title: Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis.
abstract: Artesunate, an anti-malarial drug, has been repurposed as an anticancer drug due to its induction of cell death via reactive oxygen species (ROS) production. However, the molecular mechanisms regulating cancer cell death and the resistance of cells to artesunate remain unclear. We investigated the molecular mechanisms behind the antitumor effects of artesunate and an approach to overcome artesunate resistance in head and neck cancer (HNC). The effects of artesunate and trigonelline were tested in different HNC cell lines, including three cisplatin-resistant HNC cell lines. The effects of these drugs as well as the inhibition of Keap1, Nrf2, and HO-1 were assessed by cell viability, cell death, glutathione (GSH) and ROS production, protein expression, and mouse tumor xenograft models. Artesunate selectively killed HNC cells but not normal cells. The artesunate sensitivity was relatively low in cisplatin-resistant HNC cells. Artesunate induced ferroptosis in HNC cells by decreasing cellular GSH levels and increasing lipid ROS levels. This effect was blocked by co-incubation with ferrostatin-1 and a trolox pretreatment. Artesunate activated the Nrf2-antioxidant response element (ARE) pathway in HNC cells, which contributed to ferroptosis resistance. The silencing of Keap1, a negative regulator of Nrf2, decreased artesunate sensitivity in HNC cells. Nrf2 genetic silencing or trigonelline reversed the ferroptosis resistance of Keap1-silenced and cisplatin-resistant HNC cells to artesunate in vitro and in vivo. Nrf2-ARE pathway activation contributes to the artesunate resistance of HNC cells, and inhibition of this pathway abolishes ferroptosis-resistant HNC.
  
  
  
  Our results show the effectiveness and molecular mechanism of artesunate treatment on head and neck cancer (HNC). Artesunate selectively killed HNC cells but not normal cells by inducing an iron-dependent, ROS-accumulated ferroptosis. However, this effect may be suboptimal in some cisplatin-resistant HNCs because of Nrf2-antioxidant response element (ARE) pathway activation. Inhibition of the Nrf2-ARE pathway increased artesunate sensitivity and reversed the ferroptosis resistance in resistant HNC cells.
authors.affiliation: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. Electronic address: rohjl@amc.seoul.kr.
authors.forename: Jong-Lyel
authors.initials: JL
authors.lastname: Roh
authors.affiliation: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Eun Hye
authors.initials: EH
authors.lastname: Kim
authors.affiliation: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Hyejin
authors.initials: H
authors.lastname: Jang
authors.affiliation: Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
authors.forename: Daiha
authors.initials: D
authors.lastname: Shin
mesh_terms: D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D037621:Artemisinins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000077332:Artesunate; D002839:Chromans / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D003514:Cyclohexylamines / Q000494:pharmacology; D058492:Drug Repositioning; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D051267:NF-E2-Related Factor 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010655:Phenylenediamines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction; D023041:Xenograft Model Antitumor Assays
chemical_list: D000470:Alkaloids; D000962:Antimalarials; D000970:Antineoplastic Agents; D037621:Artemisinins; D002839:Chromans; D003514:Cyclohexylamines; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D010655:Phenylenediamines; D017382:Reactive Oxygen Species; C573944:ferrostatin-1; C009560:trigonelline; D000077332:Artesunate; D007501:Iron; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; D002945:Cisplatin; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
keywords: Artensunate; Ferroptosis; Head and neck cancer; Nrf2; Reactive oxygen species; Resistance