopamine agonist at a low dose reduce ovarian overview. Mol Cell Biochem 2003; 253: 269-285. hyperstimulation syndrome in women at risk undergoing ICSI treatment cycles? A 29. Heissig B, Hattori K, Dias S, et al. Recruitment of stem and progenitor cells from the randomized controlled study. Eur J Obstet Gynaecol Reprod Biol 2012; 165: 254-258. bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 2002; 109: 625- 45. Lang AE, Lozano AM. Parkinson’s disease. First of two parts. N Engl J Med 1998; 339: 637. 1044-1053. 30. Giudice LC, Kao LC. Endometriosis. Lancet 2004; 364: 1789-1799. 46. Chase TN. Levodopa therapy: consequences of the nonphysiologic replacement of 31. Novella-Maestre E, Carda C, Noquera I, et al. Dopamine agonist administration causes a dopamine. Neurology 1998; 50 (5 suppl 5): S17-25. reduction in endometrial implants through modulation of angiogenesis in experimentally 47. Ohlin KE, Francardo V, Lindgren HS, et al. Vascular endothelial growth factor is induced endometriosis. Hum Reprod 2009; 24: 1025-1035. upregulated by L-dopa in the parkinsonian brain: implications for development of 32. Novella-Maestre E, Carda C, Ruiz-Sauri A, et al. Identification and quantification of dyskinesia. Brain 2011; 134: 2339-2357. dopamine receptor 2 in human eutopic and ectopic endometrium: a novel molecular 48. Lindgren HS, Ohlin KE, Cenci MA. Differential involvement of D1 and D2 dopamine target for endometriosis therapy. Biol Reprod 2010; 83: 866-873. receptors in L-DOPA-induced angiogenic activity in a rat model of Parkinson’s disease. 33. Delgado-Rosas F, Gómez R, Ferrero H, et al. The effects of ergot and non-ergot derived Neuropsychopharmacology 2009; 34: 2477-2488. dopamine agonists in an experimental mouse model of endometriosis. Reproduction 2011; 49. Westin JE, Lidgren HS, Gardi J, et al. Endothelial proliferation and increased blood-brain 142: 745-755. barrier permeability in the basal ganglia in a rat model of 3,4-dihydroxyphenyl-L-alanine- 34. Gómez R, Abad A, Delgado F, et al. Effects of hyperprolactinemia treatment with the induced dyskinesia. J Neurosci 2006; 26: 9448-9461. dopamine agonist quinagolide on endometriotic lesions in patients with endometriosis- 50. Faucheux BA, Bonnet AM, Agid Y, et al. Blood vessels change in the mesencephalon of associated hyperprolactinemia. Fertil Steril 2011; 95: 882-888. patients with Parkinson’s disease. Lancet 1999; 353: 981-982. 35. Delvigne A, Rozenberg S. Epidemiology and prevention of ovarian hyperstimulation 51. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999; 341: 738-746. syndrome (OHSS): a review. Hum Reprod Update 2002; 8: 559-577. 52. Shome S, Rana T, Ganguly S, et al. Dopamine regulates angiogenesis in normal dermal 36. Gómez R, Soares SR, Busso C, et al. Physiology and pathology of ovarian wound tissues. PloS One 2011; 6: e25215. hyperstimulation syndrome. Semin Reprod Med 2010; 28: 448-457. 53. Meredith EJ, Holder MJ, Rosén A, et al. Dopamine targets cycling B cells independent of 37. Chen SU, Chou CH, Lin CW, et al. Signal mechanisms of vascular endothelial growth receptors/transporter for oxidative attack: Implications for non-Hodgkin’s lymphoma.

