Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells.
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 alpha3beta2 nAChR was detected in addition to a muscle-type receptor. Protein for the alpha5 nAChR was also detected in NSCLC cells. Although, mRNA for the alpha7 nAChR subunit was observed in all cell lines, alpha7 protein was not detectable by immunoblot in NSCLC cell extracts. Immunohistochemistry (IHC) of NSCLC primary tissues from 18 patients demonstrated protein expression of nAChR alpha1 and beta1 subunits, but not alpha7 subunit, in lung tumors, indicating preferential expression of the muscle-type receptor. In addition, the beta1 subunit showed significantly increased expression in lung tumors as compared to non-tumor bronchial tissue. The alpha1 subunit also showed evidence of high expression in lung tumors. Nicotine at a concentration of 10 microM caused phosphorylation of mitogen-activated protein kinase (MAPK) (p44/42) that could be inhibited using nAChR antagonists. Inhibition was observed at 100 nM alpha-bungarotoxin (alpha-BTX) or 10 microM hexamethonium (HEX); maximal inhibition was achieved using a combination of alpha-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 alpha7 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.
Carlisle
Diane L
DL
Department of Pharmacology, University of Pittsburgh, E1340 Biomedical Science Tower, Pittsburgh, PA 15213, USA.
Liu
Xuwan
X
Hopkins
Toni M
TM
Swick
Michelle C
MC
Dhir
Rajiv
R
Siegfried
Jill M
JM
D000109:Acetylcholine / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D005731:Ganglionic Stimulants / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009538:Nicotine / Q000494:pharmacology*; D018722:Nicotinic Agonists / Q000494:pharmacology*; D010766:Phosphorylation; D021122:Protein Subunits / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D064569:alpha7 Nicotinic Acetylcholine Receptor
C523021:CHRNA1 protein, human; C519072:CHRNB1 protein, human; D020746:Calcium Channels, L-Type; C579167:Chrna7 protein, human; D005731:Ganglionic Stimulants; D018722:Nicotinic Agonists; D021122:Protein Subunits; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D009538:Nicotine; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D000109:Acetylcholine