The Effects And Mechanisms Of RIP1 And RIP3 On Shikonin Induced Glycolysis Suppression In Glioma Cells

Background Malignant glioma is the most common form of intrinsic brain tumor,and is one of the most serious diseases which endanger human health。At present,the main strategy for treating gliomas is surgery in combination with postoperative chemotherapy and radiotherapy but the average survival is not longer than one year.Thus,novel therapeutic strategy is needed urgently.Recently,accumulating evidences have shown that induction of necroptosis is an effective approach to kill glioma cells.The model of cell death especially necroptosis has been a hotspot in the field of life sciences in recent years.Considering that glioma cells tolerate chemotherapy and radiotherapy was associated with resistance to apoptosis,fortunately,recent studies have shown that induction of necroptosis is an effective way to kill glioma cells。As a new type of caspase-independent programmed cell death,necroptosis is initiated by sequential activation of receptor interacting serine-threonine protein kinases1(RIP1)and 3(RIP3).After being activated,RIP1 interacts with RIP3 through the RHIM motif to form an amyloid-like necrosome complex in which RIP3 is phosphorylated by activated RIP1.The phosphorylation of RIP1 and RIP3 stabilizes their association within the necrosome complex and activates their pronecrotic necrotic kinase activities。Then,the necrosome complex triggers a series of downstream biochemical events including cellular membrane disruption,lethal autophagy and mitochondria damage,which eventually leads to cell death.Programmed cell death was always accompanied by cellular membrane disruption,DNA double strand breaks,mitochondria damage,glycolisis suppression.Glycolysis is the primary pathway used by cancer cells to produce energy and intermediate metabolite even under aerobic conditions,but non-tumor cells acquires energy though krebs cycle.Via aerobic glycolysis,cancer cells transform glucose into ATP and pyruvate.Three key enzymes including hexokinase(HK),phosphofructokinase,and pyruvate kinase(PK)are known to be responsible for regulating the rate-limiting steps of glycolysis.In cancer cells,HK II catalyses the first irreversible reaction of glycolysis to produce glucose-6-phosphate,and PKM2 are highly expressed and their protein levels are positively correlated with glioma genesis,malignancy and growth.The agents targeting glycolysis have shown promising efficay in reversing drug resistance and are being considered as potential anticancer targets.Although RIP1 and RIP3 are demonstrated to be the primary initiators of necroptosis,it is still unclear of their roles in regulation of the glycolysis in necroptotic cancer cells.As a natural naphthoquinone isolated from Lithospermum Erythrothizon,shikonin has been proben to be a potent necroptosis-inducer for glioma cells,as well as for other types of cancer cells including breast cancer,multiple myeloma and osteosarcoma.Despite shikonin was reported to inhibit the glycolysis of cancer cells,the underlying mechanism is still needed to be clarified.It will help us further perfect the underlying mechanism of necroptosis induced by shikonin and provide new strategies and solutions to overcome the tumor resistance in clinic.Objectives In this study,we use Human and rat glioma cells and BALB/C nude mice model of xenograft glioma to investigate the roles of RIP1 and RIP3 in shikonin-induced glycolysis suppression and the underlying mechanism.Methods 1.LDH assay was used to detect the cellular death rate.2.Hoechst33342 and PI staining were used to visualize the morphological changes in glioma cells induced by shikonin through a laser scanning confocal microscope.Flow cytometry(Annexin-V-FITC and PI double staining)was used to clarify the glioma cell death model.3.Immunohistochemical staining was used to investigate the interaction between RIP1 and RIP3 in glioma cells.4.The BALB/C nude mice model of xenograft glioma was used to investigate the level of G-6-P、pyruvate、ATP、GSH and related proteins induced by shikonin.5.The redox-sensitive dye DCFH-DA was used to evaluate the levels of intracellular ROS.H2O2 assay kit was used to analyze the content of intracellular H2O2 in glioma cells and xenograft glioma tissue.6.The DTNB-GSSH reductase recycling assay kit was used to measure the intracellular total GSH.7.Cysteine assay kit was used to measure the content of intracellular cysteine in glioma cells and xenograft glioma tissue.8.G-6-P assay kit、ATP assay kit、pyruvate assay kit were used to detect the G-6-P、ATP、pyruvate of glioma cells and xenograft tissue respectively.9.Knockdown of RIP1 or RIP3 with si RNA was used to investigate the roles of RIP1 or RIP3 in shikonin-induced glycolysiss suppression of glioma cells.10.The expressional level of related proteins were analyzed by western blot.Results 1.LDH release assay showed that shikonin could kill glioma cells in a dose-dependent manner.Dual staining with Hoechst 33342 and PI observed with a contocal microscope proved that the PI positive cells due to shikonin treatment did not have apoptosis features,such as nuclear condensation or fragmentation.Flow cytometry with Annexin V/PI double staining indicated that shikonin induced necrosis but not apoptosis and supplement of sodium pyruvate markedly inhibited the death rate of glioma cells.2.Western blotting analysis showed that shikonin induced upregulation of RIP1 and RIP3 in a concentration-dependent manner,as well increased the level of p-RIP1 and p-RIP3 in a time-dependent manner.Immunoprecipitation of RIP1 with an RIP1 antibody showed that the co-precipitated RIP3 increased as shikonin concentration increased.3.The shikonin-induced reduction in G-6-P、pyruvate、ATP revealed that shikonin could induce the glycolysis suppression in glioma cells,and Nec-1,GSK872,genetic knockdown of RIP1 or RIP3 with si RNA could reverse this effect.Western blotting revealed that shikonin obviously downregulated the protein levels of HK II、PKM2 and x-CT,these changes could be inhibited by Nec-1,GSK872 or genetic knockdown of RIP1 or RIP3.4.The intracellular ROS levels detected by DCFH-DA proved that the production of ROS was also dependent on shikonin concentration.The accumulation of H2O2 and depletion of GSH in glioma cells were inhibited when the cells were pretreated with Nec-1 or GSK872.Shikonin induced the accumulation of intracellular H2O2 and upregulated the levels of G-6-P、pyruvate、HK II and PKM2,which was mitigated in the presence of GSH.Exogenous H2O2 could also lead to the same change as intracellular H2O2.5.Shikonin triggered time-dependent depletion of cystein.Nec-1 or GSK87 mitigated shikonin-induced cystein depletion.Western blotting proved that shikonin inhibited the protein levels of x-CT in a time-dependent manner,Nec-1,GSK872 and genetic knockdown of RIP1 or RIP3 with si RNA prevented the downregulation of x-CT caused by shikonin.6.The BALB/C nude mice model of glioma cells revealed that the volume of xenograft glioma in the shikonin group was smaller than that in the control mice.Shikonin induced significant reduction in G-6-P、pyruvate、ATP、GSH and cysteine,but accumulation of intracellular H2O2.Western blotting revealed that shikonin obviously downregulated the protein levels of HK II、PKM2 and x-CT,whereas upregulated the phosphorylation of RIP1 and RIP3.Conclusions 1.Shikonin could induce RIP1 and RIP3 dependent necroptosis in glioma cells.2.Shikonin induced glycolysis suppression in glioma cells via overproduction of H2O2.3.Shikonin induced RIP1 and RIP3 mediated glycolysis suppression through overproduction of H2O2 in glioma cells.