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548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 36 20. Ni W, Watts SW. 5-hydroxytryptamine in the cardiovascular system: focus on the serotonin factor and interleukin-8 in ovarian hyperstimulation syndrome: dopamine targets their transporter (SERT). Clin Exp Pharmacol Physiol 2006; 33: 575-583. common pathways. Hum Reprod 2010; 25: 757-767. 21. Da Prada M, Pletscher A. Differential uptake of biogenic amines by isolated 5- 38. Gomez R, Gonzalez-Izquierdo M, Zimmermann RC, et al. Low-dose dopamine agonist hydroxytryptamine organelles of blood platelets. Life Sci 1969; 8: 65-72. administration blocks vascular endothelial growth factor (VEGF)-mediated vascular 22. Marcus AJ, Safier LB. Thromboregulation: multicellular modulation of platelet reactivity hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a in hemostasis and thrombosis. FASEB J 1993; 7: 516-522. rat ovarian hyperstimulation model. Endocrinology 2006; 147: 5400-5411. 2 23. Italiano JE Jr, Richardson JL, Patel-Hett S, et al. Angiogenesis is regulated by a novel 39. Franks S. Polycystic ovary syndrome. N Engl J Med 1995; 333: 853-861. mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha 40. Gómez R, Ferror H, Delgado-Rosas F, et al. Evidences for the existence of a low granules and differentially released. Blood 2008; 111: 1227-1233. dopaminergic tone in polycystic ovarian syndrome: implications for OHSS development 24. Basu S, Nagy JA, Pal S, et al. The neurotransmitter dopamine inhibits angiogenesis and treatment. J Clin Endocrinol Metab 2011; 96: 2484-2492. induced by vascular permeability factor/vascular endothelial growth factor. Nat Med 2001; 41. Alvarez C, Martí-Bonmatí L, Novella-Maestre E, et al. Dopamine agonist cabergoline 7: 569-574. reduces hemoconcentration and ascites in hyperstimulated women undergoing assisted 25. Bhattacharya R, Sinha S, Yang SP, et al. The neurotransmitter dopamine modulates reproduction. J Clin Endocrinol Metab 2007; 92: 2931-2937. vascular permeability in the endothelium. J Mol Signal 2008; 3: 14. 42. Papaleo E, Doldi N, De Santis L, et al. Cabergoline influences ovarian stimulation in 26. Shome S, Dasgupta PS, Basu S. Dopamine regulates mobilization of mesenchymal stem hyperprolactinaemic patients with polycystic ovary syndrome. Hum Reprod 2001; 16: 2263- cells during wound angiogenesis. PLoS One 2012; 7: e31682. 2266. 27. Chakroborty D, Chowdhury UR, Sarkar C, et al. Dopamine regulates endothelial 43. Youssef M, Van Wely M, Hassan MA, et al. Can dopamine agonists reduce the incidence progenitor cell mobilization from mouse bone marrow in tumor vascularization. J Clin and severity of OHSS in IVF/ICSI treatment cycles? A systematic review and meta- Invest 2008; 118: 1380-1389. analysis. Hum Reprod 2010; 16: 459-466. 28. Chakraborti S, Mandal M, Das S, et al. Regulation of matrix metalloproteinases: an 44. Shaltout A, Shohyab A, Youssef MA. Can dopamine agonist at a low dose reduce ovarian overview. Mol Cell Biochem 2003; 253: 269-285. hyperstimulation syndrome in women at risk undergoing ICSI treatment cycles? A 29. Heissig B, Hattori K, Dias S, et al. Recruitment of stem and progenitor cells from the randomized controlled study. Eur J Obstet Gynaecol Reprod Biol 2012; 165: 254-258. bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 2002; 109: 625- 45. Lang AE, Lozano AM. Parkinson’s disease. First of two parts. N Engl J Med 1998; 339: 637. 1044-1053. 30. Giudice LC, Kao LC. Endometriosis. Lancet 2004; 364: 1789-1799. 46. Chase TN. Levodopa therapy: consequences of the nonphysiologic replacement of 31. Novella-Maestre E, Carda C, Noquera I, et al. Dopamine agonist administration causes a dopamine. Neurology 1998; 50 (5 suppl 5): S17-25. reduction in endometrial implants through modulation of angiogenesis in experimentally 47. Ohlin KE, Francardo V, Lindgren HS, et al. Vascular endothelial growth factor is induced endometriosis. Hum Reprod 2009; 24: 1025-1035. upregulated by L-dopa in the parkinsonian brain: implications for development of 32. Novella-Maestre E, Carda C, Ruiz-Sauri A, et al. Identification and quantification of dyskinesia. Brain 2011; 134: 2339-2357. dopamine receptor 2 in human eutopic and ectopic endometrium: a novel molecular 48. Lindgren HS, Ohlin KE, Cenci MA. Differential involvement of D1 and D2 dopamine target for endometriosis therapy. Biol Reprod 2010; 83: 866-873. receptors in L-DOPA-induced angiogenic activity in a rat model of Parkinson’s disease. 33. Delgado-Rosas F, Gómez R, Ferrero H, et al. The effects of ergot and non-ergot derived Neuropsychopharmacology 2009; 34: 2477-2488. dopamine agonists in an experimental mouse model of endometriosis. Reproduction 2011; 49. Westin JE, Lidgren HS, Gardi J, et al. Endothelial proliferation and increased blood-brain 142: 745-755. barrier permeability in the basal ganglia in a rat model of 3,4-dihydroxyphenyl-L-alanine- 34. Gómez R, Abad A, Delgado F, et al. Effects of hyperprolactinemia treatment with the induced dyskinesia. J Neurosci 2006; 26: 9448-9461. dopamine agonist quinagolide on endometriotic lesions in patients with endometriosis- 50. Faucheux BA, Bonnet AM, Agid Y, et al. Blood vessels change in the mesencephalon of associated hyperprolactinemia. Fertil Steril 2011; 95: 882-888. patients with Parkinson’s disease. Lancet 1999; 353: 981-982. 35. Delvigne A, Rozenberg S. Epidemiology and prevention of ovarian hyperstimulation 51. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999; 341: 738-746. syndrome (OHSS): a review. Hum Reprod Update 2002; 8: 559-577. 52. Shome S, Rana T, Ganguly S, et al. Dopamine regulates angiogenesis in normal dermal 36. Gómez R, Soares SR, Busso C, et al. Physiology and pathology of ovarian wound tissues. PloS One 2011; 6: e25215. hyperstimulation syndrome. Semin Reprod Med 2010; 28: 448-457. 53. Meredith EJ, Holder MJ, Rosén A, et al. Dopamine targets cycling B cells independent of 37. Chen SU, Chou CH, Lin CW, et al. Signal mechanisms of vascular endothelial growth receptors/transporter for oxidative attack: Implications for non-Hodgkin’s lymphoma.

