Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation

Abstract

Most colon cancers overexpress M 3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M 3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by ϳ40% (P Ͻ 0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5-and 2-fold, respectively (P Ͻ 0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n ϭ 25) whereas half of colon cancer specimens (n ϭ 24) exhibited moderate to strong staining (P Ͻ 0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation. autocrine signaling; choline acetyltransferase; muscarinic receptors FOR BOTH MEN AND WOMEN IN the United States colon cancer is a common, frequently lethal disease. Although endoscopic or surgical cancer resection is highly successful in treating early disease, advanced colon cancer responds poorly to surgery, chemotherapy, and radiation, thereby accounting for 30% mortality. A fundamental tenet of cancer biology is that elucidating mechanisms underlying neoplastic cell proliferation will identify therapeutic targets. Hence, identifying colon cancer growth factors and growth factor receptors is a prominent research goal. Nonetheless, although it was recognized more than 15 years ago that muscarinic receptor signaling stimulates colon cancer cell proliferation (9) and that most colon cancers overexpress M 3 muscarinic receptors (M 3 R) (29), muscarinic receptor ligands and receptors have been neglected as potential therapeutic targets.Postmuscarinic receptor signaling has long been recognized as integral to gastrointestinal physiology. Cholinergic nerve endings are present in colonic mucosa and muscarinic signaling, particularly via M 3 R, plays a prominent role in mediating intestinal epithelial fluid and electrolyte transport (10,26,28,30). Muscarinic receptor ...

Autocrine Activation of CHRM3 Promotes Prostate Cancer Growth and Castration Resistance via CaM/CaMKK–Mediated Phosphorylation of Akt

“…Considering that in the microenvironment of prostate cancer, the concentration of acetylcholine in prostate cancer tissues may be even higher than the acetylcholine secreted into the medium due to higher cell density, such autocrine acetylcholine signaling likely functions in the prostate in vivo. Further support for our autocrine cholinergic signaling model also comes from the results reported previously in other tissue adenocarcinoma cells, such as colon cancer cells and small-cell lung cancer cells (7,8).…”

“…In the 1990s, muscarinic receptor subtypes were firstly defined as conditional oncogenes when they were activated by acetylcholine in NIH-3T3 cells (6). Subsequently, muscarinic receptors have been implicated to be involved in a few types of epithelial cancers, including colorectal cancer and small-cell lung cancer (7,8). In addition, activation of muscarinic receptors was reported to promote prostate cancer cell proliferation in vitro (9).…”

Blocking M2 muscarinic receptor signaling inhibits tumor growth and reverses epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC)

“…3,4 It is well established that ACh is an important neurotransmitter in the central and peripheral nervous systems and acts via activation of ACh receptors (AChRs).Recently, it has been found that ACh is also widely synthesized by a variety of non-neuronal cell types including airway epithelial cells, 5,6 keratinocytes, 7 small and large intestine, gall bladder, 8 glial, 9 vascular endothelial, 10 immune cells and most common cancer cells such as NSCLC, SCLC, colon, glial and ovarian carcinomas. [11][12][13][14][15][16] Although the primary function has not yet been completely elucidated, the expression of ACh in many different cell types suggests that its roles will be fundamental and will present novel therapeutic targets.…”

Selective M3 Muscarinic Receptor Antagonist Inhibits Small-Cell Lung Carcinoma Growth in a Mouse Orthotopic Xenograft Model

“…ACh is also found in cancer cells such as small cell lung carcinoma (SCLC) (6), non-small cell lung carcinoma (7), and colon cancer (8). In cell lines of these particular cancers, expression of mAChRs, nAChRs, choline acetyltransferase (ChAT) that synthesizes ACh, and acetylcholine esterase (AChE) that degrades ACh to choline and acetic acid (1,9,10) indicate that ACh is locally synthesized and metabolized.…”