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Abstract：
Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E1), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI\'s catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E1 as well as cystamine (a known PDI inhibitor) against chemically induced ferroptosis in human breast cancer cells. Importantly, inhibition of PDI by 4-OH-E1 and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced ferroptosis. In conclusion, this study demonstrates that 4-OH-E1 is a novel inhibitor of PDI and can strongly inhibit ferroptosis in human breast cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.
摘要：
Ferroptosis是最近发现的一种调节细胞死亡形式，以过度的铁依赖性脂质过氧化为特征。最近的研究表明，蛋白质二硫键异构酶（PDI）是化学诱导的铁凋亡的重要介质，也是防止铁凋亡相关细胞死亡的新靶点。在本研究中,我们确定了4-羟基雌酮（4-OH-E1），内源性雌激素的代谢衍生物，是一种有效的PDI小分子抑制剂，并且可以强烈防止雌激素受体阴性MDA-MB-231人乳腺癌细胞中化学诱导的铁细胞死亡。下拉和CETSA测定表明4-OH-E1可以在体外和完整细胞中直接结合PDI。计算模型分析表明，4-OH-E1与PDIHis256形成两个氢键，这对于其结合相互作用并因此抑制PDI的催化活性至关重要。此外,PDI敲低减弱4-OH-E1以及胱胺(一种已知的PDI抑制剂)对抗人乳腺癌细胞中化学诱导的铁凋亡的保护作用。重要的是,4-OH-E1对PDI的抑制作用和半胱氨酸抑制剂或siRNA对PDI的敲除均显着降低iNOS活性和NO积累，最近已被证明在erastin诱导的铁凋亡中起重要作用。总之,这项研究表明,4-OH-E1是一种新型的PDI抑制剂,能够以不依赖雌激素受体的方式强烈抑制人乳腺癌细胞的铁凋亡.从本研究中获得的机理理解也可能有助于理解许多非癌细胞类型中内源性雌激素代谢物的雌激素受体非依赖性细胞保护作用。
