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

Eduardo C Filippi-Chiela,Emilly Schlee Villodre,Lauren L Zamin,Guido Lenz,Gian Maria Fimia

doi:10.1371/journal.pone.0020849

Eduardo C Filippi-Chiela, Emilly Schlee Villodre + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0020849

Copy DOI

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.

Highlights

Glioblastoma (GBM) are the most common primary brain tumors, with a worldwide annual incidence of around 7 cases per 100,000 individuals [1,2]
Autophagosomes were observed through Acridine Orange (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 mM for 48 h
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 phosphatidylinositol 3 kinase (PI3k) class III reduced the number of cells undergoing autophagy, and reduced the number of mature autophagosomes formed per cell (Fig. 1B - bottom)

Summary

Introduction

Glioblastoma (GBM) are the most common primary brain tumors, with a worldwide annual incidence of around 7 cases per 100,000 individuals [1,2]. GBM Cancer Stem Cells (gCSC) have received much attention in glioma biology and this type of cell is highly associated with high aggressiveness, being fundamental for the maintenance and recurrence of GBM [7]. The primary therapy for GBM consists in surgery followed by radio and chemotherapy with temozolomide (TMZ), which is in clinical use since 2005 [11,12,13,14]. Despite this multimodal approach, the prognosis has only slightly improved [1,2]

Methods

Results

Conclusion