Metformin revert insulin-induced oxaliplatin resistance by activating mitochondrial apoptosis pathway in human colon cancer HCT116 cells

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: AMPK; insulin; metformin.