Berberine, a natural alkaloid sensitizes human hepatocarcinoma to ionizing radiation by blocking autophagy and cell cycle arrest resulting in senescence

Objective: To study the radiosensitizing potential of Berberine and the underlying mechanism in human hepatocarcinoma (HepG2) cells. Methods: HepG2 cells were challenged with X-rays in combination with Berberine treatment and several in vitro assays were performed. Alteration in cell viability was determined by MTT assay. Changes in intracellular ROS levels, mitochondrial membrane potential/mass, intracellular acidic vesicular organelles as well as cell cycle arrest and apoptotic cell death were analysed by flow cytometry. Induction of autophagy was assessed by staining the cells with Monodansylcadaverine/Lysotracker red dyes and immunoblotting for LC3I/II and p62 proteins. Phase-contrast/fluorescence microscopy was employed to study mitotic catastrophe and senescence. Cellular senescence was confirmed by immunoblotting for p21 levels and ELISA for Interleukin-6. Key findings: X-rays + Berberine had a synergistic effect in reducing cell proliferation accompanied by a robust G2/M arrest. Berberine-mediated radiosensitization was associated with elevated levels of LC3II and p62 suggesting blocked autophagy that was followed by mitotic catastrophe and senescence. Treatment of cells with X-rays + Berberine resulted in increased oxidative stress, hyperpolarized mitochondria with increased mitochondrial mass and reduced ATP levels. Conclusions: The study expands the understanding of the pharmacological properties of Berberine and its applicability as a radiosensitizer towards treating liver cancer.