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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

ANO1 is upregulated at the mRNA and protein levels in all PDAC cell lines tested. a Mean ANO1 expressions measured by RT-qPCR in five PDAC cell lines (Panc-1, Mia PaCa-2, BxPC-3, AsPC-1, and Capan-1) compared to the immortalized human pancreatic ductal cell line (HPDE). Data for Capan-1 was taken from [45] and recalculated relative to the value in the HPDE cell line. b Western blot analysis in three pancreatic cancer cell lines and HPDE with β-actin as a loading control. Mean ± s.e.m. of n = 4–5 individual experiments
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Fig1: ANO1 is upregulated at the mRNA and protein levels in all PDAC cell lines tested. a Mean ANO1 expressions measured by RT-qPCR in five PDAC cell lines (Panc-1, Mia PaCa-2, BxPC-3, AsPC-1, and Capan-1) compared to the immortalized human pancreatic ductal cell line (HPDE). Data for Capan-1 was taken from [45] and recalculated relative to the value in the HPDE cell line. b Western blot analysis in three pancreatic cancer cell lines and HPDE with β-actin as a loading control. Mean ± s.e.m. of n = 4–5 individual experiments

Mentions: To study the presence of ANO1 in PDAC cells, we evaluated the expression of ANO1 RNA by RT-qPCR in several commercially available cell lines (Mia PaCa-2, Panc-1, BxPC-3, and AsPC-1). Mia PaCa-2, Panc-1, and BxPC-3 were derived from the primary tumor of an exocrine pancreatic cancer. AsPC-1 was established from a local metastatic site in the ascites of a patient with PDAC [42]. We used a normal pancreatic ductal epithelium derived cell line (HPDE) as a control (Fig. 1). All cell lines showed an increased mRNA level, ranging from 2.3 ± 0.5- to 320 ± 60-fold over that in HPDE cells, in the Panc-1 and BxPC-3 cell lines, respectively. Our earlier study compared ANO1 mRNA levels in Capan-1 PDAC cell line to Panc-1 and CFPAC-1 [45]; using those data together with the values for HPDE and Panc-1 of the present investigation, we calculated an ∼1,450-fold upregulation of ANO1 in Capan-1 with respect to HPDE cells. Capan-1 is a liver metastasis-derived cell line [42]. We selected the aggressive pancreatic cancer cell lines AsPC-1 and BxPC-3 [6], as well as Capan-1 and HPDE cells for further experiments. To validate ANO1 expression also at the protein level in PDAC cells, we used Western blot analysis (Fig. 1b). The results showed a similar trend as observed for mRNA levels, with almost undetectable ANO1 protein expression in HPDE cells and much higher levels in the PDAC cell lines.Fig. 1


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

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

ANO1 is upregulated at the mRNA and protein levels in all PDAC cell lines tested. a Mean ANO1 expressions measured by RT-qPCR in five PDAC cell lines (Panc-1, Mia PaCa-2, BxPC-3, AsPC-1, and Capan-1) compared to the immortalized human pancreatic ductal cell line (HPDE). Data for Capan-1 was taken from [45] and recalculated relative to the value in the HPDE cell line. b Western blot analysis in three pancreatic cancer cell lines and HPDE with β-actin as a loading control. Mean ± s.e.m. of n = 4–5 individual experiments
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Related In: Results  -  Collection

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Fig1: ANO1 is upregulated at the mRNA and protein levels in all PDAC cell lines tested. a Mean ANO1 expressions measured by RT-qPCR in five PDAC cell lines (Panc-1, Mia PaCa-2, BxPC-3, AsPC-1, and Capan-1) compared to the immortalized human pancreatic ductal cell line (HPDE). Data for Capan-1 was taken from [45] and recalculated relative to the value in the HPDE cell line. b Western blot analysis in three pancreatic cancer cell lines and HPDE with β-actin as a loading control. Mean ± s.e.m. of n = 4–5 individual experiments
Mentions: To study the presence of ANO1 in PDAC cells, we evaluated the expression of ANO1 RNA by RT-qPCR in several commercially available cell lines (Mia PaCa-2, Panc-1, BxPC-3, and AsPC-1). Mia PaCa-2, Panc-1, and BxPC-3 were derived from the primary tumor of an exocrine pancreatic cancer. AsPC-1 was established from a local metastatic site in the ascites of a patient with PDAC [42]. We used a normal pancreatic ductal epithelium derived cell line (HPDE) as a control (Fig. 1). All cell lines showed an increased mRNA level, ranging from 2.3 ± 0.5- to 320 ± 60-fold over that in HPDE cells, in the Panc-1 and BxPC-3 cell lines, respectively. Our earlier study compared ANO1 mRNA levels in Capan-1 PDAC cell line to Panc-1 and CFPAC-1 [45]; using those data together with the values for HPDE and Panc-1 of the present investigation, we calculated an ∼1,450-fold upregulation of ANO1 in Capan-1 with respect to HPDE cells. Capan-1 is a liver metastasis-derived cell line [42]. We selected the aggressive pancreatic cancer cell lines AsPC-1 and BxPC-3 [6], as well as Capan-1 and HPDE cells for further experiments. To validate ANO1 expression also at the protein level in PDAC cells, we used Western blot analysis (Fig. 1b). The results showed a similar trend as observed for mRNA levels, with almost undetectable ANO1 protein expression in HPDE cells and much higher levels in the PDAC cell lines.Fig. 1

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