Metformin enhances radiosensitivity in hepatocellular carcinoma by inhibition of specificity protein 1 and epithelial-to-mesenchymal transition

Objective: Radiotherapy becomes more and more important in hepatocellular carcinoma (HCC) due to the development of technology, especially in unresectable cases. Metformin has a synergistic benefit with radiotherapy in some cancers, but remains unclear in HCC. This study aims to investigate the effect of metformin on radiosensitivity of HCC cells and the roles of specificity protein 1 (Sp1) as a target of metformin. Methods: The SMMC-7721 cell line was exposed to various doses of γ-ray irradiation (0, 2, 4, 6, and 8 Gy) and with or without different concentrations of metformin (0, 1, 5, 10, and 20 mM) to measure the radiosensitivity using MTT assay. Flow cytometry was used to determine cell cycle by propidium iodide (PI) staining and apoptosis by Hoechst 33342/PI staining and Annexin V-FITC/PI staining. Real-time polymerase chain reaction and Western blotting were performed to analyze the Sp1 mRNA and protein expressions of Sp1 and epithelial-to-mesenchymal transition (EMT) marker E-cadherin and Vimentin. The invasion capability was measured by the Boyden chamber assay. Results: In SMMC-7721 cells exposed to irradiation, metformin reduced proliferation and survival cells at various concentrations (0, 1, 5, 10, and 20 mM) and induced cell cycle arrest, apoptosis, and inhibited invasion. In SMMC-7721 cells with irradiation, the mRNA and protein expressions of Sp1 were significantly decreased by metformin as well as a selective Sp1 inhibitor. Metformin attenuated transforming growth factor-β1 induced decrease of E-cadherin and increase of Vimentin proteins. Conclusion: Metformin demonstrated enhanced radiosensitivity and inhibition of EMT in HCC cells. Sp1 might be a target of metformin in radiosensitization