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G226, a novel epipolythiodioxopiperazine derivative, induces autophagy and caspase-dependent apoptosis in human breast cancer cells in vitro.

He PX, Che YS, He QJ, Chen Y, Ding J - Acta Pharmacol. Sin. (2014)

Bottom Line: G226 also induced mitochondrial outer membrane permeabilization, resulted in the caspase-9 activation.Silencing of p62 or LC3 partially diminished caspase-8 and subsequent caspase-3 activation.LC3 silencing partially reversed G226-induced apoptosis, but p62 silencing elicited a subtle effect on G226-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT

Aim: To investigate the effects of G226, a novel epipolythiodioxopiperazine derivative, on human breast cancer cells in vitro, and to explore its anticancer mechanisms.

Methods: A panel of human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, ZR-75-30, BT474, BT549, SK-BR-3, T47D and HBL100) was examined. Cell proliferation was measured using sulforhodamine B assay, and cell apoptosis was detected with flow cytometry and caspase activity assay. Western blotting, immunofluorescence and targeted gene knockdowns were used to study autophagy in the cells.

Results: G226 suppressed proliferation of the 9 breast cancer cell lines with a mean IC50 value of 48.5 nmol/L (the mean IC50 value of adriamycin, a reference compound, was 170.6 nmol/L). G226 induced dose-dependent apoptosis of MDA-MB-231 and MCF-7 cells, accompanied by markedly increased activities of caspase-8 and caspase-3/7, which were abolished by caspase inhibitors zVAD or zIETD. G226 also induced mitochondrial outer membrane permeabilization, resulted in the caspase-9 activation. Moreover, G226 dose-dependently enhanced the autophagy marker LC3-II and autophagy substrate p62 accumulation in the cells, which were co-localized with caspase-8. Silencing of p62 or LC3 partially diminished caspase-8 and subsequent caspase-3 activation. LC3 silencing partially reversed G226-induced apoptosis, but p62 silencing elicited a subtle effect on G226-induced apoptosis.

Conclusion: The novel epipolythiodioxopiperazine derivative G226 exerts potent anticancer action against human breast cancer cells in vitro, via triggering autophagy and caspase-dependent apoptosis.

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Related in: MedlinePlus

G226 induces mitochondrial outer membrane permeabilization. (A) MDA-MB-231 and MCF-7 cells were treated with 200 nmol/L G226 for 24 h, and the mitochondrial membrane potential of the cells was measured by flow cytometry after JC-1 staining. (B) MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h, and the cell lysates were collected and examined by immunoblotting with the indicated antibodies.
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fig3: G226 induces mitochondrial outer membrane permeabilization. (A) MDA-MB-231 and MCF-7 cells were treated with 200 nmol/L G226 for 24 h, and the mitochondrial membrane potential of the cells was measured by flow cytometry after JC-1 staining. (B) MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h, and the cell lysates were collected and examined by immunoblotting with the indicated antibodies.

Mentions: Many agents induce apoptosis in cells via two major pathways: the death receptor-mediated pathway and the mitochondria-mediated pathway. Caspase-8 is an initiator caspase, which not only activates executioner caspases, such as procaspase-3 and procaspase-7, to complete the death receptor signaling pathway but also cleaves the Bcl-2 family member Bid to amplify apoptosis via the mitochondrial pathway20. Our results show that G226 increased the levels of JC-1 monomers at a concentration of 200 nmol/L, indicating that mitochondrial outer membrane permeabilization (MOMP) occurred after treatment with G226 (Figure 3A). However, this compound elicited no effect on the expression of Bcl-2, Bcl-XL, or Bax at concentrations within the 25–200 nmol/L range (Figure 3B). Only activated Caspase-9 and cleaved Bid (tBid) were increased in the cells (Figure 3B). These data suggest that in G226-treated cells, activated Caspase-8 cleaved Bid, resulting in the release of cytochrome c from the mitochondria and consequential activation of Caspase-9.


