[Icaritin increases radiosensitivity of nasopharyngeal carcinoma cells by regulating iron death]

Abstract

Objective: To explore the radiosensitizing effect of icaritin on nasopharyngeal carcinoma (NPC) cells and the underlying mechanism.

Methods: MTT assay and clonal formation assay were used to evaluate the effect of icaritin on proliferation of human NPC HONE1 and HNE1 cells. The effects of icaritin treatment, γ-ray radiation, or both on production of reactive oxygen species (ROS), cell cycle distribution and apoptosis of the NPC cells were assessed using flow cytometry. The expressions of DNA damage markers γ-H2AX, cycle-related proteins CDC25C, p-CDC25C and cyclin B1, and ferroptosis markers ACSL4 and GXP4 were detected using Western blotting. A nude mouse model bearing subcutaneous HONE1 cell xenograft was used to observe the effect of icaritin and radiation on tumor growth.

Results: Icaritin dose-dependently inhibited the viability of the NPC cells and enhanced the inhibitory effect of radiation on cell proliferation. Flow cytometry and Western blotting showed that icaritin treatment prior to radiation significantly promoted ROS production and γ-H2AX expression in the NPC cells (P<0.001). Compared with radiation exposure alone, the combined treatment caused cell cycle arrest in G2 phase, down-regulated CDC25C and cyclin B1 expression, and up-regulated p-CDC25C expression in the cells (P<0.01), resulting also in increased cell apoptosis, enhanced expression of ferroptosis protein ACSL4 and lowered expression of GXP4 (P<0.001). In the tumor-bearing mice, icaritin treatment, compared with radiation alone, significantly reduced the tumor growth rate and decreased tumor weight (P<0.001).

Conclusion: Icaritin can enhance radiosensitivity of NPC cells both in vitro and in nude mice possibly by enhancing ROS production to promote iron death of the cells.

Keywords: ferroptosis; icaritin; nasopharyngeal carcinoma; radiosensitization; reactive oxygen species.

图 1

淫羊藿素对鼻咽癌细胞细胞活力的影响 Effect of icaritin on viability of nasopharyngeal carcinoma cells. A: Molecular structure of icaritin. B: Effects of icaritin at different concentrations on viability of HONE1 and HNE1 cells (n=3).

图 2

淫羊藿素联合放射加强对鼻咽癌细胞的抑制作用 Inhibitory effect of icaritin combined with radiation at different doses on nasopharyngeal carcinoma cells assessed by MTT assay (A) and plate clone formation assay (B).

图 3

淫羊藿素联合照射增加鼻咽癌细胞活性氧 Icaritin combined with radiation increases reactive oxygen species (ROS) in nasopharyngeal carcinoma cells. A: Changes in ROS levels. B: Expression of DNAdamage marker γ-H2AX protein. ^***P<0.001 vs IR group.

图 4

淫羊藿素和照射联合导致鼻咽癌细胞发生G2期阻滞 Icaritin combined with radiation causes cell cycle arrest in G2 phase of nasopharyngeal carcinoma cells. A: Effects of icaritin combined with radiation on cell cycle distribution in nasopharyngeal carcinoma cells. B: Expression of cell cycle arrest marker proteins CDC25C, pCDC25C and cyclin B1. ^**P<0.01, ^***P<0.001 vs IR group.

图 5

淫羊藿素和照射联合促进鼻咽癌细胞发生凋亡 Icaritin combined with radiation promotes apoptosis of nasopharyngeal carcinoma cells. A: Effects of icaritin combined with radiation on apoptosis of nasopharyngeal carcinoma cells. B: Expression ofACSL4, GPX4, Bax and Bcl-2 proteins. ^**P<0.01, ^***P<0.001 vs the IR group.

图 6

淫羊藿素和照射联合对裸鼠肿瘤组织生长的抑制作用 Icaritin enhances the inhibitory effect of radiation on tumor growth in nude mice. A: Timeline of treatment of the nude mice. B: Gross observation of the tumor tissues. C: Trends of changes of tumor volume. D: Tumor weight in the 3 groups. E: Curves of body weight changes of the mice in each group. F: Viscera index of the vital organs in each group. n=6. ^**P<0.01, ^***P<0.001 vs the IR group.