Battaglia Anna Martina, Sacco Alessandro, Giorgio Emanuele, Petriaggi Lavinia, Elzanowska Julia, Cruz Ana Rita, Rocha Luis, Pereira Catarina Esteves, Strano Moraes Maria Carolina, Palazzo Luca, De Vitis Claudia, Costa-Silva Bruno, Biamonte Flavia
Laboratory of Biochemistry and Cell Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Catanzaro, Italy.
Systems Oncology Laboratory, Champalimaud Foundation, Lisbon, Portugal.
Front Cell Dev Biol. 2025 Mar 10;13:1532097. doi: 10.3389/fcell.2025.1532097. eCollection 2025.
Ferroptosis is a promising new target for ovarian cancer (OVCA) treatment. However, some OVCA cell types resist the induction of ferroptosis by limiting the intracellular accumulation of the labile iron pool (LIP).
HEY, COV318 and PEO4 were treated with erastin and assessed for cell viability by using PI flow cytometry assays. Erastin-affected iron metabolism was analysed by using FerroOrange assay, Western Blot (WB) analysis of ferritin heavy chain (FtH), transferrin receptor (CD71), and ferroportin (FPN). Mitochondrial reactive oxygen species (mitROS) and lipid peroxidation were quantified via MitoSOX and BODIPY-C11 flow cytometry assays, respectively. Exosomes (EVs) were collected from cell culture media through ultracentrifugation and then enumerated and analyzed by Nanoparticale Tracking Analysis (NTA) and transmission electron microscopy (TEM). CD63 protein expression in EVs was measured through WB by using CD9 as a loading control. Loss-of-function assays for FtH and CD63 were performed by using siRNA-mediated transient transfection.
We demonstrate that erastin treatment (8 µM, 8 h) is accompanied by the release of iron-rich ferritin via EV pathway in COV318 and PEO4 OVCA cells, thus failing to exert cytotoxic effects. Mechanistically, erastin causes the upregulation of CD63, a tetraspanin involved in forming multivesicular bodies (MVBs) and EVs, and the increase of MBVs assessed by transmission electron microscopy. Consistent with these findings, EV isolation followed by nanoparticle tracking analysis revealed a significant increase in EVs/cell in erastin-treated COV318 and PEO4 cells. Notably, EVs harvested from these cells contained CD63 and FtH, a major iron-storage protein. Inhibition of EV biogenesis with GW4869 prevented FtH release and restored LIP accumulation, lipid peroxidation, and ferroptosis sensitivity in COV318 and PEO4 cells.
Overall, our results indicate that OVCA cells can utilize CD63+ EVs to secrete iron-rich ferritin as a mechanism to evade erastin-induced ferroptosis. These findings suggest that combining erastin with EV inhibitors could offer promising strategy for overcoming ferroptosis resistance in OVCA.
铁死亡是卵巢癌（OVCA）治疗中一个很有前景的新靶点。然而，一些OVCA细胞类型通过限制不稳定铁池（LIP）的细胞内积累来抵抗铁死亡的诱导。
用埃拉斯汀处理HEY、COV318和PEO4细胞，并通过碘化丙啶（PI）流式细胞术检测细胞活力。通过FerroOrange检测、铁蛋白重链（FtH）、转铁蛋白受体（CD71）和铁转运蛋白（FPN）的蛋白质免疫印迹（WB）分析来分析埃拉斯汀对铁代谢的影响。分别通过MitoSOX和BODIPY-C11流式细胞术检测线粒体活性氧（mitROS）和脂质过氧化。通过超速离心从细胞培养基中收集外泌体（EVs），然后通过纳米颗粒跟踪分析（NTA）和透射电子显微镜（TEM）进行计数和分析。以CD9作为上样对照，通过WB检测EVs中CD63蛋白的表达。通过小干扰RNA（siRNA）介导的瞬时转染对FtH和CD63进行功能缺失实验。
我们证明，在COV318和PEO4 OVCA细胞中，埃拉斯汀处理（8 μM，8小时）伴随着富含铁的铁蛋白通过EV途径释放，因此未能发挥细胞毒性作用。从机制上讲，埃拉斯汀导致四跨膜蛋白CD63上调，CD63参与多囊泡体（MVBs）和EVs的形成，并且通过透射电子显微镜评估的MVBs增加。与这些发现一致，通过纳米颗粒跟踪分析进行EV分离后发现，在埃拉斯汀处理的COV318和PEO4细胞中，每个细胞释放的EVs显著增加。值得注意的是，从这些细胞中收获的EVs含有CD63和主要的铁储存蛋白FtH。用GW4869抑制EV生物发生可防止FtH释放，并恢复COV318和PEO4细胞中的LIP积累、脂质过氧化和铁死亡敏感性。
总体而言，我们的结果表明，OVCA细胞可以利用CD63+ EVs分泌富含铁的铁蛋白，作为逃避埃拉斯汀诱导的铁死亡的一种机制。这些发现表明将埃拉斯汀与EV抑制剂联合使用可能为克服OVCA中的铁死亡抗性提供有前景的策略。