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Livingston RB, Ambus U, George SL, et al. In vitro determination of thymidine-3H migration of rat aortic smooth muscle cells through 5-HT2 receptors. Atherosclerosis 1997; labeling index in human solid tumors. Cancer Res 1974; 34: 1376-1380. 132: 139-143. 70. Basu S, Dasgupta PS. Alteration of dopamine D2 receptors in human malignant stomach 88. McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic tissue. Dig Dis Sci 1997; 42: 1260-1264. inflammation. Am J Respir Crit Care Med 2001; 164: S39-45.

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548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 38 Proc Natl Acad Sci U S A 2006; 103: 13485-13490. 71. Gemignani F, Landi S, Moreno V, et al. Polymorphisms of the dopamine receptor gene 54. Sarkar C, Chakroborty D, Chowdhury UR, et al. Dopamine increases the efficacy of DRD2 and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev 2005; 14: 1633-1638. anticancer drugs in breast and colon cancer preclinical models. Clin Cancer Res 2008; 14: 72. Murphy G, Cross AJ, Sansbury LS, et al. Dopamine D2 receptor polymorphisms and 2502-2510. adenoma recurrence in the Polyp Prevention Trial. Int J Cancer 2009; 124: 2148-2151. 55. Ganguly S, Basu B, Shome S, et al. Dopamine, by acting through its D2 receptor, inhibits 73. Campa D, Zienolddiny S, Lind H, et al. Polymorphisms of dopamine receptor/transporter insulin-like growth factor-I (IGF-I)-induced gastric cancer cell proliferation via up- genes and risk of non-small cell lung cancer. Lung Cancer 2007; 56: 17-23. 2 regulation of Krüppel-like factor 4 through down-regulation of IGF-IR and AKT 74. Meijer WG, Copray SC, Hollema H, et al. Catecholamine-synthesizing enzymes in phosphorylation. Am J Pathol 2010; 177: 2701-2707. carcinoid tumors and pheochromocytomas. Clin Chem 2003; 49: 586-593. 56. Ishibashi M, Fujisawa M, Furue H, et al. Inhibition of growth of human small cell lung 75. Iwabayashi M, Taniyama Y, Sanada F, et al. Role of serotonin in angiogenesis: induction of cancer by bromocriptine. Cancer Res 1994; 54: 3442-3446. angiogenesis by sarpogrelate via endothelial 5-HT1B/Akt/eNOS pathway in diabetic 57. Senogles SE. D2 dopamine receptor-mediated antiproliferation in a small cell lung cancer mice. Atherosclerosis 2012; 220: 337-342. cell line, NCI-H69. Anticancer Drugs 2007; 18: 801-807. 76. Matsusaka S, Wakabayashi I. 5-hydroxytryptamine as a potent migration enhancer of 58. Basu S, Sarkar C, Chakroborty D, et al. Ablation of peripheral dopaminergic nerves human aortic endothelial cells. FEBS Lett 2005; 579: 6721-6725. stimulates malignant tumor growth by inducing vascular permeability factor/vascular 77. Qin L, Zhao D, Xu J, et al. The vascular permeabilizing factors histamine and serotonin endothelial growth factor-mediated angiogenesis. Cancer Res 2004; 64: 5551-5555. induce angiogenesis through TR3/Nur77 and subsequently truncate it through 59. Sarkar C, Chakroborty D, Mitra RB, et al. Dopamine in vivo inhibits VEGF-induced thrombospondin-1. Blood 2013; 121: 2154-2164. phosphorylation of VEGFR-2, MAPK, and focal adhesion kinase in endothelial cells. Am 78. Yang M, Li K, Ng PC, et al. Promoting effects of serotonin on hematopoiesis: ex vivo J Physiol Heart Circ Physiol 2004; 287: H1554-1560. expansion of cord blood CD34+ stem/progenitor cells, proliferation on bone marrow 60. Asada M, Ebihara S, Numachi Y, et al. Reduced tumor growth in a mouse model of stromal cells, and antiapoptosis. Stem Cells 2007; 25: 1800-1806. schizophrenia, lacking the dopamine transporter. Int J Cancer 2008; 123: 511-518. 