Study On Anti-glioma Effects Of Avermectins And Its Related Mechanism

BackgroundGlioblastoma is a common primary tumor of the central nervous system,with high degree of malignancy,high incidence and poor prognosis.Chemotherapy is an important treatment for malignant tumors.However,the multidrug resistance(multidrugresistance,MDR)caused by tumor cells often leads to the failure of chemotherapy.According to the American Cancer Society Statistics,more than 90%people existed in the death of the patient,Temozolomide use to the treatment of glioblastoma approved by the FDA due to the side effects of the small body,can pass through the blood-brain barrier,has been widely used in the clinic.However,it is inevitable that the tumor cells still are resistant to the drug,so it is necessary to screen and develop the new drugs to enhance the killing effect of temozolomide.Avermectin(avermectins,AVMs)by Streptomyces avermitilis to produce a class of macrolide antibiotics.AVMs is usually used for the treatment of parasitic infections and other diseases,mainly to play on pest killing effect of the contact and stomach toxicity.Studies have found that AVMs can inhibit the P-glycoprotein on the substrate efflux,thereby reducing the multidrug resistance of AVMs,but if there is any other anti-t?Mor mechanism is not very clear.This group in the pre-experiment also found that AVMs have effects on U87 glioma cells,U251 glioma cell proliferation inhibition,and induce apoptosis in tumor cells,but the mechanism is not clear.In this paper,U87?U251and T98 glioma cells as the research object,the expression of AVMs on cell proliferation,apoptosis,mitochondrial membrane potential and mitochondrial apoptosis signaling pathway related protein levels,to explore the anti-tumor mechanism of AVMs,to provide a solid theoretical basis for the research and development of new anticancer drugs abamectin and its clinical application.Part one:Effects of avermectins on proliferation and apoptosis of glioma cells and its related mechanismObjectiveTo study the effects of AVMs on proliferation and apoptosis of glioma cells and its underlying mechanism.Methods1.The cells were treated with various doses of AVMs(0,2?M,4?M,6?M,8?M,10?M,12?M)for different time(24,48,72 hours)prior to analysis of cell viability by MTT assay.2.The cells were treated with various doses of AVMs(0,4,8 ?M)for 72h respectively to analyze apoptosis by Annexin V-FITC/PI.3.The cells were treated with various doses of AVMs(0,4,8 ?M)for 72h respectively and use JC-1 method to analyze the mitochondrial membrane potential4.The cells were treated with various doses of AVMs(0,4,8 ?M)for 72h respectively,then the total RNA reverse transcription was extracted and the expression of Bcl-2,Bax,Caspase3,caspase9 and paper were detected by Real-time PCR5.The cells were treated with various doses of AVMs(0,4,8 ?M)for 72h respectively.Caspase3,Bcl-2,Bax,caspase9,paper protein expression levels were detected by Western blotting.Results1.AVMs inhibited the proliferation of glioma U87 and U251 cells in a dose-and time-dependent manner.2.AVMs can induce the apoptosis of glioma U87 cells and U251 cells,treatment group compared with the control group the apoptosis rate were increased(P<0.05)3.AVMs U87 and U251 cell mitochondrial membrane potential,dosing group compared with the control group of red/green fluorescence intensity ratio were increased(P<0.05).4.PCR and Western bloting res?lts suggest that AVMs can promote the expression of Caspase3,Bax and proteins(P<0.01)in U87 and U251 cells,and decrease the expression level of BCL2 mRNA and proteins(P<0.01)Conclusion1.AVMs can inhibit the proliferation of glioma U87 and U251 cells2.AVMs can promote the apoptosis of glioma U87 and U251,and its mechanism may be related to triggering apoptosis through mitochondrial pathway.Part two:The effect of AVMs on temozolomide in the treatment of glioblastoma and its related mechanismObjectiveTo study the effect of AVMs on temozolomide in the treatment of glioblastoma and its related mechanism.Methods1.The effects of AVMs,TMZ and AVMs+TMZ on cell proliferation were detected by MTT assay,and their IC50.2.Evaluate the combined effect of AVMs and TMZ by Chou-Talalay.3.Real-time PCR with different concentrations of AVMs(0,4?M,8?M)was used to detect the expression levels of ABCB1,ABCG2 and MGMT mRNA in the cells for 72 hours after treatment.4.Different concentrations of AVMs(0,4?M,8?M)were used to detect the expression levels of ABCB1,ABCG2 and MGMT protein in cells after western blotting treatment.Results1.AVMs,TMZ and AVMs+TMZ co?ld inhibit the proliferation of glioma cells in a dose-and time-dependent manner.2.AVMs+TMZ in IC50,U87,U251 and T98 cell CI(combination index)were:0.85,0.72,0.34.3.PCR and Western bloting res?lts showed that AVMs can promote the expression of U87 and T98 cells in ABCB1,ABCG2 and mRNA protein(P<0.05),but also decreased the expression of T98 in MGMT cells and mRNA protein(P<0.05).ConclusionAVMs and TMZ have synergistic effect,and the possible mechanism is to reduce ABCB1,ABCG2 and MGMT protein molecular expression level to increase the killing effect of TMZ on tumor cells.