Internet Medical and System, Applications of National Engineering Laboratory, The First Afliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
Department of Pharmacy, The First Afliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
Cell Death Dis. 2022 Nov 10;13(11):951. doi: 10.1038/s41419-022-05341-3.
The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1) plays a role in the progression of various tumors, emerging as a potential therapeutic target. This study aimed to determine the role of USP1 as a therapeutic target in hepatocellular carcinoma (HCC). We detected USP1 expression in the tumor and adjacent tissues of patients with HCC using immunohistochemical staining. We evaluated the effect of the USP1 inhibitor ML-323 on HCC cell proliferation and cell cycle using a CCK-8 cell-counting kit and plate cloning assays, and propidium iodide, respectively. Apoptosis was detected by annexin V-FITC/Propidium Iodide (PI) staining and caspase 3 (casp3) activity. Transmission electron microscopy and LC3B immunofluorescence were used to detect autophagy. Western blotting was used to detect the accumulation of ubiquitinated proteins, the expression of endoplasmic reticulum (ER) stress-related proteins, and the AMPK-ULK1/ATG13 signaling pathway. We demonstrated that ML-323 inhibits the growth of HCC cells and induces G1 phase cell cycle arrest by regulating cyclin expression. ML-323 treatment resulted in the accumulation of ubiquitinated proteins, induced ER stress, and triggered Noxa-dependent apoptosis, which was regulated by the Activating Transcription Factor 4(ATF4). Moreover, active ER stress induces protective autophagy by increasing AMPK phosphorylation; therefore, we inhibited ER stress using 4-Phenylbutyric acid (4-PBA), which resulted in ER stress reduction, apoptosis, and autophagy in ML-323-treated HCC cells. In addition, blocking autophagy using the AMPK inhibitor compound C (CC), chloroquine (CQ), or bafilomycin A1 (BafA1) enhanced the cytotoxic effect of ML-323. Our findings revealed that targeting USP1 may be a potential strategy for the treatment of HCC.
去泛素化酶 USP1（泛素特异性蛋白酶 1）在各种肿瘤的进展中发挥作用，成为潜在的治疗靶点。本研究旨在确定 USP1 作为治疗靶点在肝细胞癌（HCC）中的作用。我们使用免疫组织化学染色检测了 HCC 患者肿瘤和相邻组织中的 USP1 表达。我们使用 CCK-8 细胞计数试剂盒和平板克隆测定分别评估了 USP1 抑制剂 ML-323 对 HCC 细胞增殖和细胞周期的影响，以及碘化丙啶（PI）。通过 Annexin V-FITC/碘化丙啶（PI）染色和 caspase 3（casp3）活性检测细胞凋亡。透射电子显微镜和 LC3B 免疫荧光检测自噬。Western blot 检测泛素化蛋白的积累、内质网（ER）应激相关蛋白的表达以及 AMPK-ULK1/ATG13 信号通路。我们证明 ML-323 通过调节细胞周期蛋白的表达抑制 HCC 细胞的生长并诱导 G1 期细胞周期停滞。ML-323 处理导致泛素化蛋白积累，诱导 ER 应激，并触发 Noxa 依赖性凋亡，这受激活转录因子 4（ATF4）调节。此外，通过增加 AMPK 磷酸化，活跃的 ER 应激诱导保护性自噬；因此，我们使用 4-苯丁酸（4-PBA）抑制 ER 应激，导致 ML-323 处理的 HCC 细胞中 ER 应激减少、凋亡和自噬。此外，使用 AMPK 抑制剂化合物 C（CC）、氯喹（CQ）或巴弗洛霉素 A1（BafA1）阻断自噬增强了 ML-323 的细胞毒性作用。我们的研究结果表明，靶向 USP1 可能是治疗 HCC 的一种潜在策略。
