Abstract
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 μmol/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 μmol/L) or the specific RIP3 inhibitor GSK-872 (5 μmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 μmol/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.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (81372697), the Program for New Century Excellent Talents in University (NCET-12-0233), the Changbaishan Scholar Project of Jilin Province (2013026), the Scientific Research Foundation of Jilin province (20150414013GH and 20160101127JC), and the Bethune Project B of Jilin University (No 2012203).
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Supplementary Material Figure S1
Representative images of shikonin-induced morphological changes captured with a transmission electron microscope. (JPG 298 kb)
Supplementary Material Figure S2
(A) Statistical analysis of shikonin-induced changes in the RIP1 and RIP3 protein levels at 2 h of incubation. (JPG 1039 kb)
Supplementary Material Figure S3
(A and B) Representative fluorescence microscopic images of SHG-44 cells detected by the mitochondrial superoxide probe Mitosox Red and the ROS probe DCFH-DA, respectively (20×). (JPG 282 kb)
Supplementary Material Figure S4
(A) Representative images of SHG-44 cells under a fluorescence microscope showed that MnTBAP pretreatment mitigated the rotenone-induced production of mitochondrial superoxide (20×). (JPG 333 kb)
Supplementary Material Figure S5
(A) Statistical analysis of the shikonin-induced upregulation of RIP1 and RIP3 in cells pretreated with rotenone. (JPG 238 kb)
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LU, B., GONG, X., WANG, Zq. et al. Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1/RIP3 necrosome formation. Acta Pharmacol Sin 38, 1543–1553 (2017). https://doi.org/10.1038/aps.2017.112
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DOI: https://doi.org/10.1038/aps.2017.112
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