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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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G226 triggers apoptosis via a caspase-dependent pathway. MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h (A) or with 200 nmol/L G226 for 0, 3, 6, 12, 24, or 48 h (B); apoptotic cells are indicated as Annexin V+-labeled cells. The cells were treated with increasing concentrations of G226 for 24 h and then collected for Western blotting with the indicated antibodies (C) and for determining the activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase (D). In the presence or absence of 20 μmol/L zVAD or 25 μmol/L zIETD, MDA-MB-231 cells were treated with 200 nmol/L G226 for 24 h (E-G). (E) The activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase were measured. The fold-changes were defined as the caspase activity of drug-treated cells/the caspase activity of respective DMSO-treated cells. (F) Apoptosis was detected by Annexin V+-PI staining and analyzed by flow cytometry. (G) The cell lysates were collected and examined by immunoblotting with the indicated antibodies. The data are shown as the mean±SD of independent experiments. The statistical significance was assessed by analysis of variance. bP<0.05, cP<0.01 compared with the DMSO group.
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fig2: G226 triggers apoptosis via a caspase-dependent pathway. MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h (A) or with 200 nmol/L G226 for 0, 3, 6, 12, 24, or 48 h (B); apoptotic cells are indicated as Annexin V+-labeled cells. The cells were treated with increasing concentrations of G226 for 24 h and then collected for Western blotting with the indicated antibodies (C) and for determining the activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase (D). In the presence or absence of 20 μmol/L zVAD or 25 μmol/L zIETD, MDA-MB-231 cells were treated with 200 nmol/L G226 for 24 h (E-G). (E) The activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase were measured. The fold-changes were defined as the caspase activity of drug-treated cells/the caspase activity of respective DMSO-treated cells. (F) Apoptosis was detected by Annexin V+-PI staining and analyzed by flow cytometry. (G) The cell lysates were collected and examined by immunoblotting with the indicated antibodies. The data are shown as the mean±SD of independent experiments. The statistical significance was assessed by analysis of variance. bP<0.05, cP<0.01 compared with the DMSO group.

Mentions: To investigate the detailed mechanism underlying G226-induced breast cancer cell death, we examined the ability of G226 to induce apoptosis. ER-negative MDA-MB-231 and ER-positive MCF-7 cells were used for these studies. G226 significantly increased the percentage of Annexin V+-labeled cells in a dose- and time-dependent manner, as indicated by flow cytometric analysis (Figure 2A and 2B). After treatment with 200 nmol/L G226 for 24 h, approximately 30 % cells underwent apoptosis. The apoptotic cleavage of poly (ADP-ribose) polymerase (PARP) and Caspase-8 was also increased in a dose-dependent manner by G226 (Figure 2C). Because Caspase-3 is deficient in MCF-7 cells, the increased cleaved Caspase-3 was only observed in MDA-MB-231 cells that were exposed to G226 (Figure 2C). Conversely, Caspase-Glo 8 and Caspase-3/7 activities were determined using commercially available kits. G226 significantly activated these caspases (Figure 2D). Taken together, these data indicate that caspases are involved in G226-induced apoptosis. Next, caspase inhibitors, such as zVAD and zIETD, were used to examine whether the inhibition of caspase cleavage would be sufficient to attenuate G226-induced apoptosis. As shown in Figure 2E–2G, both inhibitors abolished the activities of Caspase-8 and Caspase-3/7 and significantly attenuated G226-induced apoptosis. Collectively, these findings suggest that G226 induces caspase-dependent 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 triggers apoptosis via a caspase-dependent pathway. MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h (A) or with 200 nmol/L G226 for 0, 3, 6, 12, 24, or 48 h (B); apoptotic cells are indicated as Annexin V+-labeled cells. The cells were treated with increasing concentrations of G226 for 24 h and then collected for Western blotting with the indicated antibodies (C) and for determining the activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase (D). In the presence or absence of 20 μmol/L zVAD or 25 μmol/L zIETD, MDA-MB-231 cells were treated with 200 nmol/L G226 for 24 h (E-G). (E) The activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase were measured. The fold-changes were defined as the caspase activity of drug-treated cells/the caspase activity of respective DMSO-treated cells. (F) Apoptosis was detected by Annexin V+-PI staining and analyzed by flow cytometry. (G) The cell lysates were collected and examined by immunoblotting with the indicated antibodies. The data are shown as the mean±SD of independent experiments. The statistical significance was assessed by analysis of variance. bP<0.05, cP<0.01 compared with the DMSO group.
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fig2: G226 triggers apoptosis via a caspase-dependent pathway. MDA-MB-231 and MCF-7 cells were treated with increasing concentrations of G226 for 24 h (A) or with 200 nmol/L G226 for 0, 3, 6, 12, 24, or 48 h (B); apoptotic cells are indicated as Annexin V+-labeled cells. The cells were treated with increasing concentrations of G226 for 24 h and then collected for Western blotting with the indicated antibodies (C) and for determining the activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase (D). In the presence or absence of 20 μmol/L zVAD or 25 μmol/L zIETD, MDA-MB-231 cells were treated with 200 nmol/L G226 for 24 h (E-G). (E) The activities of the Caspase-8-like IETDase and the Caspase-3/7-like DEVDase were measured. The fold-changes were defined as the caspase activity of drug-treated cells/the caspase activity of respective DMSO-treated cells. (F) Apoptosis was detected by Annexin V+-PI staining and analyzed by flow cytometry. (G) The cell lysates were collected and examined by immunoblotting with the indicated antibodies. The data are shown as the mean±SD of independent experiments. The statistical significance was assessed by analysis of variance. bP<0.05, cP<0.01 compared with the DMSO group.
Mentions: To investigate the detailed mechanism underlying G226-induced breast cancer cell death, we examined the ability of G226 to induce apoptosis. ER-negative MDA-MB-231 and ER-positive MCF-7 cells were used for these studies. G226 significantly increased the percentage of Annexin V+-labeled cells in a dose- and time-dependent manner, as indicated by flow cytometric analysis (Figure 2A and 2B). After treatment with 200 nmol/L G226 for 24 h, approximately 30 % cells underwent apoptosis. The apoptotic cleavage of poly (ADP-ribose) polymerase (PARP) and Caspase-8 was also increased in a dose-dependent manner by G226 (Figure 2C). Because Caspase-3 is deficient in MCF-7 cells, the increased cleaved Caspase-3 was only observed in MDA-MB-231 cells that were exposed to G226 (Figure 2C). Conversely, Caspase-Glo 8 and Caspase-3/7 activities were determined using commercially available kits. G226 significantly activated these caspases (Figure 2D). Taken together, these data indicate that caspases are involved in G226-induced apoptosis. Next, caspase inhibitors, such as zVAD and zIETD, were used to examine whether the inhibition of caspase cleavage would be sufficient to attenuate G226-induced apoptosis. As shown in Figure 2E–2G, both inhibitors abolished the activities of Caspase-8 and Caspase-3/7 and significantly attenuated G226-induced apoptosis. Collectively, these findings suggest that G226 induces caspase-dependent 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