Ozone Induces BEL7402 Cell Apoptosis by Increasing Reactive Oxygen Species Production and Activating JNK.

Background: Oxidative stress is an important factor in the modulation of both tumorigenesis and anticancer responses. Ozone (O-3) is a strong oxidant that causes redox reactions and exerts anticancer effects in various types of cancer cells. However, the pathways involved in O-3-induced cell death are not well understood. Methods: In vitro human hepatocellular carcinoma (HCC) BEL7402 cells were treated with various O-3 concentrations to evaluate O-3 cytotoxicity by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The regulatory mechanisms were analyzed by western blot analysis. In vivo, an HCC model was established to evaluate the inhibition of HCC with O-3 treatment. Results: In vitro cells treated with O-3 exhibited a round and small morphology with nuclear shrinkage and fragmentation. The CCK-8 assay confirmed the potent cytotoxic activity of O-3 against BEL7402 cells (IC50 value of 5 mu g/mL). Acridine orange/ethidium bromide (AO/EB) staining revealed apoptosis of BEL7402 cells after O-3 treatment. Flow cytometry analysis showed that S phase cell cycle arrest and apoptosis increased with O-3 exposure. In addition, O-3 exposure reduced the mitochondrial membrane potential (Delta Psi m) and induced reactive oxygen species (ROS) accumulation. Western blot analysis showed that O-3 exposure reduced B-cell lymphoma 2 (BCL-2) expression and increased cleaved poly ADP-ribose polymerase (PARP), cytochrome C (Cyt-C), caspase-3, caspase-9, and p-JNK expression. In vivo, treatment with intratumor injection O-3 (20 mu g/mL) inhibited HCC growth. Conclusions: Overall, our findings showed that O-3 induces BEL7402 cell apoptosis via the intrinsic mitochondria-dependent pathway. Therefore, O-3 has therapeutic potential for HCC.