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Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress.

Shen S, Zhang Y, Wang Z, Liu R, Gong X - Int. J. Biol. Sci. (2014)

Bottom Line: Our results showed that bufalin inhibited the growth of glioma cells significantly.Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1.In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Biochemistry, Zhejiang University, Hangzhou, 310058, PR China;

ABSTRACT
Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.

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Bufalin induces autophagy through stimulation of ER stress in U87MG cells. A-C: U87MG cells were pretreated or not with 500 μM TUDC (A-B) or with 3 mM 3-MA (C) for 1 h and further treated with 40 nM bufalin for 24 h. A: Western blot analysis for the expression of LC3 and GRP78. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone. B: The number of MDC-labeled vacuoles was observed using a laser scanning confocal microscope (63X). C: Western blot analysis for the expression of p-eIF2α, GRP78 and CHOP. D-E: U87 cells transfected with sieIF2α/ siCHOP or sicontrol were treated with bufalin (40 nM) for 24 h and then harvested for protein analysis. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone.
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Figure 6: Bufalin induces autophagy through stimulation of ER stress in U87MG cells. A-C: U87MG cells were pretreated or not with 500 μM TUDC (A-B) or with 3 mM 3-MA (C) for 1 h and further treated with 40 nM bufalin for 24 h. A: Western blot analysis for the expression of LC3 and GRP78. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone. B: The number of MDC-labeled vacuoles was observed using a laser scanning confocal microscope (63X). C: Western blot analysis for the expression of p-eIF2α, GRP78 and CHOP. D-E: U87 cells transfected with sieIF2α/ siCHOP or sicontrol were treated with bufalin (40 nM) for 24 h and then harvested for protein analysis. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone.

Mentions: The close relationship between ER stress and autophagy is demonstrated by recent reports showing that ER stress is a potent inducer of autophagy 25. To investigate whether ER stress following bufalin treatment of cells generated an autophagic response, western blotting was performed to probe the expression of GRP78 and LC3-II. As shown in Fig. 6A, tauroursodeoxycholate (TUDC), an ER stress inhibitor, reduced the level of GRP78 and attenuated the expression of LC3-II level induced by bufalin. Consistent with the immunoblotting results, an obvious reduction in autophagy induction was further confirmed by MDC staining. The MDC-positive vacuoles were relatively sparse and weak in fluorescence intensity in cell cultures co-treated with bufalin+TUDC when compared with cultures treated with bufalin (24 h) (Fig. 6B), indicating that incorporation of MDC into vacuoles was inhibited by TUDC. To further investigate the relationship between autophagy and ER stress, the expression levels of p-eIF2α, GRP78 and CHOP were determined by western blotting. As shown in Figure 6C, U87MG cells treated with bufalin in combination with 3-MA upregulated the expression of p-eIF2α, GRP78 and CHOP compared with the group treated with bufalin alone. Our findings with 3-MA indicate that autophagy plays a protective role against bufalin-induced cytotoxicity, probably through relieving ER stress. These results support the conclusion that cells exposed to bufalin experience ER stress that leads to induction of autophagy and suggest that mobilization of the autophagic machinery might generate a feedback signal needed for full induction of ER stress.


Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress.

Shen S, Zhang Y, Wang Z, Liu R, Gong X - Int. J. Biol. Sci. (2014)

Bufalin induces autophagy through stimulation of ER stress in U87MG cells. A-C: U87MG cells were pretreated or not with 500 μM TUDC (A-B) or with 3 mM 3-MA (C) for 1 h and further treated with 40 nM bufalin for 24 h. A: Western blot analysis for the expression of LC3 and GRP78. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone. B: The number of MDC-labeled vacuoles was observed using a laser scanning confocal microscope (63X). C: Western blot analysis for the expression of p-eIF2α, GRP78 and CHOP. D-E: U87 cells transfected with sieIF2α/ siCHOP or sicontrol were treated with bufalin (40 nM) for 24 h and then harvested for protein analysis. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone.
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Figure 6: Bufalin induces autophagy through stimulation of ER stress in U87MG cells. A-C: U87MG cells were pretreated or not with 500 μM TUDC (A-B) or with 3 mM 3-MA (C) for 1 h and further treated with 40 nM bufalin for 24 h. A: Western blot analysis for the expression of LC3 and GRP78. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone. B: The number of MDC-labeled vacuoles was observed using a laser scanning confocal microscope (63X). C: Western blot analysis for the expression of p-eIF2α, GRP78 and CHOP. D-E: U87 cells transfected with sieIF2α/ siCHOP or sicontrol were treated with bufalin (40 nM) for 24 h and then harvested for protein analysis. Relative levels of LC3-II to LC3-I ratio are indicated in the graphs. Data were quantified using ImageJ software (mean±SD, n =3). *p<0.05 versus the bufalin treatment alone.
Mentions: The close relationship between ER stress and autophagy is demonstrated by recent reports showing that ER stress is a potent inducer of autophagy 25. To investigate whether ER stress following bufalin treatment of cells generated an autophagic response, western blotting was performed to probe the expression of GRP78 and LC3-II. As shown in Fig. 6A, tauroursodeoxycholate (TUDC), an ER stress inhibitor, reduced the level of GRP78 and attenuated the expression of LC3-II level induced by bufalin. Consistent with the immunoblotting results, an obvious reduction in autophagy induction was further confirmed by MDC staining. The MDC-positive vacuoles were relatively sparse and weak in fluorescence intensity in cell cultures co-treated with bufalin+TUDC when compared with cultures treated with bufalin (24 h) (Fig. 6B), indicating that incorporation of MDC into vacuoles was inhibited by TUDC. To further investigate the relationship between autophagy and ER stress, the expression levels of p-eIF2α, GRP78 and CHOP were determined by western blotting. As shown in Figure 6C, U87MG cells treated with bufalin in combination with 3-MA upregulated the expression of p-eIF2α, GRP78 and CHOP compared with the group treated with bufalin alone. Our findings with 3-MA indicate that autophagy plays a protective role against bufalin-induced cytotoxicity, probably through relieving ER stress. These results support the conclusion that cells exposed to bufalin experience ER stress that leads to induction of autophagy and suggest that mobilization of the autophagic machinery might generate a feedback signal needed for full induction of ER stress.

Bottom Line: Our results showed that bufalin inhibited the growth of glioma cells significantly.Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1.In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Biochemistry, Zhejiang University, Hangzhou, 310058, PR China;

ABSTRACT
Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.

Show MeSH
Related in: MedlinePlus