Study Of The Inhibiting Effect And Mechanisms Of Shikonin On Gliomas Resistance To Temozolomide

Background:Glioma is the most common primary malignant tumor of the adult central nervous system,which is mainly treated by surgery,radiotherapy and chemotherapy.However,the prognosis of glioma is not optimistic due to its resistance to temozolomide chemotherapy.Therefore,new therapeutic strategies need to be developed urgently.Shikonin,the main active component extracted from Lithospermum erythrorhizon root,has been reported to play an anti-tumor role through a variety of mechanisms,but the injury mechanisms of shikonin on gliomas remain unclear.Recent studies have found that drug-resistance in gliomas is closely related to expression of MGMT,drug-resistant related proteins and activation of protective autophagy,which provides a new strategy for reducing drug-resistance in gliomas.However,the effect of shikonin on the expression of MGMT and drug-resistance related proteins and the activation of protective autophagy in gliomas remain to be further studied.Investigate the effect and mechanisms of shikonin on the temozolomide-resistance of gliomas and the injury mechanisms of gliomas,then provide theoretical evidence for clinical application of shikonin.Objective:Contrast the differences in the expressions of MGMT,multi-drug resistance-related proteins and autophagy related proteins in primary and recurrent gliomas.Investigate the effect and mechanisms of shikonin on the temozolomide-resistance of gliomas and the injury mechanisms of gliomas by using U87,U251 and U87 TR glioma cells as models.Methods:1.Randomly select clinical primary and recurrent glioma specimens of different pathological grades(primary grade II glioma in 2 cases,primary grade III glioma in 2cases,primary glioblastoma in 4 cases,recurrent glioblastoma in 2 cases),and detect the differences in the expression levels of MGMT,multi-drug resistance-related proteins MRP1 and P-gp and autophagy related proteins LC3 I,LC3II and P62/SQSTM1 in glioma cells by Western Blotting.2.Detect the proliferative inhibition rate of temozolomide on U87 cells by MTT.Detect the variations among the expression levels of MGMT,multi-drug resistance-related proteins MRP1 and P-gp,autophagy related proteins ATG5,ATG12,Beclin-1,LC3 I,LC3II,P62/SQSTM1,lysosome related proteins LAMP1 and Cathepsin B in U87 and U251 cells treated for a certain time with temozolomide by Western Blotting.3.Detect the cytotoxic effects of shikonin and temozolomide on U87 glioma cells by LDH release assay.Detect the effects of shikonin on temozolomide-induced expression levels of MGMT,multi-drug resistance-related proteins MRP1 and P-gp,autophagy related proteins ATG5,ATG12,Beclin-1,LC3 I,LC3II,P62/SQSTM1,lysosome related proteins LAMP1 and Cathepsin B in U87 and U251 cells by Western Blotting.Detect the variations among the expression levels of multi-drug resistance-related proteins MRP1 and P-gp in U87 and U251 cells treated for a certain time with shikonin by Western Blotting.4.Culture U87 TR cells by concentration multiplication method.Detect the effects of inhibitors Nec-1 and GSK-872 on the inhibitory effect of shikonin on U87 and U87 TR cells by MTT.Detect the variations among the expression levels of necrosis related proteins RIP1,p-RIP1,RIP3 and p-RIP3 in U87 and U87 TR cells treated for a certain time with different concentrations of shikonin by Western Blotting.Results:1.The expression levels of MGMT,MRP1,P-gp,LC3 I,LC3II,P62/SQSTM1 in clinical primary gliomas showed an increasing trend with the increase of pathological grade.In addition,the expression levels of MGMT,MRP1,P-gp,LC3 I,LC3II and P62/SQSTM1 in clinical recurrent glioma cells with the same pathological grade were significantly higher than those primary ones.2.Temozolomide induced expressional upregulation of MGMT,MRP1,P-gp,ATG5,ATG12,Beclin-1,LC3 I,LC3II,LAMP1,Cathepsin B in glioma cells in a time-dependent and dose-dependent manner,at the same time,the expression level of autophagy substrate protein P62/SQSTM1 decreased.Shikonin could inhibit the above induction effect of temozolomide.And shikonin downregulated expression levels of MRP1 and P-gp in glioma cells in a time-dependent manner.3.The combined application of shikonin and temozolomide had a higher mortality in glioma cells than that of temozolomide alone.In addition,The injury effect of temozolomide and the synergistic effect of shikonin were in a dose-dependent manner.4.U87 TR cells were cultured in vitro,higher concentration of shikonin induced expressional upregulation of apoptosis related proteins RIP1,p-RIP1,RIP3 and p-RIP3 in U87 and U87 TR cells in a dose-dependent manner.Pretreated with Nec-1 or GSK-872 help both U87 and U87 TR cells with a higher survival rate after being treated with shikonin.Conclusion:1.The chemotherapy resistance of gliomas increases with the increase of pathological grade,and the chemotherapy resistance of gliomas enhances after recurrence.2.Shikonin can inhibite the chemotherapy resistance of gliomas to temozolomide.3.Shikonin can enhance the injury effect of temozolomide on glioma cells.4.Shikonin can induce programmed necrosis of glioma cells through upregulation of RIP1 and RIP3.