79. Zhao D, Qin L, Bourbon PM, et al. Orphan nuclear transcription factor TR3/Nur77 61. Cools AR, Gingras MA. Nijmegen high and low responders to novelty: a new tool in the regulates microvessel permeability by targeting endothelial nitric oxide synthase and search after the neurobiology of drug abuse liability. Pharmacol Biochem Behav 1998; 60: destabilizing endothelial junctions. Proc Natl Acad Sci U S A 2011; 108: 12066-12071. 151-159. 80. Pakala R, Benedict CR. Effect of serotonin and thromboxane A2 on endothelial cell 62. Teunis MA, Kavelaars A, Voest E, et al. Reduced tumor growth, experimental metastasis proliferation: effect of specific receptor antagonists. J Lab Clin Med 1998; 131: 527-537. formation, and angiogenesis in rats with a hyperreactive dopaminergic system. FASEB J 81. Nishikawa T, Tsuno NH, Shuno Y, et al. Antiangiogenic effect of a selective 5-HT4 2002; 16: 1465-1467. receptor agonist. J Surg Res 2010; 159: 696-704. 63. Chakroborty D, Sarkar C, Mitra RB, et al. Depleted dopamine in gastric cancer tissues: 82. Profirovic J, Strekalova E, Urao N, et al. A novel regulator of angiogenesis in endothelial dopamine treatment retards growth of gastric cancer by inhibiting angiogenesis. Clin cells: 5-hydroxytriptamine 4 receptor. Angiogenesis 2013; 16: 15-28. Cancer Res 2004; 10: 4349-4356. 83. Dimmeler S, Fleming I, Fisslthaler B, et al. Activation of nitric oxide synthase in 64. Dasgupta PS, Lahiri T. Antitumor effect of i.p. dopamine in mice bearing Ehrlich ascites endothelial cells by Akt-dependent phosphorylation. Nature 1999; 399: 601-605. carcinoma. J Cancer Res Clin Oncol 1987; 113: 363-368. 84. Zamani A, Qu Z. Serotonin activates angiogenic phosphorylation signaling in human 65. Moreno-Smith M, Lee SJ, Lu C, et al. Biologic effects of dopamine on tumor vasculature endothelial cells. FEBS Lett 2012; 586: 2360-2365. in ovarian carcinoma. Neoplasia 2013; 15: 502-510. 85. Asada M, Ebihara S, Yamanda S, et al. Depletion of serotonin and selective inhibition of 66. Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic 2B receptor suppressed tumor angiogenesis by inhibiting endothelial nitric oxide synthase therapy. Science 2005; 307: 58-62. and extracellular signal-regulated kinase 1/2 phosphorylation. Neoplasia 2009; 11: 408-417. 67. Basu S, Dasgupta PS. Decreased dopamine receptor expression and its second-messenger 86. Nemecek GM, Coughlin SR, Handley DA, et al. Stimulation of aortic smooth muscle cell cAMP in malignant human colon tissue. Dig Dis Sci 1999; 44: 916-921. mitogenesis by serotonin. Proc Natl Acad Sci U S A 1986; 83: 674-678. 68. Wick MM. The chemotherapy of malignant melanoma. J Invest Dermatol 1983; 80: 61s-62s. 87. Tamura K, Kanzaki T, Saito Y, et al. Serotonin (5-hydroxytryptamine, 5-HT) enhances 69. Livingston RB, Ambus U, George SL, et al. In vitro determination of thymidine-3H migration of rat aortic smooth muscle cells through 5-HT2 receptors. Atherosclerosis 1997; labeling index in human solid tumors. Cancer Res 1974; 34: 1376-1380. 132: 139-143. 70. Basu S, Dasgupta PS. Alteration of dopamine D2 receptors in human malignant stomach 88. McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic tissue. Dig Dis Sci 1997; 42: 1260-1264. inflammation. Am J Respir Crit Care Med 2001; 164: S39-45.

