OCTN1 mediates acetylcholine transport in the A549 lung cancer cells: possible pathophysiological implications

Abstract

A role for acetylcholine in cell proliferation, epithelial mesenchymal transition and invasion has been well assessed and related to the presence of the non-neuronal cholinergic system in lung cancer. For the operation of this non-neuronal system, acetylcholine should be released by a transporter mediated non-quantal process. OCTN1 is one of the transporters able to catalyse acetylcholine efflux in vitro and ex vivo. Using the A549 cell line as a lung cancer model, it has been found that these cells express OCTN1 at a higher level with respect to other cancer cells. The transport capacity of OCTN1 extracted from A549 and reconstituted into proteoliposomes reflects the protein expression profile. The properties of the acetylcholine transport mediated by OCTN1 of A549 in terms of specificity to ligands and ability to catalyse efflux of acetylcholine correspond to those previously described for the same transporter in other cells or to those of the human recombinant protein. OCTN1 is the major player in acetylcholine release in A549 and, therefore, may represent a target for inhibitors able to block the acetylcholine action in this type of aggressive tumors.

Keywords: Octn1; SLC; drug discovery; lung cancer; non-neuronal cholinergic system.

FIGURE 1

Expression of OCTN1 in cancer cell lines. (A) Cell extracts obtained as described in Section 2.2 were loaded on SDS-PAGE and blotted; OCTN1 or tubulin (loading control) was immunodetected by anti-OCTN1 or anti-tubulin, respectively. Representative blots are shown. (B) The histogram represents scanning densitometry (±S.D.) of four similar immunoblots reported as the Norm. Vol. (Int) compared to OCTN1 from HEK-293 cell line. Significantly different as estimated by One Way ANOVA followed by Tukey test for *p < 0.05, **p < 0.01, ***p < 0.005. (C) OCTN1 mRNA identification in A549, RT-PCR of OCTN1 and control (Actin) were performed as described in Section 2.5.

FIGURE 2

[³H]ACh transport by cancer cell line OCTN1. (A), time course of [³H]ACh in proteoliposomes reconstituted with cancer cell extracts of A549 (○), MCF-7 (●), HeLa (□), Hep G2 (■), HCT-15 (Δ), HEK-293 (▲). (B), Uptake (30 min) of 0.1 mM [³H]ACh into proteoliposomes reconstituted with A549 cell extract in the presence of anti-OCTN1 or anti-His reported as residual activity with respect to the 100% control (absence of an antibody). (C), Uptake (20 min) of 0.1 mM [³H]Ach in proteoliposomes reconstituted with protein extract from A549 cells transfected with siRNA scramble (control) or the indicated amount (pmol) of OCTN1-targeting siRNA. All data are means ± S.D. of three (B) or four (C) experiments. Significantly different as estimated by One Way ANOVA followed by Tukey test for *p < 0.05, **p < 0.01, ***p < 0.005. (D), A549 cell extracts obtained as described in Section 2.2 were loaded on SDS-PAGE and blotted; OCTN1 or tubulin (loading control) was immunodetected by anti-OCTN1 or anti-tubulin, respectively. Indicated nmol of SiRNA targeting Octn1 were used with respect to control (scramble SiRNA). Image is a representative blot of two experiments.

FIGURE 3

Characterization of [³H]ACh transport of A549 OCTN1. Uptake (20 min) of 0.1 mM [³H]ACh into proteoliposomes reconstituted with A549 cell extract. Activity measured in the absence or the presence of internal ATP and/or external NaCl (A) or at different pH (B). The results are means ± S.D. of three experiments, significantly different as estimated by One Way ANOVA followed by Tukey test for *p < 0.05, **p < 0.01, ***p < 0.005. (C) and (D) percent residual activity measured in the presence of added ligands (C) or inhibitors (D). The results are means ± S.D. of three experiments, significantly different from the control as estimated by Student's t-test for *p < 0.05, **p < 0.01, ***p < 0.005.

FIGURE 4

[³H]ACh efflux mediated by A549 OCTN1. Efflux of 0.1 mM [³H]ACh from proteoliposomes reconstituted with A549 protein extract was measured in the absence (○) or in the presence (●) of 50 mM external NaCl (A); in the presence of 50 mM internal KCl and 50 mM external NaCl (○) or in the presence of external 100 mM sucrose and the presence of 50 mM internal KCl (●) or 50 mM internal NaCl (□) (B). In (A) and (B) Residual internal radioactivity at the indicated times with respect to time zero was measured. (C) Efflux of 0.1 mM [³H]Ach in 15 min from proteoliposomes reconstituted with protein extract from A549 cells in the presence or absence of anti-OCTN1 added during reconstitution has been measured. Residual activity with respect to the 100% control (absence of an antibody) is indicated. The values are means ± S.D. from three independent experiments significantly different from the control as estimated by Student’s t-test for **p < 0.01.