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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

Effect of ATP and A438079, KN-62 on BrdU incorporation in Panc-1 and BxPC-3. Data show the BrdU incorporation in both cell lines after 60 h. The colored columns show the effect of different concentrations of added (exogenous) ATP (100 μM, 1 mM and 5 mM). The white columns show the effect of ATP and control (no exogenous ATP added) in combination with the two inhibitors A438079 (10 μM) or KN-62 (100 nM), which were added 1 h before ATP. The data were normalized to 1 % serum control (100). The graphs show data from three to six experiments (mean ± SEM); where each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**) from the respective 1 % serum control (*, **) and with/without inhibitor (#) are indicated
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Fig5: Effect of ATP and A438079, KN-62 on BrdU incorporation in Panc-1 and BxPC-3. Data show the BrdU incorporation in both cell lines after 60 h. The colored columns show the effect of different concentrations of added (exogenous) ATP (100 μM, 1 mM and 5 mM). The white columns show the effect of ATP and control (no exogenous ATP added) in combination with the two inhibitors A438079 (10 μM) or KN-62 (100 nM), which were added 1 h before ATP. The data were normalized to 1 % serum control (100). The graphs show data from three to six experiments (mean ± SEM); where each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**) from the respective 1 % serum control (*, **) and with/without inhibitor (#) are indicated

Mentions: In the next series of experiments we tested the effects of P2X7R inhibitors: the allosteric inhibitor AZ10606120 (10 μM) that affects cell proliferation; the competitive antagonist A438079 (10 μM) that inhibits pore formation; and the non-competitive antagonist KN-62 (100 nM) that also inhibits CaM Kinase II [24, 27, 39–41]. Experiments where AZ10606120 was given without exogenous ATP (i.e. basal conditions) resulted in decreased BrdU incorporation in all cell lines, with the largest decrease in Panc-1 and the lowest decrease in HPDE and Capan-1 cells (Fig. 3, colored symbols). This would indicate either that the inhibitor attenuated proliferation or that it was cytotoxic and promoted cell death. We excluded the cytotoxic effect of the inhibitor with a Trypan blue assay (Additional file 4: Figure S3). Therefore, the inhibitory effect of AZ10606120 in basal conditions was most likely related to a decrease in proliferation. Furthermore, the inhibitor in combination with added ATP concentrations (0.1–1.0 mM), resulted in a further reduction of BrdU incorporation in all PDAC cell lines. Accepting that the inhibitor is not cytotoxic, we presume it reduced BrdU incorporation because it inhibited cell proliferation, as also seen in other cells [17, 27, 41]. At about 5 mM ATP effects of the inhibitor were the smallest, indicating that cell death outweighted cell proliferation. Thus Fig. 3 shows two effects on BrdU incorporation in a population of cells—increasing ATP concentration causing cell death (see below) and AZ10606120 causing a decrease in cell proliferation. Interestingly, AZ10606120 was ineffective in HPDE cells treated with 0.1–5 mM exogenous ATP, indicating that in these conditions cell death was the predominant P2X7R response in these non-cancer cells. In addition to AZ10606120, we also studied the long-term effect of two other P2X7R inhibitors, the pore-inhibitor A438079 and KN-62, and the results are shown in Fig. 5. It appears that these inhibitors improved BrdU incorporation if cells were stimulated with 0.1–1 mM ATP. Thus these inhibitors protected cells against cell death until exogenous concentrations of ATP were 5 mM.Fig. 5


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

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

Effect of ATP and A438079, KN-62 on BrdU incorporation in Panc-1 and BxPC-3. Data show the BrdU incorporation in both cell lines after 60 h. The colored columns show the effect of different concentrations of added (exogenous) ATP (100 μM, 1 mM and 5 mM). The white columns show the effect of ATP and control (no exogenous ATP added) in combination with the two inhibitors A438079 (10 μM) or KN-62 (100 nM), which were added 1 h before ATP. The data were normalized to 1 % serum control (100). The graphs show data from three to six experiments (mean ± SEM); where each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**) from the respective 1 % serum control (*, **) and with/without inhibitor (#) are indicated
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4660609&req=5

Fig5: Effect of ATP and A438079, KN-62 on BrdU incorporation in Panc-1 and BxPC-3. Data show the BrdU incorporation in both cell lines after 60 h. The colored columns show the effect of different concentrations of added (exogenous) ATP (100 μM, 1 mM and 5 mM). The white columns show the effect of ATP and control (no exogenous ATP added) in combination with the two inhibitors A438079 (10 μM) or KN-62 (100 nM), which were added 1 h before ATP. The data were normalized to 1 % serum control (100). The graphs show data from three to six experiments (mean ± SEM); where each run was carried out in triplicates. Significant differences P < 0.05 (*, #) and P < 0.001 (**) from the respective 1 % serum control (*, **) and with/without inhibitor (#) are indicated
Mentions: In the next series of experiments we tested the effects of P2X7R inhibitors: the allosteric inhibitor AZ10606120 (10 μM) that affects cell proliferation; the competitive antagonist A438079 (10 μM) that inhibits pore formation; and the non-competitive antagonist KN-62 (100 nM) that also inhibits CaM Kinase II [24, 27, 39–41]. Experiments where AZ10606120 was given without exogenous ATP (i.e. basal conditions) resulted in decreased BrdU incorporation in all cell lines, with the largest decrease in Panc-1 and the lowest decrease in HPDE and Capan-1 cells (Fig. 3, colored symbols). This would indicate either that the inhibitor attenuated proliferation or that it was cytotoxic and promoted cell death. We excluded the cytotoxic effect of the inhibitor with a Trypan blue assay (Additional file 4: Figure S3). Therefore, the inhibitory effect of AZ10606120 in basal conditions was most likely related to a decrease in proliferation. Furthermore, the inhibitor in combination with added ATP concentrations (0.1–1.0 mM), resulted in a further reduction of BrdU incorporation in all PDAC cell lines. Accepting that the inhibitor is not cytotoxic, we presume it reduced BrdU incorporation because it inhibited cell proliferation, as also seen in other cells [17, 27, 41]. At about 5 mM ATP effects of the inhibitor were the smallest, indicating that cell death outweighted cell proliferation. Thus Fig. 3 shows two effects on BrdU incorporation in a population of cells—increasing ATP concentration causing cell death (see below) and AZ10606120 causing a decrease in cell proliferation. Interestingly, AZ10606120 was ineffective in HPDE cells treated with 0.1–5 mM exogenous ATP, indicating that in these conditions cell death was the predominant P2X7R response in these non-cancer cells. In addition to AZ10606120, we also studied the long-term effect of two other P2X7R inhibitors, the pore-inhibitor A438079 and KN-62, and the results are shown in Fig. 5. It appears that these inhibitors improved BrdU incorporation if cells were stimulated with 0.1–1 mM ATP. Thus these inhibitors protected cells against cell death until exogenous concentrations of ATP were 5 mM.Fig. 5

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