RIP1 and RIP3 contribute to shikonin-induced glycolysis suppression in glioma cells via increase of intracellular hydrogen peroxide.

RIP1 and RIP3 are necroptosis initiators, but their roles in regulation of glycolysis remain elusive. In this study, we found shikonin activated RIP1 and RIP3 in glioma cells in vitro and in vivo, which was accompanied with glycolysis suppression. Further investigation revealed that shikonin-induced decreases of glucose-6-phosphate and pyruvate and downregulation of HK II and PKM2 were significantly prevented when RIP1 or RIP3 was pharmacologically inhibited or genetically knocked down with SiRNA. Moreover, shikonin also triggered accumulation of intracellular H2O2 and depletion of GSH and cysteine. Mitigation of intracellular H2O2 via supplement of GSH reversed shikonin-induced glycolysis suppression. The role of intracellular H2O2 in regulation of glycolysis suppression was further confirmed in the cells treated with exogenous H2O2. Notably, inhibition of RIP1 or RIP3 prevented intracellular H2O2 accumulation, which was correlated with preventing shikonin-induced downregulation of x-CT and depletion of GSH and cysteine. In addition, supplement of pyruvate effectively inhibited shikonin- or exogenous H2O2-induced accumulation of intracellular H2O2 and glioma cell death. Taken together, we demonstrated in this study that RIP1 and RIP3 contributed to shikonin-induced glycolysis suppression via increasing intracellular H2O2.

Lu B ，Wang Z ，Ding Y ，Wang X ，Lu S ，Wang C ，He C ，Piao M ，Chi G ，Luo Y ，Ge P ... -  《-》

RIP1 and RIP3 contribute to shikonin-induced DNA double-strand breaks in glioma cells via increase of intracellular reactive oxygen species.

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.

Zhou Z ，Lu B ，Wang C ，Wang Z ，Luo T ，Piao M ，Meng F ，Chi G ，Luo Y ，Ge P ... -  《-》

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

Lu B ，Gong X ，Wang ZQ ，Ding Y ，Wang C ，Luo TF ，Piao MH ，Meng FK ，Chi GF ，Luo YN ，Ge PF ... -  《-》

MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis.

Chromatinolysis refers to enzymatic degradation of nuclear DNA and is regarded as one of the crucial events leading to cell death. Mixed-lineage kinase domain-like protein (MLKL) has been identified as a key executor of necroptosis, but it remains unclear whether MLKL contributes to necroptosis via regulation of chromatinolysis. In this study, we find that shikonin induces MLKL activation and chromatinolysis in glioma cells in vitro and in vivo, which are accompanied with nuclear translocation of AIF and γ-H2AX formation. In vitro studies reveal that inhibition of MLKL with its specific inhibitor NSA or knockdown of MLKL with siRNA abrogates shikonin-induced glioma cell necroptosis, as well as chromatinolysis. Mechanistically, activated MLKL targets mitochondria and triggers excessive generation of mitochondrial superoxide, which promotes AIF translocation into nucleus via causing mitochondrial depolarization and aggravates γ-H2AX formation via improving intracellular accumulation of ROS. Inhibition of nuclear level of AIF by knockdown of AIF with siRNA or mitigation of γ-H2AX formation by suppressing ROS with antioxidant NAC effectively prevents shikonin-induced chromatinolysis. Then, we found that RIP3 accounts for shikonin-induced activation of MLKL, and activated MLKL reversely up-regulates the protein level of CYLD and promotes the activation of RIP1 and RIP3. Taken together, our data suggest that MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis, and shikonin induces a positive feedback between MLKL and its upstream signals RIP1 and RIP3.

Ding Y ，He C ，Lu S ，Wang X ，Wang C ，Wang L ，Zhang J ，Piao M ，Chi G ，Luo Y ，Sai K ，Ge P ... -  《-》

The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis.

Fu Z ，Deng B ，Liao Y ，Shan L ，Yin F ，Wang Z ，Zeng H ，Zuo D ，Hua Y ，Cai Z ... -  《-》