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

Amelia Mazzone, Seth T. Eisenman, Peter R. Strege, Zhen Yao, Tamas Ordog, Simon J. Gibbons, Gianrico Farrugia

Research output: Contribution to journalArticle

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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 10μM 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.

Original languageEnglish (US)
Pages (from-to)248-253
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume427
Issue number2
DOIs
StatePublished - Oct 19 2012

Fingerprint

Interstitial Cells of Cajal
Cell proliferation
Cell Proliferation
Cells
Cell culture
Muscle
Tumors
Ki-67 Antigen
Pancreatic Neoplasms
Nuclear Antigens
Cell Line
Chloride Channels
Smooth Muscle
Clamping devices
Electric potential
T16AInh-A01
Ion Channels
Primary Cell Culture
Throughput
Tissue

Keywords

  • Cancer
  • GIST
  • ICC
  • Interstitial cells of Cajal
  • Intestine
  • TMEM16A

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Inhibition of cell proliferation by a selective inhibitor of the Ca2+-activated Cl- channel, Ano1. / Mazzone, Amelia; Eisenman, Seth T.; Strege, Peter R.; Yao, Zhen; Ordog, Tamas; Gibbons, Simon J.; Farrugia, Gianrico.

In: Biochemical and Biophysical Research Communications, Vol. 427, No. 2, 19.10.2012, p. 248-253.

Research output: Contribution to journalArticle

Mazzone, Amelia ; Eisenman, Seth T. ; Strege, Peter R. ; Yao, Zhen ; Ordog, Tamas ; Gibbons, Simon J. ; Farrugia, Gianrico. / Inhibition of cell proliferation by a selective inhibitor of the Ca2+-activated Cl- channel, Ano1. In: Biochemical and Biophysical Research Communications. 2012 ; Vol. 427, No. 2. pp. 248-253.
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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 10μM 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.",
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AU - Strege, Peter R.

AU - Yao, Zhen

AU - Ordog, Tamas

AU - Gibbons, Simon J.

AU - Farrugia, Gianrico

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N2 - 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 10μM 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.

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