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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 triggers AMPK phosphoactivation, p53 increase, and cytotoxic autophagy in SiHa cells. Expression of (A) p-AMPK, cleaved PARP, p53, and Bcl-2 and (B) p62 and LC3 II after 5 mM ATP exposure for 24, 48, and 72 h with or without 10 μM DIP pretreatment, determined by Western blot analysis as described in Materials and Methods. (C) Effect of the class III autophagy inhibitors 3MA and BAF and autophagy stimulator RAPA on AO staining after 48 h of ATP exposure. SiHa cells were pretreated with 2 mM 3MA, 100 nM BAF, or 200 nM RAPA before ATP treatment, and autophagy index was determined by AO staining as described in Materials and Methods. (D) Top, percentage of AO-positive cells after ATP exposure before or not to autophagy inhibitors or stimulator. Bottom, number of viable cells after treatment. Note that treatments that increased autophagy induced a reduction in cell number (see also Supplemental Figure S4). *p <0.05 compared with treatments without ATP; #p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest).
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Figure 7: Adenosine uptake triggers AMPK phosphoactivation, p53 increase, and cytotoxic autophagy in SiHa cells. Expression of (A) p-AMPK, cleaved PARP, p53, and Bcl-2 and (B) p62 and LC3 II after 5 mM ATP exposure for 24, 48, and 72 h with or without 10 μM DIP pretreatment, determined by Western blot analysis as described in Materials and Methods. (C) Effect of the class III autophagy inhibitors 3MA and BAF and autophagy stimulator RAPA on AO staining after 48 h of ATP exposure. SiHa cells were pretreated with 2 mM 3MA, 100 nM BAF, or 200 nM RAPA before ATP treatment, and autophagy index was determined by AO staining as described in Materials and Methods. (D) Top, percentage of AO-positive cells after ATP exposure before or not to autophagy inhibitors or stimulator. Bottom, number of viable cells after treatment. Note that treatments that increased autophagy induced a reduction in cell number (see also Supplemental Figure S4). *p <0.05 compared with treatments without ATP; #p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest).

Mentions: Extracellular ATP increased the levels of pAMPK(T172)—the active state of AMPK (Hardie et al., 2006)—in a time-dependent manner, mainly after 48 and 72 h, accompanied by PARP cleavage (Figure 7A). p53 levels reached the highest levels at 48 h and these molecular alterations were suppressed almost completely by DIP pretreatment. Furthermore, DIP pretreatment increased the levels of Bcl2, a classical antiapoptotic protein, after 72 h of ATP treatment. Taken together, these data suggest that adenosine uptake is responsible for the main molecular alterations that underlie the toxicity of extracellular ATP in cervical cancer cells.


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 triggers AMPK phosphoactivation, p53 increase, and cytotoxic autophagy in SiHa cells. Expression of (A) p-AMPK, cleaved PARP, p53, and Bcl-2 and (B) p62 and LC3 II after 5 mM ATP exposure for 24, 48, and 72 h with or without 10 μM DIP pretreatment, determined by Western blot analysis as described in Materials and Methods. (C) Effect of the class III autophagy inhibitors 3MA and BAF and autophagy stimulator RAPA on AO staining after 48 h of ATP exposure. SiHa cells were pretreated with 2 mM 3MA, 100 nM BAF, or 200 nM RAPA before ATP treatment, and autophagy index was determined by AO staining as described in Materials and Methods. (D) Top, percentage of AO-positive cells after ATP exposure before or not to autophagy inhibitors or stimulator. Bottom, number of viable cells after treatment. Note that treatments that increased autophagy induced a reduction in cell number (see also Supplemental Figure S4). *p <0.05 compared with treatments without ATP; #p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest).
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Figure 7: Adenosine uptake triggers AMPK phosphoactivation, p53 increase, and cytotoxic autophagy in SiHa cells. Expression of (A) p-AMPK, cleaved PARP, p53, and Bcl-2 and (B) p62 and LC3 II after 5 mM ATP exposure for 24, 48, and 72 h with or without 10 μM DIP pretreatment, determined by Western blot analysis as described in Materials and Methods. (C) Effect of the class III autophagy inhibitors 3MA and BAF and autophagy stimulator RAPA on AO staining after 48 h of ATP exposure. SiHa cells were pretreated with 2 mM 3MA, 100 nM BAF, or 200 nM RAPA before ATP treatment, and autophagy index was determined by AO staining as described in Materials and Methods. (D) Top, percentage of AO-positive cells after ATP exposure before or not to autophagy inhibitors or stimulator. Bottom, number of viable cells after treatment. Note that treatments that increased autophagy induced a reduction in cell number (see also Supplemental Figure S4). *p <0.05 compared with treatments without ATP; #p < 0.05 compared with control (two-way ANOVA, followed by Bonferroni posttest).
Mentions: Extracellular ATP increased the levels of pAMPK(T172)—the active state of AMPK (Hardie et al., 2006)—in a time-dependent manner, mainly after 48 and 72 h, accompanied by PARP cleavage (Figure 7A). p53 levels reached the highest levels at 48 h and these molecular alterations were suppressed almost completely by DIP pretreatment. Furthermore, DIP pretreatment increased the levels of Bcl2, a classical antiapoptotic protein, after 72 h of ATP treatment. Taken together, these data suggest that adenosine uptake is responsible for the main molecular alterations that underlie the toxicity of extracellular ATP in cervical cancer cells.

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