Study On The Inhibitory Effect And Mechanism Of Silibinin On Temozolomide Resistance In Glioma Cells

Research background:Glioma is highly aggressive and difficult to be completely resected by surgery.Although adjuvant radiotherapy and chemotherapy can prolong the survival of patients,the median overall survival of patients is still less than 15 months.Many clinical studies have shown that temozolomide(TMZ)combination therapy has a good clinical effect on the treatment of glioma.Therefore,it is necessary to further explore the mechanism of glioma drug resistance and explore new drugs to break through the limitation of drug resistance.The expression differences of glutaminase(GLS),cystinine/glutamate antiporter subunit(x CT)and nuclear factor kappa-B(NF-κB)in primary gliomas are closely related to TMZ resistance,and the expression level of NADPH oxidase(NOXs)also directly affects the killing effect of TMZ on glioma cells,which also affects the prognosis of patients to a large extent.Therefore,changes in the expression of GLS,x CT,NF-κB and NOXs in glioma may enhance the sensitivity of TMZ to glioma treatment and improve the prognosis of patients.Silibinin is a major active component of Silmyarin.It has obvious protective effect on liver by stabilizing liver cell membrane and scavenging oxygen free radicals,and has been widely used in clinical practice.In glioma,silibinin shows good antitumor activity and has a significant therapeutic effect on glioma when combined with TMZ.However,the mechanism of improving TMZ resistance in glioma needs to be further studied.Research purpose:Human glioma cell lines U87 and U251 and TMZ-resistant U87(U87TR)cell line were used as primary and resistant glioma models to explore the antitumor activity and chemosensitivity of silibinin on glioma in vitro.Research methods:1.Four glioma tissue samples with different pathological grades were selected to detect the expression of GLS protein by Western Blotting(WB).2.U87 and U87 TR cells were cultured in vitro,and the expression difference of GLS protein was detected by WB.3.U87 and U251 cells were cultured in vitro and treated with the same concentration of TMZ after pretreatment with autophagy inhibitors 3-MA and Baf A1.The expression of GLS protein in each group was detected by WB experiment.4.U87,U251 and U87 TR cells were cultured in vitro and treated with different concentrations of silibinin combined with TMZ.The expression of GLS protein in each group was detected by WB assay.5.Lactate dehydrogenase(LDH)release assay was used to determine the toxic effects of different concentrations of silibinin and TMZ on U87 cells.6.The effects of different concentrations of silibinin combined with TMZ on the expression of x CT,NF-k B,NOX4 proteins in U87 and U251 cells were detected by WB.Research results:1.The expression of GLS protein was positively correlated with the grade of glioma in selected clinicopathological specimens.2.The expression of GLS protein in U87 TR cells cultured in vitro was higher than that in normal U87 cells,and there was no significant difference in the expression of GLS between high-concentration drug resistance group and low-concentration drug resistance group.3.In U87 and U251 cells cultured in vitro,the expression of GLS protein in glioma cells decreased after TMZ treatment,and the expression of GLS protein in the autophagy inhibitor 3-MA and Baf A1 pretreatment group was significantly lower than that in the corresponding control group.4.In U87,U87 TR and U251 cells cultured in vitro,the expression level of GLS protein in silibinin combined with TMZ group was significantly lower than that in TMZ alone group,and the degree of down-regulation was positively correlated with the concentration of silibinin.5.LDH assay showed that silibinin and TMZ alone had killing effect on U87 glioma cells;After combined treatment with silibinin and TMZ,the death rate of U87 cells was further increased.6.WB showed that the expression of x CT,NF-κB,NOX4 was up-regulated in U87 and U251 cells treated with TMZ alone.When silibinin combined with TMZ,the up-regulation of x CT and NF-κB expression was inhibited,and the up-regulation of NOX4 expression was further increased.Research conclusions:1.The protein expression of GLS is positively correlated with the malignant degree of glioma cells,which is involved in the resistance of glioma cells to TMZ.2.Silibinin promotes the sensitivity of glioma cells to TMZ by down-regulating glutaminase3.Silibinin can inhibit x CT and promote the expression of NOX4 to increase the level of oxidative stress in glioma cells4.Silibinin inhibits the TMZ resistance of glioma cells by inhibiting the NF-κB pathway.