Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma.
It is well established that small cell lung carcinomas (SCLCs) express receptors for acetylcholine (ACh) and that stimulation of these receptors by nicotine or other cholinergic agonists stimulates cell growth via activation of nicotinic cholinergic receptors (nAChRs) and/or muscarinic cholinergic receptors (mAChRs). The aim of this study was to determine whether SCLC cells synthesize and secrete ACh and respond to endogenous ACh to create a functioning cholinergic autocrine loop. Reverse transcription-PCR was used to screen a panel of SCLC cell lines for components of cholinergic signaling. Choline acetyltransferase (ChAT) and the vesicular ACh transporter (VAChT), as well as alpha3, alpha5, alpha7, beta2, and beta4, nAChR subunits and M3 and M5 mAChRs, were found to be present in most of the SCLC cell lines tested. Real-time PCR showed that mRNA levels for ChAT, VAChT, and alpha7 and beta2 nAChR subunits varied significantly among different SCLC cell lines tested. The H82 cell line was found to express the highest levels of ChAT, and that cell line was chosen for additional studies of ACh release and cell proliferation. ACh was easily detectable in H82 cell culture media, and levels of ACh were increased by the acetylcholinesterase inhibitor neostigmine. Vesamicol, an inhibitor of VAChT, and hemicholinium-3, an inhibitor of choline transport, both reduced H82 cell ACh basal release in a dose-dependent manner. In parallel with the reductions of ACh release, vesamicol and hemicholinium-3 also decreased H82 cell proliferation. H82 cell proliferation was also inhibited by the muscarinic and nicotinic antagonists atropine and mecamylamine, respectively, in dose- and time-dependent manners. Finally, archival cases of SCLC were screened by immunohistochemistry for expression of ChAT. Thirteen of 26 tumors screened were positive for ChAT. These findings demonstrate that SCLC can synthesize, secrete, and degrade ACh and that released ACh stimulates SCLC cell growth. Identification of this new autocrine loop provides a potential new target for therapeutic intervention.
Song
Pingfang
P
Department of Pathology, Oregon Health and Science University, Portland, Oregon 97239, USA.
Sekhon
Harmanjatinder S
HS
Jia
Yibing
Y
Keller
Jennifer A
JA
Blusztajn
Jan Krzysztof
JK
Mark
Gregory P
GP
Spindel
Eliot R
ER
D000109:Acetylcholine / Q000096:biosynthesis* / Q000502:physiology*; D001483:Base Sequence; D018288:Carcinoma, Small Cell / Q000201:enzymology / Q000473:pathology*; D002352:Carrier Proteins / Q000235:genetics*; D002455:Cell Division; D002795:Choline O-Acetyltransferase / Q000235:genetics*; D017931:DNA Primers; D006133:Growth Substances / Q000502:physiology*; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology*; D026901:Membrane Transport Proteins; D021122:Protein Subunits / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D006133:Growth Substances; D026901:Membrane Transport Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D002795:Choline O-Acetyltransferase; D000109:Acetylcholine