5-HT promotes hepatocellular carcinoma by influencing β-catenin

Abstract

5-hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived human hepatocellular carcinoma cells (HCC) but the detailed intracellular mechanism is still unknown. As Wnt/β-catenin signaling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β-catenin signaling by 5-HT. 5-HT promoted proliferation of serum-deprived HuH-7 and HepG2 cells and also increased total β-catenin levels, and active β-catenin protein levels compared to just control cells under serum free medium without 5-HT. Furthermore, 5-HT increased β-catenin levels in the presence of cycloheximide, a protein synthesis inhibitor, suggesting increased β-catenin levels due to inhibition of β-catenin degradation. Quantitative real-time polymerase chain reaction (qPCR) showed increased β-catenin downstream target genes, Axin1, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS), in serum-deprived HCC cell lines treated with 5-HT. We next studied the expression of various 5-HT were receptors (5-HT1D, 5-HT2A, 5-HT2B, 5-HT5 and 5-HT7) by qPCR in 33 pairs of HCC tumors and corresponding adjacent non-tumor tissues. Receptors 5-HT1D (21/33, 64%), 5-HT2B (12/33, 36%) and 5-HT7 (15/33, 45%) were overexpressed in HCC tumour tissues whereas receptor 5-HT5 was reduced (30/33, 91%) in HCC tumour tissues. Receptor 5-HT2A did not show any significant statistical difference between HCC tumour and corresponding non-tumour tissues. We further investigated whether antagonists of the 5-HT receptors attenuated the activity of 5-HT both in vitro and in vivo and we narrowed our study to 5-HT7 antagonist as the expression of 5-HT7 was found to be significantly associated to liver histology and venous infiltration. Antagonist of receptor 5-HT7, SB258719, attenuated growth of serum-deprived HCC cell lines and primary tumor tissues in the presence of 5-HT. Furthermore, SB258719 reduced tumor growth in a xenograft mouse model accompanied by reduced β-catenin and increased GSK-3β levels as observed by immunohistochemical analysis. Conclusion: This study provides evidence of Wnt/β-catenin signaling activation in 5-HT induced proliferation of serum-deprived HCC cells. The proliferation is inhibited by 5-HT7 antagonist, SB258719, which may represent a potential therapeutic target for hepatocarcinogenesis.