Muscarinic receptor agonists and antagonists: effects on cancer

Abstract

Many epithelial and endothelial cells express a cholinergic autocrine loop in which acetylcholine acts as a growth factor to stimulate cell growth. Cancers derived from these tissues similarly express a cholinergic autocrine loop and ACh secreted by the cancer or neighboring cells interacts with M3 muscarinic receptors expressed on the cancer cells to stimulate tumor growth. Primary proliferative pathways involve MAPK and Akt activation. The ability of muscarinic agonists to stimulate, and M3 antagonists to inhibit tumor growth has clearly been demonstrated for lung and colon cancer. The ability of muscarinic agonists to stimulate growth has been shown for melanoma, pancreatic, breast, ovarian, prostate and brain cancers, suggesting that M3 antagonists will also inhibit growth of these tumors as well. As yet no clinical trials have proven the efficacy of M3 antagonists as cancer therapeutics, though the widespread clinical use and low toxicity of M3 antagonists support the potential role of these drugs as adjuvants to current cancer therapies.

Fig. 1

Cholinergic signaling in neurons and bronchial epithelial cells. (a) In neurons, choline for ACh synthesis is transported by the choline high-affinity transporter (CHT1). ACh is then synthesized by the action of choline acetyltransferase (ChAT), and packaged into synaptic vesicles by the action of the vesicular acetylcholine transporter (VAChT) and CHT1. ACh is then secreted by the complex processes that control synaptic release. Released ACh then interacts with postsynaptic nAChR and mAChR as well as presynaptic receptors. Signaling is terminated by acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Key signal transduction events lead to the generation of action potentials, opening of membrane and internal ion channels, muscle contraction and kinase activation. (b) In bronchial epithelial cells (BEC), though CHT1 is present, CHT1 does not appear necessary for choline transport for ACh synthesis. In BEC, as for neurons, ChAT is utilized for ACh synthesis, though since there are multiple isoforms of ChAT, different splicing products may be utilized in different cell types. Since CHT1 is not required, and BEC do not have synaptic vesicles, the role of VAChT and CHT1 in ACh secretion is unknown, though both are expressed in BEC (Proskocil et al. 2004). ACh released by BEC is inactivated by the same cholinesterases as expressed in neurons. A key difference is that released ACh is not limited just to synaptic communication, but can also signal multiple neighboring cells as a paracrine factor or more distal cells as a hormone

Fig. 2

Cholinergic signaling by lung cancer cells. Cholinergic signaling by lung cancer cells is similar to normal bronchial epithelial cells. Steps for ACh synthesis and signal transduction in lung cancer provide the potential steps to target for development of therapies. In particular, inhibition of choline transport and muscarinic receptor antagonists offer unique advantages as discussed in Sect. 5. Targeting proliferative kinase pathways such as MAPK and Akt is an area of major development for cancer therapy in general since so many growth factors activate those pathways

Fig. 3

Calcium responses to muscarinic agonists and antagonists in H82 cells. (a) A representative trace of the [Ca²⁺]_I response of H82 cells to ACh in the absence (−) or presence (+) of atropine. (b) Rank order potency of selective muscarinic antagonists to inhibit the [Ca²⁺]_I increase elicited by ACh in H82 cells. Antagonists tested were 4-DAMP (filled square, a selective M3 antagonist), pirenzepine (filled triangle, a selective M1 antagonist) and AFDX 116 (filled circle, a selective M2/M4 antagonist). The rank order potency of these antagonists is most consistent with mediation by the M3 mAChR. (c) siRNA knockdown of M3 mAChR blocked the ACh induced increase in [Ca²⁺]_I but control, M1 and M5 mAChR knockdowns had no effect. Filled circle = control siRNA, filled square = M1 siRNA, filled triangle = M3 siRNA, filled diamond = M5 siRNA. Data are presented as mean ± SE of at least 12 replicates from 3 separate experiments. Modified after Song et al. (2007)

Fig. 4

Effect of ACh on phosphorylation of MAPK and Akt in H82 SCLC cells. (a) Western blot showing increased MAPK and Akt phosphorylation induced by concentrations of ACh shown. (b) Western blot showing that phosphorylation of Akt and MAPK induced by 3 × 10⁻⁵ M ACh was decreased by the M3 antagonist 4-DAMP in a concentration-dependent fashion. (c) Western blot showing that 4-DAMP alone decreased basal phosphorylation of Akt and MAPK. Modified after Song et al. (2007)

Fig. 5

Regulation of H82 cell proliferation by mAChR subtype antagonists. The MTS assay was used to detect H82 cell growth after treatment with 4-DAMP and AFDX-116. (a) The M3 mAChR antagonist 4-DAMP inhibited H82 cell proliferation in a concentration-dependent manner. (b) The M2/M4 selective mAChR antagonist, AFDX 116 had no significant effect on cell growth. All data are expressed as the mean ± SE of 24 replicates of two separate experiments. White column, control; dotted-pattern column, 10⁻⁹ M; horizontal-pattern column, 10⁻⁸ M; diagonal-pattern column, 10⁻⁷ M; gray column, 10⁻⁶ M; black column, 10⁻⁵ M. *p < 0.001 and ^†p < 0.05 compared to control at 9 days by Tukey–Kramer multiple comparison test after 2-way ANOVA. (c) Effect of darifenacin on growth of H82 tumor xenografts in nude mice. (c) Tumor weight. *p < 0.05 compared to control by t test. Modified after Song et al. (2007)

Fig. 6

Immunohistochemistry of ChAT and M3 mAChR expression in an SCLC biopsy. (a) ChAT immunostaining (400×, chromogen = VIP), insert box = 1,000×. (b) M3R immunostaining (400×, chromogen = VIP). (c) Confocal image showing coexpression of M3 mAChR (red) and ChAT (green) in tumor cells in same sample as (a) and (b). Modified after Song et al. (2007)