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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

Cl− channel inhibitors alter ATP-induced Ca2+ signals. Intracellular Ca2+ measurements in FURA-2 loaded HPDE and BxPC-3 cells. a Representative single-cell traces of individual experiments. Different substances were applied as indicated. ATP was applied at 10 μM, T16Ainh-A01 and CaCCinh-A01 at 20 μM, and NS3728 at 10 μM. b Summary of maximum ATP-induced changes in [Ca2+]i; for CaCCinh-A01, these values may be overestimates as the peak response was not present. (n) = number of individual experiments; each containing between 5–45 single cells.***p ≤ 0.001 when compared to no inhibitor condition; ##p ≤ 0.01 when compared with HPDE no inhibitor
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Fig8: Cl− channel inhibitors alter ATP-induced Ca2+ signals. Intracellular Ca2+ measurements in FURA-2 loaded HPDE and BxPC-3 cells. a Representative single-cell traces of individual experiments. Different substances were applied as indicated. ATP was applied at 10 μM, T16Ainh-A01 and CaCCinh-A01 at 20 μM, and NS3728 at 10 μM. b Summary of maximum ATP-induced changes in [Ca2+]i; for CaCCinh-A01, these values may be overestimates as the peak response was not present. (n) = number of individual experiments; each containing between 5–45 single cells.***p ≤ 0.001 when compared to no inhibitor condition; ##p ≤ 0.01 when compared with HPDE no inhibitor

Mentions: Most importantly, Fig. 7b also shows that application of NS3728 resulted in a pronounced decrease in cellular proliferation in all cells. The most drastic effect was observed in HPDE control cells. The very low expression level of ANO1 in this cell line suggests that the inhibition of proliferation by NS3728 is not ANO1-mediated, but rather reflects inhibition of VRAC or altered [Ca2+]i signaling (Fig. 8). In agreement with this, it is long known that VRAC is involved in progression throughout the cell cycle of different cancer types [17, 40]. As expected, gemcitabine treatment caused an almost complete inhibition of proliferation in all tested cells (data not shown).Fig. 8


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

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

Cl− channel inhibitors alter ATP-induced Ca2+ signals. Intracellular Ca2+ measurements in FURA-2 loaded HPDE and BxPC-3 cells. a Representative single-cell traces of individual experiments. Different substances were applied as indicated. ATP was applied at 10 μM, T16Ainh-A01 and CaCCinh-A01 at 20 μM, and NS3728 at 10 μM. b Summary of maximum ATP-induced changes in [Ca2+]i; for CaCCinh-A01, these values may be overestimates as the peak response was not present. (n) = number of individual experiments; each containing between 5–45 single cells.***p ≤ 0.001 when compared to no inhibitor condition; ##p ≤ 0.01 when compared with HPDE no inhibitor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4464647&req=5

Fig8: Cl− channel inhibitors alter ATP-induced Ca2+ signals. Intracellular Ca2+ measurements in FURA-2 loaded HPDE and BxPC-3 cells. a Representative single-cell traces of individual experiments. Different substances were applied as indicated. ATP was applied at 10 μM, T16Ainh-A01 and CaCCinh-A01 at 20 μM, and NS3728 at 10 μM. b Summary of maximum ATP-induced changes in [Ca2+]i; for CaCCinh-A01, these values may be overestimates as the peak response was not present. (n) = number of individual experiments; each containing between 5–45 single cells.***p ≤ 0.001 when compared to no inhibitor condition; ##p ≤ 0.01 when compared with HPDE no inhibitor
Mentions: Most importantly, Fig. 7b also shows that application of NS3728 resulted in a pronounced decrease in cellular proliferation in all cells. The most drastic effect was observed in HPDE control cells. The very low expression level of ANO1 in this cell line suggests that the inhibition of proliferation by NS3728 is not ANO1-mediated, but rather reflects inhibition of VRAC or altered [Ca2+]i signaling (Fig. 8). In agreement with this, it is long known that VRAC is involved in progression throughout the cell cycle of different cancer types [17, 40]. As expected, gemcitabine treatment caused an almost complete inhibition of proliferation in all tested cells (data not shown).Fig. 8

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