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ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC).

Sauter DR, Novak I, Pedersen SF, Larsen EH, Hoffmann EK - Pflugers Arch. (2014)

Bottom Line: We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration.Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation.On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

View Article: PubMed Central - PubMed

Affiliation: Section for Cell and Developmental Biology, Department of Biology, University of Copenhagen, August Krogh Building, Universitetsparken 13, 2100, Copenhagen Ø, Denmark, Daniel.Sauter@bio.ku.dk.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca(2+)-activated Cl(-) channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca(2+) and voltage-dependent Cl(-) currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

No MeSH data available.


Related in: MedlinePlus

T16Ainh-A01 and CaCCinh-A01 do not inhibit VRAC. a, b Representative current densities over time in HPDE and BxPC-3 cells, respectively, exposed to hypotonic bath solution and pipette solution containing 0 μM Ca2+. Inhibitors were applied for 3–10 min when the maximum volume-activated current was obtained. c Quantification of the effect shown in percent of maximum VRAC current at +65 mV. Data shown are mean ± s.e.m.
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Fig5: T16Ainh-A01 and CaCCinh-A01 do not inhibit VRAC. a, b Representative current densities over time in HPDE and BxPC-3 cells, respectively, exposed to hypotonic bath solution and pipette solution containing 0 μM Ca2+. Inhibitors were applied for 3–10 min when the maximum volume-activated current was obtained. c Quantification of the effect shown in percent of maximum VRAC current at +65 mV. Data shown are mean ± s.e.m.

Mentions: In order to evaluate the inhibitors of the two Cl− channels, we studied effects of the ANO1 inhibitors, T16Ainh-A01 and CaCCinh-A01, and of the VRAC inhibitor, NS3728, on volume-induced currents. Cells were exposed to hypotonic solution in the absence of intracellular Ca2+, and after full activation of the membrane current, the HPDE and BxPC-3 cells were exposed to 20 μM T16Ainh-A01 or CaCCinh-A01 followed by NS3728 (Fig. 5a, b). Statistical analyses of currents were performed at Vm = +65 mV before and after application of the respective inhibitor (Fig. 5c). Neither T16Ainh-A01 nor CaCCinh-A01 inhibited the VRAC currents significantly. In contrast, NS3728 decreased the fully activated VRAC current to 17 ± 4 % of that in the absence of the inhibitor. NS3728 was originally found to inhibit VRAC [12], but it also inhibited CaCC (Fig. 3) as also shown in another study [17]. To elucidate a possible effect of NS3728 on ANO1, we used HEK293 cells transiently transfected with mANO1-GFP as described recently [10]. Transfection with ANO1-GFP resulted in induction of a Ca2+- and voltage-dependent, outwardly rectifying current (Fig. 6a) with a Vrev near ECl (Fig. 6b). These currents were almost completely inhibited when treated with NS3728 (10 μM free concentration). The associated IC50 calculated at Vm = +48 mV was estimated to be 1.3 μM (Fig. 6c).Fig. 5


ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC).

Sauter DR, Novak I, Pedersen SF, Larsen EH, Hoffmann EK - Pflugers Arch. (2014)

T16Ainh-A01 and CaCCinh-A01 do not inhibit VRAC. a, b Representative current densities over time in HPDE and BxPC-3 cells, respectively, exposed to hypotonic bath solution and pipette solution containing 0 μM Ca2+. Inhibitors were applied for 3–10 min when the maximum volume-activated current was obtained. c Quantification of the effect shown in percent of maximum VRAC current at +65 mV. Data shown are mean ± s.e.m.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4464647&req=5

Fig5: T16Ainh-A01 and CaCCinh-A01 do not inhibit VRAC. a, b Representative current densities over time in HPDE and BxPC-3 cells, respectively, exposed to hypotonic bath solution and pipette solution containing 0 μM Ca2+. Inhibitors were applied for 3–10 min when the maximum volume-activated current was obtained. c Quantification of the effect shown in percent of maximum VRAC current at +65 mV. Data shown are mean ± s.e.m.
Mentions: In order to evaluate the inhibitors of the two Cl− channels, we studied effects of the ANO1 inhibitors, T16Ainh-A01 and CaCCinh-A01, and of the VRAC inhibitor, NS3728, on volume-induced currents. Cells were exposed to hypotonic solution in the absence of intracellular Ca2+, and after full activation of the membrane current, the HPDE and BxPC-3 cells were exposed to 20 μM T16Ainh-A01 or CaCCinh-A01 followed by NS3728 (Fig. 5a, b). Statistical analyses of currents were performed at Vm = +65 mV before and after application of the respective inhibitor (Fig. 5c). Neither T16Ainh-A01 nor CaCCinh-A01 inhibited the VRAC currents significantly. In contrast, NS3728 decreased the fully activated VRAC current to 17 ± 4 % of that in the absence of the inhibitor. NS3728 was originally found to inhibit VRAC [12], but it also inhibited CaCC (Fig. 3) as also shown in another study [17]. To elucidate a possible effect of NS3728 on ANO1, we used HEK293 cells transiently transfected with mANO1-GFP as described recently [10]. Transfection with ANO1-GFP resulted in induction of a Ca2+- and voltage-dependent, outwardly rectifying current (Fig. 6a) with a Vrev near ECl (Fig. 6b). These currents were almost completely inhibited when treated with NS3728 (10 μM free concentration). The associated IC50 calculated at Vm = +48 mV was estimated to be 1.3 μM (Fig. 6c).Fig. 5

Bottom Line: We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration.Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation.On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

View Article: PubMed Central - PubMed

Affiliation: Section for Cell and Developmental Biology, Department of Biology, University of Copenhagen, August Krogh Building, Universitetsparken 13, 2100, Copenhagen Ø, Denmark, Daniel.Sauter@bio.ku.dk.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca(2+)-activated Cl(-) channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca(2+) and voltage-dependent Cl(-) currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

No MeSH data available.


Related in: MedlinePlus