双氢青蒿素通过促进活性氧的产生增强鼻咽癌细胞对顺铂诱导凋亡的敏感性

doi:10.12122/j.issn.1673-4254.2024.08.14

摘要/Abstract

摘要：

目的探究双氢青蒿素（DHA）与顺铂（DDP）联合应用对耐DDP鼻咽癌细胞株HNE1/DDP增殖抑制和促凋亡的作用及其机制。方法 CCK-8法检测不同浓度DHA（0、5、10、20、40、80、160 μmol/L）和不同浓度DDP（0、4、8、16、32、64、128 μmol/L）处理24 h和48 h后HNE1/DDP细胞的存活率；采用Compusyn软件计算DHA与DDP的联合指数。将HNE1/DDP细胞分为对照组、DHA组、DDP组、DHA联合DDP组，给药处理24 h后，CCK-8、EdU和集落克隆形成实验分别检测细胞活力、细胞增殖和集落克隆形成能力；流式细胞术检测细胞凋亡情况和细胞内活性氧（ROS）水平；Western blotting检测凋亡相关蛋白Cleaved PARP、Cleaved Caspase-9、Cleaved Caspase-3表达水平。ROS抑制剂N-乙酰半胱氨酸预处理后，检测其对DHA联合DDP诱导的细胞增殖、凋亡的影响。结果不同浓度DHA和不同浓度DDP均能明显抑制HNE1/DDP细胞活力，DHA（5 μmol/L）联合DDP（8、16、32、64、128 μmol/L）的联合指数均小于1。与DHA或DDP单独处理组相比，DHA联合DDP组细胞活力下降（P<0.01），集落形成数和EdU阳性染色细胞减少（P<0.01），细胞凋亡率和细胞内ROS水平升高（P<0.01），细胞凋亡相关蛋白Cleaved PARP、Cleaved Caspase-9、Cleaved Caspase-3的表达水平增加（P<0.05），而N-乙酰半胱氨酸预处理可部分逆转DHA联合DDP对HNE1/DDP细胞的增殖抑制和凋亡诱导作用（P<0.01）。结论 DHA增强DDP对HNE1/DDP细胞的增殖抑制和凋亡诱导作用，其机制可能与细胞内ROS的积累有关。

关键词: 鼻咽癌, 双氢青蒿素, 顺铂, 活性氧, 增殖, 细胞凋亡

Abstract:

Objective To investigate the effect of dihydroartemisinin (DHA) for enhancing the inhibitory effect of cisplatin (DDP) on DDP-resistant nasopharyngeal carcinoma cell line HNE1/DDP and explore the mechanism. Methods CCK-8 method was used to assess the survival rate of HNE1/DDP cells treated with DHA (0, 5, 10, 20, 40, 80, and 160 μmol/L) and DDP (0, 4, 8, 16, 32, 64, 128 μmol/L) for 24 or 48 h, and the combination index of DHA and DDP was calculated using Compusyn software. HNE1/DDP cells treated with DHA, DDP, or their combination for 24 h were examined for cell viability, proliferation and colony formation ability using CCK-8, EdU and colony-forming assays. Flow cytometry was used to detect cell apoptosis and intracellular reactive oxygen species (ROS). The expression levels of apoptosis-related proteins cleaved PARP, cleaved caspase-9 and cleaved caspase-3 were detected by Western blotting. The effects of N-acetyl-cysteine (a ROS inhibitor) on proliferation and apoptosis of HNE1/DDP cells with combined treatment with DHA and DDP were analyzed. Results Different concentrations of DHA and DDP alone both significantly inhibited the viability of HNE1/DDP cells. The combination index of DHA (5 μmol/L) combined with DDP (8, 16, 32, 64, 128 μmol/L) were all below 1. Compared with DHA or DDP alone, their combined treatment more potently decreased the cell viability, colony-forming ability and the number of EdU-positive cells, and significantly increased the apoptotic rate, intracellular ROS level, and the expression levels of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 in HNE1/DDP cells. N-acetyl-cysteine pretreatment obviously attenuated the inhibitory effect on proliferation and apoptosis-inducing effect of DHA combined with DDP in HNE1/DDP cells (P<0.01). Conclusion DHA enhances the growth-inhibitory and apoptosis-inducing effect of DDP on HNE1/DDP cells possibly by promoting accumulation of intracellular ROS.

