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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 induces caspase-dependent apoptosis accompanied by induction of autophagy. 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), and the cell lysates were collected and examined by immunoblotting with the indicated antibodies. (C) In the presence or absence of 25 μmol/L CQ, MCF-7 cells were treated with 100 nmol/L G226 for 24 h, and fluorescence deconvolution microscopy was performed using anti-LC3 (green) antibodies. DAPI (blue) was used to visualize nuclei. Representative images from three independent experiments are shown. In the presence or absence of 2 mmol/L 3-MA or 25 μmol/L CQ, the cells were treated with 200 nmol/L G226, and the cell lysates were examined by immunoblotting with the indicated antibodies (D). The apoptotic cells are indicated by Annexin V+-labeling (E). The data are expressed 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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fig4: G226 induces caspase-dependent apoptosis accompanied by induction of autophagy. 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), and the cell lysates were collected and examined by immunoblotting with the indicated antibodies. (C) In the presence or absence of 25 μmol/L CQ, MCF-7 cells were treated with 100 nmol/L G226 for 24 h, and fluorescence deconvolution microscopy was performed using anti-LC3 (green) antibodies. DAPI (blue) was used to visualize nuclei. Representative images from three independent experiments are shown. In the presence or absence of 2 mmol/L 3-MA or 25 μmol/L CQ, the cells were treated with 200 nmol/L G226, and the cell lysates were examined by immunoblotting with the indicated antibodies (D). The apoptotic cells are indicated by Annexin V+-labeling (E). The data are expressed 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: Next, we determined whether G226-induced autophagy, another important cell-killing intracellular event, occurs. LC3-II is required for the elongation and closure of autophagosomal membranes and is considered as a marker of autophagy9,21. Compared with the control-treated cells, LC3-II accumulated in MDA-MB-231 and MCF-7 cells treated with G226 in a dose- and time-dependent manner (Figure 4A and 4B). Furthermore, treatment of cells with the lysosomal inhibitor chloroquine (CQ) resulted in the increased accumulation of LC3 puncta in response to G226 in MCF-7 cells (Figure 4C). These data suggest that G226 also induces autophagy in breast cancer cells. The degradation of p62 is considered to be another marker for autophagy because p62 is a substrate of autophagy. To our surprise, G226 resulted in the accumulation of p62 at lower doses and at early timepoints of treatment (Figure 4A and 4B). Interestingly, we also observed that the increased LC3-II expression (Figure 4B) resulting from G226 treatment occurred concomitantly with an increase in Annexin V+-labeled cells (Figure 2B) and in cleaved PARP (Figure 4B), indicating that G226-induced autophagy is associated with the apoptotic process.


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 caspase-dependent apoptosis accompanied by induction of autophagy. 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), and the cell lysates were collected and examined by immunoblotting with the indicated antibodies. (C) In the presence or absence of 25 μmol/L CQ, MCF-7 cells were treated with 100 nmol/L G226 for 24 h, and fluorescence deconvolution microscopy was performed using anti-LC3 (green) antibodies. DAPI (blue) was used to visualize nuclei. Representative images from three independent experiments are shown. In the presence or absence of 2 mmol/L 3-MA or 25 μmol/L CQ, the cells were treated with 200 nmol/L G226, and the cell lysates were examined by immunoblotting with the indicated antibodies (D). The apoptotic cells are indicated by Annexin V+-labeling (E). The data are expressed 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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Related In: Results  -  Collection

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fig4: G226 induces caspase-dependent apoptosis accompanied by induction of autophagy. 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), and the cell lysates were collected and examined by immunoblotting with the indicated antibodies. (C) In the presence or absence of 25 μmol/L CQ, MCF-7 cells were treated with 100 nmol/L G226 for 24 h, and fluorescence deconvolution microscopy was performed using anti-LC3 (green) antibodies. DAPI (blue) was used to visualize nuclei. Representative images from three independent experiments are shown. In the presence or absence of 2 mmol/L 3-MA or 25 μmol/L CQ, the cells were treated with 200 nmol/L G226, and the cell lysates were examined by immunoblotting with the indicated antibodies (D). The apoptotic cells are indicated by Annexin V+-labeling (E). The data are expressed 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: Next, we determined whether G226-induced autophagy, another important cell-killing intracellular event, occurs. LC3-II is required for the elongation and closure of autophagosomal membranes and is considered as a marker of autophagy9,21. Compared with the control-treated cells, LC3-II accumulated in MDA-MB-231 and MCF-7 cells treated with G226 in a dose- and time-dependent manner (Figure 4A and 4B). Furthermore, treatment of cells with the lysosomal inhibitor chloroquine (CQ) resulted in the increased accumulation of LC3 puncta in response to G226 in MCF-7 cells (Figure 4C). These data suggest that G226 also induces autophagy in breast cancer cells. The degradation of p62 is considered to be another marker for autophagy because p62 is a substrate of autophagy. To our surprise, G226 resulted in the accumulation of p62 at lower doses and at early timepoints of treatment (Figure 4A and 4B). Interestingly, we also observed that the increased LC3-II expression (Figure 4B) resulting from G226 treatment occurred concomitantly with an increase in Annexin V+-labeled cells (Figure 2B) and in cleaved PARP (Figure 4B), indicating that G226-induced autophagy is associated with the apoptotic process.

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