Study on the mechanism of in vitro apoptosis induced with 5-aminolevulinic acid-mediated sonodynamic therapy in Hep G2 cells

Objective: To investigate the underlying mechanisms of cell apoptosis induced by 5-aminolevulinic acid-mediated sonodynamic therapy (5-ALA-SDT) in human hepatocellular carcinoma Hep G2 cells. Methods: The CCK-8 assays were used to examine the effects of 5-ALA and ultrasound intervention on the viability of Hep G2 at logarithmic growth phase. The 5-ALA level and intensity of ultrasonic irradiation with best efficacy were identified. The apoptosis rate, mortality rate, and changes of mitochondrial membrane potential (MMP) in Hep G2 cells in the control group (untreated cells), 5-ALA group, ultrasonic group, and SDT group were detected with flow cytometry assays. The production of reactive oxygen species (ROSs) were detected with the fluorescent probes. Western blotting was used to examine expressions of cytochrome C (Cyt-C) and apoptosis-associated proteins caspase-3 and caspase-9. The ultrastructural changes in cells were observed with the transmission electron microscope. Results: The SDT group had lower survival rate, higher apoptosis rate, increased ROS production, decreased MMP, and elevated expressions of caspase-3, caspase-9, and Cyt-C as compared with the control group, 5-ALA group, and ultrasonic group. The transmission electron microscope revealed changes such as reduced cell microvilli, swelling endoplasmic reticulum and mitochondria, and the production of apoptotic bodies in Hep G2 cells in the SDT group. Conclusion: Endogenous mitochondrial apoptosis is the main mechanism of 5-ALA-SDT-induced cell apoptosis in Hep G2 cells.