对于晚期和复发性子宫内膜癌（EC），免疫或靶向治疗目前仍处于临床研究阶段。我们的研究旨在探讨ESCRT机制在维持细胞膜完整性和逆转细胞焦亡中的作用。通过免疫组织化学、蛋白质印迹和免疫共沉淀来确定GSDMD、CHMP4B和VPS4A之间的表达和关系。我们采用FITC膜联蛋白V/碘化丙啶染色、Ca2荧光强度、IL-1β酶联免疫吸附测定和乳酸脱氢酶释放测定等技术来检测子宫内膜癌细胞的焦亡。通过电子和荧光共聚焦显微镜观察质膜穿孔和CHMP4B/VPS4A斑点。我们发现GSDMD、CHMP4B和VPS4A在焦亡EC异种移植小鼠模型组中存在差异表达，并且在EC中高、中和轻度表达与子宫内膜上皮细胞相比，用 LPS 和尼日利亚霉素处理的细胞。 Co-IP 证实了 GSDMD、CHMP4B 和 VPS4A 之间的相互作用。我们发现 GSDMD 敲除减少了 PI 阳性细胞、Ca2 外流、I​​L-1β 和 LDH 释放，而 CHMP4B 和 VPS4A 耗竭则增强了 HEC1A 和 AN3CA 细胞中的这些指标。电镜显示，GSDMD 失活后膜穿孔相应减少，EC 细胞中 CHMP4B 和 VPS4A 耗尽或过度表达后膜穿孔增加或减少。荧光共聚焦显微镜在GSDMDNT细胞损伤的质膜上检测到与VPS4A相关的CHMP4B蛋白点。我们初步证明CHMP4B和VPS4A通过促进子宫内膜癌的细胞膜重塑来逆转GSDMD介导的细胞焦亡。靶向 CHMP4B 相关蛋白可能促进子宫内膜肿瘤焦亡。版权所有 © 2023。由 Elsevier B.V. 出版。

In advanced and recurrent endometrial carcinoma (EC), the current state of immuno- or targeted therapy remains in the clinical research phase. Our study aimed to explore the role of the ESCRT machinery in maintaining cell membrane integrity and reversing pyroptotic cell death.Immunohistochemistry, western blotting, and co-immunoprecipitation were performed to determine the expression and relationship between GSDMD, CHMP4B, and VPS4A. We employed techniques such as FITC Annexin V/propidium iodide staining, Ca2+ fluorescence intensity, IL-1β enzyme-linked immunosorbent assay, and lactate dehydrogenase release assay to detect pyroptosis in endometrial cancer cells. Plasma membrane perforations and CHMP4B/VPS4A puncta were observed through electron and fluorescence confocal microscopy.We showed that GSDMD, CHMP4B, and VPS4A were differentially expressed in the pyroptotic EC xenograft mouse model group, as well as high, moderate, and mild expression in EC cells treated with LPS and nigericin compared to endometrial epithelial cells. Co-IP confirmed the interaction between GSDMD, CHMP4B, and VPS4A. We found that GSDMD knockdown reduced PI-positive cells, Ca2+ efflux, IL-1β, and LDH release, while CHMP4B and VPS4A depletion enhanced these indicators in HEC1A and AN3CA cells. Electron microscopy showed membrane perforations correspondingly decreased with inactivated GSDMD and increased or decreased after CHMP4B and VPS4A depletion or overexpression in EC cells. Fluorescence confocal microscopy detected CHMP4B protein puncta associated with VPS4A at the injured plasma membrane in GSDMDNT cells.We preliminary evidenced that CHMP4B and VPS4A reverses GSDMD-mediated pyroptosis by facilitating cell membrane remodeling in endometrial carcinoma. Targeting CHMP4B related proteins may promote pyroptosis in endometrial tumors.Copyright © 2023. Published by Elsevier B.V.