Effect Of Shikonin On Programmed Necrosis Of Glioma Cells By Inhibiting Proteasome Activity

Objective: It was clear that Shikonin exerted antitumor effects by modulating programmed necrosis of gliomas through the proteasome system in the human glioma U251 cell line.To study the effects of different concentrations of shikonin on apoptosis rate,proteasome activity and proteasome subunit of human glioma U251 cells.Methods:1.Routine culture of human glioma（U251、U87、T98G、A172）cell lines,then shikonin with different concentrations（0μmol / L,4μmol /L,8μmol / L,12μmol / L）was used to intervene glioma U251,U87,T98 G and A172 cell lines for 24 h.The morphological changes of U251,U87,T98 G and A172 cells were found by inverted phase contrast microscope,then the apoptosis/necrosis rate of glioma was detected by annexin Ⅴ-APC/7-AAD method.Shikonin（12μmol / L）combined with 4inhibitors（nec-1,necrosulfnide,gsk-872,z-VAD-fmk）of different concentrations co cultured U251 cells for 24 hours.The programmed necrosis rate of glioma was detected by annexin Ⅴ-APC/7-AAD method.2.After different concentrations of shikonin intervened U251 cells for 12 h,the proteasome subunits PSMB1、2、5、8、9、10,programmed necrosis related proteins RIPK1、3 and MLKL with specific expression were screened by transcriptome sequencing.Different concentrations of shikonin intervened U251 cells for 24 hours,Proteasome Glo ? Trypsin Like Cell-Based Assays Kit was used to detect the expression of proteasome activity,and Western Blot was used to verify the expression of related specific proteins in glioma U251 cells.Results:1.After different concentrations of shikonin intervened glioma U251 cells for 24 hours,the cell wall gradually ruptured,the cell morphology changed significantly,and the suspended necrotic cells and cell fragments were seen in the culture medium.With the increase of drug concentration gradient,the apoptosis/necrosis rate of glioma U251 cells increased gradually.2.The same concentration of Shikonin（12μmol/l）co-cultured U251 cells with programmed necrosis RIP1-RIP3-MLKL signaling pathway inhibitors（NEC-1,Necrosulfnamide,GSK-872）and pan caspase apoptotic inhibitor Z-VAD-FMK,showed that with the increase in the concentration of RIP1-RIP3-MLKL signaling pathway inhibitors,the cell mortality rate gradually decreased.With the apoptosis inhibitor z-VAD-fmk concentration increased,the decrease of cell death rate was not obvious,indicating that the glioma cell death caused by shikonin was programmed necrosis,and the possibility of cell apoptosis was small.3.After different concentrations of shikonin intervened U251 cells for 24 hours,Proteasome Glo ? Trypsin Like Cell-Based Assays Kit was used to detect the activity of proteasome.It was found that the activities of chymotrypsin like（CT-L）,trypsin like（T-L）and caspase like（C-L）decreased with the increase of drug concentration.4.According to the results of transcriptome sequencing,the expression of RIP1,RIP3 and MLKL was positively correlated with the concentration of shikonin;the expression of PSME1、PSME2、PSME 3、β1i、β2i、β5i was negatively correlated with the concentration of shikoninConclusion:1.Shikonin induces programmed necrosis of glioma U251 cells by regulating RIP1-RIP3-MLKL signaling pathway.2.The proteasome activities of caspase like,ttypsin like and chymotrypsin like decreased during shikonin induced programmed necrosis of glioma cells;The expression level of proteasome formation catalytic subunit β 1、2、5 increased and the expression of inducible subunit β1i、2i and 5i decreased,suggesting that shikonin can promote the programmed necrosis of glioma cells by inhibiting the activity of immune proteasome.