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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

ATP stimulates pore formation. Yo-Pro-1 (2.5 μM) uptake was monitored after stimulation with 5 mM ATP alone, and in combination with a pre-inhibition (1 h) with AZ10606120 (10 μM) or A438079 (10 μM). Fluorescence of YoPro-1 (Ex. 490 nm/Em. 510 nm) was measured in Panc-1 (a, b), AsPC-1 (c, d) and BxPC-3 (e, f). a, c, e. Fluorescence values taken every 10 min for 60 min and normalized to the average of values before administration of ATP. Representative images of Yo-Pro-1 green fluorescence for control, 5 mM ATP and ionomycin stimulated cells (used as control to check Yo-Pro-1 efficacy) are shown as inserts for each cell lines. Note that BxPC-3 and AsPC-1 cells are more affected by ATP and ionomycin, compared to Panc-1 cells, which agrees with data in Fig. 3 and the fact that the pore inhibitor has greater effect. All bars are 100 μm. b, d, f. Values of normalized fluorescence at 60 min. The lines and bargraphs show data of four experiments (mean ± SEM); each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**, ##) from the respective control (#, ##) and with/without inhibitor (*, **) are indicated
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Fig7: ATP stimulates pore formation. Yo-Pro-1 (2.5 μM) uptake was monitored after stimulation with 5 mM ATP alone, and in combination with a pre-inhibition (1 h) with AZ10606120 (10 μM) or A438079 (10 μM). Fluorescence of YoPro-1 (Ex. 490 nm/Em. 510 nm) was measured in Panc-1 (a, b), AsPC-1 (c, d) and BxPC-3 (e, f). a, c, e. Fluorescence values taken every 10 min for 60 min and normalized to the average of values before administration of ATP. Representative images of Yo-Pro-1 green fluorescence for control, 5 mM ATP and ionomycin stimulated cells (used as control to check Yo-Pro-1 efficacy) are shown as inserts for each cell lines. Note that BxPC-3 and AsPC-1 cells are more affected by ATP and ionomycin, compared to Panc-1 cells, which agrees with data in Fig. 3 and the fact that the pore inhibitor has greater effect. All bars are 100 μm. b, d, f. Values of normalized fluorescence at 60 min. The lines and bargraphs show data of four experiments (mean ± SEM); each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**, ##) from the respective control (#, ##) and with/without inhibitor (*, **) are indicated

Mentions: One of the hallmarks of P2X7R is the ability to form pores permeable to larger molecules up to 900 Da, as shown in the above experiments. Therefore, we investigated effect of high ATP concentration (5 mM) on membrane permeabilization using YoPro-1 dye in a physiological buffer and results are shown in Fig. 7. The cell lines tested showed an increase in YoPro-1 uptake after stimulation with 5 mM ATP. The maximum YoPro-1 uptake with 5 mM ATP was detected after 20 min in AsPC-1 and Panc-1 cells, and after 50–60 min in BxPC-3 cells. We tried to inhibit P2X7R and P2X7R-mediated pore formation using two inhibitors: the allosteric inhibitor of P2X7R AZ10606120 (10 μM); and the competitive antagonist A438079 (10 μM), the latter known as the pore inhibitor. All cell lines tested showed a small but significant reduction in pore formation when A438079 was used: for YoPro-1 uptake at 60 min, this inhibition was around 6 % in BxPC-3, 13 % in AsPC-1 and 4.5 % in Panc-1 cells. A438079 protected Panc-1 cells from pore-formation in the short-term experiment (Fig. 7) and from cell death in long-term experiment where BrdU incorporation was monitored (Fig. 5). The allosteric inhibitor, AZ10606120, had no significant effect on YoPro-1 uptake in Panc-1 and AsPC-1 cells and showed a small inhibition of 6 % only in BxPC-3 cells. In an additional series of experiments we also tested 1 mM ATP on YoPro-1 uptake, but effects on pore formation were even smaller than with 5 mM ATP and these are shown in Additional file 5: Figure S4. Interestingly, although 1–5 mM ATP did not cause very pronounced pore-formation in PDAC cells in given experimental conditions and A438079 had relatively small protective effects, the inhibitor could nevertheless protected PDAC cells from cell death during long-term incubation as shown in Fig. 5.Fig. 7


