Department of Medical Oncology, Jinling Hospital, Nanjing, China.
Department of Cardiothoracic Surgery, Jinling Hospital, Nanjing, China.
Thorac Cancer. 2022 Aug;13(15):2235-2247. doi: 10.1111/1759-7714.14552. Epub 2022 Jul 10.
Lung cancer (LC) is one of the most common cancers and a leading cause of cancer-related deaths worldwide. In many pathological conditions, particularly in the tumor microenvironment, cells and tissues frequently exist in a hypoxic state. Here, we evaluated Itchy E3 ubiquitin protein ligase (ITCH) expression in LC cells following hypoxia treatment.
LC cell lines were treated with hypoxic condition. Cell migration, invasion, inflammation, reactive oxygen species (ROS) production, and apoptosis of LC cells were determined by wound healing assay, Transwell invasive assay, ELISA, DCFH-DA staining, and flow cytometry, respectively. qPCR and WB were used to determine the expression of ITCH and TXNIP. Co-IP was performed to assess the interaction between ITCH and TXNIP.
ITCH expression was downregulated in LC cells under hypoxic conditions. Next, LC cells were subjected to hypoxic conditions and changes in cell viability and metastasis were determined. Hypoxic conditions resulted in increased migration and invasion abilities of LC cells. Intracellular reactive oxygen species (ROS) production, inflammation, and apoptosis were also promoted by hypoxia. We found that ITCH overexpression led to the proteasomal degradation of thioredoxin-interacting protein (TXNIP), whereas the expression of the ITCH C830A mutant did not affect TXNIP levels in LC cells. The gain-of-function experiment demonstrated that migration, invasion, ROS generation, inflammation, and apoptosis of hypoxia-conditioned LC cells were ameliorated by ITCH overexpression, whereas the ITCH C830A mutant did not cause any changes in these phenotypes. Furthermore, the contribution of TXNIP knockdown and ITCH overexpression to the hypoxia-induced features in LC cells with ITCH C830A was found to be similar.
Our results suggest a novel mechanism underlying the changes in ITCH-mediated malignant phenotypes of hypoxia-conditioned LC cells via TXNIP.
肺癌（LC）是最常见的癌症之一，也是全球癌症相关死亡的主要原因。在许多病理条件下，特别是在肿瘤微环境中，细胞和组织经常处于缺氧状态。在这里，我们评估了缺氧处理后 LC 细胞中发痒的 E3 泛素蛋白连接酶（ITCH）的表达。
用缺氧条件处理 LC 细胞系。通过划痕愈合试验、Transwell 侵袭试验、ELISA、DCFH-DA 染色和流式细胞术分别测定 LC 细胞的迁移、侵袭、炎症、活性氧（ROS）产生和细胞凋亡。qPCR 和 WB 用于测定 ITCH 和 TXNIP 的表达。用 co-IP 评估 ITCH 和 TXNIP 之间的相互作用。
LC 细胞在缺氧条件下 ITCH 表达下调。接下来，LC 细胞在缺氧条件下进行处理，测定细胞活力和转移的变化。缺氧条件导致 LC 细胞迁移和侵袭能力增强。细胞内活性氧（ROS）产生、炎症和细胞凋亡也被缺氧促进。我们发现 ITCH 过表达导致硫氧还蛋白相互作用蛋白（TXNIP）的蛋白酶体降解，而 ITCH C830A 突变体的表达对 LC 细胞中 TXNIP 水平没有影响。功能获得实验表明，ITACH 过表达改善了缺氧条件下 LC 细胞的迁移、侵袭、ROS 生成、炎症和细胞凋亡，而 ITCH C830A 突变体没有引起这些表型的任何变化。此外，发现 TXNIP 敲低和 ITCH 过表达对具有 ITCH C830A 的 LC 细胞中缺氧诱导特征的贡献与 ITCH C830A 相似。
我们的结果表明，通过 TXNIP，ITCH 介导的恶性表型在缺氧条件下 LC 细胞中的变化存在一种新的机制。
