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Glabridin induces paraptosis-like cell death via ER stress in breast cancer cells - PubMed

  • ️Sat Jan 01 2022

Glabridin induces paraptosis-like cell death via ER stress in breast cancer cells

Xiang Cui et al. Heliyon. 2022.

Abstract

Glabridin, a polyphenolic flavonoid isolated from the root of the glycyrrhiza glabra, has been demonstrated to have anti-tumor properties in human malignancies. This study found that glabridin decreased the viability of human breast cancer MDA-MB-231 and MCF7 cells in a dose-dependent manner that was not involved in the caspase-3 cascade. Glabridin promoted the formation of extensive cytoplasmic vacuolation by increasing the expression of endoplasmic reticulum (ER) stress markers BiP, XBP1s, and CHOP. The transmission electron microscopy and fluorescence with the ER chaperon KDEL suggested that the vacuoles were derived from ER. Glabridin-induced vacuolation was blocked when protein synthesis was inhibited by cycloheximide, demonstrating that protein synthesis is crucial for this process. Furthermore, we determined that glabridin causes loss of mitochondrial membrane potential as well as the production of reactive oxygen species, both of which lead to mitochondrial dysfunction. These features are consistent with a kind of programmed cell death described as paraptosis. This work reports for the first time that glabridin could induce paraptosis-like cell death, which may give new therapeutic approaches for apoptosis-resistant breast cancers.

Keywords: Breast cancer; ER stress; Glabridin; Paraptosis; Vacuolation.

© 2022 The Author(s).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Glabridin inhibits cell viability, clonogenic capacity, and cell cycle progression of breast cancer cells (A) and (B) The cytotoxicity of glabridin against MDA-MB-231 and MCF7 cells was assessed by CCK-8 assays. The cells were treated with (A) indicated doses of glabridin for 24 h or (B) 100 μM glabridin for indicated time periods. The OD values were measured in a microplate reader and the EC50 values of glabridin were calculated. The results are presented as the means ± SD of four independent experiments (C) Flow cytometry histograms (left panel) and quantification (right panel) of PI-labeled cells in each phase of the cell cycle. Cells were treated with 100 μM glabridin or 1 μM paclitaxel for 24 h and were then stained with PI for flow cytometric analysis. (D) Colony formation abilities of MDA-MB-231 and MCF7 cells treated with increasing concentrations of glabridin for 10 days. GLA, glabridin; NT, no treatment; PTX, paclitaxel; PI, propidium iodide.

Figure 2
Figure 2

Glabridin induces cytoplasmic vacuolation in breast cancer cells (A) and (B) Morphological changes of MDA-MB-231 and MCF7 cells treated with glabridin observed under an inverted light microscope. The cells were treated with (A) increasing concentrations of glabridin for 24 h or (B) 100 μM glabridin for indicated time periods. Cytoplasmic vacuoles were indicated by the red arrows.

Figure 3
Figure 3

Glabridin induces ER and mitochondrial dilation, ER stress, and proteasome inhibition (A) Representative fluorescence images of MDA-MB-231 and MCF7 cells after glabridin treatment. Cells were transiently transfected with the KDEL-GFP or COX8-RFP plasmids 24 h before treatment with glabridin (100 μM). After 24 h, the cells were observed under fluorescence microscopy (B) Transmission electron micrograph images of cells treated with glabridin (100 μM) for 6 h. The inset shows a part of a high magnification image. Scale bars, 1 μm; V, vacuole; NT, no treatment (C) and (D) The mRNA expression of ER stress markers in cells treated with glabridin. The cells were treated with (C) indicated concentrations of glabridin for 24 h or (D) 100 μM glabridin for indicated time periods. 28s was used as a loading control in RT-PCR (E) Cells treated with 100 μM glabridin or 100nM MG132 for 24 h were subjected to Western blot analysis with anti-ubiquitin or anti-GAPDH as indicated. The uncropped images of (C-E) were referred to in Supplementary Figure S7 (A-C). GLA, glabridin; XBP1u, unspliced XBP1; XBP1s, spliced XBP1; NT, no treatment.

Figure 4
Figure 4

Glabridin induces caspase-independent cell death in breast cancer cells (A) Western blot analysis of caspase-3 and PARP expression in 100 μM glabridin- or 1 μg/ml doxorubicin-treated cells. (B) Representative images of nuclear staining. MDA-MB-231 and MCF7 cells were treated with 100 μM glabridin for 24 h, and the nuclei were stained with DAPI (C) DNA fragmentation assay. Genomic DNA isolated from glabridin- or doxorubicin-treated cells subjected to agarose gel electrophoresis. (D) Cells were treated with 60 μM glabridin in the absence or presence of Z-VAD-FMK (20 μM), and the percentage of cell death was determined using PI staining (E) Cells were treated with 60 μM glabridin in the absence or presence of Z-VAD-FMK (10 μM or 20 μM), and the cell viability was determined using CCK-8 assay. The results are presented as the means ± SD of three independent experiments (vs. control: ∗P < 0.05; NS, not significant). The uncropped images of (A) and (C) were referred to in Supplementary Figure S8 (A) and (B). NT, no treatment; GLA, glabridin; Doxo, doxorubicin; Z, Z-VAD-FMK; Z10, 10μM Z-VAD-FMK; Z20, 20μM Z-VAD-FMK.

Figure 5
Figure 5

Glabridin induces caspase-independent cell death in breast cancer cells (A) and (B) Mitochondrial membrane potential (MMP) of the glabridin-treated cells was measured by (A) JC-1 fluorescence staining and (B) a fluorescence microplate reader. The graph represents the ratio of green (monomers) and red (dimers) fluorescence. CCCP treatment was used as a positive control (C) Fluorescence images of cellular ROS production in untreated or glabridin or Rosup treated cells. The results are presented as the means ± SD of three independent experiments (vs. control: ∗P < 0.05). GLA, glabridin; NT, no treatment.

Figure 6
Figure 6

Inhibition of PERK enhances glabridin-induced cytoplasmic vacuolation (A) Cells were pretreated with the indicated stimuli (500 nM CHX; 100 nM MG132; 10 μM 4μ8C; 1 μM GSK) for 20 min prior to the treatment with 60 μM glabridin for 24 h, and then the total RNA was extracted and subjected to RT-PCR (B) Cells were treated as in (A), and the cell viability was determined using CCK-8 assay. The results are presented as the means ± SD of three independent experiments (vs. control: ∗P < 0.05; NS, not significant) (C) Cells were treated as in (A), and observed under the phase-contrast microscope. Cytoplasmic vacuoles were indicated by the red arrow. The uncropped images of (A) were referred to in Supplementary Figure S9. CHX, cycloheximide; MG, MG132; GSK, GSK2656157.

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