Li Hailiang, Ma Zheng, Yang Wanfu, Zhang Yifan, Sun Jinping, Jiang Haifeng, Wang Faxuan, Hou Li, Xia Hechun
Department of Radiation Oncology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China.
Key Laboratory of Craniocerebral Diseases, Ningxia Medical University, Yinchuan, China.
Front Pharmacol. 2025 Mar 17;16:1563865. doi: 10.3389/fphar.2025.1563865. eCollection 2025.
Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Standard therapies are followed by poor patient's survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Metformin has been reported to have anti-cancer effects. However, the precise mechanism underlying these effects remains elusive. A better understanding of its underlying mechanism will inform future experimental designs exploring metformin as a potential adjuvant therapy for GBM. This research aimed to elucidate the potential molecular mechanism of metformin in GBM by integrating proteomics and transcriptomics.
The study examined the effects of metformin on GBM cell lines using various methods. The U87, U251 and HA1800 were cultured and modified through PER2 knockdown and overexpression. Cell viability was assessed using the CCK8 assay, and G6PDH activity and intracellular NADPH levels were measured with specific kits. ROS levels, mitochondrial membrane potential, cell cycle distribution and apoptosis were analyzed by flow cytometry. RNA was extracted for transcriptomic analysis through RNA sequencing, while proteomic analysis was performed on total protein from treated cells. WB detected specific proteins, and RT-qPCR quantified gene expression. In vivo experiments, GBM xenograft on nude mice treated with metformin combining radiotherapy was evaluated and received IHC and TUNEL staining for protein expression and apoptosis assessment. Statistical analyses were conducted using Prism software to identify significant group differences.
We found that differential expressional genes and proteins relating to circadian rhythm were enriched in proteomic or transcriptomic. The expression of PER2, the key circadian gene, was up-regulated in GBM cell lines when treated with metformin. Furthermore, the expression of silent information regulator 2(SIRT2) was down-regulated, while the expression of the G6PD protein just slightly increased in GBM cell lines. Meanwhile, NADPH+ production and G6PDH enzyme activity significantly decreased. Further study validated that metformin inhibited the cell growth of GBM cell lines through up-regulating and inhibited SIRT2/G6PD signaling pathway, enhancing radiotherapy(RT) sensitivity. We also found that the inhibition of SIRT2 caused by metformin is mediated by PER2.
We found the pivotal role of metformin as an effective circadian rhythm regulator. Targeting circadian clock gene to modify and rescue the dysfunctional circadian clock of GBM cells at molecular level might be an innovative way to administer cancer chronotherapy and maintain metabolic homeostasis in real world practice.
多形性胶质母细胞瘤（GBM）被认为是最致命的脑癌。标准疗法之后患者的生存结果不佳，因此必须有新颖且更有效的治疗策略来应对这一灾祸。据报道，二甲双胍具有抗癌作用。然而，这些作用背后的确切机制仍不清楚。更好地了解其潜在机制将为未来探索二甲双胍作为GBM潜在辅助治疗的实验设计提供依据。本研究旨在通过整合蛋白质组学和转录组学来阐明二甲双胍在GBM中的潜在分子机制。
该研究使用多种方法检测了二甲双胍对GBM细胞系的影响。培养U87、U251和HA1800细胞，并通过PER2基因敲低和过表达进行修饰。使用CCK8法评估细胞活力，并用特定试剂盒测量G6PDH活性和细胞内NADPH水平。通过流式细胞术分析ROS水平、线粒体膜电位、细胞周期分布和细胞凋亡。提取RNA通过RNA测序进行转录组分析，同时对处理过的细胞的总蛋白进行蛋白质组分析。WB检测特定蛋白质，RT-qPCR定量基因表达。在体内实验中，评估了用二甲双胍联合放疗治疗的裸鼠GBM异种移植模型，并进行免疫组化和TUNEL染色以评估蛋白质表达和细胞凋亡。使用Prism软件进行统计分析以确定组间的显著差异。
我们发现与昼夜节律相关的差异表达基因和蛋白质在蛋白质组学或转录组学中富集。在用二甲双胍处理时，GBM细胞系中关键昼夜节律基因PER2的表达上调。此外，沉默信息调节因子2（SIRT2）的表达下调，而GBM细胞系中G6PD蛋白的表达仅略有增加。同时，NADPH+产量和G6PDH酶活性显著降低。进一步研究证实，二甲双胍通过上调并抑制SIRT2/G6PD信号通路来抑制GBM细胞系的细胞生长，增强放疗（RT）敏感性。我们还发现二甲双胍对SIRT2的抑制作用是由PER2介导的。
我们发现了二甲双胍作为有效昼夜节律调节因子的关键作用。在分子水平上靶向昼夜节律基因来改变和挽救GBM细胞功能失调的昼夜节律可能是在实际临床实践中实施癌症时辰疗法并维持代谢稳态的一种创新方法。
