Apoptosis induction in human glioblastoma multiforme T98G cells upon temozolomide and quercetin treatment

Abstract

Glioblastoma multiforme is the most aggressive primary brain tumour. At the cellular and molecular levels, several mechanisms responsible for apoptosis or autophagy induction are blocked. Identification of molecular targets stimulating cells to initiate programmed cell death should be performed for therapeutic purposes. A promising solution is the combination of temozolomide and quercetin. The aim of our study was to evaluate the effect of both drugs, applied alone and in combinations, on apoptosis and autophagy induction in human glioblastoma multiforme T98G cells. Our results clearly indicate that quercetin and temozolomide induce apoptosis very significantly, having no effect on autophagy induction. At the molecular level, it was correlated with caspase 3 and 9 activation, cytochrome c release from the mitochondrium and a decrease in the mitochondrial membrane potential. Both drugs are also potent Hsp27 and Hsp72 inhibitors. This suggests that the apoptotic signal goes through an internal pathway. Increased expression of caspase 12 and the presence of several granules in the cytoplasm after temozolomide treatment with or without quercetin preceding appearance of apoptosis may suggest that apoptosis is initiated by ER stress. Additionally, it was accompanied by changes in the nuclear morphology from circular to 'croissant like'.

Fig. 1

The level of apoptosis, necrosis and autophagy induction in the glioblastoma multiforme T98G cell line treated with temozolomide (0–200 μM) for 24 (a), 48 (b) and 72 h (c), stained with Hoechst 33342, propidium iodide and acridine orange. *P < 0.05

Fig. 2

The time-dependent effect of quercetin (0–100 μM) on apoptosis, necrosis and autophagy induction in the T98G cell line incubated with the drug for 24 (a) and 48 h (b). c A picture of T98G cell stained with Hoechst 33342 and propidium iodide after 48 h of treatment with 50 μM of quercetin. Green arrow indicates necrotic cell, while red arrow points apoptotic cell. *P < 0.05

Fig. 3

Estimation of the level of apoptosis, necrosis and autophagy in T98G cells incubated with 50 (a–f) or 100 μM (g–i) of temozolomide and an increasing quercetin concentration (0–50 μM) for 24 h (a–c, g–i) and 48 h (d–f), stained with Hoechst 33342 and propidium iodide; a, d, g cells pre-incubated with quercetin followed by temozolomide administration; b, e, h cells treated with both drugs administered at the same time; c, f, i cells pre-incubated with temozolomide followed by quercetin administration

Fig. 4

The mitochondrial membrane potential in T98G cells stained with DiOC₆(3), incubated separately with 50 μM of quercetin (Q) and 50 μM of temozolomide (T) or in combination of both drugs for 48 h and analysed by flow cytometry. C control cells, QT cells pre-incubated with quercetin, Q + T simultaneous drug treatment, TQ pre-incubation with temozolomide. *P < 0.05

Fig. 5

Level of Hsp72 (a), Hsp27 (b), cytochrome c (c cytoplasmic, d mitochondrial fraction), caspase 12 (e) and beclin 1 (g) expression with representative blots and the activity of caspases 3, 8 and 9 (f) after temozolomide (T) and quercetin (Q) treatment for 48 h. C control cells, QT cells pre-incubated with quercetin, Q + T simultaneous drug treatment, TQ pre-incubation with temozolomide. *P < 0.05

Fig. 6

The effect of temozolomide (T) and quercetin (Q) on the shape of the nuclei expressed by the mean roundness factor (MRF) after staining with Hoechst 33342. a Control cells, b nuclei after treatment with quercetin and temozolomide, c quantitative analysis. QT cells pre-incubated with quercetin, Q + T simultaneous drug treatment, TQ pre-incubation with temozolomide. *P < 0.05

Fig. 7

The effect of temozolomide (T) and quercetin (Q) on the cytoplasmic granule formation (white arrows) in the T98G cells. a Quantitative analysis, b cells stained with OA after temozolomide treatment, c ER structure in control cells stained with DiOC₆(3), d ER structure in cells treated with temozolomide and stained with DiOC₆(3). QT cells pre-incubated with quercetin, Q + T simultaneous drug treatment, T + Q pre-incubation with temozolomide. *P < 0.05

Fig. 8

The effect of temozolomide and quercetin on Hsp72 (a, c) and Hsp27 (b, d) in untreated cells (a, b) or treated with both drugs (c, d) for 48 h