Resveratrol abrogates the Temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the Temozolomide-induced senescence in glioma cells

Abstract

BackgroundTemozolomide (TMZ) is the most widely used drug to treat glioblastoma (GBM), which is the most common and aggressive primary tumor of the Central Nervous System and one of the hardest challenges in oncotherapy. TMZ is an alkylating agent that induces autophagy, apoptosis and senescence in GBM cells. However, therapy with TMZ increases survival after diagnosis only from 12 to 14.4 months, making the development of combined therapies to treat GBM fundamental. One candidate for GBM therapy is Resveratrol (Rsv), which has additive toxicity with TMZ in several glioma cells in vitro and in vivo. However, the mechanism of Rsv and TMZ additive toxicity, which is the aim of the present work, is not clear, especially concerning cell cycle dynamics and long term effects.MethodsGlioma cell lines were treated with Rsv and TMZ, alone or in combinations, and the induction and the role of autophagy, apoptosis, cell cycle dynamics, protein expression and phosphorylation status were measured. We further evaluated the long term senescence induction and clonogenic capacity.ResultsAs expected, temozolomide caused a G2 cell cycle arrest and extensive DNA damage response. Rsv did not reduced this response, even increasing pATM, pChk2 and gammaH2Ax levels, but abrogated the temozolomide-induced G2 arrest, increasing levels of cyclin B and pRb(S807/811) and reducing levels of pWee1(S642) and pCdk1(Y15). This suggests a cellular state of forced passage through G2 checkpoint despite large DNA damage, a scenario that may produce mitotic catastrophe. Indeed, the proportion of cells with high nuclear irregularity increased from 6 to 26% in 48 h after cotreatment. At a long term, a reduction in clonogenic capacity was observed, accompanied by a large induction of senescence.ConclusionThe presence of Rsv forces cells treated with TMZ through mitosis leading to mitotic catastrophe and senescence, reducing the clonogenic capacity of glioma cells and increasing the chronic effects of temozolomide.

Ratiometric analysis of acridine orange staining in the study of acidic organelles and autophagy

“…We and others have previously shown that TMZ induces autophagy in several cell lines, including U-87 MG glioma cells (Filippi-Chiela et al, 2013;Natsumeda et al, 2011;Katayama et al, 2007) and induces cell enlargement (Filippi-Chiela et al, 2015). Autophagy induction was confirmed through a GFP-LC3 puncta formation assay (Fig.…”

“…Analysis of protein levels were performed as described previously with minor modifications (Filippi-Chiela et al, 2013). Briefly, cells were lysed and protein concentration was quantified by QuantiPro BCA Assay Kit (Thermo Scientific, #23225).…”

Resveratrol inhibits glioma cell growth via targeting oncogenic microRNAs and multiple signaling pathways

“…It has also been reported that Res sensitizes GBM tumor initiating cells to radiotherapy by inhibition of STAT3 axis (49)(50)(51)(52).…”

Synergistic anti-glioma effect of a coloaded nano-drug delivery system

“…These results corroborated with the previous studies where Res had been shown to enhance the cytotoxicity of Tem through AMPK-TSC-mTOR signaling pathway, leading to mitotic catastrophe and senescence. 8,10 Most importantly, it was observed that drug-loaded nanoparticles were much more effective in regulating the expression of related proteins than the corresponding dose of the free drug combination, which may be attributed to the efficient uptake of nanoparticles by glioma cells. As reported previously, cellular uptake of nanoparticles was more efficient than the diffusion of free drugs.…”

“…[4][5][6][7] Some previous studies also demonstrated the role of resveratrol (Res) in sensitizing tumor cells to the conventional chemotherapy, including enhancing the cytotoxicity of Tem against glioma cells. [8][9][10][11][12][13] However, hydrophobicity and poor solubility of Res severely limit its clinical application.…”