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548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 39 89. Maleki-Dizaji N, Eteraf-Oskouei T, Fakhrjou A, et al. The effects of 5HT3 receptor impact on angiogenesis and clinical follow-up. Prostate 2011; 71: 1752-1758. antagonist granisetron on inflammatory parameters and angiogenesis in the air-pouch 105. Chevalier S, Defoy I, Lacoste J, et al. Vascular endothelial growth factor and signaling model of inflammation. Int Immunopharmacol 2010; 10: 1010-1016. in the prostate: more than angiogenesis. Mol Cell Endocrinol 2002; 189: 169-179. 90. Samborski W, Stratz T, Mackiewicz S, et al. Intra-articular treatment of arthritides and 106. Soll C, Riener MO, Oberkofler CE, et al. Expression of serotonin receptors in human activated osteoarthritis with the 5-HT 3 receptor antagonist tropisestron. A double-blind hepatocellular cancer. Clin Cancer Res 2012; 18: 5902-5910. 2 study compared with methylprednisolone. Scand J Rheumatol Suppl 2004; 119: 51-54. 107. Kopparapu PK, Tinzl M, Anagnostaki L, et al. Expression and localization of 91. Alpini G, Invernizzi P, Gaudio E, et al. Serotonin metabolism is dysregulated in serotonin receptors in human breast cancer. Anticancer Res 2013; 33: 363-370. cholangiocarcinoma, which has implications for tumor growth. Cancer Res 2008; 68: 9184- 108. Chiechi A, Novello C, Magagnoli G, et al. Elevated TNFR1 and serotonin in bone 9193. metastasis are correlated with poor survival following bone metastasis diagnosis for both 92. Cattaneo MG, Palazzi E, Bondiolotti G, et al. 5-HT1D receptor type is involved in carcinoma and sarcoma primary tumors. Clin Cancer Res 2013; 19: 2473-2485. stimulation of cell proliferation by serotonin in human small cell lung carcinoma. Eur J 109. Brown PM, Drossman DA, Wood AJJ, et al. The tryptophan hydroxylase inhibitor Pharmacol 1994; 268: 425-430. LX1031 shows clinical benefit in patients with nonconstipating irritable bowel syndrome. 93. Dizeyi N, Bjartell A, Hedlund P, et al. Expression of serotonin receptors 2B and 4 in Gastroenterology 2011; 141: 507-516. human prostate cancer tissue and effects of their antagonists on prostate cancer cell lines. Eur Urol 2005; 47: 895-900. 94. Drozdov I, Kidd M, Gustafsson BI, et al. Autoregulatory effects of serotonin on proliferation and signaling pathways in lung and small intestine neuroendocrine tumor cell lines. Cancer 2009; 115: 4934-4945. 95. Liang C, Chen W, Zhi X, et al. Serotonin promotes the proliferation of serum-deprived hepatocellular carcinoma cells via upregulation of FOXO3a. Mol Cancer 2013; 12: 14. 96. Pirozhok I, Meye A, Hakenberg OW, et al. Serotonin and melatonin do not play a prominent role in the growth of prostate cancer cell lines. Urol Int 2010; 84: 452-460. 97. Siddiqui EJ, Shabbir MA, Mikhailidis DP, et al. The effect of serotonin and serotonin antagonists on bladder cancer cell proliferation. BJU Int 2006; 97: 634-639. 98. Siddiqui EJ, Shabbir M, Mikhailidis DP, et al. The role of serotonin (5- hydroxytryptamine1A and 1B) receptors in prostate cancer cell proliferation. J Urol 2006; 176: 1648-1653. 99. Soll C, Jang JH, Riener MO, et al. Serotonin promotes tumor growth in human hepatocellular cancer. Hepatology 2010; 51: 1244-1254. 100. Sonier B, Arseneault M, Lavigne C, et al. The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin. Biochem Biophys Res Commun 2006; 343: 1053-1059. 101. Müller K, Gilbertz KP, Meineke V. Serotonin and ionizing radiation synergistically affect proliferation and adhesion molecule expression of malignant melanoma cells. J Dermatol Sci 2012; 68: 89-98. 102. Vicentini LM, Cattaneo MG, Fesce R. Evidence for receptor subtype cross-talk in the mitogenic action of serotonin on human small-cell lung carcinoma cells. Eur J Pharmacol 1996; 318: 497-504. 103. Cornelius LA, Nehring LC, Harding E, et al. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol 1998; 161: 6845-6852. 104. Heinrich E, Trojan L, Friedrich D, et al. Neuroendocrine tumor cells in prostate cancer: evaluation of the neurosecretory products serotonin, bombesin, and gastrin –

