[摘 要] 目的：探讨甲硫氨酸限制对肺腺癌（LUAD）细胞增殖、凋亡及磷酸戊糖途径的影响。方法：将H1299、A549细胞分 为Met+组和Met?组，分别用含100 μmol/L或不含甲硫氨酸的培养基连续培养4 d，采用细胞计数法评估甲硫氨酸处理对H1299 和 A549 细胞增殖的影响，PI 染色法检测细胞周期分布，Annexin Ⅴ-PE/7AAD 标记细胞凋亡，利用 DCFH-DA 探针检测细胞内 ROS水平，WST-8法和DTNB法分别测定细胞内NADPH与GSH含量；通过癌症基因组图谱（TCGA）数据库分析葡萄糖-6-磷酸 脱氢酶（G6PD）和6-磷酸葡萄糖酸脱氢酶（6PGD）表达与甲硫氨酸代谢通路的关系；采用WB法检测甲硫氨酸处理及回补甲硫氨 酸下游代谢产物S-腺苷甲硫氨酸（SAM）对LUAD细胞中磷酸戊糖途径关键酶G6PD和6PGD表达的影响。结果：甲硫氨酸限制 显著抑制H1299和A549细胞增殖（均P < 0.01），将细胞周期阻滞于 G2/M 期（均 P < 0.05），显著升高细胞内总ROS水平（均 P < 0.001）并促进细胞凋亡（均P < 0.001）；同时，甲硫氨酸限制显著降低了细胞内NADPH和GSH水平（均P < 0.01），抑制DNA 合成（均P < 0.01）。分析TCAG数据发现，G6PD和6PGD表达水平与甲硫氨酸代谢通路呈正相关（均P < 0.001），甲硫氨酸限制 下调G6PD和6PGD 蛋白表达（均 P < 0.01），而回补 SAM 可部分逆转甲硫氨酸限制对 G6PD 和 6PGD 的表达的抑制（均 P < 0.01），提示甲硫氨酸通过SAM合成调控磷酸戊糖途径。结论：甲硫氨酸限制通过抑制磷酸戊糖途径抑制LUAD细胞增殖， 为甲硫氨酸限制疗法治疗LUAD提供实验依据。

[Abstract] Objective: To investigate the impact of methionine restriction on the proliferation and apoptosis of lung adenocarcinoma (LUAD) cells, and the pentose phosphate pathway. Methods: LUAD cells H1299 and A549 were assigned to the Met+ (100 μmol/L methionine) and Met? (0 μmol/L methionine) groups and cultured continuously for 4 d. Cell counting was used to evaluate the effects of methionine treatment on the proliferation of H1299 and A549 cells. Cell-cycle distribution was detected by PI staining; Cell apoptosis by Annexin Ⅴ-PE/7AAD labeling; intracellular ROS level by DCFH-DA probe; intracellular NADPH and GSH levels by WST-8 and DTNB assays, respectively. The Cancer Genome Atlas (TCGA) was used to analyze the correlations between the expressions of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) and the methioninemetabolic pathway. WB assay was employed to evaluate the effects of methionine restriction and supplementation of S-adenosylmethionine (SAM), a downstream methionine metabolic product, on the expressions of G6PD and 6PGD, key enzymes in the pentose phosphate pathway, in LUAD cells. Results: Methionine restriction significantly inhibited the proliferation of H1299 and A549 cells (both P < 0.01), arrested cells in G2/M phase (both P < 0.05), elevated total ROS levels (both P < 0.001), and induced apoptosis (both P < 0.001). Furthermore, methionine restriction significantly reduced NADPH and GSH levels (both P < 0.01) and suppressed DNA synthesis (both P < 0.01). TCGA analysis revealed positive correlations between G6PD/6PGD expression levels and the methionine metabolic pathway (both P < 0.001). Methionine restriction down-regulated the expressions of G6PD and 6PGD proteins (both P < 0.01), whereas SAM supplementation partially restored their expressions (both P < 0.01), suggesting that methionine regulated the pentose phosphate pathway through SAM. Conclusion: Methionine restriction suppresses LUAD cell proliferation by inhibiting the PPP, which provides experimental evidence for methionine-restriction therapy for LUAD.

国家自然科学基金（No. 82403321）；广州市科技计划项目（No. 2023B03J1291）；广东省基础与应用基础研究 （No. 2023A1515110036）