Therapeutic Effect Of 2,5-dimethyl-celecoxib On Glioblastoma And Its Molecular Mechanism

ObjectivesGlioblastoma multiforme(GBM)is a clinically refractory tumor of the central nerve center.The effective therapeutic drugs are relatively single and the therapeutic effect is not ideal.Therefore,it is extremely urgent to explore new therapeutic drugs.Celecoxib has obvious anti-tumor effect,but its clinical application is limited due to its inhibition of COX-2,which leads to reduced production of prostaglandin in blood vessels and further causes thrombosis events.2,5-dimethyl-celecoxib(DMC),a close derivative of Celecoxib,has the same anti-tumor effect as Celecoxib,and has an advantage in the treatment of tumors due to its lack of inhibition of cardiovascular toxicity caused by COX-2 and other side effects.The cancerous inhibitor of protein phosphatase 2A(CIP2A),an oncogene,is highly expressed in GBM and highly expressed CIP2A also predicts poor prognosis in patients.In this study,different concentrations of DMC solution were used to treat GBM cells in vitro and in vivo,and various related indicators were detected to clarify the role of DMC in the treatment of GBM,the influence on the expression of CIP2A and the therapeutic molecular mechanism.Materials and MethodsIn this study,four GBM cell lines(LN229,A172,U251 and U87MG)and normal human astrocytes(NHA)were used for related cell experiments in vitro.Different cell lines were treated with a DMC solution at a gradually increasing concentration.Cell activity was assessed with the CCK-8 kit,and the 50%concentration of inhibition(IC50)of the relevant drugs was calculated.Cell cycle distribution was detected by flow cytometry and the incidence of apoptosis was detected by Annexin-V/PI double staining assay.Fluorescence real-time quantitative PCR(RTPCR)was used to detect the expression of CIP2A mRNA,and Western blot(WB)was used to detect the expression of cell cycle arrest protein,apoptosis related protein,CIP2A,Akt,and phosphorylated Akt(pAkt).Cycloheximide(CHX)and DMC were used to treat cells to measure the level of protein degradation.The transfected plasmid and virus overexpressed pAkt and CIP2A,respectively,and the effects of overexpressed cells on the expression of related cycle and apoptotic proteins were detected.The xenotransplantation model of nude mice was established using LN229 cell line.Ten tumor forming nude mice were divided into two groups,one group was treated with DMC,the other group was not treated with any treatment as the control group.The body weight and tumor volume of the two groups of nude mice were measured,and the anti-tumor effect of DMC was evaluated in vivo.ResultsIn this study,with the increase of DMC solution concentration,the growth activity and proliferation status of GBM cells were gradually inhibited(P<0.001).After DMC treatment,flow cytometry showed that the proportion of GBM cell cycle in G0/G1 phase was significantly increased,and cyclin P21 was increased,indicating that DMC could inhibit cell activity by blocking cell cycle.GBM cells were treated with different concentrations of DMC solution.Annexin-V/PI staining showed that the higher the concentration of DMC,the higher the apoptosis rate of GBM cells was,suggesting that high concentration of DMC could significantly induce the apoptosis of GBM cells.After GBM cells were treated with DMC,Akt phosphorylation level decreased,but total Akt level did not change significantly.After plasmid transfection to overexpress pAkt,the cells were treated with DMC solution again.It was found that the overexpression of pAkt could reverse the changes of cell cycle and apoptotic protein levels induced by DMC,which further indicated that DMC inhibited cell proliferation through the Akt pathway.DMC inhibited the expression of CIP2A in concentration and time-dependent manner.DMC can inhibit PP2A activity.After treatment with CHX and DMC,DMC not only inhibited de novo synthesis of CIP2A protein,but also promoted protein degradation.Finally,in the xenograft tumor model of nude mice with LN229 cells,the tumor growth of mice treated with DMC became slow.In removed tumors,the protein expression of CIP2A and pAKT was decreased in the DMC treated group.ConclusionThis study confirmed that DMC can block the GBM cell cycle and induce apoptosis through the CIP2A/PP2A/Akt signaling pathway,and suggested that DMC may be an effective choice for the treatment of GBM.