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. 2016 Jun 22:6:28403.
doi: 10.1038/srep28403.

Dioscin strengthens the efficiency of adriamycin in MCF-7 and MCF-7/ADR cells through autophagy induction: More than just down-regulation of MDR1

Changyuan Wang  1   2 , Xiaokui Huo  1   2 , Lijuan Wang  1 , Qiang Meng  1   2 , Zhihao Liu  1   2 , Qi Liu  1   2 , Huijun Sun  1   2 , Pengyuan Sun  1   2 , Jinyong Peng  1   2 , Kexin Liu  1   2
Affiliations

Dioscin strengthens the efficiency of adriamycin in MCF-7 and MCF-7/ADR cells through autophagy induction: More than just down-regulation of MDR1

Changyuan Wang et al. Sci Rep. .

Abstract

The purpose of present study was to investigate the effect of dioscin on activity of adriamycin (ADR) in ADR-sensitive (MCF-7) and ADR-resistant (MCF-7/ADR) human breast cancer cells and to clarify the molecular mechanisms involved. Antiproliferation effect of ADR was enhanced by dioscin in MCF-7 and MCF-7/ADR cells. Dioscin significantly inhibited MDR1 mRNA and protein expression and MDR1 promoter and nuclear factor κ-B (NF-κB) activity in MCF-7/ADR cells. Additionally, inhibitor κB-α (IκB-α) degradation was inhibited by dioscin. Moreover, dioscin induced the formation of vacuoles in the cytoplasm and protein level of LC3-II in MCF-7 and MCF-7/ADR cells. Autophagy inhibitor 3-MA abolished the effect of dioscin on ADR cytotoxicity. Dioscin inhibited phosphorylation of PI3K and Akt, resulting in upregulation of LC3-II expression. In conclusion, dioscin increased ADR chemosensitivity by down-regulating MDR1 expression through NF-κB signaling inhibition in MCF-7/ADR cells. Autophagy was induced by dioscin to ameliorate the cytotoxicity of ADR via inhibition of the PI3K/AKT pathways in MCF-7 and MCF-7/ADR cells. These findings provide evidence in support of further investigation into the clinical application of dioscin as a chemotherapy adjuvant.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Dioscin at nontoxic concentration increased ADR chemosensitivity in MCF-7 and MCF-7/ADR cells.
(a) Cells were treated with dioscin (0.1–10 μM) for 24 or 48 h and the survival cells were determined by MTT assay. (b) Cells were treated with ADR (0.1–100 μM) with or without dioscin (0.4 μM) for 24 h and the survival cells were determined by MTT assay. *,#p < 0.01 versus that obtained in the absence of dioscin.
Figure 2
Figure 2. Dioscin inhibited MDR1 expression in MCF-7/ADR cells.
(a) Cells were pre-incubated with or without dioscin (0.4 μM) or verapamil (Ver, 20 μM) for 24 h and intracellular accumulation of ADR were determined by LC-MS/MS after incubation of ADR for 1 h. (b–e) Cells were treated with 0.4 μM dioscin for 0−48 h (b,d) or different concentrations of dioscin (from 0 to 0.4 μM) for 48 h (c,e). Total RNA was extracted and MDR1 expression was analyzed by qRT-PCR (b,c). Lysates of cells were electrophoresed and the expression of MDR1 was detected with an MDR1-specific antibody (d,e). The β-actin band is shown to confirm integrity and equal loading of protein. Means ± SD of three experiments are presented. ***p < 0.001 versus that obtained in control group.
Figure 3
Figure 3. Dioscin down-regulated MDR1 expression via NF-κB signaling inhibition in MCF-7/ADR cells.
Cells were transiently transfected with MDR1 (a) and NF-κB (b) promoter plasmid. After treatment with dioscin and TNF-α, luciferase activity was determined and normalized. (c) Effects of dioscin and TNF-α on MDR1 protein expression in MCF-7/ADR cells by Western blotting. (d) Effects of dioscin and TNF-α on phospho-IκB-α protein expression in MCF-7/ADR cells by Western blotting. Means ± SD of three experiments are presented. ***p < 0.001 compared to control group; ###p < 0.001 compared to TNF-α group.
Figure 4
Figure 4. Dioscin induced autophagy in MCF-7 and MCF-7/ADR cells.
(a) Cells were treated with 0.4 μM dioscin for 24 h and cellular morphology was observed by phase-contrast microscopy. Vacuoles in the cytoplasm were marked by arrowhead. Magnification: 400×. LC3-II and beclin-1 protein expression was determined by Western blotting (b). Means ± SD of three experiments are presented.***p < 0.001 versus that obtained in control group.
Figure 5
Figure 5. Inhibition of autophagy by 3-MA weakened the sensitization effect of dioscin in MCF-7 and MCF-7/ADR cells.
A, cells were treated with 3-MA, dioscin and/or ADR for 24 h and cell viability was determined by MTT assay (a) or Annexin V/PI double staining (b). **p < 0.01 versus that obtained in the corresponding control group. ##p < 0.01 versus that obtained in the corresponding ADR alone group. §p < 0.05 versus that obtained in the corresponding ADR + dioscin group.
Figure 6
Figure 6. Dioscin induced autophagy through inhibition of PI3K/AKT pathways in MCF-7 and MCF-7/ADR cells.
(a–c) Effect of dioscin (0.4 μM) on the levels of phosphorylation of PI3K and Akt in MCF-7 and MCF-7/ADR cells. (d,e) Effect of dioscin and/or PI3K/AKT inhibitor LY294002 on the levels of LC3 protein expression in MCF-7 and MCF-7/ADR cells. Cells were treated with dioscin (0.4 μM) or LY294002 (20 μM) for 24 h. Cell lysates were prepared and analyzed by Western blot analysis. ***p < 0.001, **p < 0.01 versus that obtained in the corresponding control group.
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