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Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells.

Mello Pde A, Filippi-Chiela EC, Nascimento J, Beckenkamp A, Santana DB, Kipper F, Casali EA, Nejar Bruno A, Paccez JD, Zerbini LF, Wink MR, Lenz G, Buffon A - Mol. Biol. Cell (2014)

Bottom Line: Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity.Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole.In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemical and Cytological Analysis, Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, RS 90610-000, Brazil.

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Adenosine uptake and conversion to AMP by adenosine kinase is the major mechanism of toxicity triggered by extracellular ATP in SiHa cells. (A) Extracellular ATP hydrolysis and product formation in SiHa cell line. Cells were incubated with 5 mM ATP, and levels of nucleotides in cell medium were analyzed by HPLC after treatment times of 0, 24, 48, and 72 h. A control without ATP was done for basal determination of nucleotides released by cells (Supplemental Table S1). ATP, ADP, AMP, adenosine (ADO), inosine (INO), and hypoxanthine (HYPO) contents in reaction medium were represented by exogenous (added) plus endogenous (secreted) purinergic compound as mean (nanomoles) ± SD. (B) Effect of ATP metabolites on SiHa cell death. Cells were incubated with different concentrations of ADP, AMP, and adenosine for 24 h, and the number of viable cells was determined as described in Materials and Methods. *p < 0,05 compared with control (one-way ANOVA, followed by Tukey's test). (C, D) Dipyridamole blockage of 5 mM ATP induces cell death by inhibiting extracellular adenosine uptake. SiHa cells were exposed to 10 μM dipyridamole alone or for 30 min, and then 5 mM ATP was added for 24, 48, and 72 h. (C) Number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). (D) Apoptosis and necrosis index according to annexin V/IP stain. Images were taken from SiHa cell line after the foregoing treatments. Cell nuclei were stained with Hoescht 35565665 according to manufacturer's instruction. Note the extinction of apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP only (Figure 2C). *p < 0.05 compared with dipyridamole alone (two-way ANOVA, followed by Bonferroni posttest). (E) Adenosine kinase inhibitor (ABT-702) blockage of 5 mM ATP induces cell death by inhibiting intracellularly transported adenosine phosphorylation and conversion to AMP. SiHa cells were exposed to 100 nM ABT-702 for 30 min and followed or not by 5 mM ATP for 48 and 72 h. ABT-702, 100 nM, was replaced each 24 h. Top, number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). Bottom, apoptosis and necrosis index according to annexin V/IP stain and representative images of SiHa cells after foregoing treatments. Scale bars, 20 μm; magnification, 20×.
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Figure 6: Adenosine uptake and conversion to AMP by adenosine kinase is the major mechanism of toxicity triggered by extracellular ATP in SiHa cells. (A) Extracellular ATP hydrolysis and product formation in SiHa cell line. Cells were incubated with 5 mM ATP, and levels of nucleotides in cell medium were analyzed by HPLC after treatment times of 0, 24, 48, and 72 h. A control without ATP was done for basal determination of nucleotides released by cells (Supplemental Table S1). ATP, ADP, AMP, adenosine (ADO), inosine (INO), and hypoxanthine (HYPO) contents in reaction medium were represented by exogenous (added) plus endogenous (secreted) purinergic compound as mean (nanomoles) ± SD. (B) Effect of ATP metabolites on SiHa cell death. Cells were incubated with different concentrations of ADP, AMP, and adenosine for 24 h, and the number of viable cells was determined as described in Materials and Methods. *p < 0,05 compared with control (one-way ANOVA, followed by Tukey's test). (C, D) Dipyridamole blockage of 5 mM ATP induces cell death by inhibiting extracellular adenosine uptake. SiHa cells were exposed to 10 μM dipyridamole alone or for 30 min, and then 5 mM ATP was added for 24, 48, and 72 h. (C) Number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). (D) Apoptosis and necrosis index according to annexin V/IP stain. Images were taken from SiHa cell line after the foregoing treatments. Cell nuclei were stained with Hoescht 35565665 according to manufacturer's instruction. Note the extinction of apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP only (Figure 2C). *p < 0.05 compared with dipyridamole alone (two-way ANOVA, followed by Bonferroni posttest). (E) Adenosine kinase inhibitor (ABT-702) blockage of 5 mM ATP induces cell death by inhibiting intracellularly transported adenosine phosphorylation and conversion to AMP. SiHa cells were exposed to 100 nM ABT-702 for 30 min and followed or not by 5 mM ATP for 48 and 72 h. ABT-702, 100 nM, was replaced each 24 h. Top, number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). Bottom, apoptosis and necrosis index according to annexin V/IP stain and representative images of SiHa cells after foregoing treatments. Scale bars, 20 μm; magnification, 20×.

