The risk of cancer among patients with schizophrenia has been discussed.1, 2 The majority of studies in the last decade suggested that patients with schizophrenia are protected against cancer in general, despite increased smoking3, 4 and drinking habits in this population.5
The dopamine transporter (DAT) is believed to control the temporal and spatial activity of released dopamine by the rapid uptake of neurotransmitters into presynaptic terminals. DAT−/− mice, which showed behavioral abnormalities, neuroendocrine dysfunction, and altered sensitivity to certain drugs,6, 7 was proposed as an animal model of schizophrenia8 and attention-deficit hyperactivity disorder.9
Blood supply is essential for solid tumors and tumor growth highly depends on angiogenesis, the formation of new capillaries from pre-existing blood vessels.10 Therefore, the angiogenic process is an essential early step in the progression of malignant tumors. In the conventional view, angiogenesis is mediated by the local proliferation and migration of vessel wall-associated endothelial cells that emerge from their resting state in response to angiogenic growth factor, such as vascular endothelial growth factor and basic fibroblast growth factor.10 Recently, several experimental works suggests that traditional neurotransimitters, such as dopamine, acetylcholine and noraderenaline, may also contribute to solid tumor progression by modulating tumor angiogenesis.11–14 However, it is still not clear whether the abnormally transmitted neurotransmitter in psychiatric disorders affects tumor angiogenesis or not.
Dopamine D1 and D5 receptors are classified as D1-like, and D2, D3 and D4 receptors as D2-like receptors.15 In endothelial cells, dopaminergic stimulation via dopamine D2 receptor (D2R) was reported to prevent angiogenesis.11, 16, 17 On the other hand, the stimulation of dopamine D1 receptor (D1R), which also exists in endothelial cells, release GTP-binding protein coupled βγ subunits, resulting in activation of Src kinase proteins. Src kinase proteins are known to transactivate protein kinase receptors, such as the epidermal growth factor receptor.18, 19 Therefore, the overall effect of dopaminergic stimulation on angiogenesis and endothelial cell functions is still being debated. To elucidate the relationship between the dopaminergic system and cancer progression, we investigated tumor growth in DAT knockout mice as an animal model of schizophrenia.
Material and methods
Six- to 9-week-old male mutant mice lacking DAT and littermate wild-type mice were obtained from heterozygous crosses with an Sv129/C57BL/6 mixed genetic background. The details of the generation of DAT knockout mice have been described previously.6 Four- to six-week-old male D1R−/− mice with a C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, ME). In every mutant mice group, we generated homozygous, heterozygous, and wild types by crossing adult heterozygotes. DNA extracted from tail biopsies was genotyped using PCR. Mice were group housed (2–4 per cage) with food and water ad libitum in a room maintained at 22 ± 2°C and 65 ± 5% humidity under a 12 hr light-dark cycle. The animals were killed with an overdose of urethane (20 g/kg). All animal experiments were performed according to the Animals Act (scientific procedures) 1986 and approved by the local ethics panel at Tohoku University School of Medicine.
Lewis lung carcinoma (LLC) cells were purchased from the American Type Culture Collection (Manassas, VA). LLCs were cultured in high glucose DMEM containing 10% FCS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Human umbilical vascular endothelial cells (HUVEC) were purchased from Kurabo (Osaka, Japan) and were cultured in EC growth medium (Kurabo).
In vivo tumor models
LLCs were injected (1 × 106 cells/animal) subcutaneously (s.c.) into the flank of male 6- to 9-week-old wild-type mice, DAT+/−, DAT−/−, D1R+/− and D1R−/− mice on day 0. In tumor growth rate models, saline, GBR12909 (10 mg/kg), SCH23390 (0.3 mg/kg) or domperidon (1 mg/kg) was injected intraperitoneally (i.p.) every 2 days. Tumor size was quantified daily as width2 × length × 0.52. Mice inoculated with LLCs were killed on day 22 and tumors were collected, weighed and sized.
RT-PCR/RNA was prepared from dissected tissues of adult mice or LLC or HUVEC and treated extensively with DNase. Human whole brain RNA was purchased from Ambion (Austin, TX). Reverse transcription and amplification were carried out as described previously.20, 21 The oligonucleotide primers (Invitrogen, Carlsbad, CA) used for amplification of the dopamine receptor subtypes D1 and D2 were reported previously.20 PCR was performed with cDNA prepared from 5 ng of RNA in 25-μl reactions for 37 cycles.
Immunoprecipitation and Western blot analysis
2 × 105 HUVECs were seeded in 10 cm dishes, cultured for 2 days, serum-starved (0.1 % serum) for 24 hr, and then treated with either dopamine (1 μM) and/or SCH23390 (10 nM) and/or domperidon (10 nM), followed 5 min later by the addition of 10 ng/ml vascular endothelial growth factor (VEGF) (R&D Systems, Minneapolis, MN). Cells treated with or without dopamine or SCH23390 or domperidon or VEGF were suspended in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA) containing protease inhibitors (20 mg/ml leupeptin, 1 mg/ml pepstatin A and 1 mM PMSF) and then sonicated on ice. Cell extracts, obtained by centrifugation at 16,000g for 15 min, were incubated with anti-phosphotyrosine mAb (Upstate Biotechnology, Lake Placid, NY) at 4°C for 3 hr. Protein G-Sepharose 4 Fast beads (20 μl of wet volume) incubation was performed at 4°C for 1 hr. After the beads were washed with lysis buffer, the bound proteins were eluted by boiling the beads in SDS sample buffer for 10 min. The sample was subjected to SDS-PAGE, followed by Western blotting using anti-vascular endothelial growth factor receptor-2 (VEGF-R2) Ab (Santa Cruz Biotechnology, Santa Cruz, CA).
When the diameter of the tumor was 1 cm, tumor tissues were fixed in 10% formalin, embedded in paraffin and sectioned. They were blocked with 10 % normal goat serum and incubated with polyclonal anti-human factor VIII-related Ag Ab (Dako Japan, Kyoto, Japan). Subsequently, the sections were incubated first with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and then with the ABC kit (Vector Laboratories), then detected by 3-amino-9-ethylcarbazole (Vector Laboratories), and counterstained with hematoxylin.
Determination of microvessel density (MVD)
Intratumoral microvessel density was determined as previously described.21 In brief, intratumoral vessels were stained immunohistochemically with anti-human factor VIII-related Ag Ab. The image that contained the highest number of microvessels was chosen for each section by initial scan at 100× magnification, and then the vessels were counted in the selected image at 200× magnification. At least 4 fields were counted for each section, and the highest count was taken. Two independent investigators evaluated the number of vessels.
FITC-labeled control mouse IgG1 and PE-labeled anti-human VEGF-R2/KDR mAb were purchased from BD PharMingen (San Diego, CA). To determine cell-associated VEGF-R2, 1 × 105 HUVECs were treated with 1 μM dopamine or 10 nM SCH23390 or 10 nM domperidon for 5 min at 37°C in a humidified 5% CO2 atmosphere. HUVECs were treated with trypsin-EDTA and suspended in PBS. The cells were first incubated with unlabeled anti-CD16/32 mAb (eBiosience, San Diego, CA) to block nonspecific binding to FcgR. After washing, the cells were incubated on ice with a mixture of FITC-, PE- and nonlabeled Abs. After washing again, the cells were subjected to flow cytometry on a FACScan (BD Biosciences,), and the data were analyzed with CellQuest software (BD Biosciences). For all samples, dead cells were excluded from the analysis by proridium iodide staining.
Measurement of dopamine
Dopamine was measured in the plasma of DAT−/−, DAT+/− and DAT+/+ mice. Prepared samples from blood were used for the assay of dopamine by high-performance liquid chromatography with electrochemical detection.
Dopamine, GBR12909, SCH23390 and domperidon were purchased from Sigma (St. Louis, MO).
Statistical analysis of the results was performed using ANOVA with Fisher's least significant difference test for multiple comparisons. A value of p < 0.05 was considered significant.
To investigate whether the natural differences in dopaminergic reactivity among DAT−/−, DAT+/− and DAT+/+ mice, are associated with differences in tumor development, we evaluated tumor growth using a cancer animal model, a mouse inoculated with LLCs s.c. As shown in Figure 1, tumors in DAT−/− mice were significantly smaller than tumors in DAT+/− or wild-type mice (Fig. 1a and 1b). H&E staining of the tumor tissues revealed a decrease in tumor tissue vessels from DAT−/− mice (Fig. 1c). To confirm the endothelial cells, we stained paraffin sections immunohistochemically using an Ab against factor VIII-related Ag (Fig. 1d). Factor VIII-related Ag is a well-established cell surface marker of vascular endothelial cells.22 Compared with control mice, we found a decreased number of tumor vessels in DAT−/− mice. The difference in MVD between control and DAT−/− mice was statistically significant (Fig. 1e). To get more insight into the possible contribution of changes in peripheral catecholamines to the observed effect of deletion of DAT on LLC tumors, we determined the concentration of norepinephrine, epinephrine, and dopamine in plasma from DAT−/−, DAT+/− and DAT+/+ animals. There were no differences in plasma epinephrine and norepinephrine (data not shown). In contrast, the level of plasma dopamine was dramatically reduced (p < 0.01) in DAT−/− compared with wild-type mice (Fig. 1f).
