Chenxin Xu,Yinghua Chen,Zhuoen Zhou,Ying Yan,Wenying Fu,Peng Zou,Daoyong Ni
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.
Shikonin , a naturally occurring naphthoquinone with potent anti-tumor activity, has been reported to induce cancer cell death via targeting selenoenzyme thioredoxin reductase 1 (TrxR1; TXNRD1). However, the interaction between shikonin and TrxR1 remains unclear, and the roles of the cellular antioxidant system in shikonin induced cell death are obscure. Here, we found that shikonin modified the Sec 498 residue of TrxR1 to fully inhibit its antioxidant activity , however, the shikonin-modified TrxR1 still remained intrinsic NADPH oxidase activity, which promotes superoxide anions production. Besides, TrxR1 efficiently reduced shikonin in both selenocysteine dependent and selenocysteine independent manners, and the oxygen-coupled redox cycling of shikonin also generates excessive superoxide anions. The inhibitory effects and the redox cycling of shikonin towards TrxR1 caused cancer cell ROS-dependent necroptosis. Interestingly, as we evaluated, some cancer cell lines were insensitive to shikonin, especially kelch-like ECH associated protein 1 (KEAP1)-mutant non-small cell lung cancer (NSCLC) cells, which harbor constitutive activation of the nuclear factor-erythroid 2-related factor 2 (NRF2). NADPH bankruptcy caused by glucose starvation or glucose limitation (inhibiting glucose transporter 1 by BAY-876) could efficiently overcome the resistance of KEAP1-mutant NSCLC cells to shikonin. Glucose-6-phosphate dehydrogenase (G6PD), was known as a rate-limiting enzyme in the pentose phosphate pathway , however, the pharmacological inhibition of G6PD by 6-aminonicotinamide (6-AN), enhanced the shikonin-induced cytotoxicity but has no selectivity on KEAP1-mutant NSCLC cells. This study will be helpful in applying shikonin for potential chemotherapy, and in combinational treatment of KEAP1-mutant NSCLC.
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