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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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ER stress-mediated apoptosis is involved in bufalin-induced cell death. A-C: U87MG cells were exposed to various concentrations of bufalin for 24 h. A: The effect of bufalin on ER stress sensors PERK, p-PERK, eIF2α, p-eIF2α, ATF6 f, IRE1 and p-IRE1. B: The expression levels of GRP78 and GRP94 in U87MG cells affected by bufalin. C: Western blot analysis for the expression of ER stress-associated apoptotic proteins CHOP and cleaved caspase-4. D-F: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin for another 24 h. D: CHOP and cleaved caspase-3 were examined by western blotting. E: The caspase-3 activity was determined by Caspase-3 colorimetric assay kit. The data are presented as the mean±SD, n=3. **p < 0.01 vs. sicontrol-transfected group. F: Cell viability was determined by MTT assay. Data are presented as mean ± SD, n = 3. **p < 0.01 versus control group. G: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin ( 80 nM ) for 48 h, stained with Annexin V and PI, and analyzed by flow cytometry. Results are representative of three independent experiments. H: Treatment was as described for G. The apoptosis rates are represented as the mean ± SD of three independent experiments. **p<0.01versus si-control group.
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Figure 5: ER stress-mediated apoptosis is involved in bufalin-induced cell death. A-C: U87MG cells were exposed to various concentrations of bufalin for 24 h. A: The effect of bufalin on ER stress sensors PERK, p-PERK, eIF2α, p-eIF2α, ATF6 f, IRE1 and p-IRE1. B: The expression levels of GRP78 and GRP94 in U87MG cells affected by bufalin. C: Western blot analysis for the expression of ER stress-associated apoptotic proteins CHOP and cleaved caspase-4. D-F: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin for another 24 h. D: CHOP and cleaved caspase-3 were examined by western blotting. E: The caspase-3 activity was determined by Caspase-3 colorimetric assay kit. The data are presented as the mean±SD, n=3. **p < 0.01 vs. sicontrol-transfected group. F: Cell viability was determined by MTT assay. Data are presented as mean ± SD, n = 3. **p < 0.01 versus control group. G: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin ( 80 nM ) for 48 h, stained with Annexin V and PI, and analyzed by flow cytometry. Results are representative of three independent experiments. H: Treatment was as described for G. The apoptosis rates are represented as the mean ± SD of three independent experiments. **p<0.01versus si-control group.

Mentions: Evidence has accumulated from many studies that ER stress-associated apoptosis could be an intriguing candidate to be responsible for cell death induced by anti-tumor agents 21. To identify whether ER stress was involved in bufalin induced cell death, we followed the behavior of some ER stress markers. There are three ER stress sensors including PERK, IRE1 and ATF6 that can activate the UPR response under ER dysfunction 22. We therefore performed western blots to check these proteins and found that p-PERK, p-IRE1α, p-eIF2α and ATF6 fragments increased in a concentration-dependent manner (Fig. 5A). Upon ER stress, GRP78 is released from the UPR sensors, resulting in the activation and transduction of UPR signals 23. As shown in Figure 5B, protein expression of GRP78 and GRP94 increased in a dose-dependent manner after 24 h of bufalin treatment. Next, we raised the question that whether ER stress was involved in apoptosis induced by bufalin. During ER stress, ATF 6 activates the transcription of CHOP and ultimately leads to caspase cascade activation to complete the execution of ER stress-induced apoptosis 24. Recent studies have shown that caspase-4, an ER-resident caspase, is activated in response to ER stress. Protein expression analysis showed that bufalin upregulated CHOP and accelerated cleavage of caspase-4 in U87MG cells (Fig. 5C). Accordingly, the increases in GRP78, p-PERK, p-eIF2α and CHOP expression in LN229 cells also suggest that part of the UPR is activated with bufalin (Supplementary Material: Fig. S5). Altogether, these results indicate that ER stress-associated apoptosis is also involved in bufalin-induced glioma cell death.


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)

ER stress-mediated apoptosis is involved in bufalin-induced cell death. A-C: U87MG cells were exposed to various concentrations of bufalin for 24 h. A: The effect of bufalin on ER stress sensors PERK, p-PERK, eIF2α, p-eIF2α, ATF6 f, IRE1 and p-IRE1. B: The expression levels of GRP78 and GRP94 in U87MG cells affected by bufalin. C: Western blot analysis for the expression of ER stress-associated apoptotic proteins CHOP and cleaved caspase-4. D-F: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin for another 24 h. D: CHOP and cleaved caspase-3 were examined by western blotting. E: The caspase-3 activity was determined by Caspase-3 colorimetric assay kit. The data are presented as the mean±SD, n=3. **p < 0.01 vs. sicontrol-transfected group. F: Cell viability was determined by MTT assay. Data are presented as mean ± SD, n = 3. **p < 0.01 versus control group. G: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin ( 80 nM ) for 48 h, stained with Annexin V and PI, and analyzed by flow cytometry. Results are representative of three independent experiments. H: Treatment was as described for G. The apoptosis rates are represented as the mean ± SD of three independent experiments. **p<0.01versus si-control group.
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Figure 5: ER stress-mediated apoptosis is involved in bufalin-induced cell death. A-C: U87MG cells were exposed to various concentrations of bufalin for 24 h. A: The effect of bufalin on ER stress sensors PERK, p-PERK, eIF2α, p-eIF2α, ATF6 f, IRE1 and p-IRE1. B: The expression levels of GRP78 and GRP94 in U87MG cells affected by bufalin. C: Western blot analysis for the expression of ER stress-associated apoptotic proteins CHOP and cleaved caspase-4. D-F: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin for another 24 h. D: CHOP and cleaved caspase-3 were examined by western blotting. E: The caspase-3 activity was determined by Caspase-3 colorimetric assay kit. The data are presented as the mean±SD, n=3. **p < 0.01 vs. sicontrol-transfected group. F: Cell viability was determined by MTT assay. Data are presented as mean ± SD, n = 3. **p < 0.01 versus control group. G: U87MG cells were transfected with CHOP specific siRNA and scrambled siRNA for 48 h, respectively. Then the cells were exposed to bufalin ( 80 nM ) for 48 h, stained with Annexin V and PI, and analyzed by flow cytometry. Results are representative of three independent experiments. H: Treatment was as described for G. The apoptosis rates are represented as the mean ± SD of three independent experiments. **p<0.01versus si-control group.
Mentions: Evidence has accumulated from many studies that ER stress-associated apoptosis could be an intriguing candidate to be responsible for cell death induced by anti-tumor agents 21. To identify whether ER stress was involved in bufalin induced cell death, we followed the behavior of some ER stress markers. There are three ER stress sensors including PERK, IRE1 and ATF6 that can activate the UPR response under ER dysfunction 22. We therefore performed western blots to check these proteins and found that p-PERK, p-IRE1α, p-eIF2α and ATF6 fragments increased in a concentration-dependent manner (Fig. 5A). Upon ER stress, GRP78 is released from the UPR sensors, resulting in the activation and transduction of UPR signals 23. As shown in Figure 5B, protein expression of GRP78 and GRP94 increased in a dose-dependent manner after 24 h of bufalin treatment. Next, we raised the question that whether ER stress was involved in apoptosis induced by bufalin. During ER stress, ATF 6 activates the transcription of CHOP and ultimately leads to caspase cascade activation to complete the execution of ER stress-induced apoptosis 24. Recent studies have shown that caspase-4, an ER-resident caspase, is activated in response to ER stress. Protein expression analysis showed that bufalin upregulated CHOP and accelerated cleavage of caspase-4 in U87MG cells (Fig. 5C). Accordingly, the increases in GRP78, p-PERK, p-eIF2α and CHOP expression in LN229 cells also suggest that part of the UPR is activated with bufalin (Supplementary Material: Fig. S5). Altogether, these results indicate that ER stress-associated apoptosis is also involved in bufalin-induced glioma cell death.

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