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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 SiHa cervical cancer cells. (A) Forward scatter analysis after treatment with 5 mM ATP for 24, 48, and 72 h. (B) Loss of membrane integrity measured by LDH release after treatment with 5 mM ATP for 24, 48 and 72 h. Triton X-100 was used as positive control for LDH release. (C) Top, images of SiHa cell treatment with 5 mM ATP for 24, 48, and 72 h. Cell nuclei was stained with Hoescht 35565665 according to manufacturer's instruction. Note apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP. Scale bars, 20 μm; magnification, 20×. Bottom, apoptosis and necrosis measured by annexin V– and PI-positive cells, quantified by flow cytometry in the same conditions as in C. Values refer to average of the percentage of cells in each gate of three independent experiments ± SD. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).
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Figure 2: Extracellular ATP triggers apoptosis in SiHa cervical cancer cells. (A) Forward scatter analysis after treatment with 5 mM ATP for 24, 48, and 72 h. (B) Loss of membrane integrity measured by LDH release after treatment with 5 mM ATP for 24, 48 and 72 h. Triton X-100 was used as positive control for LDH release. (C) Top, images of SiHa cell treatment with 5 mM ATP for 24, 48, and 72 h. Cell nuclei was stained with Hoescht 35565665 according to manufacturer's instruction. Note apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP. Scale bars, 20 μm; magnification, 20×. Bottom, apoptosis and necrosis measured by annexin V– and PI-positive cells, quantified by flow cytometry in the same conditions as in C. Values refer to average of the percentage of cells in each gate of three independent experiments ± SD. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).

Mentions: ATP, 5 mM, led to cell shrinkage in a time-dependent manner, as observed by forward scatter, suggesting apoptotic cell death (Figures 2A and Supplemental Figure S1). Indeed, treatment with extracellular ATP induced only a slight increase of lactate dehydrogenase (LDH) levels in the culture medium after 72 h (Figure 2B) and no increase of propidium iodide (PI) staining (Figure 2C), showing that necrosis was not the primary mechanism of ATP toxicity in SiHa cells. On the other hand, cells presented some phenotypic alterations that resemble apoptosis, including membrane blebbing, cell shrinkage, and chromatin condensation after 48 and 72 h. In agreement, ATP treatment highly increased annexin V staining (Figure 2C), confirming that ATP exerts a cytotoxic effect in SiHa cancer cells mainly through induction of apoptotic cell death.


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 SiHa cervical cancer cells. (A) Forward scatter analysis after treatment with 5 mM ATP for 24, 48, and 72 h. (B) Loss of membrane integrity measured by LDH release after treatment with 5 mM ATP for 24, 48 and 72 h. Triton X-100 was used as positive control for LDH release. (C) Top, images of SiHa cell treatment with 5 mM ATP for 24, 48, and 72 h. Cell nuclei was stained with Hoescht 35565665 according to manufacturer's instruction. Note apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP. Scale bars, 20 μm; magnification, 20×. Bottom, apoptosis and necrosis measured by annexin V– and PI-positive cells, quantified by flow cytometry in the same conditions as in C. Values refer to average of the percentage of cells in each gate of three independent experiments ± SD. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).
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Related In: Results  -  Collection

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Figure 2: Extracellular ATP triggers apoptosis in SiHa cervical cancer cells. (A) Forward scatter analysis after treatment with 5 mM ATP for 24, 48, and 72 h. (B) Loss of membrane integrity measured by LDH release after treatment with 5 mM ATP for 24, 48 and 72 h. Triton X-100 was used as positive control for LDH release. (C) Top, images of SiHa cell treatment with 5 mM ATP for 24, 48, and 72 h. Cell nuclei was stained with Hoescht 35565665 according to manufacturer's instruction. Note apoptotic features such as cell shrinkage and blebbing and fragmented nuclei when cells were treated with ATP. Scale bars, 20 μm; magnification, 20×. Bottom, apoptosis and necrosis measured by annexin V– and PI-positive cells, quantified by flow cytometry in the same conditions as in C. Values refer to average of the percentage of cells in each gate of three independent experiments ± SD. *p < 0.05 compared with control (one-way ANOVA, followed by Tukey's test).
Mentions: ATP, 5 mM, led to cell shrinkage in a time-dependent manner, as observed by forward scatter, suggesting apoptotic cell death (Figures 2A and Supplemental Figure S1). Indeed, treatment with extracellular ATP induced only a slight increase of lactate dehydrogenase (LDH) levels in the culture medium after 72 h (Figure 2B) and no increase of propidium iodide (PI) staining (Figure 2C), showing that necrosis was not the primary mechanism of ATP toxicity in SiHa cells. On the other hand, cells presented some phenotypic alterations that resemble apoptosis, including membrane blebbing, cell shrinkage, and chromatin condensation after 48 and 72 h. In agreement, ATP treatment highly increased annexin V staining (Figure 2C), confirming that ATP exerts a cytotoxic effect in SiHa cancer cells mainly through induction of apoptotic cell death.

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