ABSTRACT: Inhibitors of ataxia-telangiectasia mutated (ATM), such as KU-55933 (Ku), represent a promising class of novel anticancer drugs. In addition, the biguanide derivative phenformin exhibits antitumor activity superior to that of the AMPK activator metformin. Herein, we assessed the potential combinatorial therapeutic efficacy of phenformin and Ku when used to inhibit the growth of liver cancer cells, and we assessed the mechanisms underlying such efficacy. The Hep-G2 and SMMC-7721 liver cancer cell lines were treated with phenformin and Ku either alone or in combination, after which the impact of these drugs on cellular proliferation was assessed via 3-(4,5-dimethylthiazol) 2, 5-diphenyltetrazolium and colony formation assays, whereas Transwell assays were used to gauge cell migratory activity. The potential synergy between these two drugs was assessed using the CompuSyn software, while flow cytometry was employed to evaluate cellular apoptosis. In addition, western blotting was utilized to measure p-ATM, p-AMPK, p-mTOR, and p-p70s6k expression, while mitochondrial functionality was monitored via morphological analyses, JC-1 staining, and measurements of ATP levels. Phenformin and Ku synergistically impacted the proliferation, migration, and apoptotic death of liver cancer cells. Together, these compounds were able to enhance AMPK phosphorylation while inhibiting the phosphorylation of mTOR and p70s6k. These data also revealed that phenformin and Ku induced mitochondrial dysfunction as evidenced by impaired ATP synthesis, mitochondrial membrane potential, and abnormal mitochondrial morphology. These findings suggest that combination treatment with phenformin and Ku may be an effective approach to treating liver cancer via damaging mitochondria within these tumor cells.
