Autoregulatory effects of serotonin on proliferation and signaling pathways in lung and small intestine neuroendocrine tumor cell lines

Abstract

Background: : Survival rates for gastrointestinal (GI) and bronchopulmonary (BP) neuroendocrine tumors (NETs) have not altered significantly (5-year survival rate: GI NETs, 64.1%; BP NETs, 87%-89%) in 30 years (from 1973 to 2004). No effective or specific antineoplastic agents are available to date, although somatostatin analogs inhibit NET 5-hydroxytryptophan (5-HT) secretion. Given the expression of 5-HT receptors on NETs, the authors hypothesized that 5-HT autoregulated NET proliferation.

Methods: : Proliferation was evaluated in 3 NET cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake; in addition, real-time polymerase chain reaction analyses and enzyme-linked immunosorbent assay 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 (5-HT receptor subtypes 2A and 2C [5-HT(2A/2C)]); ondansetron (5-HT(3)); and the suicide inhibitor of the rate-limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (7-HTP) were investigated.

Results: : Exogenously added 5-HT stimulated proliferation in atypical BP NET NCI-H720 cells (+50%; half-maximal stimulatory concentration [EC(50)] = 10 nM), in typical BP NET NCI-H727 cells (+40%; EC(50) = 0.01 nM), and in GI NET KRJ-I cells (+60%; EC(50) = 25 nM). In NCI-H720 cells, proliferation was inhibited by ketanserin (half-maximal inhibitory concentration [IC(50)] = 0.06 nM) and ondansetron (IC(50) = 0.4 nM) and also was inhibited by 7-HTP (IC(50) = 0.3 nM). In NCI-H727 cells, ketanserin and 7-HTP inhibited proliferation (IC(50) = 0.3 nM and IC(50) = 2.3 nM, respectively), whereas ondansetron had no effect. In KRJ-I cells, ketanserin (IC(50) = 0.1 nM) and 7-HTP (IC(50) = 0.6 nM), but not ondansetron, inhibited proliferation. In all cell lines, 5-HT activated proliferation through extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation and c-Jun N-terminal kinase (JNK)-mediated pathways (c-JUN and Ki-67 transcription). An autoregulatory effect was indicated by the 7-HTP-mediated inhibition of extracellular 5-HT and downstream effects on NET proliferation.

Conclusions: : Lung and GI NET proliferation was autoregulated by 5-HT through alterations in ERK and JNK signaling. Targeting NET cells with 5-HT(2) receptor antagonists and 7-HTP reversed proliferation. The current results indicated that 5-HT(2) receptor subtype-specific antagonists may represent a viable antiproliferative therapeutic strategy. Cancer 2009. (c) 2009 American Cancer Society.

Figure 1. 5-HT receptor profiles in NCI-H720 (atypical BP carcinoid), NCI-H727 (typical BP carcinoid), and KRJ-I (SI NET) measured by real-time PCR

NCI-H720 was positive for 5- HT_2A/C and 5HT_3A (1A–C). NCI-H727 and KRJ-I exhibited only the 5-HT_2C receptor (1B, 1C). Mean±SEM, n=3.

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-HT_2/3 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-HT_2C 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.

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; NCI-H727: 2.3nM, 3E; KRJ-I: 0.6nM, 3F). Addition of 5-HT (100nM) reversed 7-TPH inhibition of proliferation. Mean±SEM, n=4–6.

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.

Figure 5. Real-time PCR analysis of the effect of 5-HT, ketanserin, 7-HTP and PD98059 on c-JUN 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 5-HT. 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 c-JUN 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.