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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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Rsv induces autophagy in glioma cells.(A) U87 cells were transfected with pEGFP-LC3 and 48 h later treated with Rsv 30 µM or 100 µM for 48 h. Top - Representative images of cells with cytosolic green dots, representing LC3-GFP marked autophagosomes (white arrows), in cells treated with Rsv 30 µM; scale bar: 10 µm. Lower Pannel: Percentage of cells treated with Rsv 30 or 100 µM for 48 h which presented more than five defined cytosolic green dots. *** p<0.001; (B) U87 cells were pre-incubated with buffer (top and middle) or 3MA (2 mM) (bottom) for 1 h, treated with Rsv 30 µM for 48 h, followed by acridine orange (AO) staining and flow cytometry. Top - Representative images of AO stained cells treated with Rsv 30 µM for 48 h – acidic vacuolar organelles (AVO) stain red; scale bar: 20 µm. Middle and bottom - percentage of cells with positive red fluorescence as analyzed by flow cytometry. Numbers in the quadrants refer to average of events (black) or X-mean of AO red fluorescence intensity (red) ± SEM of three independent experiments; **p<0.01, ***p<0.001 in relation to control as indicated; # p<0.01, 3MA versus C for Rsv treated cells; (C and D) U87 cells were treated as in B and western blots for the indicated proteins were performed at the indicated time. Numbers indicate the band intensity in relation to control. LC: Loading control – coomassie stained PVDF membrane.
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pone-0020849-g001: Rsv induces autophagy in glioma cells.(A) U87 cells were transfected with pEGFP-LC3 and 48 h later treated with Rsv 30 µM or 100 µM for 48 h. Top - Representative images of cells with cytosolic green dots, representing LC3-GFP marked autophagosomes (white arrows), in cells treated with Rsv 30 µM; scale bar: 10 µm. Lower Pannel: Percentage of cells treated with Rsv 30 or 100 µM for 48 h which presented more than five defined cytosolic green dots. *** p<0.001; (B) U87 cells were pre-incubated with buffer (top and middle) or 3MA (2 mM) (bottom) for 1 h, treated with Rsv 30 µM for 48 h, followed by acridine orange (AO) staining and flow cytometry. Top - Representative images of AO stained cells treated with Rsv 30 µM for 48 h – acidic vacuolar organelles (AVO) stain red; scale bar: 20 µm. Middle and bottom - percentage of cells with positive red fluorescence as analyzed by flow cytometry. Numbers in the quadrants refer to average of events (black) or X-mean of AO red fluorescence intensity (red) ± SEM of three independent experiments; **p<0.01, ***p<0.001 in relation to control as indicated; # p<0.01, 3MA versus C for Rsv treated cells; (C and D) U87 cells were treated as in B and western blots for the indicated proteins were performed at the indicated time. Numbers indicate the band intensity in relation to control. LC: Loading control – coomassie stained PVDF membrane.

Mentions: We previously showed that Rsv inhibited the growth of glioma cells through processes that included senescence and apoptosis [32]. Here we show that treatment of U87 glioma cell line with Rsv at the relatively low concentration of 30 µM increased the percentage of cells with LC3-GFP cytosolic dots representing autophagosomes (Fig. 1A). This effect was not further increased with higher concentrations of Rsv, reaching a plateau of around 50% LC3-GFP positive cells, as previously observed in other cells [53]. Autophagosomes were also observed through AO staining, which significantly increased after Rsv treatment (Fig. 1B). 3MA, an inhibitor of the enzyme phosphatidylinositol 3-kinase class III (PI3k class III), essential for the autophagic process [54], reduced the number of cells containing LC3-GFP marked autophagosomes from 19% to 9% under basal conditions and from 55% to 24% when treated with Rsv 30 µM for 48 h. Similarly, the proportion of cells with red staining and the intensity of red staining with AO increased with Rsv and was partially reverted with 3MA, indicating that inhibition of PI3k class III not only reduced the number of cells undergoing autophagy, but also reduced the number of mature autophagosomes formed per cell (Fig. 1B - bottom).