Mentions: Thus far our data suggest that P2×7 activation per se only eliminates cells with high expression levels of P2×7. To understand where the additional toxicity of ATP comes from, we turned our attention to adenine nucleotides and adenosine formed from ATP by the action of ectonucleotidases, which are expressed in human cervical cancer cells (Beckenkamp et al., 2014). Most of the extracellular ATP was degraded to its metabolites over 72 h (Figure 6A; see also Supplemental Table S1). All of the main metabolites of ATP had toxic effects in cervical cancer cell lines (SiHa, HeLa, and C33A) and a human epithelial cell line (HaCaT), with C33A and HeLa cells presenting a more resistant profile. Of importance, only adenosine significantly reduced cell viability in all cell lines (Figures 6B and Supplemental Figure S3A). Because several studies attributed a cytotoxic effect to adenosine in other cell types (Saitoh et al., 2004; Sai et al., 2006), we investigated whether uptake of extracellular adenosine formed by ATP degradation, which did not accumulate in extracellular medium (Supplemental Figure S5A), was responsible for apoptosis induction after ATP treatment. We treated SiHa cells with 10 μM dipyridamole (DIP), an inhibitor of adenosine transport, 30 min before ATP exposure (Figure 6C, left). DIP reduced cell shrinkage (Supplemental Figure S1), cell number reduction (Figure 6C, right), and annexin V staining (Figures 6D, right, and Supplemental Figure S1) induced by ATP treatment. Furthermore, phenotypic observation confirmed the reduction of apoptotic features such as cell shrinkage, membrane blebbing, and nuclear condensation and fragmentation (Figure 6D, left and middle), which were present after ATP treatment only (Figure 2C). After 72 h, there was a reduction of 20% in the cell number in DIP plus ATP treatment, which was not altered by DIP replacement each 24 h (unpublished data) but was by knockdown of the P2×7 receptor (Supplemental Figure S2), suggesting that this slight effect occurs through ATP-mediated P2×7 receptor activation and toxicity and is not due to the loss of action of DIP. Taken together, these results strongly suggest that adenosine uptake, formed by ATP degradation, is a central player in the cell death induced by extracellular ATP. In agreement, inhibition of adenosine kinase by ABT-702 completely reversed ATP-induced apoptosis (Figure 6E, bottom), indicating that intracellular adenosine phosphorylation and conversion to AMP is a key step in the toxicity of extracellular ATP. Indeed, as occurred with DIP plus ATP, there was a reduction of 20% in the number of cells after 72 h of treatment with ABT-702 plus ATP (Figure 6E, top), reinforcing the slight involvement of ATP-P2×7 in ATP-induced cell death and the importance of the metabolization of adenosine to AMP. Corroborating these data, sensitivity of cells to adenosine was strongly positively correlated (r = 0.9) with ATP cytotoxic effect in the four cell lines studied. On the other hand, ATP sensitivity at 24 h was not correlated with mRNA P2×7 levels (Supplemental Figure S3). Of interest, when cells were exposed to ATP for 48 or 72 h, the correlation between ATP sensitivity and mRNA P2×7 levels increased (unpublished data), suggesting that P2×7 activation could be important after a long exposure and thus could be involved with the cell death observed after DIP plus ATP at 72 h.


Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells.

Mello Pde A, Filippi-Chiela EC, Nascimento J, Beckenkamp A, Santana DB, Kipper F, Casali EA, Nejar Bruno A, Paccez JD, Zerbini LF, Wink MR, Lenz G, Buffon A - Mol. Biol. Cell (2014)

Adenosine uptake and conversion to AMP by adenosine kinase is the major mechanism of toxicity triggered by extracellular ATP in SiHa cells. (A) Extracellular ATP hydrolysis and product formation in SiHa cell line. Cells were incubated with 5 mM ATP, and levels of nucleotides in cell medium were analyzed by HPLC after treatment times of 0, 24, 48, and 72 h. A control without ATP was done for basal determination of nucleotides released by cells (Supplemental Table S1). ATP, ADP, AMP, adenosine (ADO), inosine (INO), and hypoxanthine (HYPO) contents in reaction medium were represented by exogenous (added) plus endogenous (secreted) purinergic compound as mean (nanomoles) ± SD. (B) Effect of ATP metabolites on SiHa cell death. Cells were incubated with different concentrations of ADP, AMP, and adenosine for 24 h, and the number of viable cells was determined as described in Materials and Methods. *p < 0,05 compared with control (one-way ANOVA, followed by Tukey's test). (C, D) Dipyridamole blockage of 5 mM ATP induces cell death by inhibiting extracellular adenosine uptake. SiHa cells were exposed to 10 μM dipyridamole alone or for 30 min, and then 5 mM ATP was added for 24, 48, and 72 h. (C) Number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). (D) Apoptosis and necrosis index according to annexin V/IP stain. Images were taken from SiHa cell line after the foregoing treatments. Cell nuclei were stained with Hoescht 35565665 according to manufacturer's instruction. Note the extinction of apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP only (Figure 2C). *p < 0.05 compared with dipyridamole alone (two-way ANOVA, followed by Bonferroni posttest). (E) Adenosine kinase inhibitor (ABT-702) blockage of 5 mM ATP induces cell death by inhibiting intracellularly transported adenosine phosphorylation and conversion to AMP. SiHa cells were exposed to 100 nM ABT-702 for 30 min and followed or not by 5 mM ATP for 48 and 72 h. ABT-702, 100 nM, was replaced each 24 h. Top, number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). Bottom, apoptosis and necrosis index according to annexin V/IP stain and representative images of SiHa cells after foregoing treatments. Scale bars, 20 μm; magnification, 20×.
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Figure 6: Adenosine uptake and conversion to AMP by adenosine kinase is the major mechanism of toxicity triggered by extracellular ATP in SiHa cells. (A) Extracellular ATP hydrolysis and product formation in SiHa cell line. Cells were incubated with 5 mM ATP, and levels of nucleotides in cell medium were analyzed by HPLC after treatment times of 0, 24, 48, and 72 h. A control without ATP was done for basal determination of nucleotides released by cells (Supplemental Table S1). ATP, ADP, AMP, adenosine (ADO), inosine (INO), and hypoxanthine (HYPO) contents in reaction medium were represented by exogenous (added) plus endogenous (secreted) purinergic compound as mean (nanomoles) ± SD. (B) Effect of ATP metabolites on SiHa cell death. Cells were incubated with different concentrations of ADP, AMP, and adenosine for 24 h, and the number of viable cells was determined as described in Materials and Methods. *p < 0,05 compared with control (one-way ANOVA, followed by Tukey's test). (C, D) Dipyridamole blockage of 5 mM ATP induces cell death by inhibiting extracellular adenosine uptake. SiHa cells were exposed to 10 μM dipyridamole alone or for 30 min, and then 5 mM ATP was added for 24, 48, and 72 h. (C) Number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). (D) Apoptosis and necrosis index according to annexin V/IP stain. Images were taken from SiHa cell line after the foregoing treatments. Cell nuclei were stained with Hoescht 35565665 according to manufacturer's instruction. Note the extinction of apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP only (Figure 2C). *p < 0.05 compared with dipyridamole alone (two-way ANOVA, followed by Bonferroni posttest). (E) Adenosine kinase inhibitor (ABT-702) blockage of 5 mM ATP induces cell death by inhibiting intracellularly transported adenosine phosphorylation and conversion to AMP. SiHa cells were exposed to 100 nM ABT-702 for 30 min and followed or not by 5 mM ATP for 48 and 72 h. ABT-702, 100 nM, was replaced each 24 h. Top, number of viable cells after treatment. *p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest). Bottom, apoptosis and necrosis index according to annexin V/IP stain and representative images of SiHa cells after foregoing treatments. Scale bars, 20 μm; magnification, 20×.
