Fig. 1.

Acetylcholine (ACh) stimulates colon cancer cell migration in a time- and concentration-dependent manner. Human colon cancer cells were plated at confluence before a linear “wound” was made. As described in materials and methods, photomicrographs were taken immediately after wounding, before test agents were added, and again as described below. A: time course for effects of ACh (100 μM), epidermal growth factor (EGF; 10 μg/ml), and carbamylcholine (Carb; 100 μM) on H508 colon cancer cell migration. Cell migration was measured at the time points indicated. B: dose-response curve for the effects of ACh on H508 cell migration. Plates were incubated with the indicated concentrations of ACh and cell migration was measured at 8 h. The data point on the vertical axis represents cell migration without ACh. C: representative photographs showing that incubation with ACh (100 μM for 8 h) induces stress fiber formation in H508 cells. Arrowheads in bottom image indicate representative stress fibers in ACh-treated cells. D: representative photographs showing cell culture wounding assay immediately after scraping with pipette tip (0 h) and 8 h after incubation with ACh (100 μM) or vehicle (control). The overlying grid used to measure cell migration is shown. E: preincubation with atropine (1 μM) blocked ACh (100 μM)-induced but not EGF (10 μg/ml)-induced H508 cell migration. Cell migration was measured 8 h after addition of test agents. F: in SNU-C4 human colon cancers that do not express muscarinic receptors, cell migration, measured 8 h after addition of test agents, was stimulated by EGF (10 μg/ml) but not ACh (100 μM). Values in A, B, D, and E are means ± SE from at least 3 separate experiments. *, ***P < 0.05 and 0.001, respectively, compared with control (treatment with vehicle alone).