We investigated whether the DAT inhibitor or dopamine agonist influenced tumor growth. LLCs were inoculated into the flank of C57BL/6 mice s.c. on day 0. From day 6 after tumor identification, we injected GBR12909, a DAT inhibitor; SCH23390, a D1R inhibitor; domperidon, a D2R inhibitor; or saline i.p. every 2 days. Compared with saline treatment, GBR12909 and domperidon treatment did not inhibit tumor growth (Figs. 2a and 2b); however SCH23390 decreased tumor growth (Figs. 2a and b). H&E staining of the tumor tissues revealed a decrease in tumor tissue vessels from mice with SCH23390 treatment (Figs. 2c). To confirm the endothelial cells, we stained paraffin sections immunohistochemically using an Ab against factor VIII-related Ag (Fig. 2d). Compared with control mice, a decreased number of tumor vessels in mice with SCH23390 treatment were found. The difference in MVD between control and SCH23390-treated mice was statistically significant (Fig. 2e).
We confirmed dopamine receptor expression in LLC and HUVEC. We used RT-PCR to analyze the mRNA expression in LLC and HUVEC. LLC expressed only D1R. HUVEC expressed D1R and D2R (Figs. 3a and 3b). Dopamine stimulation could not induce or reduce cAMP and had no effect on cell proliferation in LLC (data not shown). We could not detect the expression of DAT mRNA in LLC and HUVEC (data not shown).
Tumors from DAT−/− mice showed significantly lower levels of VEGF-R2 phosphorylation (Fig. 4a), suggesting that reduced vascularization of the tumor in DAT−/− mice was the result of the inhibited phosphorylation of VEGF-R2. We also confirmed that dopamine inhibited VEGF-induced phosphorylation of VEGF-R2 in HUVEC (Fig. 4b). However, we found that, in the absence of VEGF, 1 μM of dopamine alone induced the phosphorylation of VEGF-R2 in HUVEC (Fig. 4c). To elucidate the receptors involved in dopamine-induced phosphorylation of VEGF-R2 tyrosine kinase, we conducted a blocker study using SCH23390 and domperidon. SCH23390 inhibited the dopamine-induced phosphorylation of VEGF-R2, suggesting that dopamine-induced phosphorylation of VEGF-R2 was through D1R. Since VEGF-R2 tyrosine kinase was known to be phosphorylated from the outside of the VEGF-R2 tyrosine kinase axis by Src kinase,23 we investigated the involvement of Src kinase using PP2, a specific Src kinase antagonist. PP2 completely inhibited the phosphorylation of VEGF-R2 by dopamine stimulation (Fig. 4c). These results suggested that dopamine stimulation in peripheral vessels induced the phosphorylation of VEGF-R2 through Src via D1R. On the other hand, it has been reported the involvement of VEGF-R2 internalization by stimulation of D2R in endothelial cells.11 Therefore, we estimated endothelial cell surface VEGF-R2 expression using FACS analysis. FACS analysis revealed that in the absence of VEGF, dopamine slightly reduced the surface expression of VEGF-R2 on HUVEC. The cells treated with both dopamine and domperidon recovered the surface expression of VEGF-R2 (Fig. 4d). These results suggested that D2R stimulation induced VEGF-R2 internalization in the absence of VEGF, whereas, in the presence of VEGF, downstream of VEGF signaling was activated by D1R stimulation.
Both D1R and D2R are reported to be down-regulated in DAT−/− mice.24 Since extracellular dopamine concentration was significantly lowered in DAT−/− mice, it was speculated that reduced tumor growth in DAT−/− mice was due to reduced D1R stimulation in DAT−/− mice, resulting in the inhibition of dopamine-induced VEGF-R2 phosphorylation. Although this hypothesis is supported by SCH23390-induced tumor growth inhibition (Fig. 2a), further investigation of the hypothesis using D1R−/− mice was conducted. To investigate whether differences among D1R−/−, D1R+/− and wild-type mice are associated with differences in tumor progression, we analyzed tumor growth in these mice. Tumors from D1R−/− mice were significantly smaller than tumors from D1R+/− or wild-type mice (Figs. 5a and 5b). H&E staining of the tumor tissues revealed a decrease in tumor tissue vessels from D1R−/− mice (Fig. 5c). To confirm the vessels, we stained paraffin sections immunohistochemically using an Ab against factor VIII-related Ag (Fig. 5d). Compared with control mice, we found a decreased number of tumor vessels in D1R−/− mice. The difference in MVD between control and D1R−/− mice was statistically significant (Fig. 5e). These observations suggest that reduced tumor growth in D1R−/− mice is the result of reduced vascularization of the tumor in vivo.
