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.
Highlights
Temozolomide (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
Rsv potentiated the cytotoxic effect of TMZ on human glioma cell lines U87, U138 and U251 (Figure 1 and Additional file 1: Figure S1a)
Rsv 30 μM plus TMZ 100 μM did not induce loss of membrane integrity, large morphological alterations, increase in annexinV/PI-staining or reactive oxygen species (ROS) when cells were treated for 48 h (Additional file 2: Figure S2), indicating that apoptosis, necrosis or a major imbalance in ROS levels are not involved in the reduction in cell number induced by Resveratrol+temozolomide cotreatment (RT)
Summary
Temozolomide (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 a cytotoxic imidazotetrazine that leads to the formation of O6-methylguanine, which mismatches with thymine in subsequent DNA replication cycles This was described as leading to several cellular outcomes, such as apoptosis [5,6], autophagy [7], mitotic catastrophe and senescence-like events [5] in GBMs. In most cells, TMZ produces cell cycle arrest at G2/M through activation of ATM/ATR-Chk1/2 [8]. Impeding the cell cycle arrest in DNA-damaged cells normally leads to cell death by mitotic catastrophe (MC) [5], a failure caused by mitosis entry even in the presence of DNA damage or checkpoint activation [10,11] Some cancer types, such as GBM, are intrinsically resistant to apoptosis and may be more sensitive to other mechanisms of cell death, such as autophagy, senescence and MC [12,13]
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