G226, a novel epipolythiodioxopiperazine derivative, induces autophagy and caspase-dependent apoptosis in human breast cancer cells in vitro.

He PX, Che YS, He QJ, Chen Y, Ding J - Acta Pharmacol. Sin. (2014)

G226 induces mitochondrial outer membrane permeabilization. (A) MDA-MB-231 and MCF-7 cells were treated with 200 nmol/L G226 for 24 h, and the mitochondrial membrane potential of the cells was measured by flow cytometry after JC-1 staining. (B) MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h, and the cell lysates were collected and examined by immunoblotting with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125716&req=5

fig3: G226 induces mitochondrial outer membrane permeabilization. (A) MDA-MB-231 and MCF-7 cells were treated with 200 nmol/L G226 for 24 h, and the mitochondrial membrane potential of the cells was measured by flow cytometry after JC-1 staining. (B) MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h, and the cell lysates were collected and examined by immunoblotting with the indicated antibodies.
Mentions: Many agents induce apoptosis in cells via two major pathways: the death receptor-mediated pathway and the mitochondria-mediated pathway. Caspase-8 is an initiator caspase, which not only activates executioner caspases, such as procaspase-3 and procaspase-7, to complete the death receptor signaling pathway but also cleaves the Bcl-2 family member Bid to amplify apoptosis via the mitochondrial pathway20. Our results show that G226 increased the levels of JC-1 monomers at a concentration of 200 nmol/L, indicating that mitochondrial outer membrane permeabilization (MOMP) occurred after treatment with G226 (Figure 3A). However, this compound elicited no effect on the expression of Bcl-2, Bcl-XL, or Bax at concentrations within the 25–200 nmol/L range (Figure 3B). Only activated Caspase-9 and cleaved Bid (tBid) were increased in the cells (Figure 3B). These data suggest that in G226-treated cells, activated Caspase-8 cleaved Bid, resulting in the release of cytochrome c from the mitochondria and consequential activation of Caspase-9.

Bottom Line: G226 also induced mitochondrial outer membrane permeabilization, resulted in the caspase-9 activation.Silencing of p62 or LC3 partially diminished caspase-8 and subsequent caspase-3 activation.LC3 silencing partially reversed G226-induced apoptosis, but p62 silencing elicited a subtle effect on G226-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT

Aim: To investigate the effects of G226, a novel epipolythiodioxopiperazine derivative, on human breast cancer cells in vitro, and to explore its anticancer mechanisms.

Methods: A panel of human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, ZR-75-30, BT474, BT549, SK-BR-3, T47D and HBL100) was examined. Cell proliferation was measured using sulforhodamine B assay, and cell apoptosis was detected with flow cytometry and caspase activity assay. Western blotting, immunofluorescence and targeted gene knockdowns were used to study autophagy in the cells.

Results: G226 suppressed proliferation of the 9 breast cancer cell lines with a mean IC50 value of 48.5 nmol/L (the mean IC50 value of adriamycin, a reference compound, was 170.6 nmol/L). G226 induced dose-dependent apoptosis of MDA-MB-231 and MCF-7 cells, accompanied by markedly increased activities of caspase-8 and caspase-3/7, which were abolished by caspase inhibitors zVAD or zIETD. G226 also induced mitochondrial outer membrane permeabilization, resulted in the caspase-9 activation. Moreover, G226 dose-dependently enhanced the autophagy marker LC3-II and autophagy substrate p62 accumulation in the cells, which were co-localized with caspase-8. Silencing of p62 or LC3 partially diminished caspase-8 and subsequent caspase-3 activation. LC3 silencing partially reversed G226-induced apoptosis, but p62 silencing elicited a subtle effect on G226-induced apoptosis.

Conclusion: The novel epipolythiodioxopiperazine derivative G226 exerts potent anticancer action against human breast cancer cells in vitro, via triggering autophagy and caspase-dependent apoptosis.

Show MeSH
Related in: MedlinePlus