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

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Abstract

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, T16Ainh-A01 as a specific inhibitor of Ano1.

Aim

To investigate the effect of the T16Ainh-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 T16Ainh-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

T16Ainh-A01 inhibited Ca2+-activated Cl currents by 60% at 10 μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16Ainh-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16Ainh-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 T16Ainh-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.

Highlights

► T16Ainh-A01 blocked Ano1 currents in HEK cells expressing Ano1. ► T16Ainh-A01 reduced proliferation in ICC primary cultures and CFPAC-1 cell line. ► T16Ainh-A01 reduced proliferation of ICC in intact smooth muscle strips.

Introduction

Alteration of membrane potential through changes in extracellular ion concentration is a crucial regulator of proliferation, performing important roles in the progression of cell cycle at multiple key checkpoints (reviewed in [1]). Many ions are involved in these processes; the role of chloride ions in regulating cell cycle has been studied mostly as a driving force in cytoplasmic condensation [2], [3]. Ano1 is a Ca2+ activated Cl channel expressed in secretory epithelia of lung, salivary glands and kidney [4], [5], [6]. Expression of Ano1 is up-regulated in several cancers including esophageal cancer [7] and gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors in the gastrointestinal tract [8], [9]. In the muscle layers of the gastrointestinal tract Ano1 expression is restricted to interstitial cells of Cajal (ICC [10]), pacemaker and neuromodulator cells of the gut. Ano1 appears to be required for normal gastrointestinal function [11], [12] and has been proposed to play a key role in the pacemaker activity of ICC [12], [13]. Ano1 is expressed in all classes of ICC, including those that do not generate slow waves [10] suggesting that Ano1 may have other functions. A possible role of Ano1 in regulation of proliferation was suggested by its expression in GISTs. We investigated this possibility and showed that Ano1 activity contributes to the proliferation of Ano1 positive cells by acting at the G1/S phase of cell cycle. This was demonstrated by showing reduced proliferation of ICC in Ano1(−/−) mice, in the presence of chloride depleted culture medium and by the use of three structurally different chloride channel blockers [14]. Also, in a human head and neck squamous cell carcinoma cell line (UM-SCC1) knockdown of Ano1 resulted in a decrease in xenograft growth in nude mice [15].

A significant problem in investigating the role of Ano1 in proliferation is the lack of selectivity of many Cl channel inhibitors for Cl channels and transporters and for Ano1. Compounds such as DIDS, niflumic acid and tamoxifen decrease ICC proliferation in culture; however, they are not selective and have multiple effects on other targets that can indirectly alter chloride secretion [16], [17], [18], [19].

Recently, a high throughput screen of 110,000 compounds revealed a novel small molecule, an aminophenylthiazole named T16Ainh-A01 as a specific inhibitor of Ano1 [20]. This compound was reported to inhibit Cl efflux due to Ano1 (human) with an IC50 of 1.1 μM but to have little effect on CFTR and no effect on cytoplasmic calcium [20]. However, direct effects on Ano1-mediated Cl currents have not been reported.

The aim of this study was to confirm T16Ainh-A01’s inhibitory effect on Ano1-mediated Cl currents in an heterologous expression system and to investigate the effect of the inhibitor on the proliferation of ICC and an Ano1-expressing human pancreatic cancer cell line.

Section snippets

Animals

BALB/c mice were obtained from Harlan (Indianapolis, IN). Mice were killed by CO2 inhalation and cervical dislocation at post-natal day 3 (PND 3). The mice were maintained and the experiments were performed with approval from the Institutional Animal Care and Use Committee of the Mayo Clinic.

Cell cultures

Primary cultures enriched in ICC were obtained by enzymatic dissociation of the mouse small intestines and co-cultured in the presence of Sl/Sl4 mSCF248, murine stem cell factor—secreting fibroblasts as

T16Ainh-A01 inhibits Ano1 currents in transfected cells

To test if the T16Ainh-A01 inhibitor blocked Ano1 currents we recorded whole cell patch clamp currents from HEK293 cells transfected with a plasmid containing the full-length human Ano1 in the presence or absence of the inhibitor. As previously shown in Mazzone et al. [22], transfection with the Ano1 vector resulted in expression of channels that produced Ca2+- and voltage-dependent, outwardly rectifying currents (30.2 ± 9.1 pA/pF at 100 mV, Fig. 1(A)). When the transfected cells were treated with

Discussion

In the present study we show that the inhibitor T16Ainh-A01 blocked human Ano1 mediated Cl currents and that this more selective inhibitor decreased proliferation of Ano1-expressing mouse ICC in dissociated culture and intact tissue. Furthermore, the compound also inhibited proliferation in the human pancreatic cancer cell line CFPAC-1. This supports the work published previously using non-selective Cl channel inhibitors, modulation of Cl concentration and Ano1 knockout mice to interrogate

Acknowledgments

We would like to thank Dr. Verkman from UCSF for providing the T16Ainh-A01 compound. We would also like to thank Kristy Zodrow for secretarial assistance and Gary Stoltz for technical assistance. This work was supported by NIH grant DK57061.

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