title: RIP1 and RIP3 contribute to shikonin-induced DNA double-strand breaks in glioma cells via increase of intracellular reactive oxygen species.
abstract: Shikonin has been reported to induce glioma cell death via necroptosis, a type of programmed necrosis primarily mediated by RIP1 and RIP3. Although RIP1 and RIP3 are found to regulate some features of necrosis such as energy depletion and cellular membrane disruption, it remains unclear whether RIP1 and RIP3 could modulate DNA double strand breaks (DSBs), which is a crucial event leading to chromatinolysis. In this study, we used glioma cell lines and mice model of xenograft glioma to investigate the roles of RIP1 and RIP3 in shikonin-induced DNA DSBs. We found that shikonin induced upregulation of RIP1 and RIP3, necrosome formation and DNA DSBs in vitro and in vivo. In vitro investigation showed that the DNA DSBs and the reduction of cellular viabilities induced by shikonin were both prevented when RIP1 or RIP3 was pharmacologically inhibited by specific inhibitor or genetically knocked down with siRNA. Then, we proved that suppression of intracellular ROS with antioxidant NAC inhibited DNA DSBs caused by either hydrogen peroxide or shikonin, suggesting that ROS played a crucial role in regulation of DNA DSBs of glioma cells induced by shikonin. Further, we found that RIP1 and RIP3 regulated shikonin-induced overproduction of ROS via causing excessive generation of mitochondrial superoxide and depletion of GSH, indicating that ROS was the downstream signal of RIP1 and RIP3. Taken together, we demonstrated that RIP1 and RIP3 contributed to shikonin-induced DNA DSBs in glioma cells via increase of intracellular ROS levels.
authors.affiliation: Department of Neurosurgery, First Hospital of Jilin University, Changchun 130021, China; Research Center of Neuroscience, First Hospital of Jilin University, Changchun 130021, China.
authors.forename: Zijian
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Department of Neurosurgery, First Hospital of Jilin University, Changchun 130021, China; Research Center of Neuroscience, First Hospital of Jilin University, Changchun 130021, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Lu
authors.affiliation: Department of Neurosurgery, First Hospital of Jilin University, Changchun 130021, China; Research Center of Neuroscience, First Hospital of Jilin University, Changchun 130021, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, First Hospital of Jilin University, Changchun 130021, China; Research Center of Neuroscience, First Hospital of Jilin University, Changchun 130021, China.
authors.forename: Zongqi
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Research Center of Neuroscience, First Hospital of Jilin University, Changchun 130021, China; Department of Neurology, First Hospital of Jilin University, Changchun 130021, China.
authors.forename: Tianfei
authors.initials: T
authors.lastname: Luo
authors.affiliation: Department of Anesthesiology, First Hospital of Jilin University, Changchun 130021, China.
authors.forename: Meihua
authors.initials: M
authors.lastname: Piao
authors.affiliation: Department of Neurosurgery, People's Hospital of Jilin Province, Changchun 130021, China.
authors.forename: Fankai
authors.initials: F
authors.lastname: Meng
authors.affiliation: Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130021, China.
authors.forename: Guangfan
authors.initials: G
authors.lastname: Chi
authors.affiliation: Department of Neurosurgery, First Hospital of Jilin University, Changchun 130021, China.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Neurosurgery, First Hospital of Jilin University, Changchun 130021, China; Research Center of Neuroscience, First Hospital of Jilin University, Changchun 130021, China. Electronic address: gepf@jlu.edu.cn.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Ge
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004195:Disease Models, Animal; D005910:Glioma / Q000188:drug therapy*; D064593:Heterografts; D051379:Mice; D009285:Naphthoquinones / Q000494:pharmacology; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C119724:AGFG1 protein, human; D009285:Naphthoquinones; D028861:Nuclear Pore Complex Proteins; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; C016101:shikonin; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases
keywords: DNA double strand breaks; Glioma cell; RIP1 and RIP3; ROS; Shikonin