ABSTRACT Metformin is a widely used drug for the treatment of type 2 diabetes. Antidiabetic drugs are also known to influence cancer progression, as high glucose levels affect both cancer and diabetes. Metformin induces cell cycle arrest in cancer cells, but the underlying mechanism remains unclear in cervical cancer system. Here, we examined how metformin affects cell cycle arrest and apoptosis in cervical cancer cells. Western blot analysis showed that levels of O-linked N-acetylglucosamine (O-GlcNAc) and O-GlcNAc transferase (OGT) were increased in cervical cancer cells; these effects were reversed by metformin treatment. Immunoprecipitation analysis was used to examine the interplay between O-GlcNAcylation and phosphorylation in HeLa cells, revealing that metformin decreased O-GlcNAcylated AMP-activated protein kinase (AMPK) and increased levels of phospho-AMPK compared to untreated cells. These results were associated with decreased cell cycle arrest and apoptotic cell death in HeLa cells, as shown by flow cytometry. Moreover, 6-diazo-5-oxo-L-norleucine (a glutamine fructose-6-phosphate aminotransferase inhibitor) or thiamet G (an O-GlcNAcase inhibitor) decreased or increased levels of O-GlcNAcylated AMPK, and increased or decreased levels of phosphorylated AMPK, respectively, suggesting that O-GlcNAc modification affects AMPK activation. Of note, we found that metformin treatment of HeLa cells increased the levels of p21 and p27 (which are AMPK-dependent cell cycle inhibitors), leading to increased cell cycle arrest and apoptosis in HeLa cells compared to untreated cells. These findings suggest that metformin may serve as a useful antiproliferative drug in cervical cancer cells, with potential therapeutic benefit.

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二甲双胍通过降低 AMPK O-GlcNAcylation 抑制宫颈癌细胞增殖

摘要 二甲双胍是一种广泛用于治疗 2 型糖尿病的药物。众所周知，抗糖尿病药物会影响癌症进展，因为高血糖水平会影响癌症和糖尿病。二甲双胍在癌细胞中诱导细胞周期停滞，但在宫颈癌系统中的潜在机制仍不清楚。在这里，我们研究了二甲双胍如何影响宫颈癌细胞的细胞周期停滞和凋亡。蛋白质印迹分析显示宫颈癌细胞中O-连接的N-乙酰氨基葡萄糖（O-GlcNAc）和O-GlcNAc转移酶（OGT）的水平增加；这些影响被二甲双胍治疗逆转。免疫沉淀分析用于检查 HeLa 细胞中 O-GlcNAcylation 和磷酸化之间的相互作用，揭示与未处理的细胞相比，二甲双胍降低了 O-GlcNAcylated AMP 活化蛋白激酶 (AMPK) 并增加了磷酸化 AMPK 的水平。如流式细胞术所示，这些结果与 HeLa 细胞中细胞周期停滞和细胞凋亡减少有关。此外，6-diazo-5-oxo-L-norleucine（一种谷氨酰胺果糖-6-磷酸氨基转移酶抑制剂）或 thiamet G（一种 O-GlcNAcase 抑制剂）降低或增加了 O-GlcNAcylated AMPK 的水平，并增加或降低了分别磷酸化 AMPK，表明 O-GlcNAc 修饰影响 AMPK 活化。值得注意的是，我们发现二甲双胍处理 HeLa 细胞会增加 p21 和 p27（它们是 AMPK 依赖性细胞周期抑制剂）的水平，与未处理的细胞相比，导致 HeLa 细胞的细胞周期停滞和凋亡增加。