Modulatory roles of muscarinic cholinergic receptor 3 in proliferation and migration of small cell lung cancer

AIM: To study the expression and roles of muscarinic cholinergic receptor 3 (M3R) in human small cell lung cancer (SCLC) cells. METHODS: Human SCLC cell lines SBC3 and H82 were cultured in vitro. RT-PCR and Western blotting were used to investigate the expression of M3R. MTT assay and Boyden chamber assay were carried out to determine the roles of cholinergic receptor agonist acetylcholine iodide (ACh) and M3R antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) in the proliferation and migration of SBC3 cells. RESULTS: M3R was expressed in SBC3 and H82 cells. The relative protein expression of M3R normalized with β-actin in SBC3 was 2.65-fold higher than that in H82. ACh stimulated SBC3 cell proliferation in a dose-dependent manner. Treatment with ACh at concentrations of 10^-4 and 10^-3 mol/L significantly stimulated SBC3 cell growth at 48 h and 72 h (P＜0.01). SBC3 cell proliferation induced by ACh was inhibited by 4-DAMP in a dose-dependent manner. Pretreatment of the cells with 10^-5 mol/L 4-DAMP suppressed the effect of ACh at 48 h (P＜0.05). Pretreatment with 4-DAMP at concentrations of 10^-6, 10^-7 (P＜0.05) and 10^-5 mol/L (P＜0.01) inhibited the effect of ACh at 72 h. Treatment with 10^-5 or 10^-6 mol/L 4-DAMP alone inhibited the cell proliferation at 48 h (P＜0.01) and the inhibitory effect of 4-DAMP at concentration of 10^-5 mol/L was stronger than that of 4-DAMP at concentration of 10^-6 mol/L at 72 h. ACh increased the cell migration towards fibronectin (Fn) in a dose-dependent manner and ACh at concentration of 10^-4 mol/L enhanced the cell migration by about 3 folds. The cell migration stimulated by 10^-4 mol/L ACh was almost completely blocked by pretreatment with 4-DAMP at concentration of 10^-6 or 10^-5 mol/L (P＜0.01). CONCLUSION: M3R is expressed in human SCLC cells. The M3R antagonist inhibits SBC3 cell proliferation and migration.