Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1/RIP3 necrosome formation

• Published in: Acta pharmacologica Sinica Vol. 38; no. 11; pp. 1543 - 1553
• Main Authors: LU, Bin, GONG, Xu, WANG, Zong-qi, DING, Ye, WANG, Chen, LUO, Tian-fei, PIAO, Mei-hua, MENG, Fan-kai, CHI, Guang-fan, LUO, Yi-nan, GE, Peng-fei
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-11-2017; Nature Publishing Group
• Subjects: Animals; Antineoplastic Agents - pharmacology; Apoptosis - drug effects; Biomedical and Life Sciences; Biomedicine; Brain Neoplasms - drug therapy; Brain Neoplasms - enzymology; Brain Neoplasms - pathology; Brain tumors; Cell Line, Tumor; Dose-Response Relationship, Drug; Gangrene; Glioma; Glioma - drug therapy; Glioma - enzymology; Glioma - pathology; Glioma cells; Humans; Immunology; Internal Medicine; Intracellular; Kinases; Medical Microbiology; Mitochondria; Mitochondria - drug effects; Mitochondria - enzymology; Mitochondria - pathology; Naphthoquinones - pharmacology; Necroptosis; Necrosis; Nuclear Pore Complex Proteins - metabolism; Original; original-article; Oxidative Stress - drug effects; Pharmacology/Toxicology; Protein kinase; Protein Serine-Threonine Kinases - metabolism; Rats; Reactive oxygen species; Reactive Oxygen Species - metabolism; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; RNA-Binding Proteins - metabolism; Rotenone; Shikonin; Signal Transduction - drug effects; Superoxide; Time Factors; Vaccine; mitochondrial superoxide; MnTBAP; ROS; GSK-872; rotenone; glioma; Nec-1; necrosome; RIP3; RIP1; shikonin
• ISSN: 1671-4083; 1745-7254
• DOI: 10.1038/aps.2017.112

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 （RIP1） and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species （ROS）, but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin （2-10 pmol/L） dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose- dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 （100 pmol/L） or the specific RIP3 inhibitor GSK-872 （5 pmol/L） not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intraceliular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP （40 pmol/L）, which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.