title: The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis.
abstract: BACKGROUND
  Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers.
  
  
  METHODS
  MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo.
  
  
  RESULTS
  The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001).
  
  
  CONCLUSIONS
  Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.
authors: Zeze
authors: Z
authors: Fu
authors: Biyong
authors: B
authors: Deng
authors: Yuxin
authors: Y
authors: Liao
authors: Liancheng
authors: L
authors: Shan
authors: Fei
authors: F
authors: Yin
authors: Zhuoying
authors: Z
authors: Wang
authors: Hui
authors: H
authors: Zeng
authors: Dongqing
authors: D
authors: Zuo
authors: Department of Orthopedics, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, Shanghai 200072, China. hua_yingqi@163.com.
authors: Yingqi
authors: Y
authors: Hua
authors: Zhengdong
authors: Z
authors: Cai
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis; D001859:Bone Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D004365:Drugs, Chinese Herbal / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000556:secondary; D051379:Mice; D008807:Mice, Inbred BALB C; D009285:Naphthoquinones / Q000494:pharmacology* / Q000627:therapeutic use; D009336:Necrosis; D009368:Neoplasm Transplantation; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D012516:Osteosarcoma / Q000188:drug therapy* / Q000378:metabolism / Q000556:secondary; D016601:RNA-Binding Proteins / Q000378:metabolism*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C119724:AGFG1 protein, human; D000970:Antineoplastic Agents; D004365:Drugs, Chinese Herbal; D009285:Naphthoquinones; D028861:Nuclear Pore Complex Proteins; D016601:RNA-Binding Proteins; C016101:shikonin; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases