NIH Public Access Author Manuscript Cancer. Author manuscript; available in PMC 2010 November 1. NIH-PA Author Manuscript Published in final edited form as: Cancer. 2009 November 1; 115(21): 4934–4945. doi:10.1002/cncr.24533. Auto-regulatory Effects of Serotonin on Proliferation and Signaling Pathways In Lung and Small Intestine Neuroendocrine Tumor Cell Lines Ignat Drozdov*, Mark Kidd*, Bjorn I Gustafsson*, B. Svejda*,#, Richard Joseph*, Roswitha Pfragner#, and Irvin M Modlin* * Gastrointestinal Pathobiology Research Group, Yale University School of Medicine New Haven, Connecticut 06520-8062 # Institute of Pathophysiology and Immunology, Centre for Molecular Medicine, Medical University of Graz, Austria NIH-PA Author Manuscript Abstract Introduction—Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not significantly altered (5yr survival: 64.1% and 87–89%) in thirty years (1973–2004). No effective or specific anti-neoplastic agent(s) is available although somatostatin analogs inhibit NET serotonin (5-HT) secretion. Given the expression of 5-HT receptors on NETs, we hypothesized that 5-HT autoregulated NET proliferation. Methods—Proliferation was evaluated in three NET cell lines using MTT uptake while real-time PCR and ELISA studies were performed to delineate 5-HT-mediated signaling pathways. To determine the receptor and role of endogenous 5-HT production, the effects of ketanserin (5HT2A/C receptor antagonist), ondansetron (5-HT3 antagonist) and the suicide inhibitor 7Hydroxytryptophan (7-HTP) were investigated. NIH-PA Author Manuscript Results—Exogenously added 5-HT stimulated proliferation in the atypical BP-NET, NCI-H720 (+50%, EC50=10nM), the typical BP-NET, NCI-H727 (+40%, EC50=0.01nM), and the GI-NET, KRJ-I (+60%, EC50=25nM). In NCI-H720, proliferation was inhibited by ketanserin (IC50=0.06nM) and ondansetron (IC50=0.4nM) as well as 7-HTP (IC50=0.3nM). In NCI-H727, ketanserin and 7-HTP inhibited proliferation (IC50=0.3nM and 2.3nM respectively) while ondansetron had no effect. In KRJ-I, ketanserin (IC50=0.1nM) and 7-HTP (IC50=0.6nM) but not ondansetron inhibited proliferation. In all cell lines, 5-HT activated proliferation through ERK1/2 phosphorylation and JNK-mediated pathways (c-JUN and Ki67 transcription). An auto-regulatory effect was indicated by 7-HTP-mediated inhibition of extracellular 5-HT and downstream effects on NET proliferation. Conclusions—Lung and GI-NET proliferation is autoregulated by 5-HT through alterations in ERK and JNK signaling. Targeting NET cells with 5-HT2 receptor antagonists and 7-HTP reversed proliferation. 5-HT2 receptor subtype-specific antagonists may represent a viable antiproliferative therapeutic strategy. Keywords bronchopulmonary; carcinoid; gastrointestinal; serotonin; ERK; H720; H727; KRJ-I; neuroendocrine tumor; proliferation Correspondence address to: Irvin M. Modlin, M.D. Ph.D, D.Sc, F.R.C.S. (Eng & Ed), Yale University School of Medicine, 333 Cedar Street, P.O. Box 208062, New Haven, Connecticut 06520-8062, Tel: (203) 785-5429, Fax: (203) 737-4067, imodlin@optonline.net. Drozdov et al. Page 2 Introduction NIH-PA Author Manuscript Neuroendocrine tumors (NETs) – previously termed “carcinoids” – account for 0.66% of all malignancies. They are generally considered “slow-growing” and of minor oncological importance but their incidence has increased 3–10% per year over the last 30 years 1–3 and the majority of gastrointestinal (GI) tract lesions are non-localized 1–3. They are predominantly found in the GI tract (66%: the majority of which - ~60% - are small intestinal NETs) and bronchopulmonary (BP) system (31%) 1–3. It is now recognized that the highly specialized serotonin (5-HT)-producing EC cells and 5-HT-secreting pulmonary neuroendocrine (PNE) cells are the progenitor cells of the majority of GI and BP-NETs 2, 4. The role of 5-HT in the manifestation of “carcinoid syndrome” (cutaneous flushing, bronchospasm, and diarrhea) is well-known 5, while both 5-HT-mediated flushing and diarrhea and nausea associated with cancer therapy can be inhibited by the 5-HT2 receptor antagonist ketanserin 6 and the 5-HT3 receptor antagonist, ondansetron, respectively 7, 8. Additionally, somatostatin analogs have become an important part of the treatment of carcinoid syndrome 9. In general, these agents reduce urine 5-hydroxyindole acetic acid (5HIAA) by ~25–50% 10 suggesting inhibition of 5-HT secretion. NIH-PA Author Manuscript Apart from its role in flushing, nausea and diarrhea, 5-HT has mitogenic effects in fibroblasts 11, smooth muscle cells12, osteoblasts 13, mesangial cells 14, and endothelial cells 15. While the exact mechanism of mitogenic 5-HT action is unknown, it has been shown that some subtypes (5-HT1B, 5-HT2A) of 5-HT receptors can directly facilitate activation of the extracellular signal-regulated kinase (ERK) pathway 16–18 and JNK activation. A role for 5-HT in mediating NET proliferation has not previously been studied but is suggested by the recently published PROMID phase III study which demonstrated that tumor growth and the development of hepatic metastases could be inhibited by Sandostatin LAR ® 19. As this agent is a known inhibitor of 5-HT release but is not recognized as a direct anti-proliferative agent, there is a need to delineate whether 5-HT secreted from tumors is a proliferative regulator as well as the mitogenic mechanisms regulating aminemediated proliferation in this tumor type. We analyzed the proliferative effects of 5-HT on three human NET (carcinoid) cell lines (atypical BP NET: NCI-H720, a typical BP NET: NCI-H727, and the small intestinal NET: KRJ-I 20, 21) to investigate whether NET cells auto-regulate proliferation through 5-HT production and secretion. In addition, we examined the effect of ketanserin (5-HT2A/c receptor antagonist), ondansetron (5-HT3 receptor antagonist), 7-HTP (suicide inhibitor of the rate-limiting enzyme for 5-HT synthesis, tryptophan hydroxylase: Tph-1) and PD98059 (MAPK pathway inhibitor) on the mechanisms of proliferation and cellular transcription. NIH-PA Author Manuscript Materials and Methods Culture conditions KRJ-I and NCI-H720 cells were cultured as floating aggregates at 37°C with 5% CO2. KRJI cells were kept in Ham’s F12 medium (Gibco™) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich) 21, 22. For NCI-H720, a 1:1 solution of Ham’s F12 and Dulbecco’s minimal essential medium (DMES) supplemented with final concentrations of FBS (5%), insulin (0.005mg/ml), transferrin (0.01 mg/ml), sodium selenite (30nM), hydrocortisone (10nM), β-estradiol (10nM), HEPES medium (10mM) and L-glutamine (2mM) 20. The adhesive growing NCI-H727 cells were kept at 37°C in RPMI 1640 medium containing final concentrations of FBS (10%), L-glutamine (2mM), sodium pyruvate (1mM) and glucose (2.5g/l) 20. For further processing, NCI-H727 cells were washed in PBS before TrypsinEDTA was applied. Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 3 Proliferation studies NIH-PA Author Manuscript After being spun down (5 mins – 1,500g), the pellets of the three cell lines were resuspended in each medium to 5×105 cells/ml and seeded in 96 well plates at 100μl at 5×104 cells/well (2 plates/experimental condition) 22. Lane 1 and 2 contained negative (media only) and positive (pure cell suspension) controls. 5-HT, ketanserin, and 7-HTP were diluted in respective cell media and applied in final concentrations from 10−12 to 10−6. Chemicals 5-HT, ketanserin (5-HT2A/C antagonist), ondansetron (5-HT3 antagonist) and PD98059 were purchased from Sigma-Aldrich and 7-HTP (suicide inhibitor) was obtained from SYNCHEM Laborgemeinschaft (Kassel, Germany). MTT assay After 72 hrs of incubation (37°C; 5% CO2), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) was added to cell lines with a final concentration of 0.5mg/ml per well followed by additional incubation for 3 hrs 23. The reaction was stopped and the formazan dye solubilized by adding an equal volume (100μl) of acid-isopropanol (0.01N HCl in isopropanol). The optical density was read at 595nm (Microplate reader: Bio-Rad 3500) 22. NIH-PA Author Manuscript ELISA (CASE) Assay The effects of 5-HT, ketanserin, 7-HTP and PD98059 on the ERK1/2 signaling pathway were measured in NET cells as described 22. Cells were stimulated with 5-HT or 7-HTP or combinations of 5-HT and ketanserin, 7-HTP and PD98059 for 30 mins. EC50 or IC50 concentrations were used except for PD98059 where 0.1μM was used. ERK1/2 phosphorylation was measured using an ELISA (SuperArray CASE™) per the manufacturer’s protocol. Briefly, stimulated cells were fixed (4% formaldehyde), and stained with either primary antibodies against the non-phosphorylated (α-ERK (P42-44/ MAPK polyclonal antibody) or phosphorylated forms (α-P-ERK: phospho-P44/42 MAPK targeting Thr202/Tyr204: E10 monoclonal antibody) of each protein (60 min, RT). After washing and secondary antibody application (60 min, RT), cells were incubated with color developer (10 min, RT) and plates read at 450 nm. Thereafter, protein was assayed in each well (protein development – reading at 595nm). Results were calculated as antibody reading (at 450nm)/protein concentration (measured at 595nm) and normalized to unstimulated cells. Phosphorylated signal was compared to total non-phosphorylated signal. Real-Time PCR NIH-PA Author Manuscript RNA was extracted from 2×106 NCI-H720, NCI-H727, andKRJ-I cells in log phase growth (n=3) (TRIZOL®, Invitrogen, USA) and cleaned (Qiagen RNeasy kit and DNeasy Tissue kit, Qiagen Inc., USA) to minimize contaminating genomic DNA. RNA (2μg) was converted to cDNA (High Capacity cDNA Archive Kit, Applied Biosystems) 24, 25. Real time RT-PCR analysis was performed using Assays-on-Demand™ products and the ABI 7900 Sequence Detection System according to the manufacturer’s suggestions24, 25. Cycling was performed under standard conditions (TaqMan® Universal PCR Master Mix Protocol) and data normalized using geNorm 26 and NET house-keeping gene, ALG9, TFCP2 and ZNF41025, expression. 1. Receptor profile—Transcript levels of 5-HT2A/C and 5-HT3 receptors were measured in RNA from each cell line 25. Transcript levels <0.1 geNorm-normalized were considered absent. Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 4 NIH-PA Author Manuscript 2. Cell cycle markers—c-JUN and Ki67 transcriptions were measured in response to 5HT or 7-HTP or combinations of 5-HT and ketanserin, 7-HTP and PD98059. EC50 or IC50 concentrations were used except for PD98059 (0.1μM). Cells were cultured for 24 hrs prior to RNA extraction. RNA was isolated using the standard PCR protocol 24, 25. Extracellular 5-HT content 100μl of NCI-H720, NCI-H727, and KRJ-I cells (2×104 cells/100μl) were seeded into 96well plates and an ELISA assay used to measure 5-HT released into media from untreated cells after incubation period of 0 hrs, 24 hrs and 48 hrs as per manufacturer’s instructions (Serotonin EIA, Rocky Mountain Diagnostics). Similar measurements were performed after 24 hrs and 48 hrs following the administration of 7-HTP (IC50 concentration). Statistical Evaluation All statistical analyses were performed using Prism 4 (GraphPad Software, San Diego, CA). Sigmoidal dose responses and non-linear regression analyses were calculated to identify half-maximal stimulatory (EC50) and inhibitory (IC50) concentrations for each agent. Alterations in signal transduction and transcriptional activation were assessed using 2-tailed, paired t-tests. NIH-PA Author Manuscript Results 1. Receptor profile by PCR Initially, we used a real-time PCR approach to analyze 5-HT receptor subtypes in each of the NET cell lines. The NCI-H720 cell line was derived from an “atypical” BP NET 20; transcripts for 5-HT receptors 2A, 2C, and 3A were identified (Figure 1A). NCI-H727 is derived from a “typical” BP NET 20 and expresses transcripts for the 5-HT2C receptor (Figure 1B). KRJ-I is derived from a “typical” small intestinal NET 21; the receptor profile in this cell line is 5-HT2C (Figure 1C). 2. Proliferative effects of 5-HT NIH-PA Author Manuscript Having identified that NET cell lines expressed 5-HT receptors, we next evaluated whether exogenously added 5-HT stimulated proliferation and if this could be inhibited by selective 5-HT receptor antagonists. 5-HT induced proliferation in a concentration-dependent manner (+50%, EC50=10 nM) in NCI-H720 cells (Figure 2A). This 5-HT-mediated effect was reversed by ketanserin (IC50=0.06nM) and ondansetron (IC50=0.4nM) while each of these agents also inhibited basal (unstimulated proliferation: ketanserin: −30%, IC50=0.1nM; ondansetron: −10%, IC50=0.01 nM – Figure 2D). These results demonstrate that 5-HT stimulates proliferation of this atypical lung cell line through activation of both 5-HT2 and 5-HT3 receptors. In the typical lung cell line (NCI-H727), 5-HT induced proliferation with an estimated EC50=0.01nM (Figure 2B). This effect was reversed by ketanserin (IC50=0.3nM) which also inhibited basal (unstimulated) proliferation (−15%, IC50=0.50nM – Figure 2E). Ondansetron had no measurable effect on either 5-HT-stimulated proliferation or on basal proliferation consistent with the absence of a 5-HT3 receptor on this cell line (Figure 2B, Table 1). In KRJ-I cells, proliferation was activated by 5-HT (+40%, EC50=25nM) (Figure 2C). Inhibitory effects on both 5-HT stimulated proliferation as well as to basal proliferation were observed in response to ketanserin (IC50=0.1nM and 0.3nM, respectively (Figures 2C, 2F). Ondansetron had little measurable effect consistent with the 5-HT receptor profile on this small intestinal cell (Figure 2C, Table 1). Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 5 3. Effects of 7-HTP on NET cells NIH-PA Author Manuscript After demonstrating a role for exogenous 5-HT in the regulation of NET cell proliferation, we next evaluated whether inhibition of endogenous 5-HT production and secretion with 7HTP inhibited NET proliferation. In initial studies, we evaluated the effect of 7-HTP on 5HT secretion. In NCI-H720 cells, the mean extracellular levels of 5-HT in untreated cells measured at 0, 24, and 48 hrs were 11.3ng/ml, 43.9ng/ml, and 36.1ng/ml respectively. Administration of 7HTP significantly inhibited 5-HT secretion to concentrations of 23.6ng/ml (24 hrs, p<0.05) and 18.5ng/ml (48 hrs, p<0.05) respectively (Figure 3A). In NCI-727 cells, the extracellular 5-HT content remained elevated and constant (133ng/ml) after 24 and 48 hrs compared to initial reading (66.7ng/ml, p<0.01). Stimulation of these cells with 7-HTP resulted in inhibition of 5-HT secretion after 24 and 48 hrs (61.3ng/ml and 61.1ng/ml respectively, p<0.05) (Figure 3B). In KRJ-I cells, the extracellular 5-HT content increased in a timedependant manner; 173.9ng/ml, 278.2ng/ml, and 318.1ng/ml concentrations were observed at 0 hrs, 24 hrs, and 48 hrs. At 24 hrs and 48 hrs, 7-HTP inhibited 5-HT release to 117.5ng/ ml (p<0.01) and 191.1ng/ml (p<0.05) (Figure 3C). These results demonstrate that 7-HTP effectively inhibited NET cell 5-HT secretion. NIH-PA Author Manuscript We next evaluated whether this decrease in secreted 5-HT was associated with a decrease in cell proliferation. When NCI-H720 cells were incubated with 7-HTP, inhibition of basal proliferation was noted at low concentrations of this substrate (−20%, IC50=0.3nM) (Figure 3D, Table 1). In NCI-H727 cells, basal proliferation was reversed by 7-HTP (−20%, IC50=2.3nM, Figure 3E) while in KRJ-I cells, the inhibitory effect was observed to be ~ −20%, IC50=0.6nM (Figure 3F). In the presence of exogenously added excess 5-HT (1μM), 7-HTP had no inhibitory effect on any cell line proliferation (Figure 3D–F). MTT uptake in these cells were not different to untreated cells demonstrating that the inhibitory effect of 7HTP was not due to a direct cytotoxic effect on the cells but reflected inhibition of 5-HT production. 4. Effects of 5-HT, ketanserin, and 7-HTP on ERK1/2 phosphorylation NIH-PA Author Manuscript After we demonstrated a role for 5-HT in mediating NET cell proliferation, we next investigated whether the MAPK pathway was involved in amine-mediated signal transduction as this pathway is a well-characterized growth factor regulated pathway in NET cells 27, 28. As we were interested in pathways common to all three cell types, we focused on the 5-HT2C receptor and the effects of 7-HTP on ERK signaling. For these studies, we used an ELISA assay to quantitate the effects of 5-HT (100nM), 5-HT+ketanserin (100nM), 7-HTP (100nM), 7-HTP+5-HT (both 100nM) as well as PD98059+5-HT (both 100nM) on ERK1/2 phosphorylation. In NCI-H720, 5-HT phosphorylated ERK 1/2 (48.8%, p<0.01 vs. control). This effect was reduced to 22.1% (p<0.01 vs. control) when cells were treated with ketanserin followed by 5-HT (5-HT+ketanserin). Inhibition of ERK phosphorylation was observed in response to 7HTP (−22.6%, p<0.01 vs. control) an effect that was reversed in the presence of exogenously added 5-HT. The ERK inhibitor, PD98059, also significantly reduced 5-HTmediated ERK phosphorylation to 10% confirming amine-mediated growth signaling occurred through the MAPK pathway (Figure 4A, Table 1). In NCI-H727 cells, 5-HT increased ERK1/2 phosphorylation (+3%, p=0.07) while 5-HT+ketanserin inhibited ERK1/2 phosphorylation, −7% (p<0.01 vs. control). Similarly, in response to 7-HTP, phosphorylation of ERK1/2 was decreased (−17%, p<0.01 vs. control), an effect not observed after 5-HT was added to cells. As for NCI-H720 cells, PD98059 inhibited 5-HTmediated ERK phosphorylation (Figure 4B, Table 1). In KRJ-1 cells, 5-HT induced Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 6 NIH-PA Author Manuscript phosphorylation of ERK1/2 (31.4%, p<0.01 vs. control). 5-HT+ketanserin decreased phosphorylated ERK1/2 levels to 23.6% (p<0.01 vs. control), while treatment of this cell line with 7-HTP inhibited ERK1/2 phosphorylation by −10% (p<0.01 vs. control) (Figure 4C, Table 1). The latter effect was reversed when exogenous 5-HT was added. In contrast, PD98059 inhibited 5-HT mediated phosphorylation. 5. Effects of 5-HT, ketanserin, and 7-HTP on downstream transcriptional targets Amine-mediated signal transduction in each of the cell lines is mediated via the 5-HT2 receptor through ERK1/2 phosphorylation. As signaling through this pathway is associated with activation of c-Jun and Ki-67 transcripts (markers of cell proliferation) 27, 28, we next evaluated whether 5-HT stimulated transcription of these ERK targets in each of the cell lines. NIH-PA Author Manuscript NIH-PA Author Manuscript In NCI-H720 cells, real-time PCR analysis (5-HT: 100nM, for 24 hr) demonstrated that cJUN transcript levels were significantly increased (72%) compared to unstimulated cells (p<0.01) (Figure 5A, Table 1). Ki67 levels were noted to be elevated 13.4% compared to control (p<0.01 vs. control) (Figure 5B, Table 1), which is consistent with the observation that 5-HT increased NCI-H720 cell proliferation. In 5-HT-stimulated cells co-incubated with ketanserin, c-JUN and Ki67 transcript levels were decreased to 16.6% and 3% respectively (p<0.01 vs. 5-HT alone), confirming that this compound inhibited 5-HT-mediated NCIH720 cell proliferation. A similar observation was noted when 7-HTP decreased c-JUN and Ki67 transcript levels by −74% and −95% (p<0.01 vs. control). This effect was reversed in the presence of excess exogenous 5-HT. Addition of PD98059 was noted to inhibit 5-HT mediated c-JUN and Ki67 transcripts confirming ERK signaling coupled 5-HT2 receptor activation and JNK transcription. In NCI-H727 cells, 5-HT increased c-JUN transcript (3.2%, p=0.06 vs. control) (Figure 5C, Table 1), while Ki67 transcription was elevated 17.9% compared to control (p<0.01) (Figure 5D, Table 1) indicating that the proliferative effect of this amine principally occurred through activation of Ki67 transcription. In the presence of exogenous 5-HT, ketanserin had no effect on c-JUN transcript levels, while Ki67 transcript levels were decreased (2.4%, p<0.05). 7-HTP inhibited c-JUN and Ki67 transcripts by −91% and −57% respectively, consistent with its anti-proliferative effects but no effect was identified after exogenous 5-HT was applied to the cells (Table 1). PD98059 reversed 5-HT mediated transcription. In the KRJ-I cell line, c-JUN and Ki67 transcript levels in cells treated with 5-HT demonstrated upregulation of 57.7% and 33.5% respectively (p<0.01 vs. control) (Figure 5E, Table 1). Ketanserin inhibited 5-HT-stimulated c-JUN transcription (2.1%, p<0.05), and also inhibited Ki67 (−23.2%, p<0.01) (Figure 5F, Table 1). 7-HTP inhibited c-JUN and Ki67 transcripts −26% and −61.5% respectively (p<0.01 vs. control), effects reversed by exogenous 5-HT. As for NCI-H720 and H727 cells, PD98059 reversed 5-HT stimulated JNK transcription. Discussion This study demonstrates that 5-HT plays an autocrine proliferative role in BP and GI 5-HTsecreting neoplastic neuroendocrine cell lines through phosphorylation of ERK1/2 and activation of the JNK pathway. Targeting these NET cells with 5-HT receptor subtypespecific antagonists and 7-HTP reversed proliferation through inhibition of 5-HT synthesis, release and 5-HT2 receptor activation. A real-time PCR assessment of the 5-HT receptor subtypes (5HT2A/C, 5HT3A) in NET cell lines identified that 5-HT2C was commonly expressed on both BP and GI NET cell lines. In addition, the atypical BP-NET, NCI-H720, also expressed transcripts for 5-HT2A and 5HT3. In this study, the mitogenic effects of 5-HT in NET cell lines was concentrationdependant with EC50’s in the nM range. These ranges are similar to those identified in 5-HT Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 7 NIH-PA Author Manuscript secreting prostate cancer cell line (PC3) (E50=10 nM 29) but significantly lower than in the 5-HT2A expressing MCF-7 breast cancer cell line (10 mM) 30. Of note is that the endogenous 5-HT concentration in the cell culture media was low (<0.1 nM). Secretion of this amine over the experimental period resulted in concentrations as high as ~0.1–1μM to which the cells were exposed. Decreasing secreted 5-HT by addition of 7-TPH (usually by 50–70%) inhibited NET proliferation. This effect was reversed by adding 5-HT (10−7M: ~20ng/ml) indicating that 5-HT was required to autoregulate proliferation. These reductions in 5-HT are similar to those caused by somatostatin analogs 10 and suggests one mechanism by which these agents inhibit NET growth. It is also of interest to note that 5-HT concentrations in normal blood range between 10−7 – 10−6M and can be 15–20x higher in patients with NETs 31. In addition, tissue levels of 5-HT, particularly in carcinoid tumors are at similar levels 32 suggesting that systemic or local 5-HT concentrations are sufficient to regulate NET growth while decreasing them may reverse proliferation. NIH-PA Author Manuscript The proliferative effects of 5-HT on NET cells was inhibited by the 5-HT2A/C antagonist ketanserin in all cell lines and the 5-HT3 antagonist, ondansetron, in NCI-H720. These findings are consistent with the 5-HT receptor profile in these cell lines. Additionally, although ondansetron was a potent inhibitor in all NET cell lines, NCI-H720 was ~10x more sensitive to ketanserin. This is consistent with observations that 5-HT2C receptors have the highest affinity for 5-HT but also suggests that targeting multiple 5-HT receptor subtypes (when expressed) may be an effective strategy for inhibiting NET proliferation at different sites. To investigate the subcellular mechanisms that regulate the mitogenic effects of 5-HT in NET cell lines, we measured phosphorylation of ERK and performed real-time PCR to assess the effects of this amine on transcript levels of c-JUN and the proliferation marker Ki67. Previous studies have shown that Ras-dependent ERK activation as well as c-JUN transcriptional regulation is involved in NET cell proliferation 27, 28 and is a common feature of neoplastic cell proliferation pathways 33–35. Additionally, recent studies have demonstrated that 5-HT receptor subtypes induce cellular proliferation through direct coupling with ERK 17. Coupling of 5-HT2C receptors to ERK activation has been reported in studies utilizing Chinese hamster ovary fibroblasts (CHO) 36. The 5-HT3 receptor, a ligand-gated ion channel, has not been previously shown to couple to ERK; it appears, however, that this 5-HT receptor subtype may be involved in neuroprotective pathways 17, 37 through inhibition of cytosolic Ca2+ concentration and caspase-3 activity – an indicator of apoptosis 38. NIH-PA Author Manuscript In the current study, NCI-H720 and KRJ-I cell line proliferation was induced by 5-HT through the phosphorylation of ERK1/2, followed by the activation of c-JUN transcription, and upregulation of Ki67, a cyclin marker of cellular proliferation. Consistent with its antiproliferative effects, ketanserin inhibited transcription of c-JUN and Ki67 as well as phosphorylation of ERK suggesting that the 5-HT2C receptor may be activated in the presence of basally released 5-HT. These observations suggest that 5-HT2C receptor subtypes are coupled to the ERK-JNK pathway in NET cell lines. It is, however, of note that although 5-HT induced proliferation in the typical BP cell line NCI-H727 (Ki67 transcript was elevated), it did not significantly phosphorylate ERK and no effect on c-JUN transcription was noted. However, ketanserin reversed 5-HT-mediated proliferation largely through inhibition of Ki67 transcription suggesting that the 5-HT2C receptor in this cell line is coupled to proliferation; the exact signaling pathway remains to be defined. To investigate whether newly synthesized 5-HT was involved in regulation of NET cell proliferation, 7-hydroxytryptophan (7-HTP) was used to inhibit the biosynthesis of 5-HT. 7HTP is a competitive inhibitor of Tph-1, the rate-limiting enzyme in the biosynthesis of 5- Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 8 NIH-PA Author Manuscript HT, and is converted by Tph-1 and the aromatic amino acid decarboxylase (AAAD) into 5,7-dihydroxytryptamine (5,7-DHT) 39,40. Addition of 7-HTP significantly inhibited 5-HT levels in each of the cell lines >50–70% which was associated with a decrease in proliferation through inhibition of ERK1/2 phosphorylation and down-regulation of c-Jun and Ki67 transcription. These effects could be reversed by addition of exogenous 5-HT (~0.1μM). We interpret these data to suggest an autocrine role for 5-HT in NET cell proliferation. Of note was that 7-HTP was a more potent inhibitor of the downstream targets than ketanserin in all cell lines. This suggests that an aspect of the inhibitory effects of this agent may be consistent with cytotoxicity demonstrated in earlier studies 40. In conclusion, our investigations demonstrate that lung and gastrointestinal NET cell proliferation is auto-regulated by endogenously produced 5-HT. These amine-mediated proliferative effects are associated with ERK1/2 and JNK signaling through c-JUN and Ki67 expression and can be inhibited by 5-HT2/3-receptor targeting and through inhibition of 5HT synthesis. Given the relationship between somatostatin-mediated inhibition of 5-HT secretion and the reduction in NET growth in a recent study 19, our observations suggest that the 5-HT receptor pathway plays a role in regulating tumor growth and may provide one explanation for these observations. 