Investigation On Correlation Between MTOR Transduction Pathway And Arsenious Acid Response

PrefaceArsenious acid has an obvious anti-tumor activity in vitro and in vivo.the anti-tumor mechanisms of arsenious acid include decreasing mitochondrial transmembrane potential,activating caspases,increasing intracellular reactive oxygen species(ROS),reducing telomerase activity,increasing cytoplasmic Ca²⁺ concentration, regulating key enzymes of calcium-sensitive,such as protein kinases,phospholipase, endonuclease,transglutaminase and so on,to induce apoptosis.Arsenious acid can degradate PML/RARαfusion protein,reduce the BCR-ABL protein content and reduce its activity as protein tyrosine kinase,during the period when the anti-tumor mechanism of arsenious acid was gradually understand,people found that a variety of kinases in some signal transduction pathways were activated in a negative feedback regulatory manner.In this study we investigate the correlation between phosphorylation level of the kinases in mTOR transduction pathway and arsenious acid response on protein level. Objective of this study is to observe the expressions of pmTOR,pAKT and pP70S6K in K562/DNR cells treated by arsenic acid;to observe the apoptosis rate of K562/DNR cells treated by the combination of arsenic acid and PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin.Methods1,The expression of pmTOR,pAKT,pP70S6K in K562/DNR cells was detected by western blot after the cells had been treated with arsenic acid for 10 minutes,30 minutes,60 minutes,120 minutes and when the cells have not been treated with arsenic acid.The signals for the different bands shown in blots were quantitated by densitometry analysis,and the ratio of the expressions of pmTOR,pAKT and pP70S6K to the expression ofβ-actin was calculated by ScionImaging software.2,The apoptosis rate of K562/DNR cells were assayed by flow cytometry(FCM) with Annexin V-FITC Apoptosis Detection Kit after the cells were incubated for 120 hours in the presence or absence of arsenic acid,with or without PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin.Results1,The expression of pmTOR in K562/DNR cells having been treated with arsenic acid for 60 minutes(0.843±0.006) or 120 minutes(1.030±0.017) was higher than which having not been treated with arsenic acid(control group)(0.477±0.015)(p＜0.01). Comparing the expression of pmTOR in K562/DNR cells having been treated with arsenic acid for 10 minutes(0.510±0.010) or 30 minutes(0.527±0.006) with control group,there was no significant difference between them(p＞0.01).The expression of pAKT in K562/DNR cells having been treated with arsenic acid for 30 minutes(0.960±0.010) or 60 minutes(1.023±0.025) was higher than control group(0.443±0.012)(p＜0.01).Comparing the expression of pAKT in K562/DNR cells having been treated with arsenic acid for 10 minutes(0.423±0.021) or 120 minutes(0.560±0.036) with control group,there was no significant difference between them(p＞0.01).The expression of pP70S6K in K562/DNR cells having been treated with arsenic acid for 60 minutes(1.017±0.015)was higher than control group(0.503±0.021)(p＜0.01). Comparing the expression of pP70S6K in K562/DNR cells having been treated with arsenic acid for 10 minutes(0.533±0.025),30 minutes(0.760±0.030) or 120 minutes(0.613±0.047) with control group,there was no significant difference between them(p＞0.01).2,The apoptosis rates of K562/DNR cells treated with combination of arsenic acid and Ly294002(18.0±1.0) or rapamycin(8.4±0.7) were higher than the untreated group(control group)(4.5±0.8),arsenic acid-treated group(3.1±1.1),rapamycin-treated group(3.5±0.6) and Ly294002- treated group(3.4±0.5)(p＜0.01).Comparing the apoptosis rates of K562/DNR cells in arsenic acid-treated group,rapamycin-treated group or Ly294002-treated group with the control group,under the condition of the giving treating concentration of arsenic acid,rapamycin or Ly294002 in this experimental,there was no significant difference between them(p＞0.01). Conclusion1,mTOR signaling pathway is activated in K562/DNR cells by arsenic acid of a certain concentration.2,mTOR signaling pathway inhibitors can promote arsenic acid to trigger apoptosis of K562/DNR cells.