5-Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing β-catenin

Abstract

5-Hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived hepatocellular carcinoma (HCC) cells but the detailed intracellular mechanism is unknown. As Wnt/β-catenin signalling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β-catenin signalling by 5-HT. The expression of various 5-HT receptors was studied by quantitative real-time polymerase chain reaction (qPCR) in HCC cell lines as well as in 33 pairs of HCC tumours and corresponding adjacent non-tumour tissues. Receptors 5-HT1D (21/33, 63.6%), 5-HT2B (12/33, 36.4%) and 5-HT7 (15/33, 45.4%) were overexpressed whereas receptors 5-HT2A (17/33, 51.5%) and 5-HT5 (30/33, 90.1%) were reduced in HCC tumour tissues. In vitro data suggests 5-HT increased total β-catenin, active β-catenin and decreased phosphorylated β-catenin protein levels in serum deprived HuH-7 and HepG2 cells compared to control cells under serum free medium without 5-HT. Activation of Wnt/β-catenin signalling was evidenced by increased expression of β-catenin downstream target genes, Axin2, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS) by qPCR in serum-deprived HCC cell lines treated with 5-HT. Additionally, biochemical analysis revealed 5-HT disrupted Axin1/β-catenin interaction, a critical step in β-catenin phosphorylation. Increased Wnt/β-catenin activity was attenuated by antagonist of receptor 5-HT7 (SB-258719) in HCC cell lines and patient-derived primary tumour tissues in the presence of 5-HT. SB-258719 also reduced tumour growth in vivo. This study provides evidence of Wnt/β-catenin signalling activation by 5-HT and may represent a potential therapeutic target for hepatocarcinogenesis.

Keywords: 5-HT; SB-258719; Wnt/β-catenin signalling.

Figure 1

Expression of different 5‐HT receptors on HCC cell lines and tissues. qPCR analysis of 5‐HT receptor subtypes in (A) HCC cell lines and (B) 33 pairs of HCC tissues (T) and their corresponding adjacent non‐tumour tissues (NT). Receptor expression of each sample is presented by the fold ratio against MIHA or all NT tissues (*P < 0.05, **P < 0.01, ns denotes no significant difference, t‐test).

Figure 2

5‐HT can promote proliferation of serum‐deprived HuH‐7 and HepG2 cells and upregulate β‐catenin. (A) The relative viability of HuH‐7 and HepG2 cells cultured in 10% FBS, or SFM with or without 5‐HT (50 μM and 100 μM 5‐HT) was detected by MTT. 5‐HT increased proliferation of serum‐deprived HuH‐7 and HepG2 cells (SFM vs SFM + 5‐HT, ***P < 0.001, two‐way ANOVA). (B) HuH‐7 and HepG2 cells were cultured in SFM for 48 h followed by addition of 5‐HT for indicated time points. 5‐HT increased total β‐catenin, active β‐catenin and decreased p‐β‐catenin protein levels. Axin1, total GSK3β and p‐GSK3β protein levels remain unchanged. (C) Relative mRNA expression of β‐catenin downstream targets was assessed by qPCR. 5‐HT increased mRNA levels of Axin2, cyclin D1, DKK1, and GS in HuH‐7 and HepG2 cells (*P < 0.05, **P < 0.01, t‐test). (D) 5‐HT increased cytoplasmic β‐catenin protein levels in serum‐deprived HuH‐7 and HepG2 cells.

Figure 3

5‐HT enhances β‐catenin protein level via decreased degradation and disrupts Axin1/β‐catenin interaction. (A) Serum‐deprived HuH‐7 cells and HepG2 cells were first cultured with 5‐HT for 2 h followed by 100 μM CHX treatment for the indicated time points. β‐Catenin levels decreased in SFM cells in the presence of CHX only but treatment with CHX and 5‐HT in serum‐deprived cells resulted in increased β‐catenin protein levels as did 5‐HT treatment alone. (B) Serum‐deprived HuH‐7 and HepG2 cells were pre‐treated with 0.5 μM MG132 for 2 h and followed by 5‐HT for another 24 h 5‐HT increased β‐catenin protein levels in the presence of MG312. (C) Protein from HuH‐7 cells under SFM and 5‐HT treatment was immunoprecipitated with Axin1 or β‐catenin antibodies and immunoblotted for Axin1, β‐catenin and GSK3β by western blot.

Figure 4

5‐HT7 receptor antagonist, SB‐258719, reduces proliferation of HuH‐7 and HepG2 cells and also down regulates β‐catenin. (A) Serum deprived HuH‐7 and HepG2 cells were subjected to different concentrations of SB‐258719 for 2 h followed by incubation with 5‐HT. Cell viability was detected by MTT. SB‐258719 inhibited the proliferative effect of 5‐HT (5‐HT vs 5‐HT + SB‐258719, ***P < 0.001, two‐way ANOVA). (B) SB‐258719 reduced β‐catenin protein levels in serum‐deprived HCC cells in the presence of 5‐HT compared to 5‐HT treatment alone in both HuH‐7 and HepG2 cells.

Figure 5

The effect of SB‐258719 on patient‐derived primary HCC cultures. (A) SB‐258719 attenuated the growth of patient‐derived primary HCC cultures in the presence of 5‐HT and cell viability was detected by MTT. (B) SB‐258719 down‐regulated the expression of β‐catenin in patient‐derived primary HCC cultures compared to 5‐HT treatment only.

Figure 6

SB‐258719 suppresses tumorigenicity of HCC xenografts in vivo. (A) HuH‐7 cells were subcutaneously injected into BALB/c nude mice. The xenograft tumours were treated with vehicle or SB‐258719 20 mg/kg twice a day subcutaneously for 10 days and tumour growth was measured every day. Compared to the control group, SB‐258719 treatment reduced (B) tumour growth (**P < 0.01, two‐way ANOVA) and (C) tumour weight (ns denotes no significant difference, t‐test). (D) Immunohistochemical analysis of β‐catenin and GSK3β in the SB‐258719 treatment and control group. Tumour xenografts from SB‐258719 treatment group had reduced accumulation of β‐catenin and increased accumulation of GSK3β.