ML385, an Nrf2 Inhibitor, Synergically Enhanced Celastrol Triggered Endoplasmic Reticulum Stress in Lung Cancer Cells

Lung cancer is one of the leading causes of death. Celastrol is a natural product that has shown anticancer activity but has not yet been applied in clinical settings due to its narrow therapeutic window. In this study, we discovered that celastrol stimulates an abnormal rise in the reactive oxygen species (ROS) level in lung cancer cells and that the ROS scavenger N-acetylcysteine (NAC) could counteract the cell death caused by celastrol. At the same time, celastrol upregulated the expression of cytoprotective transcription factor Nrf2 and its downstream proteins, which are effective in preventing the oxidative damage caused by ROS accumulation. Notably, we found that the overexpression of Nrf2 enhances the tolerance of lung cancer cells to celastrol and that lung cancer cells H460 with a Keap1 mutation are insensitive to celastrol. This indicates that the increase in Nrf2 contributes to the survival of lung cancer cells. Thus, we brought in an Nrf2 inhibitor ML385 to suppress the activation of Nrf2. We found that when ML385 and celastrol were added together the survival rates of lung cancer cells decreased more and the detected ROS level became much higher compared to treatment with celastrol alone. We also discovered that ML385 suppressed the expression of HO-1 and GCLC, which amplified celastrol-induced ATF4/CHOP-dependent endoplasmic reticulum stress (ER stress). Above all, our study found that ML385 enhanced celastrol-induced increases in ROS and ER stress, leading to lung cancer cell death. This research provides a potential strategy for the preclinical investigation of celastrol.