Yajie Li1, Xinliu Zeng1, Dingheng Lu2, Minuo Yin1, Meirong Shan3, Ying Gao1. 1. Department of Gynecology and Obstetrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China. 2. Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China. 3. The Key Laboratory of Cardiovascular Remodeling and Function Research, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, Qilu Hospital of Shandong University, Jinan, China.
Abstract
STUDY QUESTION: Could erastin activate ferroptosis to regress endometriotic lesions? SUMMARY ANSWER: Erastin could induce ferroptosis to regress endometriotic lesions in endometriosis. WHAT IS KNOWN ALREADY: Ectopic endometrial stromal cells (EESCs) are in an iron overloading microenvironment and tend to be more sensitive to oxidative damage. The feature of erastin-induced ferroptosis is iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION: Eleven patients without endometriosis and 21 patients with endometriosis were recruited in this study. Primary normal and ectopic endometrial stromal cells were isolated, cultured and subjected to various treatments. The in vivo study involved 10 C57BL/6 female mice to establish the model of endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The markers of ferroptosis were assessed by cell viability, lipid peroxidation level and morphological changes. The cell viability was measured by colorimetric method, lipid peroxidation levels were measured by flow cytometry, and morphological changes were observed by transmission electron microscopy. Immunohistochemistry and western blot were used to detect ferroportin (FPN) expression. Prussian blue staining and immunofluorescent microscopy of catalytic ferrous iron were semi-quantified the levels of iron. Adenovirus-mediated overexpression and siRNA-mediated knockdown were used to investigate the role of FPN on erastin-induced ferroptosis in EESCs. MAIN RESULTS AND THE ROLE OF CHANCE: EESCs were more susceptible to erastin treatment, compared to normal endometrial stromal cells (NESCs) (P<0.05). Treatment of cultured EESCs with erastin dramatically increased the total ROS level (P<0.05, versus control), lipid ROS level (P<0.05, versus NESCs) and intracellular iron level (P<0.05, versus NESCs). The cytotoxicity of erastin could be attenuated by iron chelator, deferoxamine (DFO), and ferroptosis inhibitors, ferrostatin-1 and liproxstatin-1, (P<0.05, versus erastin) in EESCs. In EESCs with erastin treatment, shorter and condensed mitochondria were observed by electron microscopy. These findings together suggest that erastin is capable to induce EESC death by ferroptosis. However, the influence of erastin on NESCs was slight. The process of erastin-induced ferroptosis in EESCs accompanied iron accumulation and decreased FPN expression. The overexpression of FPN ablated erastin-induced ferroptosis in EESCs. In addition, knockdown of FPN accelerated erastin-induced ferroptosis in EESCs. In a mouse model of endometriosis, we found ectopic lesions were regressed after erastin administration. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was mainly conducted in primary human endometrial stromal cells. Therefore, the function of FPN in vivo need to be further investigated. WIDER IMPLICATIONS OF THE FINDINGS: Our findings reveal that erastin may serve as a potential therapeutic treatment for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare no conflict of interest.
STUDY QUESTION: Could erastin activate ferroptosis to regress endometriotic lesions? SUMMARY ANSWER: Erastin could induce ferroptosis to regress endometriotic lesions in endometriosis. WHAT IS KNOWN ALREADY: Ectopic endometrial stromal cells (EESCs) are in an iron overloading microenvironment and tend to be more sensitive to oxidative damage. The feature of erastin-induced ferroptosis is iron-dependent accumulation of lethal lipidreactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION: Eleven patients without endometriosis and 21 patients with endometriosis were recruited in this study. Primary normal and ectopic endometrial stromal cells were isolated, cultured and subjected to various treatments. The in vivo study involved 10 C57BL/6 female mice to establish the model of endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The markers of ferroptosis were assessed by cell viability, lipid peroxidation level and morphological changes. The cell viability was measured by colorimetric method, lipid peroxidation levels were measured by flow cytometry, and morphological changes were observed by transmission electron microscopy. Immunohistochemistry and western blot were used to detect ferroportin (FPN) expression. Prussian blue staining and immunofluorescent microscopy of catalytic ferrous iron were semi-quantified the levels of iron. Adenovirus-mediated overexpression and siRNA-mediated knockdown were used to investigate the role of FPN on erastin-induced ferroptosis in EESCs. MAIN RESULTS AND THE ROLE OF CHANCE: EESCs were more susceptible to erastin treatment, compared to normal endometrial stromal cells (NESCs) (P<0.05). Treatment of cultured EESCs with erastin dramatically increased the total ROS level (P<0.05, versus control), lipidROS level (P<0.05, versus NESCs) and intracellular iron level (P<0.05, versus NESCs). The cytotoxicity of erastin could be attenuated by iron chelator, deferoxamine (DFO), and ferroptosis inhibitors, ferrostatin-1 and liproxstatin-1, (P<0.05, versus erastin) in EESCs. In EESCs with erastin treatment, shorter and condensed mitochondria were observed by electron microscopy. These findings together suggest that erastin is capable to induce EESC death by ferroptosis. However, the influence of erastin on NESCs was slight. The process of erastin-induced ferroptosis in EESCs accompanied iron accumulation and decreased FPN expression. The overexpression of FPN ablated erastin-induced ferroptosis in EESCs. In addition, knockdown of FPN accelerated erastin-induced ferroptosis in EESCs. In a mouse model of endometriosis, we found ectopic lesions were regressed after erastin administration. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was mainly conducted in primary human endometrial stromal cells. Therefore, the function of FPN in vivo need to be further investigated. WIDER IMPLICATIONS OF THE FINDINGS: Our findings reveal that erastin may serve as a potential therapeutic treatment for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare no conflict of interest.
Authors: Łukasz Milewski; Aneta Ścieżyńska; Joanna Ponińska; Marta Soszyńska; Ewa Barcz; Piotr I Roszkowski; Paweł Kamiński; Paweł Włodarski; Rafał Płoski; Jacek Malejczyk Journal: Cells Date: 2021-03-21 Impact factor: 6.600