The Study Mechanisms Of Apoptosis Induced By Arsenic Trioxide And Amifostine In HL-60 Cells In Vitro

Objective:To explore the mechanisms of apoptosis induced by arsenic trioxide and amifostine in human acute myelocytic leukemia cell lines Hi-60 in vitro.Methods:(1)The object was divided into ten groups:control(D),2μmol/l As₂O₃(A₁),5μmol/l As₂O₃(A₂),10μmol/l As₂O₃(A₃),1μmol/l AMI(B₁),10μmol/l AMI(B₂),1000μmol/l AMI(B₃),and the combinated groups(As₂O₃+AMI)2μmol/l+1000μmol/l(C₁),5μmol/l+1000μmol/l(C₂),10μmol/l+1000μmol/l(C₃).The time was divided into three phases:24 hours,48 hours and 72 hours.(2)Cell morphology was obsevered by light microscope after Wright-Giemsa staining the cells treated with different concentration groups at indicated time (24h,48h,72h);(3)The proliferating activity of the cells was measured with a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromide(MTT)assay;(4)Cell cycle was examined by flow cytometry(FCM);(5)The expression of Survivin mRNA was detected by semi-quantitate reverse transcription polymers chain reaction(RT-PCR).Result:(1)morphological observation showed characteristic apoptosis changes including cell shrinkage chromatin condensation DNA fragmentation,membrane blebbing and formation of apoptotic bodies.(2)The-outcome of MTT assay showed that In different time,compared to the control, Proliferation of HL-60 cells exposed to every drug groups significantly dropped down with increasing dose of the drug(P＜0.01).This inhibit effect was much higher when arsenic trioxide was used in combination with amifostine than arsenic trioxide only(P＜0.01).The inhibit effect of arsenic trioxid to HL-60 cells reach the peak in 48 hours;The inhibit effect of amifostine and combinated groups to HL-60 ceils was in time-dependent manner(P＜0.05),their effect reached the inhibitary peak in 72 hours.(3)Flow cytometry(FCM)analysis showed that As203 induced HE-60 cells arrrest at G₁ phase and decrease significantly in S phase after 24 hours and 48 hours(P＜0.05); Amifostine induced HL-60 cells arrrest at G₁ phase and decrease significantly in S phase after 48 hours and 72 hours(P＜0.05);The cells exposured to arsenic trioxide combinated with amifostine was arrrested at G₂/M+G₁ phase after 24 hours and G₁ phase after 48 hours and 72 hours(P＜0.05).(4)Semi-quantitate reverse transcription polymers chain reaction(RT-PCR) disclosed that arsenic trioxide down regulate the expression of Survivin in concentration-dependent manner(P＜0.01)and the expression was minimum in 48 hours; Amifostine down regulate the expression of Survivin in a dose-dependent manner as well as a time-dependent manner(P＜0.01)and the expression was minimum in 72 hours;there was a more significant decrease in Survivin expression in HL-60 cells trated with arsenic trioxide in combination with amifostine as compared to the cells trated only with arsenic trioxide or amifostine(P＜0.01).there were interaction between the drugs and time(P＜0.01).Conclusions:(1)Arsenic trioxide induced HL-60 cells form apoptosis by arrresting at G₂/M phase and down regulating the expression of Survivin.(2)Amifostine enhanced the sensitivity of HL-60 cells to arsenic trioxide by arrresting at G,phase(perhaps this is the non-expression phase of Survivin),thus inhibited proliferation of the cells and educed its promoting apoptosis effect.(3)The promoting apoptosis effect of arsenic trioxide in combination with amifostine to HL-60 cells was even better than of one drug only.