Nicotine enhances hepatocyte growth factor-mediated lung cancer cell migration by activating the α7 nicotine acetylcholine receptor and phosphoinositide kinase-3-dependent pathway

Abstract

Cigarette smoking not only promotes lung carcinogenesis, but it has also been demonstrated to promote the progression of lung cancer. Despite nicotine being a major component of cigarette smoke, it is not carcinogenic when acting alone. Instead, it is believed to function as a tumor promoter. Due to the fatal consequences of lung cancer being primarily associated with the processes of invasion and metastasis, the present study aimed to determine the effect of nicotine on the migratory activity of lung cancer cells. The effect of nicotine on the migration of lung cancer A549 cells was evaluated by a wound healing assay. Hepatocyte growth factor (HGF) was used as a pro-migratory stimulus. During several of the experiments, specific inhibitors of α7-nicotine acetylcholine receptor (α7-nAchR), phosphoinositide kinase-3 (PI3K) and extracellular signal-related kinase (ERK)1/2 were included. The phosphorylation levels of Akt and ERK1/2 were examined using a cell-based protein phosphorylation assay. It was observed that nicotine did not induce cell migration by itself, but that it instead promoted HGF-induced cell migration. The effects of nicotine were inhibited by the pretreatment of the cells with the α7-nAchR inhibitor, methyllycaconitine, and the PI3K inhibitor, LY294002. The mitogen-activated protein kinase/ERK kinase kinase inhibitor exerted modest, but non-significant inhibitory activity on the effect of nicotine. Nicotine did not induce Akt phosphorylation by itself, but instead promoted the HGF-induced phosphorylation of Akt. It was also observed that nicotine had no effect on ERK1/2 phosphorylation. The results from the present study indicate that nicotine, when alone, does not have a pro-migratory function, but instead enhances responsiveness to the pro-migratory stimulus emitted by HGF. The current study provides an insight into the mechanism of tumor promotion by demonstrating that nicotine and α7-nAchRs act in synergy with the HGF-induced PI3K/Akt signaling pathway, increasing the sensitivity of lung cancer cells to HGF, and thereby promoting cell migration, a vital step in invasion and metastasis.

Keywords: hepatocyte growth factor; lung cancer; migration; nicotine; phosphoinositide kinase-3; α7 nicotine acetylcholine receptor.

Figure 1.

Nicotine promotes migration of HGF-treated A549 cells. A549 cell monolayers were scratched and subsequently incubated in serum-free nicotine-containing medium (0.1, 1 and 10 µM), either in the presence or absence of HGF (20 ng/ml). (A) Representative photomicrographs of cell monolayers 18 h after wounding. Broken lines indicate the leading edge of the wound repopulating cells. Scale bar, 500 µm. (B) Time-course of the cell migration. The migration of cells towards the wounds was expressed as the percentage of wound closure. The data points represent the mean ± SEM of 6 wells. (C) Dose-dependent effect of nicotine on cell migration 18 h post-wounding. The data points represent the mean ± SEM of 6 wells. HGF, hepatocyte growth factor.

Figure 2.

Nicotine promotes HGF-induced cell migration through the activation of α7-nAchR. A549 cells were pretreated for 1 h with the α7-nAchR antagonist, methyllycaconitine (10 µM), prior to inflicting the scratch. The cells were subsequently incubated for 18 h in serum-free medium, containing or lacking 10 µM nicotine in the presence of of 20 ng/ml HGF. The bars represent the mean ± standard error of the mean of 6 wells. HGF, hepatocyte growth factor; SEM, standard error of the mean; α7-nAchR, α7-nicotine acetylcholine receptor.

Figure 3.

Effects of PI3K and ERK1/2 inhibition on HGF-induced cell migration. A549 cells were pretreated with a pharmacological inhibitor of PI3K, LY294002, or ERK kinase, PD98059, for 1 h. The cells were subsequently scratched and incubated for 18 h in serum-free medium, either containing or lacking 20 ng/ml HGF. The bars represent the mean ± standard error of the mean of 6 wells. PI3K, phosphoinositide kinase-3; ERK, extracellular signal-related kinase; HGF, hepatocyte growth factor.

Figure 4.

Effects of nicotine on the phosphorylation of Akt and ERK1/2. A549 cells were incubated for 2 h in a serum-free medium containing 10 µM nicotine, either in the presence or absence of 20 ng/ml HGF. Protein phosphorylation was detected using either (A) the phospho-AKT antibody or (B) the phospho-ERK1/2 antibody, from Fast Activated Cell-based ELISA kits. The protein phosphorylation levels were corrected for the cell numbers. The bars represent the mean ± standard error of the mean of 6 wells. Akt, protein kinase B; ERK, extracellular signal-related kinase; HGF, hepatocyte growth factor.