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Estrogen Signaling Induces Mitochondrial Dysfunction-Associated Autophagy and Senescence in Breast Cancer Cells - PubMed

  • ️Wed Jan 01 2020

Estrogen Signaling Induces Mitochondrial Dysfunction-Associated Autophagy and Senescence in Breast Cancer Cells

Khuloud Bajbouj et al. Biology (Basel). 2020.

Abstract

Previous work has shown that although estrogen (E2) disrupts cellular iron metabolism and induces oxidative stress in breast and ovarian cancer cells, it fails to induce apoptosis. However, E2 treatment was reported to enhance the apoptotic effects of doxorubicin in cancer cells. This suggests that E2 can precipitate anti-growth effects that render cancer cells more susceptible to chemotherapy. To investigate such anti-growth non-apoptotic, effects of E2 in cancer cells, MDA-MB-231 and MCF-7 cells were evaluated for the expression of key autophagy and senescence markers and for mitochondrial damage following E2 treatment. Treated cells experienced mitochondrial membrane depolarization along with increased expression of LC3-I/II, Pink1 and LAMP2, increased LC3-II accumulation and increased lysosomal and mitochondrial accumulation and flattening. E2-treated MCF-7 cells also showed reduced P53 and pRb780 expression and increased Rb and P21 expression. Increased expression of the autophagy markers ATG3 and Beclin1 along with increased levels of β-galactosidase activity and IL-6 production were evident in E2-treated MCF-7 cells. These findings suggest that E2 precipitates a form of mitochondrial damage that leads to cell senescence and autophagy in breast cancer cells.

Keywords: Estrogen; MCF-7; MDA-MB-231; autophagy; mitochondria; senescence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Mitochondrial accumulation and autophagy in E2-treated MCF-7 cells. (A) Cells treated with 20 nM E2 or left untreated for 24 or 48 h were stained for DNA (DAPI; blue), mitochondrial accumulation (mitotracker; red) and LC3 (green); images were taken at 40X magnification; scale bar (white) represents 10 μm. Presence of SAHF (senescence-associated heterochromatin foci) in the nuclear region of senescent cells is indicated by white arrows. (B) Expression of cell senescence and autophagy markers P53, P21, and LC3-I/II in lysates of cells treated with 20 nM E2 for 24 and 48 h; RQ refer to LC3-II quantity under different treatment conditions relative to control. (C) Mean + SD fold change in protein expression in treated and untreated cells based on three separate experiments.

Figure 2
Figure 2

Mitochondrial accumulation and autophagy in E2-treated MDA-MB-231 cells. (A) Cells treated with 20 nM E2 or left untreated for 24 or 48 h were stained for DNA (DAPI; blue), mitochondrial accumulation (mitotracker; red) and LC3 (green); images were taken at 40X magnification; scale bar (white) represents 10 μm. Presence of SAHF (senescence-associated heterochromatin foci) in the nuclear region of senescent cells is indicated by white arrows. (B) Expression of cell senescence and autophagy markers P53, P21 and LC3-I/II in lysates of cells treated with 20 nM E2 for 24 and 48 h; RQ refer to LC3-II quantity under different treatment conditions relative to control. (C) Mean + SD fold change in protein expression in treated and untreated cells based on three separate experiments.

Figure 3
Figure 3

Expression pattern of autophagy-, cell cycle- and senescence-associated proteins in E2-treated MCF-7 cells. (A) Expression of autophagy related proteins ATG3, ATG5, ATG7, ATG12, ATG16, and Beclin1 in MCF-7 and MDA-MB-231 cells treated with 20 nM E2 for 24 and 48 hr. Data shown is representative of two separate experiments. (B) Expression of Rb and its phosphorylated form p-Rb780 was assessed by Western blotting in lysates of MCF-7 and MDA-MB-231 cells treated with 20 nM E2 for 6, 12, 24 or 48 h. (C) Mean + SD fold change in Rb and p-Rb780 expression levels in treated and untreated cells based on three separate experiments.

Figure 4
Figure 4

Mitochondrial membrane potential (Δψm) in E2-treated cells. (A) Mean + SD of MMP in MCF-7 and MDA-MB-231 cells treated with 20 nM E2 for 24 and 48 h. FCCP-treated cells served as a positive control and untreated cells served as a negative control. Data shown is based on three separate experiments/cell type. (B) Expression of the autophagy proteins LC3-I/II and Beclin1, the mitochondrial integrity regulator Pink1, and the lysosome-associated membrane protein 2 (LAMP2) in MCF-7 and MDA-MB-231 cells treated with 20 nM bafilomycin, 20 nM E2 or both for 24 hr. (C) Expression of LAMP2, Pink1 and NADPH oxidase 4 (NOX4) in mitochondrial protein lysates isolated from MCF-7 and MDA-MB-231 cells at 24 and 48 h post treatment with E2. Data shown in B and C is representative of three separate experiments each.

Figure 5
Figure 5

Functional assessment of autophagy in E2-treated cells. MCF-7 (A) and MDA-MB-231 (B) cells treated with 20 nM E2 or left untreated for 24 or 48 h were stained for DNA (DAPI; blue) or lysosomal accumulation (lysotracker; red). Data shown is representative of three independent experiments. (C) Qualitative assessment of senescence in MCF-7 and MDA-MB-231 cells treated with 20 nM E2 for 24 and 48 was done by observing the formation of green-metallic color following the addition of SA-β-Gal reagent. (D) Mean + SD of manually counted senescent MCF-7 and MDA-MB-231 cells at 24 and 48 h post E2 treatment in three separate experiments. (E) Cells treated with E2 (20 nM) or left untreated were assayed for the expression of cytoplasmic IL-6 and TNF-1α at 24 and 48 hr post E2 treatment. Data shown represent the average + SD of MFI based on three separate experiments.

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