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MUSCARINIC RECEPTOR LIGANDS important MR ligands. In 1998, we made the serendipitous
IN CRC (ACETYLCHOLINE AND BILE observation that bile acids interact functionally with
ACIDS) muscarinic receptors on gastric epithelial cells.
[26]
Subsequently, we made several novel observations: (1)
Role of acetylcholine conjugated secondary bile acids interact selectively and
Acetylcholine (ACh) was traditionally regarded solely functionally with choline containing compounds (Cho)
[27]
as a neurotransmitter that functioned exclusively in the cells expressing muscarinic receptors; (2) molecular
central and peripheral nervous systems. However, over modeling revealed a strikingly similar structural alignment
the past decade emerging evidence indicates that ACh is of the geometry and surface electrostatic charges of bile
[27]
also produced and released by normal and neoplastic non- acids and ACh; (3) bile acid binding triggers appropriate
neuronal cells including human keratinocytes, small cell post-M3R signaling; (4) hybrid molecules created
[27]
lung cancer cells, immune cells, and intestinal epithelial from bile acids and ACh are MR ligands; and (5)
[28]
cells. [16-21] Choline acetyltransferase (ChAT) plays a critical lithocholic and deoxycholic acid conjugates interact with
catalytic role in the biosynthesis of both neuronal and M3R on human colon cancer cells, thereby stimulating
non-neuronal ACh. Homozygous ChAT mutant embryos post-receptor signaling and cell proliferation. Because
[29]
lack detectable ACh. Also, ACh released from nerve muscarinic effects on colon cancer cell proliferation are
[22]
endings is rapidly hydrolyzed by choline esterases thereby mediated by transactivation of EGFR, [30,31] results of
limiting its actions to immediately-neighboring cells. As a bile acid interaction with M3R depend on the cell type
consequence, ACh production by non-neuronal cells can examined. In Cho cells that express M3R but not EGFR,
play a key role in regulating the actions of cells or tissues deoxycholic acid conjugates are MR antagonists. In
[32]
that are not directly innervated by cholinergic neurons. H508 and HT-29 colon cancer cells that express both
M3R and EGFR deoxycholyltaurine (DCT) is a MR
ACh can be released by enteric neurons or produced by agonist whose effects are mediated by transactivation of
colon cancer cells. Although ACh release from intestinal EGFR. [30,33] To our knowledge, no endogenous mammalian
non-neuronal cells was identified more than a decade cholinergic agonists other than ACh and bile acids have
ago, its physiological relevance remains poorly been identified.
[23]
understood. We showed that human colon cancer cells
produce and release ACh that acts as an autocrine growth The observation that bile acids interact selectively and
factor to stimulate cell proliferation. We demonstrated functionally with plasma membrane muscarinic receptors
[24]
that: (1) basal colon cancer cell proliferation is inhibited prompted us to examine their actions on intestinal
by the cholinergic antagonist atropine, cholinesterases, epithelial cells. In particular, we studied H508 human
and inhibitors of choline transport; (2) ChAT is expressed colon cancer cells that co-express M3R and EGFR, and
at low levels by mouse and human intestinal mucosa, SNU-C4 cells that express EGFR but not muscarinic
and at high levels by mouse colon tumors, a majority receptors. DCT caused dose-dependent increases in
[14]
of human colon cancer cell lines, and CRC surgical M3R signaling and H508 cell proliferation that were not
specimens; and (3) human colon cancer cell lines produce observed in SNU-C4 cells. These proliferative effects
[30]
and release ACh as demonstrated by high-performance of bile acids are mediated by interaction with plasma
liquid chromatography with electrochemical detection. membrane M3R, not by interaction with bile acid nuclear
These findings strongly support a role for ACh in CRC receptors (i.e. the farnesoid X receptor) that regulate bile
progression. acid metabolism.
We proposed an important role for MR activation We demonstrated that efficacious concentrations of pro-
in ultrarapid growth of CRC in a patient with proliferative bile acids are achieved in the intestine.
[34]
pheochromocytoma. This elderly man with long- Because H508 cells derive from a moderately well-
[25]
standing, unresectable pheochromocytoma experienced differentiated cecal adenocarcinoma, cecal contents
rapid development of rectal adenocarcinoma despite were obtained immediately post-mortem from 19
close endoscopic surveillance. We determined that persons. Using internal controls, bile acid spectrum
the patient’s CRC overexpressed M3R, whereas his and concentration were determined by an enzymatic
pheochromocytoma expressed ChAT. These findings assay and gas-chromatography/mass spectrometry.
suggested that ACh release from the pheochromocytoma Total 3α-hydroxy bile acids were 400 ± 200 μmol/L
stimulated rapid growth of the rectal neoplasm. As proof- (mean ± SD) and deoxycholic acid conjugates were
of-principle we found that culture media conditioned 12 ± 28 μmol/L (maximum, 104 μmol/L). [33] Overall,
by pheochromocytoma cells stimulates proliferation of in one-third of subjects, cecal conjugated deoxycholic
a human colon cancer cell line, an effect attenuated by acid achieved levels (10-100 μmol/L) that stimulate

adding the MR antagonist atropine. colon cancer cell proliferation in vitro. [30,33,34] Cecal bile
acid concentrations in persons with ileal disease, ileal
Role of bile acids resection, and colon cancer are not known. Additional
Besides ACh, bile acids and its derivatives are also factors suggest that bile acid interaction with GI epithelial
196
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ June 15, 2016 ¦