Inhibition of cell proliferation by a selective inhibitor of the Ca²⁺-activated Cl^- channel, Ano1

Background: Ion channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca²⁺-activated Cl^- channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16A_inh-A01 as a specific inhibitor of Ano1. Aim: To investigate the effect of the T16A_inh-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. Methods: Inhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16A_inh-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. Results: T16A_inh-A01 inhibited Ca²⁺-activated Cl^- currents by 60% at 10μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16A_inh-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16A_inh-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC. Conclusions: The selective Ano1 inhibitor T16A_inh-A01 inhibited Ca²⁺-activated Cl^- currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.