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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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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
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Fig5: 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

Mentions: Apoptosis and autophagy at the molecular level are characterised by changes in the expression of typical marker proteins. Using the immunoblot technique, we studied the effect of temozolomide and quercetin applied separately or in combinations (Fig. 5) on the level of expression of pro-apoptotic cytochrome c [in the cytoplasmic (Fig. 5c] and mitochondrial (Fig. 5d) fractions], anti-apoptotic molecular chaperones Hsp27 (Fig. 5b) and Hsp72 (Fig. 5a) and pro-autophagic beclin 1 (Fig. 5g). Additionally, we analysed fluorimetrically the activity of caspases 3, 8 and 9, i.e. aspartate-specific cysteine proteases being part of the caspase cascade system, which plays a vital role in apoptosis (Fig. 5f). Using the immunoblot technique, we also studied the level of expression of caspase 12, a well-known enzyme mediating apoptosis under ER stress (Fig. 5e).Fig. 5


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)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: 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
Mentions: Apoptosis and autophagy at the molecular level are characterised by changes in the expression of typical marker proteins. Using the immunoblot technique, we studied the effect of temozolomide and quercetin applied separately or in combinations (Fig. 5) on the level of expression of pro-apoptotic cytochrome c [in the cytoplasmic (Fig. 5c] and mitochondrial (Fig. 5d) fractions], anti-apoptotic molecular chaperones Hsp27 (Fig. 5b) and Hsp72 (Fig. 5a) and pro-autophagic beclin 1 (Fig. 5g). Additionally, we analysed fluorimetrically the activity of caspases 3, 8 and 9, i.e. aspartate-specific cysteine proteases being part of the caspase cascade system, which plays a vital role in apoptosis (Fig. 5f). Using the immunoblot technique, we also studied the level of expression of caspase 12, a well-known enzyme mediating apoptosis under ER stress (Fig. 5e).Fig. 5

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