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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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Proliferation and viability curves of NCI/ADR-RES cells treated with PE5 or onconase. Cells were treated with 0 (black circle), 2 (white circle), 7 (black triangle), 14 (white triangle) and 35 μM (black square) of PE5 (A and C) or 0 (black circle), 0.3 (white circle), 1 (black triangle), 2 (white triangle) and 5 μM (black square) of onconase (B and D). Trypan blue dye exclusion cell viability was estimated at 0, 24, 48 and 72 h after administration of the RNases. The plotted points represent means of at least three independent experiments.
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Figure 1: Proliferation and viability curves of NCI/ADR-RES cells treated with PE5 or onconase. Cells were treated with 0 (black circle), 2 (white circle), 7 (black triangle), 14 (white triangle) and 35 μM (black square) of PE5 (A and C) or 0 (black circle), 0.3 (white circle), 1 (black triangle), 2 (white triangle) and 5 μM (black square) of onconase (B and D). Trypan blue dye exclusion cell viability was estimated at 0, 24, 48 and 72 h after administration of the RNases. The plotted points represent means of at least three independent experiments.

Mentions: The MTT assay does not allow discerning whether the drug exerts a cytostatic or a cytotoxic effect and therefore we investigated the effect of both proteins on cell growth and cell viability. We focused this study on the NCI/ADR-RES cell line because it was the most sensitive to PE5. Figure 1 shows the effect on NCI/ADR-RES proliferation and viability of incubating four different concentrations of PE5 or onconase for up to 72 h. Even at 24 h of intoxication an effect of both RNases on proliferation could be observed at the highest concentrations assayed. At this same time the viability of NCI/ADR-RES cells was reduced only by treatment with 14 or 35 μM PE5. Consistently with the cytotoxicity results (Table 1), cells treated with 7 μM PE5 or 1 μM onconase for 72 h (which correspond to the IC50 values described in Table 1) decreased the proliferation to around 50% respective to untreated cells (Figures 1A and 1B). Therefore, PE5 is mainly cytotoxic although at low concentrations a minimal cytostatic effect can be observed. On the contrary, onconase arrests proliferation at low concentrations (below to 1 μM) but begins to be cytotoxic at higher concentrations, as it has been previously described for other cell lines [2]. Interestingly, although a seven-fold lower concentration of onconase was necessary to reduce cell growth to 50%, the percentage of viable cells at each incubation time was similar when using either PE5 or onconase. This shows that both RNases induce cell death at the same extent but that onconase presents an additional capacity of arresting cell proliferation.


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)

Proliferation and viability curves of NCI/ADR-RES cells treated with PE5 or onconase. Cells were treated with 0 (black circle), 2 (white circle), 7 (black triangle), 14 (white triangle) and 35 μM (black square) of PE5 (A and C) or 0 (black circle), 0.3 (white circle), 1 (black triangle), 2 (white triangle) and 5 μM (black square) of onconase (B and D). Trypan blue dye exclusion cell viability was estimated at 0, 24, 48 and 72 h after administration of the RNases. The plotted points represent means of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Proliferation and viability curves of NCI/ADR-RES cells treated with PE5 or onconase. Cells were treated with 0 (black circle), 2 (white circle), 7 (black triangle), 14 (white triangle) and 35 μM (black square) of PE5 (A and C) or 0 (black circle), 0.3 (white circle), 1 (black triangle), 2 (white triangle) and 5 μM (black square) of onconase (B and D). Trypan blue dye exclusion cell viability was estimated at 0, 24, 48 and 72 h after administration of the RNases. The plotted points represent means of at least three independent experiments.
Mentions: The MTT assay does not allow discerning whether the drug exerts a cytostatic or a cytotoxic effect and therefore we investigated the effect of both proteins on cell growth and cell viability. We focused this study on the NCI/ADR-RES cell line because it was the most sensitive to PE5. Figure 1 shows the effect on NCI/ADR-RES proliferation and viability of incubating four different concentrations of PE5 or onconase for up to 72 h. Even at 24 h of intoxication an effect of both RNases on proliferation could be observed at the highest concentrations assayed. At this same time the viability of NCI/ADR-RES cells was reduced only by treatment with 14 or 35 μM PE5. Consistently with the cytotoxicity results (Table 1), cells treated with 7 μM PE5 or 1 μM onconase for 72 h (which correspond to the IC50 values described in Table 1) decreased the proliferation to around 50% respective to untreated cells (Figures 1A and 1B). Therefore, PE5 is mainly cytotoxic although at low concentrations a minimal cytostatic effect can be observed. On the contrary, onconase arrests proliferation at low concentrations (below to 1 μM) but begins to be cytotoxic at higher concentrations, as it has been previously described for other cell lines [2]. Interestingly, although a seven-fold lower concentration of onconase was necessary to reduce cell growth to 50%, the percentage of viable cells at each incubation time was similar when using either PE5 or onconase. This shows that both RNases induce cell death at the same extent but that onconase presents an additional capacity of arresting cell proliferation.

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