岩藻黄质通过AKT/mTOR途径调节糖酵解代谢促进人急性淋巴细胞白血病细胞凋亡的作用研究

Fucoxanthin Promotes Hunman Acute Lymphoblastic LeukemiaApoptosis by Suppressing Glycolysis via the AKT/mTOR Pathway

为阐明岩藻黄质促进人急性淋巴细胞白血病(CEM/C1)细胞凋亡的作用机制,本研究采用MTT法检测岩藻黄质处理后的CEM/C1细胞活力;利用流式细胞仪测定CEM/C1细胞的早期与晚期凋亡率以及细胞周期的分布;采用试剂盒检测岩藻黄质对CEM/C1细胞中丙酮酸激酶(PK)和己糖激酶(HK)活性以及葡萄糖摄取量、三磷酸腺苷(ATP)生成量、乳酸生成量的影响;通过蛋白免疫印迹法(WB)测定CEM/C1细胞相关蛋白表达水平,并通过分子对接进行验证。结果表明,岩藻黄质对CEM/C1细胞活力具有显著的抑制效果,并呈剂量和时间依赖性;随着岩藻黄质浓度的增加,CEM/C1细胞的早期凋亡率和晚期凋亡率明显增加,G1/G2细胞的比例显著或极显著下降,S期细胞的比例显著或极显著上升;岩藻黄质显著抑制糖酵解相关酶(PK、HK)的活性以及蛋白表达;岩藻黄质显著降低磷酸化mTOR和磷酸化AKT蛋白的表达水平;岩藻黄质与AKT和mTOR蛋白对接的结合能均小于-5 kcal·mol^(-1),主要通过氢键作用于酶活中心。综上,岩藻黄质能显著促进CEM/C1细胞凋亡,其作用机制可能与AKT/mTOR信号通过调控的糖酵解有关。研究结果为羊栖菜高附加值产品开发及天然、安全抗白血病药物前体的筛选提供了一定的理论依据。

To elucidate the mechanism by which fucoxanthin promotes the apoptosis in human acute lymphoblastic leukemia(CEM/C1)cells,the following experimental approaches were employed.Cell viability of CEM/C1 cells after fucoxanthin treatment was assessed using the MTT assay.Early and late apoptosis rates,as well as cell cycle distribution of CEM/C1 cells,were determined using flow cytometry.Fucoxanthin’s impact on pyruvate kinase(PK)and hexokinase(HK)activity,glucose uptake,adenosine triphosphate(ATP)production,and lactate production in CEM/C1 cells was measured using assay kits.The expression levels of relevant proteins in CEM/C1 cells were assessed through Western blot analysis.Molecular docking was performed to validate the interactions.Results indicated that fucoxanthin significantly inhibited the viability of CEM/C1 cells in a dose-and time-dependent manner.With increasing fucoxanthin concentration,the early and late apoptosis rates of CEM/C1 cells increased,accompanied by a significant or extremely significant decrease in the proportion of cells in the G1/G2 phase and significant or extremely significant increase in the proportion of cells in the S phase.Fucoxanthin markedly inhibited the activity and expression levels of glycolytic enzymes(PK,HK),and also reduced the expression levels of phosphorylated mTOR and phosphorylated AKT proteins.Molecular docking analysis revealed that the binding energy between fucoxanthin and AKT and mTOR proteins was less than-5 kcal·mol^(-1),primarily through hydrogen bonding at the enzyme’s active site.In conclusion,fucoxanthin significantly promoted the apoptosis in CEM/C1 cells,and its mechanism of action may be associated with the regulation of glycolysis through the AKT/mTOR signal pathway.These findings provide a theoretical basis for the development of high-value-added products from Sargassum fusiforme and the screening of natural,safe anti-leukemia drug precursors.