Anti-mitogenic and apoptotic effects of 5-HT1B receptor antagonist on HT29 colorectal cancer cell line

Abstract

Purpose: There is lack of evidence about impact of 5-HT receptors on colorectal cancers. The current study was designed to investigate the role of serotonin and its receptors in colorectal cancer cell line and tissues.

Methods: In cell cultures, we investigated the effects of 5-HT and 5-HT(1A,1B,1D) agonists and antagonists on proliferation of HT29 cells. We also tested apoptosis for the receptor antagonists. The expression of 5-HT1(A,B,D) receptor subtypes was examined by immunohistochemistry and western blotting.

Results: Our data indicated that 5-HT(1B) receptor was fully expressed in HT29 cell line and tumor tissues. MTT proliferation assay also revealed that serotonin and CP93129 dihydrochloride, a selective 5-HT(1B) receptor agonist, stimulated growth of HT29 cells. Further, SB224289 hydrochloride (that is a selective 5-HT(1B) receptor antagonist) had anti-proliferative and apoptotic effects on HT29 cells.

Conclusions: The findings of this study provide evidence for the potential role of 5-HT(1B) receptor in colorectal cancer. Further investigation is required to explore the effect of receptor antagonists on the prevention, prognosis and treatment of patients with colorectal cancer.

Fig. 1

a MTT assay results effect of different concentrations of 5-HT (serotonin) and 5-HT receptor agonists on proliferation index of HT29 cells after 48-h incubation period (BP554 maleate = selective 5-HT_1A agonist, CP93129 dihydrochloride = selective 5-HT_1B agonist, L-694,247 = selective 5-HT_1Dagonist.), *P < 0.05; **P < 0.01. b Dose–response curve for the effect of different concentrations of 5-HT_1A,1B,1D receptors antagonists (anta1A, 1B, 1D) on proliferation index of HT29 cells after 48-h incubation period; **P < 0.01; ***P < 0.001. c Cell cycle kinetics by flowcytometry for HT29 cells incubated (for 48 h) with 5-HT_1B,1A,1D receptor selective antagonists (5 μM). Cells were processed by FACS analysis to determine the cell cycle phase kinetics (A G₀; B–D cells in G₁, S and G₂/M phases, respectively); I flowcytometry of HT29, cells incubated with 5-HT_1B antagonist, II flowcytometry of HT29, cells incubated with 5-HT_1D antagonist, III flowcytometry of HT29 cells incubated with 5-HT_1A antagonist

Fig. 1

a MTT assay results effect of different concentrations of 5-HT (serotonin) and 5-HT receptor agonists on proliferation index of HT29 cells after 48-h incubation period (BP554 maleate = selective 5-HT_1A agonist, CP93129 dihydrochloride = selective 5-HT_1B agonist, L-694,247 = selective 5-HT_1Dagonist.), *P < 0.05; **P < 0.01. b Dose–response curve for the effect of different concentrations of 5-HT_1A,1B,1D receptors antagonists (anta1A, 1B, 1D) on proliferation index of HT29 cells after 48-h incubation period; **P < 0.01; ***P < 0.001. c Cell cycle kinetics by flowcytometry for HT29 cells incubated (for 48 h) with 5-HT_1B,1A,1D receptor selective antagonists (5 μM). Cells were processed by FACS analysis to determine the cell cycle phase kinetics (A G₀; B–D cells in G₁, S and G₂/M phases, respectively); I flowcytometry of HT29, cells incubated with 5-HT_1B antagonist, II flowcytometry of HT29, cells incubated with 5-HT_1D antagonist, III flowcytometry of HT29 cells incubated with 5-HT_1A antagonist

Fig. 2

Immunohistochemistry (IHC) (avidin–biotin peroxidase staining procedure) of tumor tissues from a 45 years old patient with grade II colorectal cancer; positivity is illustrated as brown areas resulted from precipitation of DAB-Biotinylated conjugate complex in cell membrane. a High positive IHC result for 5-HT1B receptor of tumor tissue, b moderate positive IHC result for 5-HT_1A receptor, c mild positive IHC result for 5-HT_1D receptor, d comparison of IHC immunoreactivity between patients with three kinds of receptors (5-HT_{1A, 1B, 1D}) and negative control; *P < 0.05; **P < 0.01

Fig. 3

Western blotting analysis of HT29 cells with 5-HT receptor antibodies; positive control was homogenate proteins of human brain cell line (A172) and negative control was proteins of normal human colon cell line (LS174). Weight ranges of protein bands were determined by comparing the size markers of proteins

Fig. 4

Apoptosis assay for HT29 cells with TUNEL procedure after 48-h incubation of cells with 5-HT receptor antagonists (5 μM). Apoptosis was detected by fluorescence microscopy (excitation wavelength in the range of 450–500 nm, detection in the range of 515–565 nm). a Apoptotic HT29 cell after incubation with 5-HT_1D antagonist (5 μM) for 48 h (green fluorescence of cell nucleus). b Apoptotic HT29 cell after incubation with 5-HT_1A antagonist (5 μM) for 48 h. c Apoptotic HT29 cell after incubation with 5-HT_1B antagonist (5 μM) for 48 h. d Comparison of apoptotic index between three test groups and negative control; *P < 0.05; **P < 0.01