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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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Extracellular ATP triggers apoptosis in a caspase- and calcium influx–independent way. (A) SiHa was exposed or not for 30 min to 0.6 mM EGTA or 50 μM zAsp, and then 5 mM ATP or 100 μM BzATP was added for 48 or 24 h, respectively, and apoptosis and necrosis was measured according to annexin V/PI binding (see Materials and Methods). Right, average values measured in each gate. NORMAL cells, annexin V–/IP–; NECR cells annexin, V–/IP+; EARLY APO cells, annexin V+/IP–; LATE APO cells, annexin V+/IP+. (B) Dose–response curve of BzATP treatment for 24 h measured with number of viable cells using trypan blue dye exclusion. (C) Number of viable cells not stained with trypan blue, after exposure of SiHa cells to 300 μM nondegradable ATP, ATPyS, for 24 h with or without previous exposure to 0.6 mM EGTA or 50 μM zAsp. (D) Apoptosis and necrosis induction at these same conditions. *p < 0.05 compared with other treatments (two-way ANOVA, followed by Bonferroni posttest).
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Figure 3: Extracellular ATP triggers apoptosis in a caspase- and calcium influx–independent way. (A) SiHa was exposed or not for 30 min to 0.6 mM EGTA or 50 μM zAsp, and then 5 mM ATP or 100 μM BzATP was added for 48 or 24 h, respectively, and apoptosis and necrosis was measured according to annexin V/PI binding (see Materials and Methods). Right, average values measured in each gate. NORMAL cells, annexin V–/IP–; NECR cells annexin, V–/IP+; EARLY APO cells, annexin V+/IP–; LATE APO cells, annexin V+/IP+. (B) Dose–response curve of BzATP treatment for 24 h measured with number of viable cells using trypan blue dye exclusion. (C) Number of viable cells not stained with trypan blue, after exposure of SiHa cells to 300 μM nondegradable ATP, ATPyS, for 24 h with or without previous exposure to 0.6 mM EGTA or 50 μM zAsp. (D) Apoptosis and necrosis induction at these same conditions. *p < 0.05 compared with other treatments (two-way ANOVA, followed by Bonferroni posttest).

Mentions: The main mechanism of P2×7-mediated cell death in normal human ectocervical epithelial cells (hECEs) involves pore formation, calcium influx, and apoptosis induction through caspase activation (Wang et al., 2004). Surprisingly, neither ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) nor the pancaspase inhibitor zAsp-CH(2)-DCB (zAsp) was able to reduced ATP toxicity (Figure 3A). Of interest, BzATP, a P2×7 agonist, showed a cytotoxic effect in a dose-dependent manner, with EC50 ≈ 100 μM (Figure 3B), and this cytotoxicity was apoptotic and completely blocked by EGTA and zAsp (Figure 3A), suggesting that P2×7 is functional in inducing cell death but does not respond to concentrations of ATP above the described concentration needed for activation of this receptor. In addition, when SiHa cells were treated with ATPγS, a nondegradable ATP form, only ∼20% of cells died (Figure 3C), which was completely blocked by calcium chelation and caspase inhibition (Figure 3, C and D), indicating that a degradation product of ATP plays a role in its cytotoxicity.


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)

Extracellular ATP triggers apoptosis in a caspase- and calcium influx–independent way. (A) SiHa was exposed or not for 30 min to 0.6 mM EGTA or 50 μM zAsp, and then 5 mM ATP or 100 μM BzATP was added for 48 or 24 h, respectively, and apoptosis and necrosis was measured according to annexin V/PI binding (see Materials and Methods). Right, average values measured in each gate. NORMAL cells, annexin V–/IP–; NECR cells annexin, V–/IP+; EARLY APO cells, annexin V+/IP–; LATE APO cells, annexin V+/IP+. (B) Dose–response curve of BzATP treatment for 24 h measured with number of viable cells using trypan blue dye exclusion. (C) Number of viable cells not stained with trypan blue, after exposure of SiHa cells to 300 μM nondegradable ATP, ATPyS, for 24 h with or without previous exposure to 0.6 mM EGTA or 50 μM zAsp. (D) Apoptosis and necrosis induction at these same conditions. *p < 0.05 compared with other treatments (two-way ANOVA, followed by Bonferroni posttest).
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Figure 3: Extracellular ATP triggers apoptosis in a caspase- and calcium influx–independent way. (A) SiHa was exposed or not for 30 min to 0.6 mM EGTA or 50 μM zAsp, and then 5 mM ATP or 100 μM BzATP was added for 48 or 24 h, respectively, and apoptosis and necrosis was measured according to annexin V/PI binding (see Materials and Methods). Right, average values measured in each gate. NORMAL cells, annexin V–/IP–; NECR cells annexin, V–/IP+; EARLY APO cells, annexin V+/IP–; LATE APO cells, annexin V+/IP+. (B) Dose–response curve of BzATP treatment for 24 h measured with number of viable cells using trypan blue dye exclusion. (C) Number of viable cells not stained with trypan blue, after exposure of SiHa cells to 300 μM nondegradable ATP, ATPyS, for 24 h with or without previous exposure to 0.6 mM EGTA or 50 μM zAsp. (D) Apoptosis and necrosis induction at these same conditions. *p < 0.05 compared with other treatments (two-way ANOVA, followed by Bonferroni posttest).
Mentions: The main mechanism of P2×7-mediated cell death in normal human ectocervical epithelial cells (hECEs) involves pore formation, calcium influx, and apoptosis induction through caspase activation (Wang et al., 2004). Surprisingly, neither ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) nor the pancaspase inhibitor zAsp-CH(2)-DCB (zAsp) was able to reduced ATP toxicity (Figure 3A). Of interest, BzATP, a P2×7 agonist, showed a cytotoxic effect in a dose-dependent manner, with EC50 ≈ 100 μM (Figure 3B), and this cytotoxicity was apoptotic and completely blocked by EGTA and zAsp (Figure 3A), suggesting that P2×7 is functional in inducing cell death but does not respond to concentrations of ATP above the described concentration needed for activation of this receptor. In addition, when SiHa cells were treated with ATPγS, a nondegradable ATP form, only ∼20% of cells died (Figure 3C), which was completely blocked by calcium chelation and caspase inhibition (Figure 3, C and D), indicating that a degradation product of ATP plays a role in its cytotoxicity.

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