Metformin transiently inhibits colorectal cancer cell proliferation as a result of either AMPK activation or increased ROS production

Angela Mogavero,Maria Valeria Maiorana,Antonino Belfiore,Chiara C Volpi,Susanna Zanutto,Fabio Bozzi,Annunziata Gloghini,Luca Varinelli,Manuela Gariboldi,Marco A Pierotti

doi:10.1038/s41598-017-16149-z

Angela Mogavero, Maria Valeria Maiorana + Show 8 more

Open Access

https://doi.org/10.1038/s41598-017-16149-z

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Abstract

Metformin is a widely used and well-tolerated anti-diabetic drug that can reduce cancer risk and improve the prognosis of certain malignancies. However, the mechanism underlying its anti-cancer effect is still unclear. We studied the anti-cancer activity of metformin on colorectal cancer (CRC) by using the drug to treat HT29, HCT116 and HCT116 p53−/− CRC cells. Metformin reduced cell proliferation and migration by inducing cell cycle arrest in the G0/G1 phase. This was accompanied by a sharp decrease in the expression of c-Myc and down-regulation of IGF1R. The anti-proliferative action of metformin was mediated by two different mechanisms: AMPK activation and increase in the production of reactive oxygen species, which suppressed the mTOR pathway and its downstream targets S6 and 4EBP1. A reduction in CD44 and LGR5 expression suggested that the drug had an effect on tumour cells with stem characteristics. However, a colony formation assay showed that metformin slowed the cells’ ability to form colonies without arresting cell growth, as confirmed by absence of apoptosis, autophagy or senescence. Our finding that metformin only transiently arrests CRC cell growth suggests that efforts should be made to identify compounds that combined with the biguanide can act synergistically to induce cell death.

Highlights

The methods used for the early diagnosis of colorectal cancer (CRC) are insufficiently sensitive and specific and, despite major advances in surgical techniques and adjuvant treatment, there is still no effective therapy for advanced disease
In order to investigate the effects of metformin on CRC, the HT29, HCT116 and HCT116 p53−/− cell lines were treated with 5 mM metformin as reported by Zakikhani et al.[5]
Metformin inhibited mammalian target of rapamycin (mTOR) (Ser2448), ribosomal protein S6 kinase (RPS6K, Thr389) and 4E-binding protein 1 (4EBP1, Thr37/46) in all of the cell lines (Fig. 6). These findings indicate that inhibition of the mTOR/S6/4EBP1 pathway in CRC cell lines is only partially mediated by mitochondrial depolarisation and AMPK activation, and suggest that the cellular effects induced by metformin could be at least partially due to the mTOR-mediated reduction in protein synthesis

Summary

Introduction

The methods used for the early diagnosis of colorectal cancer (CRC) are insufficiently sensitive and specific and, despite major advances in surgical techniques and adjuvant treatment, there is still no effective therapy for advanced disease. Metformin (1,1-dimethylbiguanide hydrochloride) is frequently prescribed to reduce hepatic gluconeogenesis and increase skeletal muscle glucose uptake in patients with type 2 diabetes. It directly inhibits the growth of various tumour types in vitro and in vivo[1], and seems to reduce the growth of tumour initiating cells or cancer stem cells (CSCs)[2]. We analysed the activity of metformin in three CRC cell lines, one with BRAF as driver mutation, the others with KRAS mutation, one of which is an isogenic p53 null form and found for the first time that the drug transiently inhibited their growth and mTOR pathway activation by means of AMPK-dependent and -independent mechanisms

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