Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: a target molecule for lung cancer therapy

Abstract

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.

Keywords: 2-(N-morpholino)ethanesulfonic acid; 2-amino-2-(hydroxymethyl)-1,3-propanediol; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 4-DAMP; 4-diphenylacetoxy-N-methylpiperidine methiodide; 4′,6-diamidino-2-phenylindole; ACh; AChE; Acetylcholine; Apoptosis; BuChE; CHT1; CTL1; Cell proliferation; ChAT; Choline; D-PBS; DAPI; DFP; DMA; Dulbecco's phosphate-buffered saline; GAPDH; HC-3; HEPES; MES; N-methyl-d-glucamine; NHE; NMDG; Na(+)/H(+) exchanger; OCT; PAH; PCho; PET; SCLC; SNRI; SSRI; Small cell lung carcinoma; TCA; TEA; Transporter; Tris; VAChT; acetylcholine; acetylcholinesterase; butyrylcholinesterase; choline acetyltransferase; choline transporter-like protein 1; diisopropyl fluorophosphates; dimethylamiloride; glyceraldehydes-3-phosphate dehydrogenase; hemicholinium-3; high-affinity choline transporter 1; mAChR3; muscarinic cholinergic receptor 3; organic cation transporter; p-aminohippuric acid; phosphocholine; positron emission tomography; selective serotonin reuptake inhibitor; serotonin and norepinephrine reuptake inhibitor; siRNA; small cell lung carcinoma; small interfering RNA; tetraethylammonium chloride; tricyclic antidepressant; vesicular acetylcholine transporter.