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. 2024 Oct 22;19(5):606-621.
doi: 10.4103/RPS.RPS_96_24. eCollection 2024 Oct.

The activation of the G-protein-coupled estrogen receptor promotes the aggressiveness of MDA-MB231 cells by targeting the IRE1α/TXNIP pathway

Maryam Mohammad-Sadeghipour  1 , Mohammad Hadi Nematollahi  2 , Hassan Ahmadinia  3 , Mohammad Reza Hajizadeh  4   5 , Mehdi Mahmoodi  1   4
Affiliations

The activation of the G-protein-coupled estrogen receptor promotes the aggressiveness of MDA-MB231 cells by targeting the IRE1α/TXNIP pathway

Maryam Mohammad-Sadeghipour et al. Res Pharm Sci. .

"VSports" Abstract

Background and purpose: This study investigated modulating the G protein-coupled estrogen receptor (GPER) on the IRElα/TXNIP pathway and its role in drug resistance in MDA-MB231 cells. VSports手机版.

Experimental approach: To determine the optimal concentrations of G1 and 4-hydroxytamoxifen (TAM), GPER expression and ERK1/2 phosphorylation were analyzed using qRT-PCR and western blotting, respectively. Cells were treated with individual concentrations of G1 (1000 nM), G15 (1000 nM), and TAM (2000 nM), as well as combinations of these treatments (G1 + G15, TAM + G15, and G1 + TAM) for 24 and 48 h V体育安卓版. The expression levels of GPER, IRE1α, miR-17-5p, TXNIP, ABCB1, and ABCC1 genes and TXNIP protein expression were evaluated. Finally, apoptosis and cell migration were examined using flow cytometry and the wound-healing assay, respectively. .

Findings/results: Activating GPER with its specific agonist G1 and TAM significantly increased IRE1α levels in MDA-MB231 cells. IRE1α through splicing XBP1 led to unfolded protein response V体育ios版. In addition, decreased TXNIP gene and protein expression reduced apoptosis, increased migration, and upregulated the genes associated with drug resistance. .

Conclusion and implication: Our investigation revealed that blocking the GPER/IRE1α/TXNIP pathway in MDA-MB231 cells could enhance treatment efficacy and improve chemotherapy responsiveness. The distinct unfolded protein response observed in MDA-MB231 cells may stem from the unique characteristics of these cells, which lack receptors for estrogen, progesterone, and HER2/neu hormones, possessing only the GPER receptor (ER-/PR-/HER2-/GPER+). This study introduced a new pathway in TNBC cells, indicating that targeting GPER could be crucial in comprehensive therapeutic strategies in TNBC cells. VSports最新版本.

Keywords: Breast cancer; Drug resistance; G protein-coupled estrogen receptor; Thioredoxin interacting protein; Unfolded protein response; miR-17-5P V体育平台登录. .

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Conflict of interest statement

The authors declared no conflicts of interest in this study.

V体育官网 - Figures

Fig. 1
Fig. 1
The percentage of viable MDA-MB231 cells after 24 and 48 h of treatment with (A) G1, (B) tamoxifen (TAM), and (C) G15 was evaluated using the MTT assay at various concentrations. The data are presented as mean ± SD, n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the control group (concentration zero).
Fig. 2
Fig. 2
G1 and TAM induce mRNA gene expression of GPER and phosphorylation of ERK1/2 in the MDA-MB231 cell line. GPER mRNA expression of the cells treated with (A) G1 (0, 10, 100, and 1000 nM) or (B) with TAM (0, 100, 1000, and 2000 nM) for 12 h was evaluated by qRT-PCR; and the ERK1/2 phosphorylation in the cells treated with (C) G1 (0, 10, 100, and 1000 nM) for 30 min or (D) with TAM (0, 100, 1000, and 2000 nM) for 5 min was also evaluated using western blotting. The data represents the mean ± SD, n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the control group. GPER, G-protein-coupled estrogen receptor; ERK, endoplasmic reticulum kinase; TAM, tamoxifen.
Fig. 3
Fig. 3
Evaluating the effect of G1 and TAM (as GPER agonists) and G15 (as a GPER antagonist) on the gene expression of (A) GPER, (B) IRE1α, (C) TXNIP, and (D) TXNIP protein expression after 24 and 48 h of treatment in the MDA-MB231 cells. The data represent means ± SD, n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the control group; #P < 0.05 against G1 group; $P < 0.05 versus TAM group; and @P < 0.05 indicate significant differences between the designated groups. TAM, Tamoxifen; GPER, G-protein-coupled estrogen receptor; IRE, inositol-requiring enzyme; TXNIP, thioredoxin interacting protein.
Fig. 4
Fig. 4
Evaluating the relevance between the GPER/IRE1α/TXNIP signaling pathway and miR-17-5P, ABCB1, and ABCC1 gene expression in MDA-MB231 cells. MDA-MB231 cells were treated with G1, TAM, G15, G1 + G15, TAM + G15, and G1 + tAm at 24 and 48 h. Then, the relative expression of (A) miR-17-5P, (B) ABCB1, and (C) ABCC1 genes were assessed by qRT-PCR. Data represent the mean ± SD, n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the control group; #P < 0.05 versus G1 group; $P < 0.05 against TAM group; and @P < 0.05 indicate significant differences between the designated groups. TAM, Tamoxifen; GPER, G-protein-coupled estrogen receptor; IRE, inositol-requiring enzyme; TXNIP, thioredoxin interacting protein; ABCB1, ATP binding cassette subfamily B member 1; ABCC1, ATP binding cassette subfamily C member 1.
Fig. 5
Fig. 5
Evaluating the relevance between the GPER/IRE1α/TXNIP signaling pathway and apoptosis in MDA-MB231 cells. The flow cytometry assay was used to assess cell death after 48 h of treatment with G1, TAM, G15, G1 + G15, TAM + G15, and G1 + TAM. The amount of apoptosis in cells is reported based on the sum of early and late apoptosis (Q2 + Q3). ***P < 0.001 indicates significant differences compared to the control group; #P < 0.05 versus G1 group; $P < 0.05 in contrast to the TAM group. TAM, Tamoxifen; GPER, G-protein-coupled estrogen receptor; IRE, inositol-requiring enzyme; TXNIP, thioredoxin interacting protein.
Fig. 6
Fig. 6
Evaluating the relevance between the GPER/IRE1α/TXNIP signaling pathway and cell migration in MDA-MB231 cells. (A) The cell migration ability was determined using the wound-healing assay after 24 h of treatment with G1, TAM, G15, G1 + G15, TAM + G15, and G1 + TAM. (B) The data represent means ± SD, n = 3. *P < 0.05 and ***P < 0.001 indicate significant differences compared to the control group; #P < 0.05 versus G1 group; $P < 0.05 in contrast to the TAM group).
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