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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 exerts acute and chronic toxicity in cervical cancer cells. (A) Comparison of P2×7 mRNA expression in SiHa, HeLa, C33A, and HaCaT cell lines by quantitative real-time PCR analysis. Results are presented as the ratio cDNA/GAPDH. (B) SiHa cell viability after exposure to increased ATP concentration for 24 h using MTT assay. (C) Bottom, time curve of SiHa cells treated with 5 mM ATP for 24, 48, and 72 h, determined by number of viable cells not marked by trypan blue. Top, 100 viable cells were seeded in clonogenic assay, and colony formation was evaluated. Numbers at bottom are survival fraction according to clonogenic assay. C, control; ATP, treatment with 5mM ATP. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).
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Figure 1: Extracellular ATP exerts acute and chronic toxicity in cervical cancer cells. (A) Comparison of P2×7 mRNA expression in SiHa, HeLa, C33A, and HaCaT cell lines by quantitative real-time PCR analysis. Results are presented as the ratio cDNA/GAPDH. (B) SiHa cell viability after exposure to increased ATP concentration for 24 h using MTT assay. (C) Bottom, time curve of SiHa cells treated with 5 mM ATP for 24, 48, and 72 h, determined by number of viable cells not marked by trypan blue. Top, 100 viable cells were seeded in clonogenic assay, and colony formation was evaluated. Numbers at bottom are survival fraction according to clonogenic assay. C, control; ATP, treatment with 5mM ATP. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).

Mentions: Because lower levels of P2×7 were described in epithelial cancer cells than in normal tissue (Li et al., 2009), we investigated the levels of P2×7 mRNA in different cervical cancer cell lines and in an immortalized human epithelial cell line (HaCaT) used as nontumorigenic control cells (Figure 1A). Cervical cancer cell lines (SiHa, HeLa, and C33A) and HaCaT exhibited different amounts of P2×7 mRNA. Among them, the SiHa cell line presented the highest levels of P2×7 mRNA, and therefore we chose it to investigate the role of P2×7 in the response of cervical cancer to extracellular ATP.


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 exerts acute and chronic toxicity in cervical cancer cells. (A) Comparison of P2×7 mRNA expression in SiHa, HeLa, C33A, and HaCaT cell lines by quantitative real-time PCR analysis. Results are presented as the ratio cDNA/GAPDH. (B) SiHa cell viability after exposure to increased ATP concentration for 24 h using MTT assay. (C) Bottom, time curve of SiHa cells treated with 5 mM ATP for 24, 48, and 72 h, determined by number of viable cells not marked by trypan blue. Top, 100 viable cells were seeded in clonogenic assay, and colony formation was evaluated. Numbers at bottom are survival fraction according to clonogenic assay. C, control; ATP, treatment with 5mM ATP. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230581&req=5

Figure 1: Extracellular ATP exerts acute and chronic toxicity in cervical cancer cells. (A) Comparison of P2×7 mRNA expression in SiHa, HeLa, C33A, and HaCaT cell lines by quantitative real-time PCR analysis. Results are presented as the ratio cDNA/GAPDH. (B) SiHa cell viability after exposure to increased ATP concentration for 24 h using MTT assay. (C) Bottom, time curve of SiHa cells treated with 5 mM ATP for 24, 48, and 72 h, determined by number of viable cells not marked by trypan blue. Top, 100 viable cells were seeded in clonogenic assay, and colony formation was evaluated. Numbers at bottom are survival fraction according to clonogenic assay. C, control; ATP, treatment with 5mM ATP. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).
Mentions: Because lower levels of P2×7 were described in epithelial cancer cells than in normal tissue (Li et al., 2009), we investigated the levels of P2×7 mRNA in different cervical cancer cell lines and in an immortalized human epithelial cell line (HaCaT) used as nontumorigenic control cells (Figure 1A). Cervical cancer cell lines (SiHa, HeLa, and C33A) and HaCaT exhibited different amounts of P2×7 mRNA. Among them, the SiHa cell line presented the highest levels of P2×7 mRNA, and therefore we chose it to investigate the role of P2×7 in the response of cervical cancer to extracellular ATP.

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