Nrf2 mediates mitochondrial and NADPH oxidase-derived ROS during mild heat stress at 40 °C

Hyperthermia is an adjuvant to chemotherapy and radiotherapy and sensitizes tumors to these treatments. However, repeated heat treatments result in acquisition of heat resistance (thermotolerance) in tumors. Thermotolerance is an adaptive survival response that appears to be mediated by upregulated cellular defenses. However, the mechanisms of activation remain unclear. When HeLa cells were exposed to mild heat shock at 40 °C for 3 h, levels of superoxide and peroxides increased. Cells were treated with mitochondrial antioxidant MitoQ and NADPH oxidase (NOX) inhibitor apocynin to characterize the contribution of these two sources to the total reactive oxygen species (ROS) pool. We found that both mitochondria and NOX are sources of ROS during mild heat shock at 40 °C. Heat-derived ROS are thought to activate the adaptive survival response at 40 °C. Nrf2, the master regulator of the cellular antioxidant response, is thought to play a pivotal role in establishing the adaptive survival response. Nrf2 was overexpressed or knocked down to assess its role. Moreover, Nrf2 levels correlate with the cellular redox state, and do so via scavenging of mitochondria- and NOX-derived ROS. Knockdown of Nrf2 markedly increased levels of ROS that were scavenged by either apocynin or MitoQ. Finally, critical defense proteins such as DJ-1 and PGAM5 seemed to require a two-key activation system mediated by Nrf2 and mitochondrial ROS. Our study characterized mitochondrial and NOX-derived ROS as being essential in activating cellular defenses alongside Nrf2 and underlines potential therapeutic targets that may contribute to the acquisition of thermotolerance.