Mentions: Thus far our data suggest that P2×7 activation per se only eliminates cells with high expression levels of P2×7. To understand where the additional toxicity of ATP comes from, we turned our attention to adenine nucleotides and adenosine formed from ATP by the action of ectonucleotidases, which are expressed in human cervical cancer cells (Beckenkamp et al., 2014). Most of the extracellular ATP was degraded to its metabolites over 72 h (Figure 6A; see also Supplemental Table S1). All of the main metabolites of ATP had toxic effects in cervical cancer cell lines (SiHa, HeLa, and C33A) and a human epithelial cell line (HaCaT), with C33A and HeLa cells presenting a more resistant profile. Of importance, only adenosine significantly reduced cell viability in all cell lines (Figures 6B and Supplemental Figure S3A). Because several studies attributed a cytotoxic effect to adenosine in other cell types (Saitoh et al., 2004; Sai et al., 2006), we investigated whether uptake of extracellular adenosine formed by ATP degradation, which did not accumulate in extracellular medium (Supplemental Figure S5A), was responsible for apoptosis induction after ATP treatment. We treated SiHa cells with 10 μM dipyridamole (DIP), an inhibitor of adenosine transport, 30 min before ATP exposure (Figure 6C, left). DIP reduced cell shrinkage (Supplemental Figure S1), cell number reduction (Figure 6C, right), and annexin V staining (Figures 6D, right, and Supplemental Figure S1) induced by ATP treatment. Furthermore, phenotypic observation confirmed the reduction of apoptotic features such as cell shrinkage, membrane blebbing, and nuclear condensation and fragmentation (Figure 6D, left and middle), which were present after ATP treatment only (Figure 2C). After 72 h, there was a reduction of 20% in the cell number in DIP plus ATP treatment, which was not altered by DIP replacement each 24 h (unpublished data) but was by knockdown of the P2×7 receptor (Supplemental Figure S2), suggesting that this slight effect occurs through ATP-mediated P2×7 receptor activation and toxicity and is not due to the loss of action of DIP. Taken together, these results strongly suggest that adenosine uptake, formed by ATP degradation, is a central player in the cell death induced by extracellular ATP. In agreement, inhibition of adenosine kinase by ABT-702 completely reversed ATP-induced apoptosis (Figure 6E, bottom), indicating that intracellular adenosine phosphorylation and conversion to AMP is a key step in the toxicity of extracellular ATP. Indeed, as occurred with DIP plus ATP, there was a reduction of 20% in the number of cells after 72 h of treatment with ABT-702 plus ATP (Figure 6E, top), reinforcing the slight involvement of ATP-P2×7 in ATP-induced cell death and the importance of the metabolization of adenosine to AMP. Corroborating these data, sensitivity of cells to adenosine was strongly positively correlated (r = 0.9) with ATP cytotoxic effect in the four cell lines studied. On the other hand, ATP sensitivity at 24 h was not correlated with mRNA P2×7 levels (Supplemental Figure S3). Of interest, when cells were exposed to ATP for 48 or 72 h, the correlation between ATP sensitivity and mRNA P2×7 levels increased (unpublished data), suggesting that P2×7 activation could be important after a long exposure and thus could be involved with the cell death observed after DIP plus ATP at 72 h.

Bottom Line: Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity.Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole.In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemical and Cytological Analysis, Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, RS 90610-000, Brazil.

Show MeSH
Related in: MedlinePlus