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Autophagy interplay with apoptosis and cell cycle regulation in the growth inhibiting effect of resveratrol in glioma cells.

Filippi-Chiela EC, Villodre ES, Zamin LL, Lenz G - PLoS ONE (2011)

Bottom Line: Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1.Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs.In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.

ABSTRACT
Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.

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Inhibition of Rsv-induced autophagy causes apoptosis.(A) U87 cells were pre-incubated with 3MA (2 mM) for 1 h before treatment with Rsv 30 µM for 48 h, followed by annexin V-FLUOS/PI cell staining evaluated by flow cytometry. Numbers in quadrants represents the respective percentage of cells ± SEM of three independent experiments. a p>0.01 and b p>0.01 in relation to Rsv and 3MA, respectively; *** p<0.001 in relation to control. (B) U87 cells were treated as in A and lysed 24 or 48 h later and western blots for the indicated proteins were performed. Numbers indicate the band intensity in relation to control. LC: Loading control. (C) U87 cells were pre-incubated with 3MA (2 mM) and/or zAsp (100 µM) for 1 h followed by treatment with Rsv (30 µM) for 48 h and number of cells was determined in a hemocytometer. Data are given as media ± SEM and are expressed as percentage of control, considered 100%; * p>0.05; n.s. not significant.
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pone-0020849-g003: Inhibition of Rsv-induced autophagy causes apoptosis.(A) U87 cells were pre-incubated with 3MA (2 mM) for 1 h before treatment with Rsv 30 µM for 48 h, followed by annexin V-FLUOS/PI cell staining evaluated by flow cytometry. Numbers in quadrants represents the respective percentage of cells ± SEM of three independent experiments. a p>0.01 and b p>0.01 in relation to Rsv and 3MA, respectively; *** p<0.001 in relation to control. (B) U87 cells were treated as in A and lysed 24 or 48 h later and western blots for the indicated proteins were performed. Numbers indicate the band intensity in relation to control. LC: Loading control. (C) U87 cells were pre-incubated with 3MA (2 mM) and/or zAsp (100 µM) for 1 h followed by treatment with Rsv (30 µM) for 48 h and number of cells was determined in a hemocytometer. Data are given as media ± SEM and are expressed as percentage of control, considered 100%; * p>0.05; n.s. not significant.

Mentions: As cited above, Rsv did not significantly change cell morphology or induce necrosis in GBM cells (Fig. 2C). Treatment with 30 µM of Rsv also did not increase phosphatidylserine (PS) externalization, evaluated by annexin V-staining (Fig. 3A), but induced a small increase in Bax expression and caspase cleavage (Fig. 3B). When Rsv-induced autophagy was inhibited with 3MA, a strong increase in the proportion of annexin V-positive cells was observed (Fig. 3A), together with a significant increase in Bax expression and caspase 3 cleavage (Fig. 3B). This was accompanied by phenotypic alterations indicating apoptosis, i.e. rounded morphology and loss of surface adhesion (Fig. S1). Inhibiting apoptosis by using a pan caspase inhibitor (zAsp) or inhibiting autophagy with 3MA alone did not block the cytotoxicity induced by Rsv. However, when both autophagy and apoptosis inhibitors were present, the Rsv-induced reduction in cell number was significantly inhibited (Fig. 3C).


Autophagy interplay with apoptosis and cell cycle regulation in the growth inhibiting effect of resveratrol in glioma cells.

Filippi-Chiela EC, Villodre ES, Zamin LL, Lenz G - PLoS ONE (2011)

Inhibition of Rsv-induced autophagy causes apoptosis.(A) U87 cells were pre-incubated with 3MA (2 mM) for 1 h before treatment with Rsv 30 µM for 48 h, followed by annexin V-FLUOS/PI cell staining evaluated by flow cytometry. Numbers in quadrants represents the respective percentage of cells ± SEM of three independent experiments. a p>0.01 and b p>0.01 in relation to Rsv and 3MA, respectively; *** p<0.001 in relation to control. (B) U87 cells were treated as in A and lysed 24 or 48 h later and western blots for the indicated proteins were performed. Numbers indicate the band intensity in relation to control. LC: Loading control. (C) U87 cells were pre-incubated with 3MA (2 mM) and/or zAsp (100 µM) for 1 h followed by treatment with Rsv (30 µM) for 48 h and number of cells was determined in a hemocytometer. Data are given as media ± SEM and are expressed as percentage of control, considered 100%; * p>0.05; n.s. not significant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113895&req=5

pone-0020849-g003: Inhibition of Rsv-induced autophagy causes apoptosis.(A) U87 cells were pre-incubated with 3MA (2 mM) for 1 h before treatment with Rsv 30 µM for 48 h, followed by annexin V-FLUOS/PI cell staining evaluated by flow cytometry. Numbers in quadrants represents the respective percentage of cells ± SEM of three independent experiments. a p>0.01 and b p>0.01 in relation to Rsv and 3MA, respectively; *** p<0.001 in relation to control. (B) U87 cells were treated as in A and lysed 24 or 48 h later and western blots for the indicated proteins were performed. Numbers indicate the band intensity in relation to control. LC: Loading control. (C) U87 cells were pre-incubated with 3MA (2 mM) and/or zAsp (100 µM) for 1 h followed by treatment with Rsv (30 µM) for 48 h and number of cells was determined in a hemocytometer. Data are given as media ± SEM and are expressed as percentage of control, considered 100%; * p>0.05; n.s. not significant.
Mentions: As cited above, Rsv did not significantly change cell morphology or induce necrosis in GBM cells (Fig. 2C). Treatment with 30 µM of Rsv also did not increase phosphatidylserine (PS) externalization, evaluated by annexin V-staining (Fig. 3A), but induced a small increase in Bax expression and caspase cleavage (Fig. 3B). When Rsv-induced autophagy was inhibited with 3MA, a strong increase in the proportion of annexin V-positive cells was observed (Fig. 3A), together with a significant increase in Bax expression and caspase 3 cleavage (Fig. 3B). This was accompanied by phenotypic alterations indicating apoptosis, i.e. rounded morphology and loss of surface adhesion (Fig. S1). Inhibiting apoptosis by using a pan caspase inhibitor (zAsp) or inhibiting autophagy with 3MA alone did not block the cytotoxicity induced by Rsv. However, when both autophagy and apoptosis inhibitors were present, the Rsv-induced reduction in cell number was significantly inhibited (Fig. 3C).

Bottom Line: Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1.Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs.In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.

ABSTRACT
Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.

Show MeSH
Related in: MedlinePlus