| Background and objective: Glioma is the most common primary malignant brain tumor in central nervous system in adults.Glioblastoma(GB),WHO grade IV glioma,is the most common and most malignant primary brain tumor.Even after optimized multimodal treatment with maximal surgical resection,radiotherapy and chemotherapy used in a combinatorial approach,the tumor recurs and the prognosis remains very poor.Currently,the methylating agent temozolomide(TMZ)is the drug of choice for the first line therapy of gliomas and with a proven effectiveness.However,chemoresistance of glioma remains one of the major problems and the incurability of glioma is attributable to its profound therapy resistance.Skp2 is an F-Box protein that forms a Skp2 SCF complex with Skp1,Cullin-1(Cul-1),andRBx1 to constitute an E3 ligase activity that triggers protein ubiquitination and degradation.Skp2 displays oncogenic activities by regulating cell cycle progression,senescence,and metastasis.Many research show that SKP2 is a novel regulator for cancer stem cells(CSC).Moreover,SKP2 is an important regulator for double strand break(DSB).CSC,indicated as glioma stem cells(GSCs),is responsible for tumor initiation,maintenance and recurrence and they appear to be more resistant owing to their enhanced DNA repair capacity.DSBs are produced as ultimate lesions following damage induced by alkylating agents.Therefore,we predicted that gliomas with negative SKP2 expression are more sensitive to TMZ chemotherapy.Methods: Glioma cell lines(U87)with negative SKP2 expression are built by shRNA.Cell proliferation of these glioma cell lines after treatment was measured by using CCK-8 assay.Xenograft models with subcutaneous implantation of different glioma cell lines were established in nude mice.Animals were randomly divided to groups treated with SP combined with TMZ.Tumors were measured every 3-4 days and the tumor repression rates were analyzed at the fourth week.The variability of SKP 2 protein expression levels in gliomas was detected by Western blot.Results:(1)Western blot showed that SKP 2 protein expression were declined in shSkp2-530 and shSkp2-532 whereas positive in shLuc,which shows that Glioma cell line U87 with negative SKP2 expression are built by shRNA.(2)The knockdown of SKP2 inhibits the proliferation of U138 and U87 to some extent.(3)The proliferation analysis demonstrated that IC50 of shLuc,shSkp2-530 and shSkp2-532 of U87 were 1540.71±29.69?M,1000.37±92.86?M and 1071.42±23.82?M,respectively.IC50 of shLuc,shSkp2-530 and shSkp2-532 of U138 were 1678.93±55.44?M,1150.20±26.27?M and 1367.01±51.31?M,respectively.Statistically significant differences(p<0.05)were observed compared with the TMZ alone.(4)Western blot showed that SKP2 protein expression was the lowest with the concentration of 40?M of SP by treating 48 h.(5)The proliferation analysis demonstrated that the inhibition rates of TMZ,SP+TMZ of U87 were 21.44%±1.36%,50.11%±5.09% respectively.The inhibition rates of TMZ,SP+TMZ of U138 were 15.33%±3.35%,45.49%±6.57% respectively.Statistically significant differences(p<0.05)were observed compared with the TMZ alone.(6)shSkp2-530 of U87 possessed the capability to initiate tumors.(7)The tumor growth repression efficacy of shSkp2-530+TMZ?SP+TMZ of U87 were significantly higher than TMZ alone in xenograft models(?<0.05).Conclusion:Inhibition of SKP2 by different ways can enhance the antitumor effect of TMZ on glioma cell lines both in vitro and in vivo. |
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