title: Shikonin kills glioma cells through necroptosis mediated by RIP-1.
abstract: OBJECTIVE
  Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.
  
  
  METHODS
  Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.
  
  
  RESULTS
  Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.
  
  
  CONCLUSIONS
  We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.
authors.affiliation: Department of Neurosurgery, First Bethune hospital of Jilin University, Changchun, China.
authors.forename: Chuanjiang
authors.initials: C
authors.lastname: Huang
authors.forename: Yinan
authors.initials: Y
authors.lastname: Luo
authors.forename: Jingwei
authors.initials: J
authors.lastname: Zhao
authors.forename: Fuwei
authors.initials: F
authors.lastname: Yang
authors.forename: Hongwei
authors.initials: H
authors.lastname: Zhao
authors.forename: Wenhai
authors.initials: W
authors.lastname: Fan
authors.forename: Pengfei
authors.initials: P
authors.lastname: Ge
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D045744:Cell Line, Tumor; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005910:Glioma; D006801:Humans; D008516:Medicine, Chinese Traditional; D009285:Naphthoquinones / Q000494:pharmacology*; D009336:Necrosis; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: C119724:AGFG1 protein, human; D000970:Antineoplastic Agents; D009285:Naphthoquinones; D028861:Nuclear Pore Complex Proteins; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; C016101:shikonin