Abstract
Background and Purpose: The anti-diabetic biguanide drugs metformin and phenformin exhibit antitumor activity in various models. However, their radiomodulatory effect under hypoxic conditions, particularly for phenformin, is largely unknown. This study therefore examines whether metformin and phenformin as mitochondrial complex I blockades could overcome hypoxic radioresistance through inhibition of oxygen consumption.Materials and Methods: A panel of colorectal cancer cells (HCT116, DLD-1, HT29, SW480, and CT26) was exposed to metformin or phenformin for 16 h at indicated concentrations. Afterward, cell viability was measured by MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) was examined by western blot. Mitochondria complexes activity and oxygen consumption rate (OCR) were measured by seahorse analyzer. The radiosensitivity of tumor cells was assessed by colony formation assay under aerobic and hypoxic conditions. The in vitro findings were further validated in colorectal CT26 tumor model.Results: Metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced OCR in a dose-dependent manner starting at 3 mM and 30 μM, respectively. As a result, the hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin with an enhancement ratio about 2 at 9 mM and 100 μM, respectively. Regarding intrinsic radioresistance, both of them did not exhibit any effect although there was an increase of phosphorylation of AMPK and ROS production. In tumor-bearing mice, metformin or phenformin alone did not show any anti-tumor effect. While in combination with radiation, both of them substantially delayed tumor growth and enhanced radioresponse, respectively, by 1.3 and 1.5-fold.Conclusion: Our results demonstrate that metformin and phenformin overcome hypoxic radioresistance through inhibition of mitochondrial respiration, and provide a rationale to explore metformin and phenformin as hypoxic radiosensitizers.
Highlights
Metformin, an oral biguanide, is the most widely used drug to treat type II diabetes
Metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced oxygen consumption rate (OCR) in a dose-dependent manner starting at 3 mM and 30 μM, respectively
The hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin with an enhancement ratio about 2 at 9 mM and 100 μM, respectively
Summary
An oral biguanide, is the most widely used drug to treat type II diabetes. Repurposing metformin as an anti-cancer drug is receiving considerable attention with over 100 clinical trials, wherein its anti-cancer effect is explored alone or combined with other therapies (Chae et al, 2016). The enthusiasm toward repurposing metformin is extended to another biguanide anti-diabetic drug, namely phenformin. While as a potential anti-cancer drug, phenformin showed much higher cytotoxic activity toward tumor- and tumor-supporting cells compared with metformin in various preclinical models (Shackelford et al, 2013; Gravel et al, 2014; Velez et al, 2016). The anti-diabetic biguanide drugs metformin and phenformin exhibit antitumor activity in various models. Their radiomodulatory effect under hypoxic conditions, for phenformin, is largely unknown. This study examines whether metformin and phenformin as mitochondrial complex I blockades could overcome hypoxic radioresistance through inhibition of oxygen consumption
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