In vitro radiosensitizing effect of targeting PI4K IIIα using anti-HCV agent

Abstract

Introduction: Phosphatidylinositol 4-kinase (PI4K) catalyzes the phosphorylation of phosphoinositide to produce PI4-phosphate (PI4P), a common and essential substrate of both the Phospholipase C (PLC)/Protein Kinase C (PKC) and PI3K/Akt pathways. Inhibition of PI4K simultaneously inactivates these PI4P-dependent pathways. In this study, we tried to identify that which isotype of PI4K may affect a radiosensitivity using RNA interference (RNAi) and to investigate whether anti-hepatitis C viral (HCV) agents, some of which have been shown to inhibit PI4K IIIα activity, could be repositioned as an anti-cancer agent and/or a radiosensitizer in human glioblastoma, breast, and hepatocellular carcinoma. Methods: A panel of human cancer cell lines including U251 malignant glioma cells, BT474 breast cancer cells, and HepG2 hepatocellular carcinoma cells were used. RNAi was used to specific inhibition of each isotype of PI4K and clonogenic assay was performed to assess the radiosensitizing effect of each isotype. To select an anti-HCV agent for pharmacologic inhibition of PI4K, IC50s of nine commercial antiviral agents were determined. Specific inhibitory effect on PI4K isotype was determined by in vitro kinase assay. Radiosensitizing effect of the selected anti-HCV agents was tested by clonogenic assay in vitro. Immunoblotting and immunocytochemistry were performed to identify the mechanism of radiosensitization. Results: A clonogenic assay showed that inhibition of PI4K IIIα using siRNA resulted in a significant increase in radiation-induced death of both U251 cells and BT474 cells. In contrast, inhibition of the other PI4K isotypes did not affect radiosensitivity. These results suggest that PI4K IIIα plays an important role in modulation of the response of malignant glioma cells and breast cancer cells to radiation. For pharmacologic inhibition of PI4K IIIα, simeprevir was selected based on the results of IC50 assays, and its inhibition of PI4K IIIα activity was confirmed in in vitro kinase assay. Combination of simeprevir treatment and radiation significantly attenuated expression of p-PKC and p-Akt and increased radiation-induced cell death in tested cell lines. Pretreatment with simeprevir prolonged γH2AX foci and downregulation of p-DNA-PKcs, indicating impairment of nonhomologous end-joining repair. Cells pretreated with simeprevir exhibited mixed modes of cell death, including apoptosis and autophagy. Invasion, migration, and vascular mimicry of tumor cells were markedly inhibited by simeprevir. Conclusions: Targeting PI4K IIIα using anti-HCV agent is a viable approach to enhance the therapeutic efficacy of radiotherapy in various human cancers, such as glioma, breast, and hepatocellular carcinoma.