Role of α7-nicotinic acetylcholine receptor in nicotine-induced invasion and epithelial-to-mesenchymal transition in human non-small cell lung cancer cells

Abstract

Nicotine via nicotinic acetylcholine receptors (nAChRs) stimulates non-small cell lung cancer (NSCLC) cell invasion and epithelial to mesenchymal transition (EMT) which underpin the cancer metastasis. However, the receptor subtype-dependent effects of nAChRs on NSCLC cell invasion and EMT, and the signaling pathway underlying the effects remain not fully defined. We identified that nicotine induced NSCLC cell invasion, migration, and EMT; the effects were suppressed by pharmacological intervention using α7-nAChR selective antagonists or by genetic intervention using α7-nAChR knockdown via RNA inference. Meanwhile, nicotine induced activation of MEK/ERK signaling in NSCLC cells; α7-nAChR antagonism or MEK/ERK signaling pathway inhibition suppressed NSCLC cell invasion and EMT marker expression. These results indicate that nicotine induces NSCLC cell invasion, migration, and EMT; the effects are mediated by α7-nAChRs and involve MEK/ERK signaling pathway. Delineating the effect of nicotine on the NSCLC cell invasion and EMT at receptor subtype level would improve the understanding of cancer biology and offer potentials for the exploitation of selective ligands for the control of the cancer metastasis.

Keywords: EMT; NSCLC; invasion; nicotine; α7-nAChR.

Figure 1. Functional expression of α7-nAChR in NSCLC cells

A. RT-PCR analysis of nAChR subunits in A549, H1299, and PC9 cells. B-I. Measurement of calcium influx in A549 cells. The cells were treated with 1 mM nicotine and this effect was suppressed by the simultaneous administration of α-BTX (B and C) or by the knockdown of the α7 receptor subunit (D and E). Nicotine in 30 μM also can induce calcium influx and the effect was suppressed by the administration of α-BTX 60 min prior to the nicotine treatment (F and G) or by the knockdown of the α7 receptor subunit (H and I). The representative responses of the nicotine-induced increase of calcium influx measured by fluorescence intensity were shown (B, D, F, and H). Quantifications of the mean relative fluorescence peak calculated by [(F-F₀)/F₀]×100% are shown as means ± S.E.M from three independent experiments (C, E, G, I). ^* P < 0.05, ^** P < 0.01, ^*** P< 0.001.

Figure 2. Dependence of α7-nAChR of nicotine-induced NSCLC cell invasion and migration

A. Dose-dependence effect of nicotine on A549 cell invasion. The cells were treated for 48 h. B. Induction of A549 cell invasion by nicotine and α7-nAChR selectively agonist TC-5619 and the abrogation of the effect by α-BTX. *, P < 0.05 compared with the control group; #, P < 0.05 compared with the nicotine alone group; $, P < 0.05 compared with the TC5619 alone group. C and D. Blockade of nicotine-induced A549 cell invasion by α7-nAChR selectively antagonist α-BTX. When the antagonist was added simultaneously with the agonist, 1 μM of the antagonist was needed to attenuate the agonist-induced cell invasion (C); when the antagonist was added 1 h prior to the agonist, α-BTX at 0.1 μM fully blocked the effect (D). ^* P < 0.05, ^*** P< 0.001 compared with the control group; #, P < 0.05, ###, P < 0.001 compared with the nicotine alone group. E. Attenuation of nicotine-induced A549 cell invasion by the knockdown of α7-receptor subunit. ***, P < 0.001 compared with the control group; ##, P < 0.01 compared with the nicotine-treated Control shRNA group. F. Abrogation of nicotine-induced A549 cell mobility by α7-nAChR selectively antagonist α-BTX and MLA. Images were taken with 10× objective lens. * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05 compared with nicotine alone group. MLA, mecamylamine. TGF-β at 5 ng/mL as the migration-inducing positive control. The cells were treated by TGF-β or nicotine for 20 h; the antagonist α-BTX at 1 μM or MLA 1 μM was added 5 minutes prior to the agonists. G. Non-induction of PC9 cell mobility by nicotine. Images were taken with 10× objective lens. * P < 0.05 compared with control group. The cells were treated by TGF-β at 5 ng/mL or nicotine for 20 h; the antagonist α-BTX at 1 μM was added 5 minutes prior to the agonists. BTX, α-BTX. The cells were treated by 3 μM nicotine for 48 h in invasion assay and 1 μM nicotine for 20 h in migration assay unless otherwise indicated. Quantifications in bar graphs are shown as means ± S.E.M from at least three independent experiments.

Figure 3. Dependence of α7-nAChR of nicotine-induced NSCLC cell EMT

A. RT-PCR analysis of epithelial/mesenchymal markers in A549 and H1299 cells. B. Mesenchymal transition of A549 and H1299 cells stimulated by nicotine and the attenuation of the effect by α-BTX assayed by morphology analysis. TGF-β at 5 ng/mL as the EMT-inducing positive control. Images were taken with 10 × objective lens. C. Mesenchymal transition of A549 cells stimulated by nicotine and the attenuation of the effect by α-BTX assayed by immunofluorescence analysis of EMT protein markers. TGF-β at 5 ng/mL as the EMT-inducing positive control. Fibronectin and vimentin as the mesenchymal markers and E-cadherin as the epithelial marker. D. Down-regulation of vimentin expression in A549 cell by α7-nAChR antagonism assayed by western blot analysis. E. Attenuation of nicotine-induced upregulation of vimentin expression in A549 cells by knockdown of α7-nAChR subunit. The cells were treated by TGF-β or nicotine for 48 h; the antagonist α-BTX was added 5 minutes prior to the agonist.

Figure 4. Involvement of MEK/ERK signaling in nicotine-induced invasion/migration

A. Induction of ERK phosphorylation by nicotine and the attenuation of the effect by α7-nAChR antagonism. B. Attenuation of nicotine-induced A549 cell invasion by MEK-ERK selective inhibitor U0126. * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05 compared with nicotine alone group. C. Attenuation of nicotine-induced vimentin upregulation by MEK-ERK selective inhibitor U0126. The cells were treated by 3 μM nicotine for 30 min in western blot analysis and for 48 h in invasion and HCA assay; U0126 at 50 μM was added 5 minutes prior to the agonist. Quantifications in bar graphs are shown as means ± S.E.M from at least three independent experiments.