Sublethal hyperthermia enhances anticancer activity of doxorubicin in chronically hypoxic HepG2 cells through ROS-dependent mechanism

Thermal ablation in combination with transarterial chemoembolization (TACE) has been reported to exert a more powerful antitumor effect than thermal ablation alone in hepatocellular carcinoma patients. However, the underlying mechanisms remain unclear. The purpose of the present study was to evaluate whether sublethal hyperthermia encountered in the periablation zone during thermal ablation enhances the anticancer activity of doxorubicin in chronically hypoxic (encountered in the tumor area after TACE) liver cancer cells and to explore the underlying mechanisms. In the present study, HepG2 cells precultured under chronic hypoxic conditions (1% oxygen) were treated in a 42°C water bath for 15 or 30 min, followed by incubation with doxorubicin. Assays were then performed to determine intracellular uptake of doxorubicin, cell viability, apoptosis, cell cycle, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and total antioxidant capacity. The results confirmed that sublethal hyperthermia enhanced the intracellular uptake of doxorubicin into hypoxic HepG2 cells. Hyperthermia combined with doxorubicin led to a greater inhibition of cell viability and increased apoptosis in hypoxic HepG2 cells as compared with hyperthermia or doxorubicin alone. In addition, the combination induced apoptosis by increasing ROS and causing disruption of MMP. Pretreatment with the ROS scavenger N-acetyl cysteine significantly inhibited the apoptotic response, suggesting that cell death is ROS-dependent. These findings suggested that sublethal hyperthermia enhances the anticancer activity of doxorubicin in hypoxic HepG2 cells via a ROS-dependent mechanism.