Sulfasalazine-induced ferroptosis in breast cancer cells is reduced by the inhibitory effect of estrogen receptor on the transferrin receptor

Abstract

The aim of the present study was to clarify the activation of ferroptosis in different breast cancer cells by sulfasalazine (SAS) and to explore the relationship between the estrogen receptor (ER) and the transferrin receptor (TFRC). MDA-MB-231 and T47D cells were treated with SAS for 24 h. Changes in cell morphology were observed under a microscope. CCK-8 was used to detect the proliferation inhibition rate and determine the IC50 values. Western blotting was used to detect the expression of glutathione peroxidase 4 (GPX4) and xCT. Flow cytometry was used to identify changes in the production of reactive oxygen species (ROS). Mitochondrial morphological changes in T47D were observed using transmission electron microscopy. Changes in the mitochondrial membrane potential (MMP) were observed using confocal fluorescence microscopy. RT-PCR was used to detect the mRNA expression levels of TFRC and divalent metal transporter 1 (DMT1). Bioinformatics analysis was performed on TFRC expression in 1,208 breast cancer samples and its relationship with ER. TFRC expression was detected in various breast cancer tissues using immunohistochemistry and in various breast cancer cells using western blotting. Small interfering RNA (siRNA) knocked down ER expression in T47D cells, and changes in the TFRC mRNA and protein levels were observed. RT-PCR was used to detect TFRC expression in 87 clinical specimens. The results of the present study revealed that SAS could inhibit breast cancer cell viability, which was accompanied by an abnormal increase in ROS and a depletion of GPX4 and system xc-. Liproxstatin-1 reversed the SAS-induced increase in ROS. The cells treated with SAS had shrunken mitochondria and decreased MMP. SAS upregulated TFRC and DMT1. Knockdown of the ER increased TFRC expression in breast cancer cells. Immunohistochemistry indicated that TFRC expression was lower in ER+ tissues than in ER- tissues. After confirmation with RT-PCR in 87 clinical specimens, TFRC expression in ER- tissue was revealed to be significantly higher than that of ER+ tissue. In conclusion SAS could trigger ferroptosis in breast cancer cells, especially in cells with low ER expression. Therefore, SAS is a potential agent for breast cancer treatment.