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NCI-H727 and KRJ-I exhibited only the 5-HT2C receptor (1B, 1C). Mean±SEM, n=3. NIH-PA Author Manuscript NIH-PA Author Manuscript Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 12 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 2. Effect of 5-HT and specific antagonists on NET proliferation 5-HT stimulated proliferation in all cell lines (NCI-H720: EC50=10nM; NCI-H727: EC50=0.01nM; KRJ-I: EC50=25nM) (2A–C). In the 5-HT2/3 expressing NCI-H720 cell line, 5-HT stimulated proliferation was inhibited by ketanserin (IC50=0.06nM) and ondansetron (IC50=0.4nM) (2A) while these agents also inhibited basal proliferation (IC50=0.1nM and 0.01nM respectively, 2D). In the 5-HT2 expressing NCI-H727 cell line, ketanserin inhibited both 5-HT stimulated (IC50=0.3nM, 2B) and basal proliferation (IC50=0.50nM, 2E) while ondansetron had no effect on either parameter. In the 5-HT2C expressing KRJ-I cell, ketanserin inhibited both 5-HT stimulated (IC50=0.1nM, 2C) and basal proliferation (IC50=0.3nM, 2E) while ondansetron had no effect consistent with the receptor profile of this cell. Mean±SEM, n=6. ○: 5-HT alone, ▲: 5HT+ketanserin, ▼: 5-HT+ondansetron, △: ketanserin alone, ▽: ondansetron alone. NIH-PA Author Manuscript Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 13 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 3. Effect of 7-HTP on extracellular 5-HT content and proliferation Extracellular concentration of 5-HT was measured in cell media alone and in media with cultured cells. Media 5-HT ranged between 0.7ng/ml and 7.3ng/ml. Concentration of 5-HT (ng/ml) increased over time (24 hrs, 48 hrs) in all NET cell lines. 5-HT secretion was significantly inhibited by 7-HTP (3A–C). Mean±SEM, n=4, *p<0.05, **p<0.001. In each cell line, only basal proliferation was inhibited by 7-HTP (NCI-H720: 0.3nM, 3D; NCIH727: 2.3nM, 3E; KRJ-I: 0.6nM, 3F). Addition of 5-HT (100nM) reversed 7-TPH inhibition of proliferation. Mean±SEM, n=4–6. NIH-PA Author Manuscript Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 14 NIH-PA Author Manuscript Figure 4. Effects of 5-HT, ketanserin, 7-HTP and PD98059 on ERK1/2 phosphorylation 5-HT induced phosphorylation of ERK in NCI-H720, NCI-H727 and KRJ-I cell lines (4A– C), effects that were inhibited by Ketanserin. 7-HTP alone reduced basal ERK phosphorylation in all cell lines and inhibited 5-HT-mediated ERK phosphorylation (4A–C). PD98059 also inhibited 5-HT mediated phosphorylation. Mean ±SEM, n=4, *p<0.01 vs. basal, #p<0.05 vs. 5-HT, **p<0.05 vs. 7-HTP alone. NIH-PA Author Manuscript NIH-PA Author Manuscript Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 15 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 5. Real-time PCR analysis of the effect of 5-HT, ketanserin, 7-HTP and PD98059 on cJUN and Ki67 transcripts In NCI-H720, 5-HT activated c-JUN and Ki67 transcription (5A, 5B), which were inhibited by ketanserin and PD98059. 7-TPH alone inhibited transcription, an effect reversed by 5HT. In NCI-H727, 5-HT had no effect on c-JUN transcription but Ki67 transcription was activated (5C, 5D). Ketanserin activated transcription of c-Jun but inhibited Ki67 in this cell line. Transcripts were inhibited by 7-HTP and PD98059. 5-HT activated transcription of cJUN and Ki67 in KRJ-I (5E, 5F), effects reversed by ketanserin and PD98059. 7-TPH alone inhibited transcription, an effect reversed by 5-HT. Mean±SEM, n=4, *p<0.01 vs. basal, #p<0.05 vs. 5-HT, **p<0.05 vs. 7-HTP alone. Cancer. Author manuscript; available in PMC 2010 November 1. Drozdov et al. Page 16 Table 1 Effects of 5-HT on NET cell line proliferation and gene transcription NIH-PA Author Manuscript H720 H727 KRJ-I 5-HT2A/2C/3A 5-HT2C 5-HT2C 5-HT EC50=10nM EC50=0.01nM EC50=25nM 5-HT+Ketanserin IC50=0.06nM IC50=0.3nM IC50=0.1nM 5-HT+Ondansetron IC50=0.4nM - - 7-HTP IC50=0.3nM IC50=2.3nM IC50=0.6nM ** ** ** 5-HT ↑ pERK - ↑ pERK 5-HT+Ketanserin ↓ pERK ↓ pERK ↓ pERK 7-HTP ↓ pERK ↓ pERK ↓ pERK ** ** ** ↓ pERK ↓ pERK ↓ pERK 5-HT ↑ Ki67 ↑ Ki67 ↑ Ki67 5-HT+Ketanserin ↓ Ki67 ↑ Ki67 ↓ Ki67 7-HTP ↓ Ki67 ↓ Ki67 ↓ Ki67 - - - ↓ Ki67 ↓ Ki67 ↓ Ki67 Receptor Profile Proliferation 5-HT+7-HTP ERK Phosphorylation NIH-PA Author Manuscript 5-HT+7-HTP 5-HT+PD98059 Ki67 Transcription 5-HT+7-HTP 5-HT+PD98059 -= no effect, ↑ upregulated, ↓ downregulated, ** effect reversed NIH-PA Author Manuscript Cancer. Author manuscript; available in PMC 2010 November 1.