Liu Chao, Liu Qianqian, Yan Aiwen, Chang Hui, Ding Yuyin, Tao Junye, Qiao Chen
Department of Pharmacy, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China.
Cancer Med. 2020 Jun;9(11):3875-3884. doi: 10.1002/cam4.3029. Epub 2020 Apr 5.
Several studies have suggested that drug resistance in colon cancer patients with diabetes may be associated with long-term insulin administration, which in turn decreases the survival rate. Metformin is a commonly used drug to treat diabetes but has been recently demonstrated to have a potential therapeutic effect on colon cancer. This study aimed to elucidate the underlying mechanism by which metformin reverts insulin-induced oxaliplatin resistance in human colon cancer HCT116 cells.
Two colon cancer cell lines (HCT116 and LoVo) were used to verify whether the expression of insulin receptor substrate 1 (IRS-1) could impact the half maximal inhibitory concentration (IC50) of oxaliplatin after chronic insulin treatment. The IC50 of oxaliplatin in both cell lines was measured to identify metformin sensitization to oxaliplatin. The adenosine monophosphate-activated protein kinase (AMPK) inhibitor, namely AMPK small interfering RNA, was used to block AMPK activation to identify critical proteins in the AMPK/Erk signaling pathway. Bcl-2 is a vital antiapoptotic protein involved in the mitochondrial apoptosis pathway. Finally, immunofluorescence and electron microscopy were performed to investigate how metformin affects the ultrastructural integrity of mitochondria.
The IC50 of oxaliplatin in HCT116 cells was noticeably increased. After the cells were treated with metformin, oxaliplatin resistance was reversed. According to the results of the western blotting assay of vital proteins involved in the classical apoptosis pathway, cleaved caspase-9 was noticeably upregulated, suggesting that the mitochondrial apoptosis pathway was activated. These results were verified by imaging of mitochondria using electron microscopy. The AMPK/Erk signaling pathway experiments revealed that the upregulation of Bcl-2 induced by insulin through Erk phosphorylation was inhibited by metformin and that such inhibition could be mitigated by the inhibition of AMPK.
Insulin-induced oxaliplatin resistance was reversed by metformin-mediated AMPK activation. Accordingly, metformin is likely to sensitize patients with diabetes to chemotherapeutic drugs used to treat colon cancer.
多项研究表明，糖尿病结肠癌患者的耐药性可能与长期使用胰岛素有关，而这反过来又会降低生存率。二甲双胍是一种常用的治疗糖尿病的药物，但最近已证明其对结肠癌具有潜在的治疗作用。本研究旨在阐明二甲双胍逆转人结肠癌HCT116细胞中胰岛素诱导的奥沙利铂耐药性的潜在机制。
使用两种结肠癌细胞系（HCT116和LoVo）来验证胰岛素受体底物1（IRS-1）的表达是否会影响慢性胰岛素治疗后奥沙利铂的半数最大抑制浓度（IC50）。测量两种细胞系中奥沙利铂的IC50，以确定二甲双胍对奥沙利铂的增敏作用。使用腺苷单磷酸活化蛋白激酶（AMPK）抑制剂，即AMPK小干扰RNA，来阻断AMPK激活，以确定AMPK/Erk信号通路中的关键蛋白。Bcl-2是一种参与线粒体凋亡途径的重要抗凋亡蛋白。最后，进行免疫荧光和电子显微镜检查，以研究二甲双胍如何影响线粒体的超微结构完整性。
HCT116细胞中奥沙利铂的IC50显著增加。在用二甲双胍处理细胞后，奥沙利铂耐药性得到逆转。根据经典凋亡途径中关键蛋白的蛋白质印迹分析结果，裂解的caspase-9显著上调，表明线粒体凋亡途径被激活。这些结果通过电子显微镜对线粒体的成像得到验证。AMPK/Erk信号通路实验表明，胰岛素通过Erk磷酸化诱导的Bcl-2上调被二甲双胍抑制，并且这种抑制可以通过抑制AMPK来减轻。
二甲双胍介导的AMPK激活逆转了胰岛素诱导的奥沙利铂耐药性。因此，二甲双胍可能会使糖尿病患者对用于治疗结肠癌的化疗药物敏感。
