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

Jakubowicz-Gil J, Langner E, Bądziul D, Wertel I, Rzeski W - Tumour Biol. (2013)

Bottom Line: 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.Additionally, it was accompanied by changes in the nuclear morphology from circular to 'croissant like'.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Anatomy and Anthropology, Maria Curie-Skłodowska University, Akademicka 19, 20-033, Lublin, Poland. jjgil@poczta.umcs.lublin.pl

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'.

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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
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Fig7: 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

Mentions: Staining of the cells with Hoechst 33342 revealed that temozolomide alone and together with quercetin changed the morphology of glioblastoma nuclei from circular, as observed in the control (Fig. 6a), to a more curved ‘croissant-like’ shape (Fig. 6b). The mean roundness factor used for the histomorphometrical analysis of the shape of the nuclei after TMZ with or without the flavonoid decreased from 0.86 (control) to about 0.6 (Fig. 6c). Quercetin did not change the shape of the nuclei and the mean roundness factor was similar to that in the control (0.85) (Fig. 6c). Staining of the T98G cells treated with temozolomide with orange acridine facilitated distinguishing round-shaped granules, which were not stained with the fluorochrome used (Fig. 7). The percentage of cells where such structures were noticed was the highest after 24 h of incubation (11 %) and decreased gradually with a longer incubation time. Granules were observed in about 9 % of cells after a 48-h-long incubation and 6 % after 72 h after incubation with only TMZ and simultaneously with quercetin (Fig. 7a, b, d). No granules in the cytoplasm were noticed in the control nontreated cells and cells incubated with quercetin (data not shown). Additional staining of the T98G cells treated with the alkylating drug with or without flavonoid with DiOC6(3), a fluorochrome that binds to ER, revealed the presence of round bulky structures within the reticulum that were similar in shape and size to the granules observed in the cytoplasm (Fig. 7d).Fig. 6


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

Jakubowicz-Gil J, Langner E, Bądziul D, Wertel I, Rzeski W - Tumour Biol. (2013)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3713258&req=5

Fig7: 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
Mentions: Staining of the cells with Hoechst 33342 revealed that temozolomide alone and together with quercetin changed the morphology of glioblastoma nuclei from circular, as observed in the control (Fig. 6a), to a more curved ‘croissant-like’ shape (Fig. 6b). The mean roundness factor used for the histomorphometrical analysis of the shape of the nuclei after TMZ with or without the flavonoid decreased from 0.86 (control) to about 0.6 (Fig. 6c). Quercetin did not change the shape of the nuclei and the mean roundness factor was similar to that in the control (0.85) (Fig. 6c). Staining of the T98G cells treated with temozolomide with orange acridine facilitated distinguishing round-shaped granules, which were not stained with the fluorochrome used (Fig. 7). The percentage of cells where such structures were noticed was the highest after 24 h of incubation (11 %) and decreased gradually with a longer incubation time. Granules were observed in about 9 % of cells after a 48-h-long incubation and 6 % after 72 h after incubation with only TMZ and simultaneously with quercetin (Fig. 7a, b, d). No granules in the cytoplasm were noticed in the control nontreated cells and cells incubated with quercetin (data not shown). Additional staining of the T98G cells treated with the alkylating drug with or without flavonoid with DiOC6(3), a fluorochrome that binds to ER, revealed the presence of round bulky structures within the reticulum that were similar in shape and size to the granules observed in the cytoplasm (Fig. 7d).Fig. 6

Bottom Line: 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.Additionally, it was accompanied by changes in the nuclear morphology from circular to 'croissant like'.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Anatomy and Anthropology, Maria Curie-Skłodowska University, Akademicka 19, 20-033, Lublin, Poland. jjgil@poczta.umcs.lublin.pl

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'.

Show MeSH
Related in: MedlinePlus