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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 the formation of a complex containing Caspase-8, p62 and LC3. MDA-MB-231 and MCF-7 cells were treated with 100 nmol/L G226 or vehicle for 24 h. (A) Fluorescence deconvolution microscopy was performed; Caspase-8 is shown in green, whereas p62 is shown in red. (B) LC3 is shown in green and p62 is shown in red; (C) LC3 is green and Caspase-8 is red; Nuclei labeled with DAPI are shown in blue. Representative images of three independent experiments are shown.
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fig5: G226 induces the formation of a complex containing Caspase-8, p62 and LC3. MDA-MB-231 and MCF-7 cells were treated with 100 nmol/L G226 or vehicle for 24 h. (A) Fluorescence deconvolution microscopy was performed; Caspase-8 is shown in green, whereas p62 is shown in red. (B) LC3 is shown in green and p62 is shown in red; (C) LC3 is green and Caspase-8 is red; Nuclei labeled with DAPI are shown in blue. Representative images of three independent experiments are shown.

Mentions: Because p62 and LC3 promote the formation of the intracellular death-inducing signaling complex (iDISC), which mediates Caspase-8 activation22,23, we further explored whether the activation of Caspase-8 by G226 requires p62 or LC3. Immunofluorescence analysis revealed that endogenous Caspase-8, LC3, and p62 co-localized and interacted in G226-treated cells (Figure 5). As expected, the silencing of p62 or LC3 partially diminished Caspase-8 activation and subsequent Caspase-3 activation (Figure 6A). Moreover, the silencing of LC3 partially reversed G226-induced apoptosis, although p62 silencing only elicited a subtle effect (Figure 6B and 6C). Nrf2 is considered as a target gene of p62 and can upregulate the transcription of cytoprotective genes24,25. We found that Nrf2 was increased when p62 was overexpressed in response to G226. Moreover, knockdown of p62 also reversed the G226-induced Nrf2 upregulation (Figure 6D). This finding might explain why the silencing of p62 is insufficient to block G226-induced apoptosis.


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 the formation of a complex containing Caspase-8, p62 and LC3. MDA-MB-231 and MCF-7 cells were treated with 100 nmol/L G226 or vehicle for 24 h. (A) Fluorescence deconvolution microscopy was performed; Caspase-8 is shown in green, whereas p62 is shown in red. (B) LC3 is shown in green and p62 is shown in red; (C) LC3 is green and Caspase-8 is red; Nuclei labeled with DAPI are shown in blue. Representative images of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: G226 induces the formation of a complex containing Caspase-8, p62 and LC3. MDA-MB-231 and MCF-7 cells were treated with 100 nmol/L G226 or vehicle for 24 h. (A) Fluorescence deconvolution microscopy was performed; Caspase-8 is shown in green, whereas p62 is shown in red. (B) LC3 is shown in green and p62 is shown in red; (C) LC3 is green and Caspase-8 is red; Nuclei labeled with DAPI are shown in blue. Representative images of three independent experiments are shown.
Mentions: Because p62 and LC3 promote the formation of the intracellular death-inducing signaling complex (iDISC), which mediates Caspase-8 activation22,23, we further explored whether the activation of Caspase-8 by G226 requires p62 or LC3. Immunofluorescence analysis revealed that endogenous Caspase-8, LC3, and p62 co-localized and interacted in G226-treated cells (Figure 5). As expected, the silencing of p62 or LC3 partially diminished Caspase-8 activation and subsequent Caspase-3 activation (Figure 6A). Moreover, the silencing of LC3 partially reversed G226-induced apoptosis, although p62 silencing only elicited a subtle effect (Figure 6B and 6C). Nrf2 is considered as a target gene of p62 and can upregulate the transcription of cytoprotective genes24,25. We found that Nrf2 was increased when p62 was overexpressed in response to G226. Moreover, knockdown of p62 also reversed the G226-induced Nrf2 upregulation (Figure 6D). This finding might explain why the silencing of p62 is insufficient to block G226-induced apoptosis.

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