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

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

ATP stimulates pore formation. Yo-Pro-1 (2.5 μM) uptake was monitored after stimulation with 5 mM ATP alone, and in combination with a pre-inhibition (1 h) with AZ10606120 (10 μM) or A438079 (10 μM). Fluorescence of YoPro-1 (Ex. 490 nm/Em. 510 nm) was measured in Panc-1 (a, b), AsPC-1 (c, d) and BxPC-3 (e, f). a, c, e. Fluorescence values taken every 10 min for 60 min and normalized to the average of values before administration of ATP. Representative images of Yo-Pro-1 green fluorescence for control, 5 mM ATP and ionomycin stimulated cells (used as control to check Yo-Pro-1 efficacy) are shown as inserts for each cell lines. Note that BxPC-3 and AsPC-1 cells are more affected by ATP and ionomycin, compared to Panc-1 cells, which agrees with data in Fig. 3 and the fact that the pore inhibitor has greater effect. All bars are 100 μm. b, d, f. Values of normalized fluorescence at 60 min. The lines and bargraphs show data of four experiments (mean ± SEM); each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**, ##) from the respective control (#, ##) and with/without inhibitor (*, **) are indicated
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4660609&req=5

Fig7: ATP stimulates pore formation. Yo-Pro-1 (2.5 μM) uptake was monitored after stimulation with 5 mM ATP alone, and in combination with a pre-inhibition (1 h) with AZ10606120 (10 μM) or A438079 (10 μM). Fluorescence of YoPro-1 (Ex. 490 nm/Em. 510 nm) was measured in Panc-1 (a, b), AsPC-1 (c, d) and BxPC-3 (e, f). a, c, e. Fluorescence values taken every 10 min for 60 min and normalized to the average of values before administration of ATP. Representative images of Yo-Pro-1 green fluorescence for control, 5 mM ATP and ionomycin stimulated cells (used as control to check Yo-Pro-1 efficacy) are shown as inserts for each cell lines. Note that BxPC-3 and AsPC-1 cells are more affected by ATP and ionomycin, compared to Panc-1 cells, which agrees with data in Fig. 3 and the fact that the pore inhibitor has greater effect. All bars are 100 μm. b, d, f. Values of normalized fluorescence at 60 min. The lines and bargraphs show data of four experiments (mean ± SEM); each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**, ##) from the respective control (#, ##) and with/without inhibitor (*, **) are indicated
Mentions: One of the hallmarks of P2X7R is the ability to form pores permeable to larger molecules up to 900 Da, as shown in the above experiments. Therefore, we investigated effect of high ATP concentration (5 mM) on membrane permeabilization using YoPro-1 dye in a physiological buffer and results are shown in Fig. 7. The cell lines tested showed an increase in YoPro-1 uptake after stimulation with 5 mM ATP. The maximum YoPro-1 uptake with 5 mM ATP was detected after 20 min in AsPC-1 and Panc-1 cells, and after 50–60 min in BxPC-3 cells. We tried to inhibit P2X7R and P2X7R-mediated pore formation using two inhibitors: the allosteric inhibitor of P2X7R AZ10606120 (10 μM); and the competitive antagonist A438079 (10 μM), the latter known as the pore inhibitor. All cell lines tested showed a small but significant reduction in pore formation when A438079 was used: for YoPro-1 uptake at 60 min, this inhibition was around 6 % in BxPC-3, 13 % in AsPC-1 and 4.5 % in Panc-1 cells. A438079 protected Panc-1 cells from pore-formation in the short-term experiment (Fig. 7) and from cell death in long-term experiment where BrdU incorporation was monitored (Fig. 5). The allosteric inhibitor, AZ10606120, had no significant effect on YoPro-1 uptake in Panc-1 and AsPC-1 cells and showed a small inhibition of 6 % only in BxPC-3 cells. In an additional series of experiments we also tested 1 mM ATP on YoPro-1 uptake, but effects on pore formation were even smaller than with 5 mM ATP and these are shown in Additional file 5: Figure S4. Interestingly, although 1–5 mM ATP did not cause very pronounced pore-formation in PDAC cells in given experimental conditions and A438079 had relatively small protective effects, the inhibitor could nevertheless protected PDAC cells from cell death during long-term incubation as shown in Fig. 5.Fig. 7

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