Limits...
The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells.

Giannuzzo A, Pedersen SF, Novak I - Mol. Cancer (2015)

Bottom Line: We found higher expression of P2X7R protein in PDAC compared to HPDE cells.P2X7R had notable disparate effects on PDAC survival.AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section of Cell Biology and Physiology, University of Copenhagen, August Krogh Building, Universitetsparken 13, DK-2100, Copenhagen, Denmark. andrea.giannuzzo@bio.ku.dk.

ABSTRACT

Background: Pancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour.

Methods: We determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a "normal" human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays).

Results: We found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R-pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion.

Conclusions: PDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus

Immunolocalization of P2X7R in PDACs and HPDE cells. HPDE and Panc-1 cells were grown on coverslips. P2X7R was stained with a polyclonal antibody against the extracellular domain and Alexa Fluor 568 (red) and DAPI was used to stain the nucleus (blue). All bars are 25 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4660609&req=5

Fig2: Immunolocalization of P2X7R in PDACs and HPDE cells. HPDE and Panc-1 cells were grown on coverslips. P2X7R was stained with a polyclonal antibody against the extracellular domain and Alexa Fluor 568 (red) and DAPI was used to stain the nucleus (blue). All bars are 25 μm

Mentions: Localization of P2X7R was detected by immunofluorescence and confocal microscopy and the results are shown in Fig. 2. The immunofluorescence was observed in HPDE and Panc-1 cells and the immunofluorescence signal resulting from the polyclonal antibody recognizing the extracellular loop, hence the A and B and potentially other isoforms, was localized to the plasma membrane, and weakly also in the cytoplasmic compartment (Fig. 2).Fig. 2


The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells.

Giannuzzo A, Pedersen SF, Novak I - Mol. Cancer (2015)

Immunolocalization of P2X7R in PDACs and HPDE cells. HPDE and Panc-1 cells were grown on coverslips. P2X7R was stained with a polyclonal antibody against the extracellular domain and Alexa Fluor 568 (red) and DAPI was used to stain the nucleus (blue). All bars are 25 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4660609&req=5

Fig2: Immunolocalization of P2X7R in PDACs and HPDE cells. HPDE and Panc-1 cells were grown on coverslips. P2X7R was stained with a polyclonal antibody against the extracellular domain and Alexa Fluor 568 (red) and DAPI was used to stain the nucleus (blue). All bars are 25 μm
Mentions: Localization of P2X7R was detected by immunofluorescence and confocal microscopy and the results are shown in Fig. 2. The immunofluorescence was observed in HPDE and Panc-1 cells and the immunofluorescence signal resulting from the polyclonal antibody recognizing the extracellular loop, hence the A and B and potentially other isoforms, was localized to the plasma membrane, and weakly also in the cytoplasmic compartment (Fig. 2).Fig. 2

Bottom Line: We found higher expression of P2X7R protein in PDAC compared to HPDE cells.P2X7R had notable disparate effects on PDAC survival.AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section of Cell Biology and Physiology, University of Copenhagen, August Krogh Building, Universitetsparken 13, DK-2100, Copenhagen, Denmark. andrea.giannuzzo@bio.ku.dk.

ABSTRACT

Background: Pancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour.

Methods: We determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a "normal" human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays).

Results: We found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R-pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion.

Conclusions: PDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus