Dihydroartemisinin enhances sensitivity of nasopharyngeal carcinoma HNE1/DDP cells to cisplatin-induced apoptosis by promoting ROS production

doi:10.12122/j.issn.1673-4254.2024.08.14

Abstract

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

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.

Figures/Tables 6

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

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.

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.

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.

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.

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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