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A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation.

Castro J, Ribó M, Navarro S, Nogués MV, Vilanova M, Benito A - BMC Cancer (2011)

Bottom Line: PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK.Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP CONCLUSIONS: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK.This mechanism is significantly different from that found for onconase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n E-17071 Girona, Spain.

ABSTRACT

Background: Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.

Methods: Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.

Results: We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP CONCLUSIONS: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.

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Expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 in 7 μM PE5-treated cells. 72 h after the treatments, cells were harvested and analyzed by Western blot using the antibodies described in the Methods section. A) Western blot of a representative experiment. B) Densitometric analysis of the GAPDH-normalized immunoblots of each treatment respective to that of untreated cells. White bars indicate untreated cells (control) and black bars indicate PE5-treated cells.
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Figure 3: Expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 in 7 μM PE5-treated cells. 72 h after the treatments, cells were harvested and analyzed by Western blot using the antibodies described in the Methods section. A) Western blot of a representative experiment. B) Densitometric analysis of the GAPDH-normalized immunoblots of each treatment respective to that of untreated cells. White bars indicate untreated cells (control) and black bars indicate PE5-treated cells.

Mentions: To further characterize the effect of PE5 on the cell cycle phase distribution we used western blot to also analyze the expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 (Figure 3). PE5 increased the expression of cyclin E to a 209% ± 25 of that of untreated cells and also that p21WAF1/CIP1 to a minor extent (to 175% ± 37 of untreated cells). The amounts of cyclin D1 and of p27KIP1 remained nearly unchanged respective to untreated cells.


A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation.

Castro J, Ribó M, Navarro S, Nogués MV, Vilanova M, Benito A - BMC Cancer (2011)

Expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 in 7 μM PE5-treated cells. 72 h after the treatments, cells were harvested and analyzed by Western blot using the antibodies described in the Methods section. A) Western blot of a representative experiment. B) Densitometric analysis of the GAPDH-normalized immunoblots of each treatment respective to that of untreated cells. White bars indicate untreated cells (control) and black bars indicate PE5-treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3025972&req=5

Figure 3: Expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 in 7 μM PE5-treated cells. 72 h after the treatments, cells were harvested and analyzed by Western blot using the antibodies described in the Methods section. A) Western blot of a representative experiment. B) Densitometric analysis of the GAPDH-normalized immunoblots of each treatment respective to that of untreated cells. White bars indicate untreated cells (control) and black bars indicate PE5-treated cells.
Mentions: To further characterize the effect of PE5 on the cell cycle phase distribution we used western blot to also analyze the expression of cyclin D1, cyclin E, p21WAF1/CIP1 and p27KIP1 (Figure 3). PE5 increased the expression of cyclin E to a 209% ± 25 of that of untreated cells and also that p21WAF1/CIP1 to a minor extent (to 175% ± 37 of untreated cells). The amounts of cyclin D1 and of p27KIP1 remained nearly unchanged respective to untreated cells.

Bottom Line: PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK.Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP CONCLUSIONS: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK.This mechanism is significantly different from that found for onconase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n E-17071 Girona, Spain.

ABSTRACT

Background: Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.

Methods: Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.

Results: We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP CONCLUSIONS: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.

Show MeSH
Related in: MedlinePlus