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)

Rsv induces autophagy in glioma cells.(A) U87 cells were transfected with pEGFP-LC3 and 48 h later treated with Rsv 30 µM or 100 µM for 48 h. Top - Representative images of cells with cytosolic green dots, representing LC3-GFP marked autophagosomes (white arrows), in cells treated with Rsv 30 µM; scale bar: 10 µm. Lower Pannel: Percentage of cells treated with Rsv 30 or 100 µM for 48 h which presented more than five defined cytosolic green dots. *** p<0.001; (B) U87 cells were pre-incubated with buffer (top and middle) or 3MA (2 mM) (bottom) for 1 h, treated with Rsv 30 µM for 48 h, followed by acridine orange (AO) staining and flow cytometry. Top - Representative images of AO stained cells treated with Rsv 30 µM for 48 h – acidic vacuolar organelles (AVO) stain red; scale bar: 20 µm. Middle and bottom - percentage of cells with positive red fluorescence as analyzed by flow cytometry. Numbers in the quadrants refer to average of events (black) or X-mean of AO red fluorescence intensity (red) ± SEM of three independent experiments; **p<0.01, ***p<0.001 in relation to control as indicated; # p<0.01, 3MA versus C for Rsv treated cells; (C and D) U87 cells were treated as in B and western blots for the indicated proteins were performed at the indicated time. Numbers indicate the band intensity in relation to control. LC: Loading control – coomassie stained PVDF membrane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113895&req=5

pone-0020849-g001: Rsv induces autophagy in glioma cells.(A) U87 cells were transfected with pEGFP-LC3 and 48 h later treated with Rsv 30 µM or 100 µM for 48 h. Top - Representative images of cells with cytosolic green dots, representing LC3-GFP marked autophagosomes (white arrows), in cells treated with Rsv 30 µM; scale bar: 10 µm. Lower Pannel: Percentage of cells treated with Rsv 30 or 100 µM for 48 h which presented more than five defined cytosolic green dots. *** p<0.001; (B) U87 cells were pre-incubated with buffer (top and middle) or 3MA (2 mM) (bottom) for 1 h, treated with Rsv 30 µM for 48 h, followed by acridine orange (AO) staining and flow cytometry. Top - Representative images of AO stained cells treated with Rsv 30 µM for 48 h – acidic vacuolar organelles (AVO) stain red; scale bar: 20 µm. Middle and bottom - percentage of cells with positive red fluorescence as analyzed by flow cytometry. Numbers in the quadrants refer to average of events (black) or X-mean of AO red fluorescence intensity (red) ± SEM of three independent experiments; **p<0.01, ***p<0.001 in relation to control as indicated; # p<0.01, 3MA versus C for Rsv treated cells; (C and D) U87 cells were treated as in B and western blots for the indicated proteins were performed at the indicated time. Numbers indicate the band intensity in relation to control. LC: Loading control – coomassie stained PVDF membrane.
Mentions: We previously showed that Rsv inhibited the growth of glioma cells through processes that included senescence and apoptosis [32]. Here we show that treatment of U87 glioma cell line with Rsv at the relatively low concentration of 30 µM increased the percentage of cells with LC3-GFP cytosolic dots representing autophagosomes (Fig. 1A). This effect was not further increased with higher concentrations of Rsv, reaching a plateau of around 50% LC3-GFP positive cells, as previously observed in other cells [53]. Autophagosomes were also observed through AO staining, which significantly increased after Rsv treatment (Fig. 1B). 3MA, an inhibitor of the enzyme phosphatidylinositol 3-kinase class III (PI3k class III), essential for the autophagic process [54], reduced the number of cells containing LC3-GFP marked autophagosomes from 19% to 9% under basal conditions and from 55% to 24% when treated with Rsv 30 µM for 48 h. Similarly, the proportion of cells with red staining and the intensity of red staining with AO increased with Rsv and was partially reverted with 3MA, indicating that inhibition of PI3k class III not only reduced the number of cells undergoing autophagy, but also reduced the number of mature autophagosomes formed per cell (Fig. 1B - bottom).

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