Inhibitory Effect And Mechanism Of Nimustine Combined With Temozolomide On Glioma C6 Cells

Background: Glioma is a malignant tumor originating from glial cells,which is characterized by rapid tumor growth,high invasiveness,high degree of vascularization and high heterogeneity,high recurrence rate and low cure rate,and is a serious threat to human health.At present,the main treatment of glioma is radiotherapy and chemotherapy after maximum surgical resection.However,the prognosis of clinical treatment is poor and easy to relapse,and surgery has some problems such as high risk,difficulty,high incidence of postoperative complications and expensive treatment,which has not significantly improved the survival time of patients.The invasion and infiltration of tumor,the existence of blood-brain barrier and the drug resistance of tumor cells are the main reasons for the poor therapeutic effect of glioma.TMZ(temozolomide,TMZ)is the first-line chemotherapy drug for glioma,while ACNU(nimustine,ACNU)is the second-line chemotherapy drug for glioma.Some studies have found that ACNU and other chemotherapeutic drugs can be used as salvage treatment after TMZ resistance.In addition,the combination of drugs can reduce the toxic side effects by reducing the dose.Therefore,this study will discuss the effect and interaction of the combined application of ACNU and TMZ on glioma C6 cells and explore the possible mechanism,in order to provide experimental basis for the treatment of gliomas as well as the CED(convection enhancement delivery,CED)as reference drug dosage.Objective: To explore the effects of ACNU combined with TMZ on the growth inhibition effect,cell morphology and migration ability of glioma C6 cells,as well as the possible mechanism of action,so as to provide drug reference and experimental basis for the clinical treatment of glioma.Methods: Glioma C6 cells were used as the research object in this study.The experiment was divided into DMSO control group,ACNU group,TMZ group and ACNU combined TMZ group.(1)CCK-8 method was used to detect the proliferation of glioma C6 cells treated with different concentrations of ACNU(10,40,80,120,160,200 μM)and TMZ(200,400,600,800,1000 and 1200 μM),and IC25 and IC50 values were calculated.(2)The morphological changes of glioma C6 cells induced by ACNU,TMZ and their combination were observed under inverted microscope.(3)The effects of ACNU,TMZ and their combination on the migration ability of C6 glioma cells were detected by wound healing experiment.(4)Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the effects of ACNU,TMZ and their combination on the expression of the apoptosis gene(Bcl-2 and Bax)and the drugresistant gene MGMT in glioma C6 cells.(5)western blotting was used to detect the effects of ACNU,TMZ and their combination on the expression of apoptosis related protein(Bcl-2 and Bax)and drug-resistant protein(MGMT)in glioma C6 cells.Results:(1)The CCK-8 experimental results showed that compared with the control group,both ACNU and TMZ could inhibit the growth of glioma C6 cells.With the increase of time and drug concentration,the growth inhibition effect was more significant,which was time-and drug concentration-dependent.The combination of ACNU and TMZ significantly inhibited the growth and proliferation of glioma C6 cells compared with ACNU and TMZ monotherapy group,indicating that the combination of ACNU and TMZ had a synergistic effect.(2)The results of cell morphology showed that compared with other drug treatment groups,DMSO control group had more cells,faster proliferation,higher growth density and good adherent growth state.Compared with DMSO control group,ACNU monotherapy group and TMZ monotherapy group had fewer cells,less growth density and less cell-to-cell connections,and there was no significant difference between the two monotherapy groups.The cell volume of ACNU and TMZ group was significantly reduced and rounded,the growth density was the sparsest,and the connections between cells were the least.(3)The results of wound healing experiment showed that compared with the control group,the cell healing rate of the other drug treatment group was decreased(P < 0.05 or P < 0.01);and the migration inhibition ability of the combined drug group was more significant than that of any single drug group,with statistical significance(P < 0.05).(4)The Real-time quantitative PCR experiment results showed that compared with DMSO control group,the ratio of Bax/Bcl-2 in other drug treatment groups increased,and the ratio of ACNU combined with TMZ group increased most significantly,with statistical significance(P< 0.01).Compared with DMSO control group,MGMT expression was up-regulated in other drug treatment groups,and the expression in ACNU group and combination group increased significantly(P < 0.001),and the up-regulation effect was most obvious in combination group.(5)Western Blot experiment showed that compared with DMSO control group,the ratio of Bax/Bcl-2 in other drug treatment groups increased,and the ratio of ACNU combined with TMZ group increased most significantly,with statistical significance(P < 0.01).Compared with DMSO control group,MGMT expression could be up-regulated in other drug treatment groups,and the expression level increased most significantly in combination treatment group(P < 0.01).but there was no statistical significance in ACNU and TMZ monotherapy groups(P > 0.01).Conclusion:(1)Compared with the single drug treatment group,ACNU combined with TMZ showed higher cytotoxic effect on glioma C6 cells,stronger growth inhibition effect and synergistic effect.(2)ACNU combined with TMZ can affect the morphology and migration ability of glioma C6 cells significantly.(3)ACNU combined with TMZ can enhance the apoptosis of glioma C6 cells by up-regulating the ratio of Bax/Bcl-2.(4)Glioma C6 cell line is a MGMT positive cell line,and ACNU,TMZ and their combination can up-regulate the expression of MGMT,and the combination has a stronger up-regulation effect,providing a possible target for the treatment of glioma.