According to the hypothetical role of dopamine in schizophrenia,25 we investigated tumor growth in DAT−/− mice, which are a genetic model of persistent hyperdopaminergia.26 Tumor growth was inhibited in DAT−/− mice that also had less peripheral dopamine; moreover, the DAT inhibitor could not inhibit tumor growth. We revealed that dopamine stimulation reduced the surface expression of VEGF-R2 on HUVEC via D2R but induced the phosphorylation of VEGF-R2 through D1R via Src in vitro. Finally, we investigated whether D1R expression in tumor angiogenesis influenced tumor growth in vivo; in D1R−/− mice, tumor growth was reduced. These results showed that it was important for reduced tumor growth not only to induce D2R stimulation but also to prevent D1R stimulation.
In our study, we focused on DAT−/− mice as a schizophrenic model, which receive the most attention and, in our opinion, hold the most promise for yielding insights into the complex nature between cancer and schizophrenia. Contrary to our expectations, DAT−/− mice, which have hyperdopaminergia in the central nervous system (CNS), had less peripheral serum dopamine. Moreover, it has been reported that DAT−/− mice have less D1R in the CNS.24 Although patients with schizophrenia may have a hyperdopaminergic brain, systemic D1R density was reduced in schizophrenia.27 These observations suggest that not only the hyperdopaminergic state but also less D1R might reduce tumor growth, resulting in the possible protection of patients with schizophrenia against cancer.
D1R−/− mice exhibit normal coordination and locomotion, although they displayed significantly decreased behavior.28D1R−/− mice are growth retarded and die shortly after weaning age.28 The distribution of peripheral D1R was exhibited in blood vessels, kidney and adrenal gland.29 However, the precise roles of D1R are not really elucidated in peripheral organs. In neural cells, receptors coupled to the Gs family of G proteins, such as D1R, are characterized by their abilities to trigger adenyl cyclase-mediated cAMP formation.30 Activation of Gs-coupled D1R in SK-N-MC human neuroblastoma cells increased JNK activity in a cAMP and PKA-dependent manner.31 There was a report that Gs-linked receptors are also capable of stimulating this kinase via an alternative pathway, in which Gβγ subunits serve as the primary players in the signal transduction. In COS-7 transfected with D1R, the Gβγ subunits released from Gs and Gi cooperated, using a Gβγ/Src-dependent pathway to mediate the JNK activation. On the other hand, D1R signaling suppressed the gastrin-releasing peptide-preferring bombesin receptor (GRPR) and mediated JNK activation by down-regulating D1R signaling the cAMP-dependent protein kinase in the phospholipase C pathway.32 In HUVEC, the release of Gβγ subunits activated Src kinase proteins, which, in turn, transactivate protein kinase receptors.33, 34
Src, a proto-oncogene, has been strongly implicated in the growth, progression and metastasis of a number of human cancers.34 Activation of Src stimulates VEGF protein production from various types of cell lines, and Src cooperates with VEGF receptors (KDR/Flk-1) in endothelial cells, resulting in stimulation of endothelial proliferation.35 Thus, efforts to reduce the growth and spread of cancers have recently focused on inhibiting Src activity.36 Activated G protein via a neurotransmitter such as dopamine activates an Src family kinase.33 We also showed this pathway could contribute to a dopamine-induced signaling pathway to phosphorylate VEGF-R2 in endothelial cells. Then the tumor growth might be accelerated by angiogenesis which was induced by activation of Src through D1R signaling. Although dopamine stimulation through D2R reduced the surface expression of VEGFR-2,11 our data showed that D2R antagonist treatment did not influence tumor growth. There was a report that internalization of VEGF-R2 was mediated by a distinct mechanism involving PKC.37 Our results suggest that D2R stimulation with the concerned PKC led to the down regulation of VEGF-R2, but the down regulation of VEGF-R2 might discontinue later.
Our study showed that the stimulation of D1R might accelerate tumor angiogenesis in patients with solid tumors; therefore, peripheral D1R could be a molecular target for cancer therapy.
The authors thank Dr. T. Nagase (Tokyo University) for useful comments. They also thank Dr. S. Freeman for the English correction.