Autophagy Interplay with Apoptosis and Cell Cycle Regulation in the Growth Inhibiting Effect of Resveratrol in Glioma Cells

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

Resveratrol and quercetin cooperate to induce senescence-like growth arrest in C6 rat glioma cells

Glioma is the most frequent and malignant primary human brain tumor with dismal prognosis despite multimodal therapy. Resveratrol and quercetin, two structurally related and naturally occurring polyphenols, are proposed to have anticancer effects. We report here that resveratrol and quercetin decreased the cell number in four glioma cell lines but not in rat astrocytes. Low doses of resveratrol (10 mu M) or quercetin (25 mu M) separately had no effect on apoptosis induction, but had a strong effect on caspase 3/7 activation when administered together. Western blot analyses showed that resveratrol (10 mu M) and quercetin (25 mu M) caused a reduction in phosphorylation of Akt, but this reduction was not sufficient by itself to mediate the effects of these polyphenols. Most important, resveratrol and quercetin chronically administered presented a strong synergism in inducing senescence-like growth arrest. These results suggest that the combination of polyphenols can potentialize their antitumoral activity, thereby reducing the therapeutic concentration needed for glioma treatment. (Cancer Sci 2009; 100: 1655-1662).CNPqFAPERGSPROPESQ/UFRG

Nuclear Morphometric Analysis (NMA): Screening of Senescence, Apoptosis and Nuclear Irregularities

<div><p>Several cellular mechanisms affect nuclear morphology which can therefore be used to assess certain processes. Here, we present an analytic tool to quantify the number of cells in a population that present characteristics of senescence, apoptosis or nuclear irregularities through nuclear morphometric analysis. The tool presented here is based on nuclear image analysis and evaluation of size and regularity of adhered cells in culture. From 46 measurements of nuclear morphometry, principal component analysis filtered four measurements that best separated regular from irregular nuclei. These measurements, namely aspect, area box, radius ratio and roundness were combined into a single nuclear irregularity index (NII). Normal nuclei are used to set the parameters for a given cell type, and different nuclear phenotypes are separated in an area versus NII plot. The tool was validated with β-gal staining for senescence and annexin or caspases inhibitor for apoptosis as well as several treatments that induce different cellular phenotypes. This method provides a direct and objective way of screening normal, senescent, apoptotic and nuclear irregularities which may occur during failed mitosis or mitotic catastrophe, which may be very useful in basic and clinical research.</p> </div

NMA of senescent glioma cells.

<p>C6 glioma cells were treated with DMSO as vehicle control (<b>A</b>) or resveratrol (Rsv) and quercetin (Quer) for 12 days (<b>B</b>) for senescence induction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042522#pone.0042522-Dimri1" target="_blank">[6]</a>. Light blue diamonds are nuclei from β-galactosidase positive cells. Numbers denote β-gal positive and total nuclei in the different categories and the percentage of nuclei in each category. Inserts show examples of nuclear and β-gal staining (a–g positive and h-j negative).</p

Morphological appearances and putative biological meanings of the different NMA populations presented in Figure 1.

<p>Morphological appearances and putative biological meanings of the different NMA populations presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042522#pone-0042522-g001" target="_blank">Figure 1</a>.</p

NMA ofcells treated with vincristine.

<p>U87 cells were treated with DMSO as vehicle control (<b>A</b>) or vincristine 50 nM for 24 h (<b>B</b>). Numbers in boxes denote the percentage of nuclei in each category. Inserts show examples of representative nuclei. Images were obtained directly from IPP6 software (with the boundary marking and the number of nuclei in the photo), to exemplify the marking established by the software. All nuclei are from single cells, as defined by direct visualization.</p

Distribution of nuclei in a plot of area versus NII.

<p>N: Normal nuclei (crosses represent nuclei used to establish the reference population and the ellipse that represents the conjoint distribution for area and NII for normal nuclei); I: irregular, LR: Large Regular; LI: Large Irregular; SR: Small and Regular; S: Small; SI: Small and Irregular. Squares denote the averages of the different populations. Pictures (a–r) show examples of nuclei and their localization in the graph.</p

Comparison of NMA and annexin apoptosis quantification.

<p>(<b>A</b>) HeLa cells were pre-treated (1 h) with the caspases inhibitor zAsp-CH2-DCB (100 µM), followed by treatment with Cisplatin (40 µM), DAPI staining and NMA analysis. Top: NMA graphs; Middle: phase contrast; Bottom: DAPI fluorescence. Arrows point to cells with apoptotic phenotype. (<b>B</b>) HeLa cells were treated as in B, followed by annexin V-FITC staining and flow cytometry. Left: Flow cytometry plots. Green Fluorescence: annexin V; Red Fluorescence: PI. Region enclosed in red: Annexin-positive, PI-negative cells (<i>i.e.</i> apoptotic cells). Right: comparative percentage of apoptotic cells measured through NMA or annexin analysis.</p