目的 以过氧麦角甾醇为先导化合物，基于分子杂合思想设计合成新型过氧麦角甾醇-3-(3,4,5-三甲氧基苯基)-丙烯酸酯（EP-TTB）并探究其对肺癌A549细胞的作用机制。方法 通过酯化反应合成目标化合物EP-TTB，采用MTT法检测不同浓度（0、2.5、5、10、20、40、80μmol/L）EP-TTB作用于A549细胞后增殖抑制情况。将细胞分为对照组，EP-TTB（1、2、4μmol/L）组及过氧麦角甾醇4 μmol/L组，采用流式细胞术检测细胞凋亡率、活性氧（ROS）水平、线粒体膜电位变化（JC-1）以及细胞周期分布；采用微管绿色荧光染色试剂盒进行染色，通过激光共聚焦观察细胞核状态；采用Western blotting法检测A549细胞B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、细胞色素C（Cyt-C）、磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶B（Akt）、p-PI3K、p-AKT、β-tubulin及TrxR的蛋白表达水平。结果 EP-TTB的细胞毒活性显著优于过氧麦角甾醇。EP-TTB可显著降低A549细胞增殖率（P＜0.01、0.001）；细胞周期阻滞在G₀/G₁期。与对照组相比，EP-TTB组细胞凋亡率明显增加；ROS水平明显升高；线粒体膜电位明显升高；Bax、Cyt-C蛋白表达显著上调，p-PI3K、p-Akt、Bcl-2、β-tubulin、TrxR蛋白表达显著下调（P＜0.01、0.001）。结论 EP-TTB能同时靶向微管蛋白与硫氧还蛋白还原酶，从而有效抑制肺癌细胞增殖、诱导其凋亡，揭示其双靶点抗肿瘤作用机制。

Objective To design and synthesize a novel peroxide ergosterol-3-(3,4,5-trimethoxyphenyl)-propionic acid ester derivative (EP-TTB) based on the concept of molecular heterogeneity, and to investigate its mechanism of action on lung cancer A549 cells. Methods The target compound EP-TTB was synthesized through esterification reaction. The proliferation inhibition of different concentrations (0, 2.5, 5, 10, 20, 40, 80 μmol/L) of EP-TTB on A549 cells was detected by MTT assay. The cells were divided into control group, EP-TTB (1, 2, 4 μmol/L) group and peroxide ergosterol 4 μmol/L group. The apoptosis rate, ROS level, mitochondrial membrane potential change (JC-1), and cell cycle distribution of the cells were detected by flow cytometry. The cell nucleus state was observed by green fluorescent staining with the microtubule staining kit and laser confocal microscopy. The protein expression levels of Bcl-2, Bax, Cyt-C, PI3K, Akt, p-PI3K, p-AKT, β-tubulin, and TrxR in A549 cells were detected by Western blotting. Results The cytotoxic activity of EP-TTB was significantly superior to that of peroxide ergosterol. EP-TTB significantly reduced the proliferation rate of A549 cells (P < 0.01, 0.001), the cell cycle was arrested in G₀/G₁ phase. Compared with the control group, the apoptosis rate of the EP-TTB group was significantly increased; the ROS level was significantly elevated; the mitochondrial membrane potential was significantly increased; the protein expression of Bax and Cyt-C was significantly upregulated, and the protein expression of Bcl-2, β-tubulin, TrxR, p-PI3K, p-Akt, and p-PI3K was significantly downregulated (P < 0.01, 0.001). Conclusion EP-TTB can simultaneously target tubulin and thioredoxin reductase, thereby effectively inhibiting the proliferation of lung cancer cells and inducing their apoptosis. This preliminary study reveals its dual-target anti-tumor mechanism.

R965

黑龙江省省属本科高校基本科研业务费科研项目（2024-KYYWF-0342）