血根碱通过调控STUB1/GPX4诱导直肠癌细胞发生铁死亡

doi:10.12122/j.issn.1673-4254.2024.08.12

摘要/Abstract

摘要：

目的探究血根碱（SAN）对结直肠癌细胞增殖及铁死亡的影响。方法 SW620和HCT-116细胞体外培养，在SW620细胞加入浓度分别为0、0.5、1、1.5、2、2.5、3 μmol/L的SAN，HCT-116细胞加入0、0.5、0.75、1、1.25、1.5、1.75、2 μmol/L的SAN。采用CCK8法检测检测细胞活力，并计算出IC₅₀；检测SAN对细胞的增殖抑制作用采用集落克隆实验；Transwell实验观测SAN对细胞侵袭迁移力的影响；ROS检测试剂盒通过流式细胞仪分析细胞ROS含量的变化；丙二醛（MDA）检测盒来检测脂质过氧化物的产生。氧化型谷胱甘肽/还原型谷胱甘肽定量试剂盒测定谷胱甘肽（GSH）水平。Western blotting法检测铁死亡相关蛋白（STUB1、GPX4）的表达。结果 CCK8、集落克隆的结果显示，SAN可显著抑制SW620和HCT-116细胞增殖（P<0.05）；Transwell实验结果显示，SAN可抑制SW620和HCT-116细胞侵袭迁移能力（P<0.05）；流式细胞术检测显示：SAN显著促进SW620和HCT-116细胞内ROS的产生（P<0.05）；MDA检测结果显示，SAN可使SW620和HCT-116细胞内MDA含量增加（P<0.05）；GSH检测结果显示，SAN使SW620和HCT-116细胞内GSH下降（P<0.05）；Western blotting结果显示，SAN可通过调控STUB1下游蛋白GPX4的下调（P<0.05）。结论 SAN可通过调节 STUB1/GPX4诱导结直肠癌细胞发生依赖性铁死亡，提供了新治疗结直肠癌的靶点以及实验依据。

关键词: 血根碱, 结直肠癌, 铁死亡, STUB1, GPX4

Abstract:

Objective To investigate the effect of sanguinarine (SAN) on proliferation and ferroptosis of colorectal cancer cells. Methods SW620 and HCT-116 cells treated with different concentrations of SAN were examined for cell viability changes using CCK8 assay to determine the IC₅₀ of SAN in the two cells. The inhibitory effects of SAN on proliferation, invasion and migration of the cells were evaluated using colony-forming assay and Transwell assays. ROS production in the treated cells was analyzed with flow cytometry, and lipid peroxide production was assessed by detecting malondialdehyde (MDA) level. Glutathione (GSH) levels in the cells were detected, and Western blotting was used to detect the expressions of ferroptosis-related proteins STUB1 and GPX4. Results SAN significantly inhibited the proliferation, invasion and migration of SW620 and HCT-116 cells. SAN treatment significantly promoted ROS production, increased intracellular MDA level, and lowered GSH level in the two cells (P<0.05). Western blotting showed that SAN significantly upregulated the expression of STUB1 and down-regulated the expression of its downstream protein GPX4 (P<0.05). Conclusion SAN induces ferroptosis in colorectal cancer cells by regulating STUB1/GPX4, which may serve as a new therapeutic target for colorectal cancer.

Key words: sanguinarine, colorectal cancer, ferroptosis, STUB1, GPX4

张银亮, 骆泽谭, 赵睿, 赵娜, 徐志东, 奥迪, 丛古一, 刘新宇, 郑海伦. 血根碱通过调控STUB1/GPX4诱导直肠癌细胞发生铁死亡[J]. 南方医科大学学报, 2024, 44(8): 1537-1544.

Yinliang ZHANG, Zetan LUO, Rui ZHAO, Na ZHAO, Zhidong XU, Di AO, Guyi CONG, Xinyu LIU, Hailun ZHENG. Sanguinarine induces ferroptosis of colorectal cancer cells by upregulating STUB1 and downregulating GPX4[J]. Journal of Southern Medical University, 2024, 44(8): 1537-1544.

图/表 6

图1 SAN抑制SW620、HCT-116细胞存活并改变其形态及密度, 但Fer-1可恢复部分形态及密度

Fig.1 SAN represses colorectal cancer (CRC) cell viability and induces cell death. A, B: CCK-8 assay of SW620 and HCT-116 cells treated with different concentrations of SAN for 24 h. C, D: SW620 and HCT-116 cell viability following treatment with different concentrations of SAN for 24 and 72 h. E, F: Morphologies of CRC cells treated with SAN in the presence or absence of Fer-1 (1 μmol/L) (Original magnification: ×10). *P<0.05, **P<0.01, ****P<0.0001 vs control group; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.05 vs 24 h group.

图2 SAN抑制SW620 和 HCT-116细胞集落克隆形成并且Fer-1减弱其抑制作用

Fig.2 SAN inhibits colony formation ability of SW620 and HCT-116 cells. A, B: Representative cell colonies after 14 days of SAN treatment in the presence or absence of Fer-1 (1 μmol/L). C, D: Colonies numbers in the 4 groups, demonstrating that SAN-induced SW620 and HCT-116 cell death was effectively blocked by Fer-1. ****P<0.0001 vs control group; #P<0.05, ##P<0.01 vs SAN group.

图3 SAN抑制SW620 及HCT-116细胞迁移及侵袭,且Fer-1可显著逆转此现象

Fig.3 SAN inhibits migration and invasion ability of SW620 and HCT-116 cells A, B: Transwell migration assay of SAN-treated cells (×10). C, D: Number of migrated SW620 and HCT-116 cells. E, F: Transwell invasion assay of SAN-treated cells in the presence or absence of Fer-1 (×10). G, H: Number of migrated SW620 and HCT-116 cells. ****P<0.0001 vs control group; #P<0.05, ##P<0.01, ####P<0.0001 vs SAN group.

图4 SAN干预SW620、HCT116细胞后ROS骤升, Fer-1可显著逆转此作用

Fig.4 SAN induces ROS generation and contributes to ferroptosis in CRC cells. A, B: DCFH-DA probe was used to detected ROS levels in SW620 cells by flow cytometry and statistical analysis by Graphpad Prism. C, D: Flow cytometry was used to detected ROS levels in HCT116 cells and statistical analysis by Graphpad Prism. ****P<0.0001 vs control group; ##P<0.01, ###P<0.001 vs SAN group.

图5 SAN处理后LIP及MDA可上升, 而GSH下降, 且均可被Fer-1阻断

Fig.5 Levels of MDA and GSH in the CRC cells treated with SAN in the presence or absence of Fer-1. A, B: LIP levels in SW620 and HCT-116 cells at 24 h. C, D: MDA content in the cells. E, F: Intracellular GSH content of the cells. **P<0.01, ***P<0.001, ****P<0.0001 vs control group; #P<0.05, ##P<0.01 vs SAN group.

图6 SAN治疗后SW620 和 HCT-116 STUB1表达上调, GPX4下调, 并且此现象均可被Fer-1部分阻断

Fig.6 Effects of SAN on STUB1 and GPX4 expressions in SW620 and HCT-116 cells with or without Fer-1 treatment. A, B: Western blotting for STUB1 and GPX4 expressions in SW620 (A) and HCT-116 cells (B). C-F: Quantitative analysis of protein expressions. *P<0.05, **P<0.01 vs control group; #P<0.05 vs SAN group.

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