Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells

Diane L. Carlisle, Xuwan Liu, Toni M. Hopkins, Michelle C. Swick, Rajiv Dhir, Jill M. Siegfried

Research output: Contribution to journalArticlepeer-review

80 Scopus citations

Abstract

Nicotinic acetylcholine receptors (nAChR) are expressed on non-neuronal cell types, including normal bronchial epithelial cells, and nicotine has been reported to cause Akt activation in cultured normal airway cells. This study documents mRNA and protein expression of subunits known to form a muscle-type nAChR in non-small cell lung cancer (NSCLC) cell lines. In one NSCLC examined, mRNA and protein for a heteropentamer neuronal-type α3β2 nAChR was detected in addition to a muscle-type receptor. Protein for the α5 nAChR was also detected in NSCLC cells. Although, mRNA for the α7 nAChR subunit was observed in all cell lines, α7 protein was not detectable by immunoblot in NSCLC cell extracts. Immunohistochemistry (IHC) of NSCLC primary tissues from 18 patients demonstrated protein expression of nAChR α1 and β1 subunits, but not α7 subunit, in lung tumors, indicating preferential expression of the muscle-type receptor. In addition, the β1 subunit showed significantly increased expression in lung tumors as compared to non-tumor bronchial tissue. The α1 subunit also showed evidence of high expression in lung tumors. Nicotine at a concentration of 10 μM caused phosphorylation of mitogen-activated protein kinase (MAPK) (p44/42) that could be inhibited using nAChR antagonists. Inhibition was observed at 100 nM α-bungarotoxin (α-BTX) or 10 μM hexamethonium (HEX); maximal inhibition was achieved using a combination of α-BTX and HEX. Akt was also phosphorylated in NSCLC cells after exposure to nicotine; this effect was inhibited by the PI3K inhibitor LY294002 and antagonists to the neuronal-type nAChR, but not to the muscle-type receptor. Nicotine triggered influx of calcium in the 273T NSCLC cell line, suggesting that L-type calcium channels were activated. 273T cells also showed greater activation of p44/42 MAPK than of Akt in response to nicotine. Cultures treated with nicotine and the EGFR tyrosine kinase inhibitor gefitinib showed a significant increase in the number of surviving cells compared to gefitinib alone. These data indicate that the muscle-type nAChR, rather than the α7 type, is highly expressed in NSCLC and leads to downstream activation of the p44/42 MAPK pathway. Neuronal-type receptors are also present and functional, as evidenced by antagonist studies, although, the expression levels are lower than muscle-type nAChR. They also lead to downstream activation of MAPK and Akt. Nicotine may play a role in regulating survival of NSCLC cells and endogenous acetylcholine released locally in the lung and/or chronic nicotine exposure might play a role in NSCLC development. In addition, exposure of NSCLC patients to nicotine through use of nicotine replacement products or use of tobacco products may alter the efficacy of therapy with EGFR inhibitors.

Original languageEnglish (US)
Pages (from-to)629-641
Number of pages13
JournalPulmonary Pharmacology and Therapeutics
Volume20
Issue number6
DOIs
StatePublished - Dec 2007

Bibliographical note

Funding Information:
We gratefully acknowledge Z.Z. Wang, Ph.D. for the gift of anti-delta nAChR subunit antibody. This work was supported by P50 CA090440, SPORE in Lung Cancer from the National Cancer Institute to JMS and by the Flight Attendants Medical Research Institute (FAMRI) grant to DLC.

Keywords

  • Acetylcholine receptor
  • Lung cancer
  • Nicotine
  • Non-small cell lung cancer
  • Signaling

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