尤文肉瘤（ES）是儿童中最常见的恶性肿瘤之一。 ES 细胞的活跃代谢状态为治疗干预提供了新的潜在目标。作为细胞稳态的主要调节剂，碳酸酐酶（CA；EC 4.2.1.1）已成为抗癌药物开发的有前景的分子靶标。在本研究中，我们测试了商业药物乙酰唑胺和我们之前发现的针对 CAII 亚型的抑制剂，该亚型过度表达并与 ES 患者复发呈正相关。我们采用分子生物学测试来鉴定 CAII 的有效抑制剂，该抑制剂可以通过下调 ES 细胞中 FTH1 的表达来诱导铁死亡。在体外，我们还证明了它们具有减少细胞增殖、减少侵袭和诱导细胞凋亡或自噬相关细胞死亡的能力。使用蛋白质印迹法，我们证实了用 CA 抑制剂处理的细胞中组织蛋白酶 B 的诱导。研究发现，治疗过程中组织蛋白酶 B 表达的抑制会降低所选 CAII 抑制剂的抗癌功效。这些实验强调了 CAII 抑制剂的深远抗肿瘤活性，归因于它们在不造成实质性宿主损伤的情况下触发尤文肉瘤细胞铁死亡的卓越能力。所获得的结果表明，胞质CAII可能是ES治疗的潜在靶点，CAII抑制剂可以被认为是潜在的单药或联合抗肿瘤药物用于治疗ES。

Ewing sarcoma (ES) is one of the most frequent types of malignant tumors among children. The active metabolic state of ES cells presents a new potential target for therapeutic interventions. As a primary regulator of cellular homeostasis, carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as promising molecular targets for the development of anticancer drugs. Within the present study, we tested the commercial drug acetazolamide and our previously discovered inhibitors to target the CAII isoform, which was overexpressed and positively correlated with ES patient relapse. We employed molecular biology tests to identify effective inhibitors of CAII that can induce ferroptosis by downregulating FTH1 expression in ES cells. In vitro, we have also demonstrated their ability to reduce cell proliferation, decrease invasion, and induce apoptosis- or autophagy-related cell death. Using Western blotting, we confirmed the induction of cathepsin B in cells treated with CA inhibitors. It was found that the suppression of cathepsin B expression during the treatment reduces the anticancer efficacy of selected CAII inhibitors. These experiments highlighted profound antitumor activity of CAII inhibitors attributive to their remarkable ability to trigger ferroptosis in Ewing sarcoma cells without causing substantial host damage. The obtained results suggest that cytosolic CAII may be a prospective target for ES treatment, and CAII inhibitors can be considered as potential single-agent or combination antitumor agents to be used in the treatment of ES.