Key words: nasopharyngeal carcinoma, dihydroartemisinin, cisplatin, reactive oxygen species, proliferation, apoptosis

从小凡, 陈腾, 李硕, 王媛媛, 周龙云, 李小龙, 张配, 孙小锦, 赵素容. 双氢青蒿素通过促进活性氧的产生增强鼻咽癌细胞对顺铂诱导凋亡的敏感性[J]. 南方医科大学学报, 2024, 44(8): 1553-1560.

Xiaofan CONG, Teng CHEN, Shuo LI, Yuanyuan WANG, Longyun ZHOU, Xiaolong LI, Pei ZHANG, Xiaojin SUN, Surong ZHAO. Dihydroartemisinin enhances sensitivity of nasopharyngeal carcinoma HNE1/DDP cells to cisplatin-induced apoptosis by promoting ROS production[J]. Journal of Southern Medical University, 2024, 44(8): 1553-1560.

图/表 6

图1 DDP对HNE1和HNE1/DDP细胞活力的影响

Fig.1 Effect of dihydroartemisinin (DDP) on viability of HNE1 and HNE1/DDP cells. **P<0.01 vs 0 μmol/L.

图2 DHA或/和DDP抑制HNE1/DDP细胞活力和增殖

Fig.2 Inhibitory effects of DHA, DDP, and their combination on viability and proliferation of HNE1/DDP cells. A, B: CCK-8 assay for detecting viability of HNE1/DDP cells treated with DHA and DDP for 24 and 48 h. C: CCK-8 assay for detecting viability of HNE1/DDP cells treated with DHA (5 μmol/L) combined with DDP (0, 4, 8, 16, 32, 64, 128 μmol/L) for 24 and 48 h. *P<0.05, **P<0.01 vs 0 μmol/L. D, E: Combination index (CI) of combination treatment with DHA and DDP for 24, 48 h. F: Colony formation ability of cells treated with DHA or/and DDP for 24 h. G: EdU test for detecting proliferation of HNE1/DDP cells treated with DHA or/and DDP for 24 h (Original magnification: ×10). *P<0.05, **P<0.01 vs Control group; ##P<0.01 vs DHA group; &&P<0.01 vs DDP group.

图3 DHA联合DDP诱导HNE1/DDP细胞凋亡

Fig.3 DHA combined with DDP more potently induces apoptosis in HNE1/DDP cells. A: Apoptosis of HNE1/DDP cells treated with DHA or/and DDP for 24 h detected by flow cytometry. B: Protein levels of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 in HNE1/DDP cells detected by Western blotting. *P<0.05, **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs DHA group; &P<0.05, &&P<0.01 vs DDP group.

图4 DHA联合DDP促进HNE1/DDP细胞内ROS的产生

Fig.4 DHA combined with DDP enhances ROS production in HNE1/DDP cells. **P<0.01 vs Control group; ##P<0.01 vs DHA group; &&P<0.01 vs DDP group.

图5 DHA联合DDP抑制HNE1/DDP细胞增殖作用依赖于ROS的积累

Fig.5 DHA combined with DDP more strongly inhibits proliferation of HNE1/DDP cells by promoting ROS accumulation. **P<0.01 vs Control group; ##P<0.01 vs DHA+DDP group.

图6 抑制ROS生成对HNE1/DDP细胞凋亡和凋亡相关蛋白表达水平的影响

Fig.6 Effects of inhibiting ROS production on apoptosis and expression of apoptosis-related proteins in HNE1/DDP cells following combined treatment with DHA and DDP for 24 h. A: Apoptosis of HNE1/DDP cells treated with NAC (5 mmol/L) followed by combined treatment with DHA and DDP for 24 h was detected by flow cytometry. B: Protein levels of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 in HNE1/DDP cells detected by Western blotting. **P<0.01 vs Control group; #P<0.05 vs DHA+DDP group.

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