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 (IC(50) value of 5 µg/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, O3 exposure reduced the mitochondrial membrane potential (ΔΨm) 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 µg/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.