目的 探讨穿心莲内酯对头颈鳞癌的作用，并通过脂质组学从铁自噬角度揭示其潜在的抗肿瘤作用机制。方法 体外培养人头颈鳞癌CAL27细胞，通过CCK-8实验、克隆形成实验考察穿心莲内酯对细胞增殖的抑制作用，流式细胞术检测细胞凋亡。构建人头颈鳞癌患者来源异种移植（patient-derived xenograft，PDX）小鼠模型，设置对照组和穿心莲内酯（10 mg/kg）组，给药干预后，动态观察肿瘤生长情况，检测肿瘤组织中增殖相关指标Ki67的表达。采用脂质组学技术分析穿心莲内酯处理后CAL27细胞的代谢谱变化，并进行京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析；通过Western blotting检测细胞中铁自噬相关蛋白的表达。结果 体外实验结果显示，穿心莲内酯可显著抑制CAL27细胞增殖并诱导细胞凋亡（P＜0.01、0.001），且呈剂量相关性；体内PDX模型实验结果显示，穿心莲内酯能显著抑制肿瘤体内生长（P＜0.001），且肿瘤组织中Ki67阳性表达较对照组显著降低（P＜0.001）；脂质组学分析及KEGG通路富集结果显示，穿心莲内酯处理后细胞的差异代谢通路富集在铁死亡、自噬相关通路；Western blotting结果证实，穿心莲内酯可显著调控铁自噬相关蛋白的表达（P＜0.05、0.01、0.001），表明其能有效诱导CAL27细胞发生铁自噬。通过核受体共激活因子4（nuclear receptor coactivator 4，NCOA4）靶向降解剂及铁死亡抑制剂干预验证，二者可显著逆转穿心莲内酯的作用（P＜0.05、0.01、0.001），证实其抗肿瘤效应依赖铁自噬信号通路。结论 穿心莲内酯可在体内外显著抑制头颈鳞癌增殖并诱导其凋亡，其抗肿瘤作用机制与诱导肿瘤细胞发生铁自噬相关。

Objective To investigate the effect of andrographolide on head and neck squamous cell carcinoma, and reveal its potential anti-tumor mechanism from the perspective of ferroptophagy through lipidomics. Methods CAL27 cells were cultured in vitro. CCK-8 assay and colony formation assay were used to detect the inhibitory effect of andrographolide on cell proliferation, and flow cytometry was used to detect the cell apoptosis. A patient-derived xenograft (PDX) mouse model of human head and neck squamous cell carcinoma was established. Control group and andrographolide (10 mg/kg) group were set up, and after intervention with administration, the tumor growth was observed dynamically, and the expression of proliferation-related index Ki67 in tumor tissues was detected. Lipidomics technology was used to analyze the metabolic profile changes of CAL27 cells after andrographolide treatment, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was performed. Western blotting was used to detect the expressions of ferroptophagy-related proteins in cells. Results The in vitro experimental results showed that andrographolide could significantly inhibit the proliferation of CAL27 cells and induce cell apoptosis (P < 0.01, 0.001), and showed a dose-dependent effect. The results of in vivo PDX model experiment showed that andrographolide could significantly inhibit the growth of tumors in vivo (P < 0.001), and the positive expression of Ki67 in tumor tissues was significantly reduced compared to the control group (P < 0.001). The lipidomics analysis and KEGG pathway enrichment results showed that the differential metabolic pathways of cells treated with andrographolide were enriched in ferroptosis and autophagy-related pathways. Western blotting results confirmed that andrographolide could significantly regulate the expressions of ferroptophagy-related proteins (P < 0.05, 0.01, 0.001), indicating that andrographolide could effectively induce ferroptophagy in CAL27 cells. Further validation with the nuclear receptor coactivator 4 (NCOA4) targeted degrader and ferroptosis inhibitor demonstrated that both agents markedly reversed the effects of andrographolide (P < 0.05, 0.01, 0.001), verifying that its antitumor effect depends on the ferroptophagy signaling pathway. Conclusion Andrographolide could significantly inhibit the proliferation and induce apoptosis of head and neck squamous cell carcinoma in vitro and in vivo. Its anti-tumor mechanism is related to inducing ferroptophagy in tumor cells.

R285.5

国家自然科学基金资助项目（82072950）；浙江省自然科学基金资助项目（ZCLMS25H1602）