Mechanism of Hypericin Inducing Ferroptosis in Breast Cancer Cells
JIANG Shuangyan, XIANG Siqi, LIU Shi, CUI Ruxia, PENG Hang, XIANG Mingjun*
This study was aimed to investigate the mechanism of hypericin inducing ferroptosis in breast cancer cells MCF-7 and MDA-MB-231. CCK-8 assay was used to detect the effects of various concentrations of hypericin on the viability of MCF-7, MDA-MB-231 and MCF-10A cells, test the recovery effect of Fer-1 (ferrostatin-1) on the cell viability affected by hypericin. Clonogenic assay was employed to test the inhibitory effect of hypericin on the clonal formation of MCF-7 and MDA-MB-231 cells. DCFH-DA probe was used to detect the accumulation of intracellular ROS (reactive oxygen species). LDH (lactate dehydrogenase) release assay was conducted to measure changes in LDH. Tissue iron assay kit was used to detect the accumulation of intracellular Fe2+. Reduced GSH (glutathione) assay kit was employed to measure GSH levels. And immunoblotting was performed to assess the expression of SLC7A11/xCT (solute carrier family 7 member 11) and GPX4 (glutathione peroxidase 4) proteins in breast cancer cells after hypericin treatment. The results showed that hypericin had a semi-inhibitory concentration (IC50) of 10 µmol/L for breast cancer cells MCF-7 and 15 µmol/L for MDA-MB-231 cells, and had no significant effect on normal cells MCF-10A. Hypericin could cause the accumulation of ROS and LDH release in MCF-7 and MDA-MB-231 cells. Additionally, the ferroptosis inhibitor Fer-1 could reverse the impact of hypericin on the viability of MCF-7 and MDA-MB-231 cells. At the same time, hypericin could also increase the intracellular Fe2+ content in MCF-7 and MDA-MB-231 cells, decrease the levels of GSH, and downregulate the expression of xCT and GPX4. The results indicated that hypericin might inhibit the proliferation of breast cancer cells by inducing ferroptosis.
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