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548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 40 89. Maleki-Dizaji N, Eteraf-Oskouei T, Fakhrjou A, et al. The effects of 5HT3 receptor impact on angiogenesis and clinical follow-up. Prostate 2011; 71: 1752-1758. antagonist granisetron on inflammatory parameters and angiogenesis in the air-pouch 105. Chevalier S, Defoy I, Lacoste J, et al. Vascular endothelial growth factor and signaling model of inflammation. Int Immunopharmacol 2010; 10: 1010-1016. in the prostate: more than angiogenesis. Mol Cell Endocrinol 2002; 189: 169-179. 90. Samborski W, Stratz T, Mackiewicz S, et al. Intra-articular treatment of arthritides and 106. Soll C, Riener MO, Oberkofler CE, et al. Expression of serotonin receptors in human activated osteoarthritis with the 5-HT 3 receptor antagonist tropisestron. A double-blind hepatocellular cancer. Clin Cancer Res 2012; 18: 5902-5910. study compared with methylprednisolone. Scand J Rheumatol Suppl 2004; 119: 51-54. 107. Kopparapu PK, Tinzl M, Anagnostaki L, et al. Expression and localization of 2 91. Alpini G, Invernizzi P, Gaudio E, et al. Serotonin metabolism is dysregulated in serotonin receptors in human breast cancer. Anticancer Res 2013; 33: 363-370. cholangiocarcinoma, which has implications for tumor growth. Cancer Res 2008; 68: 9184- 108. Chiechi A, Novello C, Magagnoli G, et al. Elevated TNFR1 and serotonin in bone 9193. metastasis are correlated with poor survival following bone metastasis diagnosis for both 92. Cattaneo MG, Palazzi E, Bondiolotti G, et al. 5-HT1D receptor type is involved in carcinoma and sarcoma primary tumors. Clin Cancer Res 2013; 19: 2473-2485. stimulation of cell proliferation by serotonin in human small cell lung carcinoma. Eur J 109. Brown PM, Drossman DA, Wood AJJ, et al. The tryptophan hydroxylase inhibitor Pharmacol 1994; 268: 425-430. LX1031 shows clinical benefit in patients with nonconstipating irritable bowel syndrome. 93. Dizeyi N, Bjartell A, Hedlund P, et al. Expression of serotonin receptors 2B and 4 in Gastroenterology 2011; 141: 507-516. human prostate cancer tissue and effects of their antagonists on prostate cancer cell lines. Eur Urol 2005; 47: 895-900. 94. Drozdov I, Kidd M, Gustafsson BI, et al. Autoregulatory effects of serotonin on proliferation and signaling pathways in lung and small intestine neuroendocrine tumor cell lines. Cancer 2009; 115: 4934-4945. 95. Liang C, Chen W, Zhi X, et al. Serotonin promotes the proliferation of serum-deprived hepatocellular carcinoma cells via upregulation of FOXO3a. Mol Cancer 2013; 12: 14. 96. Pirozhok I, Meye A, Hakenberg OW, et al. Serotonin and melatonin do not play a prominent role in the growth of prostate cancer cell lines. Urol Int 2010; 84: 452-460. 97. Siddiqui EJ, Shabbir MA, Mikhailidis DP, et al. The effect of serotonin and serotonin antagonists on bladder cancer cell proliferation. BJU Int 2006; 97: 634-639. 98. Siddiqui EJ, Shabbir M, Mikhailidis DP, et al. The role of serotonin (5- hydroxytryptamine1A and 1B) receptors in prostate cancer cell proliferation. J Urol 2006; 176: 1648-1653. 99. Soll C, Jang JH, Riener MO, et al. Serotonin promotes tumor growth in human hepatocellular cancer. Hepatology 2010; 51: 1244-1254. 100. Sonier B, Arseneault M, Lavigne C, et al. The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin. Biochem Biophys Res Commun 2006; 343: 1053-1059. 101. Müller K, Gilbertz KP, Meineke V. Serotonin and ionizing radiation synergistically affect proliferation and adhesion molecule expression of malignant melanoma cells. J Dermatol Sci 2012; 68: 89-98. 102. Vicentini LM, Cattaneo MG, Fesce R. Evidence for receptor subtype cross-talk in the mitogenic action of serotonin on human small-cell lung carcinoma cells. Eur J Pharmacol 1996; 318: 497-504. 103. Cornelius LA, Nehring LC, Harding E, et al. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol 1998; 161: 6845-6852. 104. Heinrich E, Trojan L, Friedrich D, et al. Neuroendocrine tumor cells in prostate cancer: evaluation of the neurosecretory products serotonin, bombesin, and gastrin –

- 40 - - 41 -

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9 Ref 94 97 100 91 9 106 95 -

and and - - - - -

7 7

2 say s Prolifera tion assay MTT assay Crystal violet colori- metric assay MTT assay MTT assay MTT a MTT assay in (only Huh HepG2) ND

PCR PCR, PCR PCR PCR - - - - - Receptor Receptor expression assay RT blot Western RT blot Western RT blot Western RT RT

- - -

HTR HTR2A, HTR2A, HTR HTR2A HTR1A, HTR1B, 5 HTR1D, HTR2B, 5 HTR2C, HTR4, HTR6, HTR7 5HTR2A, HTR2, 5HTR7 HTR2B, HTR1A, HTR1B, HTR2B, HTR7 HTR2C, HTR2C, HTR3A HTR1B, HTR1D/2, 5 HTR1D/2, HTR4 HTR expression and the capacity of the most of the most and the capacity expression HTR ------5 antagonist tested 5 5 5 5 5 5 5 5 5 HTR2A, 5 5 HTR3, 5 5 5 5HTR1, 5 5 5 5 5 5 ND 5 HTR3, HTR3, 5

