Relationship Between Leukemia Cell Apoptosis Induced By Arsenic Trioxide And The Expression Of IκB-α, Nuclear Factor-κB

BackgroundFrom 70's, it has been shown in China that venous infusion of arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL) with a high complete remission rate. Recently, it has become evident that the apoptotic effects of As2O3 are not restricted to APL cells but may also be observed in leukemia cells of non-APL origin such as chronic myeloid leukemia and other subtype of acute myeloblastic leukemia. Its effects on leukemic cells mainly include induction of apoptosis and differentiation. The mechanism of apoptosis induction by As2O3 was not known thoroughly. Nuclear Factor-KB (NF-KB) is an ubiquitous transcription factor and regulates the expression of several genes involved in inflammatory and immune responses, positively regulate cell proliferation and apoptosis. Recent evidence indicates that NF-KB and the signaling pathways that are involved in its activation are also important for tumor cell apoptosis. In this study we investigated the effect of As2O3 on leukemia cells in vitro and the relationship between leukemia cell apoptosis induced by arsenic trioxide and their expression of IkB-a, Nuclear Factor-KB.Materials and methodsThe human chronic myeloid leukemia cell line K562 cells, its adriamycin resistance cell line K562/adr cells were provided by Zhejiang University Cancer Research Institution and human acute granulocytic leukemia cell line U937 cells was provided by Zhejiang University Hematology Institution. The concentration of As2O3 used in cell culture system was from 1 to 16uM and 24 hour was selected as the termination point of cell culture. Cell culture without As2O3 or first-antibody-dismissed test were enrolled as controls.All of the three cells were treated with various concentration As2O3. MTT was used to examine the effect of As2O3 on cell growth and IC50 was calculated respectively. Apoptosis and changes of kB-a protein was observed by flow cytomerty. Western blot was also used to analyze the expression of NF-KB in nuclear and kB-a in cytoplasm.ResultsAs2O3 could inhibit the cell proliferation in K562 cells, K562/ADR cells and U937 cells and its IC50 in 24 hour was 9.06uM, 19.07uM and 16.39uM, respectively. After exposure to AS2O3, all of the three cells were induced to apoptosis.During 0-8uM the apoptosis rate of K562, K562/ADR and U937 was concertration-dependent measured by FCM. After 24 hours treated with 8uM As2O3, the apoptosis rate of K562 cells was 48.12% while that of K562/ADR and U937 was 7.32% , 16.32% each. When the concentration of As2O3 increased to 16uM the apoptosis of U937 was also raised to 55.20%. It was seemed that K562 cell was the most sensitive to As2O3.In K562 cell the level of kB-a in cytoplasm was downregulated after As2O3 stimulation, while NF-KB in nuclear was upregulated. Flow cytometry also confirm the downregulated of kB-a from 93.03% to 27.07% treated with 8uMAs2O3(p<0.001) and the effect was concentration-dependent. While the kB-a in K562/ADR and U937 was not affected after treated with 8uM As2O3(p>0.05). An higher primary expression of NF-KB in nuclear was showed in K562/ADR cells and U937 cells and As2O3 had no effect on NF-KB in the nuclear of K562/ADR but downregulated NF-KB in the nuclear of U937.Conclusions1. As2O3 could inhibit the growth of K562, K562/ADR, U937 cells and also induce apoptosis with the following rank order of potency: K562 cells > U937cells > K562/ADR cells. These results suggested that As2O3 could inhibit the proliferation of leukemia cells through induction of apoptosis.2. As2O3 could upregulate the P65 subunit in the nuclear as observed in K562 cells which didn't express the constitutive activation of NF-KB, and could downregulate the P65 subunit in the nuclear of the U937 cells expressing the constitutive activation of NF-KB. But As2O3 had no effect on NF-KB in the nuclear of K562/ADR cells in which NF-KB was activated by adriamycin. All the above data suggested that the constitutive activation of NF-KB had important effects on the apopt...