Limits...
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

ANO1 is the major contributor of CaCC currents in PDAC cells and is sensitive to Cl− channel inhibitors and siRNA. Whole-cell patch-clamp recordings with 1 μM Ca2+ in pipette solution. a Representative current traces measured in Capan-1 cells upon exposure to different inhibitors. b Quantification of CaCC currents at +67 mV recorded with inhibitors compared to currents in presence of DMSO; currents of siANO1 cells were compared to those of MOCK transfected cells. c Steady-state I–V relationships of cells transfected with either scrambled siRNA (MOCK) or siRNA targeting ANO1. d Densitometric quantification of Western blot analysis of siRNA knockdown. β-Actin was used as a loading control. (n) = number of individual experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4464647&req=5

Fig3: ANO1 is the major contributor of CaCC currents in PDAC cells and is sensitive to Cl− channel inhibitors and siRNA. Whole-cell patch-clamp recordings with 1 μM Ca2+ in pipette solution. a Representative current traces measured in Capan-1 cells upon exposure to different inhibitors. b Quantification of CaCC currents at +67 mV recorded with inhibitors compared to currents in presence of DMSO; currents of siANO1 cells were compared to those of MOCK transfected cells. c Steady-state I–V relationships of cells transfected with either scrambled siRNA (MOCK) or siRNA targeting ANO1. d Densitometric quantification of Western blot analysis of siRNA knockdown. β-Actin was used as a loading control. (n) = number of individual experiments

Mentions: To quantify the ANO1-mediated part of the total CaCC current, we used siRNA knockdown, as well as the three Cl− channel inhibitors NS3728 (10 μM), CaCCinh-A01 (20 μM), and T16Ainh-A01 (20 μM). NS3728 is an inhibitor of VRAC but was also found to inhibit CaCC [17]. CaCCinh-A01 is known to potently inhibit CaCC current but was further shown to alter [Ca2+]i signals [18]. T16Ainh-A01 is the latest developed inhibitor of ANO1; no unspecific effects have been identified so far. Results are shown in Fig. 3. Application of T16Ainh-A01 attenuated the CaCC current significantly in both Capan-1 (43 ± 8 % inhibition at +67 mV) and AsPC-1 (29 ± 2 %) cells, but was ineffective on BxPC-3 cells (Fig. 3b). CaCCinh-A01 inhibited the current more effectively in all tested cell lines with 56 ± 6 % inhibition in Capan-1, 46 ± 6 % in AsPC-1, and 29 ± 3 % in BxPC-3 cells, respectively (all calculated at +67 mV). Application of NS3728 resulted in the most pronounced inhibition in all cell lines with 77 ± 26 % in Capan-1, 67 ± 9 % in AsPC-1, and 54 ± 8 % in BxPC-3 cells at +67 mV. Using siRNA targeting ANO1, the CaCC currents decreased by 88 ± 42 % in Capan-1, 89 ± 51 % in AsPC-1, and 84 ± 23 % in BxPC-3 cells when compared to currents measured in cells transfected with scrambled siRNA (MOCK) (Fig. 3c). Successful knockdown of ANO1 protein expression was confirmed using Western blot (Fig. 3d). Protein expression was decreased to 37 ± 8 % in Capan-1, 54 ± 14 % in AsPC-1, and 20 ± 7 % in BxPC-3 cells.Fig. 3


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

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

ANO1 is the major contributor of CaCC currents in PDAC cells and is sensitive to Cl− channel inhibitors and siRNA. Whole-cell patch-clamp recordings with 1 μM Ca2+ in pipette solution. a Representative current traces measured in Capan-1 cells upon exposure to different inhibitors. b Quantification of CaCC currents at +67 mV recorded with inhibitors compared to currents in presence of DMSO; currents of siANO1 cells were compared to those of MOCK transfected cells. c Steady-state I–V relationships of cells transfected with either scrambled siRNA (MOCK) or siRNA targeting ANO1. d Densitometric quantification of Western blot analysis of siRNA knockdown. β-Actin was used as a loading control. (n) = number of individual experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4464647&req=5

Fig3: ANO1 is the major contributor of CaCC currents in PDAC cells and is sensitive to Cl− channel inhibitors and siRNA. Whole-cell patch-clamp recordings with 1 μM Ca2+ in pipette solution. a Representative current traces measured in Capan-1 cells upon exposure to different inhibitors. b Quantification of CaCC currents at +67 mV recorded with inhibitors compared to currents in presence of DMSO; currents of siANO1 cells were compared to those of MOCK transfected cells. c Steady-state I–V relationships of cells transfected with either scrambled siRNA (MOCK) or siRNA targeting ANO1. d Densitometric quantification of Western blot analysis of siRNA knockdown. β-Actin was used as a loading control. (n) = number of individual experiments
Mentions: To quantify the ANO1-mediated part of the total CaCC current, we used siRNA knockdown, as well as the three Cl− channel inhibitors NS3728 (10 μM), CaCCinh-A01 (20 μM), and T16Ainh-A01 (20 μM). NS3728 is an inhibitor of VRAC but was also found to inhibit CaCC [17]. CaCCinh-A01 is known to potently inhibit CaCC current but was further shown to alter [Ca2+]i signals [18]. T16Ainh-A01 is the latest developed inhibitor of ANO1; no unspecific effects have been identified so far. Results are shown in Fig. 3. Application of T16Ainh-A01 attenuated the CaCC current significantly in both Capan-1 (43 ± 8 % inhibition at +67 mV) and AsPC-1 (29 ± 2 %) cells, but was ineffective on BxPC-3 cells (Fig. 3b). CaCCinh-A01 inhibited the current more effectively in all tested cell lines with 56 ± 6 % inhibition in Capan-1, 46 ± 6 % in AsPC-1, and 29 ± 3 % in BxPC-3 cells, respectively (all calculated at +67 mV). Application of NS3728 resulted in the most pronounced inhibition in all cell lines with 77 ± 26 % in Capan-1, 67 ± 9 % in AsPC-1, and 54 ± 8 % in BxPC-3 cells at +67 mV. Using siRNA targeting ANO1, the CaCC currents decreased by 88 ± 42 % in Capan-1, 89 ± 51 % in AsPC-1, and 84 ± 23 % in BxPC-3 cells when compared to currents measured in cells transfected with scrambled siRNA (MOCK) (Fig. 3c). Successful knockdown of ANO1 protein expression was confirmed using Western blot (Fig. 3d). Protein expression was decreased to 37 ± 8 % in Capan-1, 54 ± 14 % in AsPC-1, and 20 ± 7 % in BxPC-3 cells.Fig. 3

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