, ,

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R6 42 43

- - HTR HTR2A, HTR2A, HTR1A, HTR2A HTR1A, HTR2A, HTR2B, HTR4, H HTR2, HTR2B HTR1B HTR2B HTR HTR1B HTR3A ntagonist ------Effective Effective 5 a 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ND 5

gated i st e v HTR HTR2A, HTR2A, HTR1A, HTR2A HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR2C, HTR3, HTR4, HTR6, HTR7 HTR2B HTR1A, HTR1B, HTR2A, HTR2B, HTR4, HTR5, HTR7 HTR2B HTR2C, HTR2C, HTR3A HTR1B, HTR1D, HTR1D, HTR2A, HTR2B, HTR2C, HTR2C, HTR3, HTR4 ------5 in 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

pressed HTR2B HTR HTR2A, HTR2A, HTR2C, HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR2A HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR2C, HTR3, HTR4, HTR6, HTR7 HTR2B HTR1A, HTR1B, HTR2B, HTR7 HTR3A HTR2C ------5 5 ex 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 , ,

- - A spectrum of tumor cell lines tested for 5 for cell tested lines A of tumor spectrum

1, 1,

cell

T1,

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- CLP Tumor cell cell Tumor tissue line or sample NCI Human line cell H720 Human cell HT1376 line MCF Human line 7 cell Human Mz HuH HuCC C and SG231, TFK lines Huh7 Human line cell Huh7, Human HepG2, and Hep3B cell lines Huh7 Human HepG2, and Hep3B line

HTR antagonists to inhibit tumor cell proliferation. to inhibit tumor cell proliferation. antagonists HTR

- - gio

Supplementary Table Supplementary Table 1. 5 promising cell Tumor type Atypical broncho pulmonary neuroendocrine tumor cancer Bladder Breast cancer Cholan carcinoma Hepatocellular carcinoma Hepatocellular carcinoma Hepatocellular carcinoma

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9 Ref 94 97 100 91 9 106 95 -

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------

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2 say s Prolifera tion assay MTT assay Crystal violet colori metric assay MTT assay MTT assay MTT a MTT assay in (only Huh HepG2) ND

PCR PCR, PCR PCR PCR - - - - - Receptor Receptor expression assay RT blot Western RT blot Western RT blot Western RT RT

- - -

HTR HTR2A, HTR2A, HTR2C, HTR3A HTR HTR1B, 5 HTR1D/2, HTR4 HTR2A HTR1A, 5HTR2A, HTR2, HTR1A, HTR1B, HTR1B, 5HTR7 HTR2B, HTR1B, HTR1D, 5 HTR1D, HTR2B, HTR2B, HTR2B, HTR7 HTR2C, 5 HTR2C, HTR4, HTR4, HTR6, HTR6, HTR7 HTR expression and the capacity of the most of the most and the capacity expression HTR ------5 antagonist tested 5 5 5 5 5 5 HTR3, 5 5 5 5 5HTR1, 5 5 5 5 ND 5 5 HTR2A, HTR2A, 5 5 5 HTR3, HTR3, 5 5 5

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- - HTR HTR2A, HTR2A, HTR HTR3A HTR1A, HTR1B HTR2A HTR1A, HTR2, HTR1B HTR2A, HTR2A, HTR2B HTR2B HTR2B, HTR2B, HTR4, HTR4, H ntagonist ------Effective Effective 5 a 5 5 5 5 5 5 5 5 5 5 5 5 ND 5 5 5

gated i st e v HTR HTR2A, HTR2A, HTR2C, HTR3A HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR2C, HTR3, HTR4 HTR2A HTR1A, HTR2B HTR1A, HTR2B HTR1B, HTR1B, HTR1B, HTR1D, HTR1D, HTR2A, HTR2B, HTR2A, HTR2B, HTR2C, HTR2C, HTR3, HTR4, HTR4, HTR4, HTR5, HTR6, HTR7 HTR7 ------5 in 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

pressed HTR2B HTR HTR2A, HTR2A, HTR2C, HTR3A HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR2C HTR2A HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR2C, HTR2B HTR1A, HTR1B, HTR2B, HTR3, HTR3, HTR7 HTR4, HTR4, HTR6, HTR6, HTR7 ------5 5 ex 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 , ,

- - A spectrum of tumor cell lines tested for 5 for cell tested lines A of tumor spectrum

1, 1,

cell

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ChA

s - CLP Tumor cell cell Tumor tissue line or sample NCI Human line cell H720 Human cell HT1376 line MCF Human line 7 cell Human Mz Huh7 Human line cell Huh7, Human HepG2, and Hep3B cell lines Huh7 Human HepG2, and Hep3B line HuH HuCC C and SG231, TFK lines

HTR antagonists to inhibit tumor cell proliferation. to inhibit tumor cell proliferation. antagonists HTR

- - gio

548127-L-bw-Peters Processed on: 17-9-2020 PDF page: 43

101 98 93 96 93 93 92 94 - - -

- -

1) - ity - U -

il 2 est ND MTT assay BRD incorpora tion assay Cellular viab t (WST in BRDU corpora tion assay in BRDU corpora tion assay [³H]thymi dine incorpora tion assay MTT assay

n blot, CR

P PCR PCR PCR PCR - - - - - Flow cytometry blot, Western Immunohisto chemistry. blot, Western RT RT Wester RT blot, Western RT ND RT

HTR1A, HTR1A, HTR2B, HTR1A, HTR2B, HTR2B, HTR1A, HTR1D HTR2A, 5 HTR2C, HTR1B, HTR1B, HTR4 HTR1B HTR4 HT HTR1D/2, HTR3, HTR3, HTR4 ------ND 5 5 5 5 5 5 5 5 5 5 5 5 5 5 HTR3A 5 5 5

- -

44 45

- - HTR1A, HTR1A, HTR2B HTR1B HTR4 HTR1A, HTR1D HTR2C HTR1B ------ND 5 5 5 5 None 5 5 5 5

HTR2A, HTR2A, HTR1A, HTR2B, HTR1A, HTR2B, HTR2B, HTR2 HTR2C, HTR3A HTR2B, HTR2B, HTR1B, HTR4 HTR1B HTR4 HTR4 HTR2C HTR1D, HTR2A, HTR2B, HTR3, HTR3, HTR4 ------5 5 5 5 5 5 5 5 5 5 5 5 ND 5 5 5 5 5 5 5 5 5

HTR2A, HTR2A, HTR2B, HTR1A, HTR1B, HTR1D, HTR2A, HTR2B HTR1A, HTR2B, HTR2B, HTR2C HTR2C HTR2B, HTR1B HTR HTR4 HTR2C ------5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ND 5 5

C3 ell ell P 145

U ne D li

man ell Human Human c IPC298 line Hu line cell PC3 Human line cell PC3 Human and Human cell DU145 line Human cell LNCaP line GLC8 Human line cell KRJ1 Human c

cancer

ations: ND = Not determined. ations: i te

sta mor ro Melanoma cancer. Prostate P cancer Prostate cancer Prostate cancer Prostate Small cell lung lung cell Small cancer cancer small Typical intestinal neuroendocrine tu Abbrev

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101 98 93 96 93 93 92 94 - - -

- -

1) - ity - U -

il 2 est ND MTT assay BRD incorpora tion assay Cellular viab t (WST in BRDU corpora tion assay in BRDU corpora tion assay [³H]thymi dine incorpora tion assay MTT assay

n blot, CR

PCR P PCR PCR PCR - - - - - Flow cytometry blot, Western Immunohisto chemistry. blot, Western RT RT Wester RT blot, Western RT ND RT

HTR1A, HTR1A, HTR1B, HTR1D/2, HTR3, HTR4 HTR2B, HTR4 HTR1A, HTR1B HTR2B, HTR4 HTR2B, HT HTR1A, HTR2A, HTR1D 5 HTR2C, ------ND 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 HTR3A

- -

44 45

- - HTR1A, HTR1A, HTR1B HTR2B HTR1B HTR4 HTR1A, HTR2C HTR1D ------ND 5 5 5 5 None 5 5 5 5

HTR2A, HTR2A, HTR2B, HTR2C HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR3, HTR4 HTR2B, HTR4 HTR1A, HTR1B HTR2B, HTR4 HTR2B, HTR4 HTR2 HTR2C, HTR2C, HTR3A ------5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ND 5 5 5

HTR2A, HTR2A, HTR2B, HTR2C HTR1A, HTR1B, HTR1D, HTR2A, HTR2B, HTR2C HTR2B HTR1A, HTR1B HTR2B, HTR HTR2B, HTR4 HTR2C ------5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ND 5

C3 ell ell P 145

U ne D li

man ell Human Human c IPC298 line Hu line cell PC3 Human line cell PC3 Human and Human cell DU145 line Human cell LNCaP line GLC8 Human line cell KRJ1 Human c

cancer

ations: ND = Not determined. ations: i te sta mor ro Melanoma cancer. Prostate P cancer Prostate cancer Prostate cancer Prostate lung cell Small cancer cancer small Typical intestinal neuroendocrine tu Abbrev

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CHAPTER 3

The dopamine receptor D2 agonist quinpirole inhibits angiogenesis in an in ovo ovarian cancer model

Marloes A.M. Peters,1 Coby Meijer,1 Annemiek M.E. Walenkamp,1 Ido P. Kema,2 Elisabeth G.E. de Vries,1 Sjoukje F. Oosting1

Submitted

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