The Study Mechanisms Of Apoptosis Induced By Bortezomib And Arsenic Trioxide In NB4 Cells In Vitro

Objective:In this experiment,we choose proteasome inhibitor Bortezomib(BOR) and Arsenic Trioxide(ATO),observe the depressant effect of proteasome inhibitor and ATO on human APL NB4 cells,study if proteasome inhibitor can increase NB4 cells' sensibility to ATO and co-induct NB4 cells' apoptosis,offer reference to bolting new anti-cancer agent.Methods:The object was divided into nine groups:control(D);5 nmol/L BOR (A1),10 nmol/L BOR(A2);0.5μmol/LATO(B1),1.0μmol/LATO(B2); 5 nmol/L BOR +0.5μmol/L ATO(C1),5 nmol/L BOR+ 1.0μmol/L ATO(C2), 10 nmol/L BOR+0.5μmol/L ATO(C3),10 nmol/L BOR+1.0μmol/L ATO (C4).The time was divided into three phases:24 hours,48 hours,72 hours.To detect the change of correlated index:(1) Cell morphology was observed by optics microscope after staining the cells treated with different drug groups by Wright-Giemsa;(2) The cell proliferating activity was assessed with MTT assay;(3) Cell apoptotic rate and cell cycle were examined by flow cytometry(FCM);(4) The expression of survivin mRNA was detected by semi-quantitate reverse transcription polymers chain reaction(RT-PCR).Result:(1) In morphological observation of apoptotic cells using Giemsa staining technique,ceils displayed classic apoptotic changes treated with BOR 5 nmol/L or 10 nmol/L,with the drugs' consistency rising up,the apoptotic cells were more and more.after using 1μmol/L ATO plus 10 nmol/L BOR,the quantity of apoptotic cells is bigger than that treated with 1μmol/L ATO alne.(2) MTT assay displayed that:At the same time,compared with contral group, both single drug group and combination group can decrease the NB4 cells' survival rate(P＜0.01).At the different time point,the effect of same consistency drug group is obviously different(P＜0.01),the suppressant effect of combination group obviously outweigh the single group.The survival rate is lowest at 72h with 1μmol/L ATO and 10 nmol/L BOR together(P＜0.01). (3) Flow cytometry(FCM) analysis showed that NB4 cells were treated with BOR group after 48h,the ratio of S phase gradrually degraded,G1 phase did not change obviously,While G2/M phase gradrually steped up,thus cells were arrested at G2/M phase(P＜0.01),the cells were also arrested at G2/M phase treated with ATO group or combination group(P＜0.01),the blockage rate of 10 nmol/L BOR plus 1μmol/L ATO is highest.Compared with the same dose ATO,the ratio of G2/M phase increased in combination group(P＜0.01).Cells apoptosis analyzed that after treated with BOR,cells' apoptotic rate appeared dose dependent manner(P＜0.01).BOR can strengthen the apoptotic effect of ATO.Compared with the same dose' ATO,the apoptotic rate increased in combination group(P＜0.05).(4) Reverse transcription polymers chain reaction(RT-PCR) disclosed that both BOR and ATO can downregulate the levels of survivin mRNA in a dose and time dependent manner(P＜0.01);BOR can also cooperate with ATO to decrease the expression levels of survivin mRNA.There were interaction between the drugs and time(P＜0.01),the levels of survivin mRNA is lowest treated with 1μmol/L ATO plus 10 nmol/L BOR after 72h.Conclusion:(1) BOR or ATO alone can induce NB4 cells' apoptosis in a dose and time dependent manner.They can arrest NB4 cells in G2/M phase and oppose the expression of survivin mRNA,which may be the mechanism of drugs' induction apoptosis.(2) BOR can strengthen the lethal effect of ATO.The combination of them has additional effects.(3) BOR can strengthen the apoptotic effect of ATO.The elevation of block rate in G2/M phase and the drop of survivin mRNA may be the mechanism. The combination of them has additional or synergistic effects.