Mechanism Of Arsenid Trioxide-Induced Apoptosis On Multipie Myeloma Cells

BackgroundThe incidence of multiple myeloma ( MM ), a presently incurable B-cell malignancy, lists the second of common hematologic malignancy. High-dose chemotherapy with stem cell support has achieved higher response rates than conventional therapy, but few patients remain in long-term remission, highlighting the urgent need for novel therapeutic strategies. The dysregulation of the apoptotic mechanism in plasma cells is considered a major underlying factor in the pathogeneses and subsequent chemoresistance in MM. Arsenic trioxide (As203) is a very effective treatment for acute promyelocytic leukemia (APL) with a high complete remission rate. It has become evident that theanti-tumor effects of As2O3 are not restricted to leukemia. Currently, clinical studies are investigating arsenic as a therapeutic agent for a variety of malignancies. Its effects on leukemic cells mainly include induction of apoptosis and differentiation. However, these mechanisms cannot explain the induction of apoptosis in many other cell types. The mechanism of cytotoxicity inducted by arsenic trioxide has not been known thoroughly. Nuclear factor- K B(NF- K B) proteins comprise a family of transcription factors, which regulates the expression of several genes involved in inflammatory , immune responses, cell proliferation and apoptosis. The maladjustment of NF-B can induce accurrence of tumour, inflammation and other human diseases. Constitutively activated NF-B is a characteristic feature of different tumour entities including MM clones and malignant plasma cells from MM patients. It plays an important role in maintaining tumour cell proliferation and protecting cell from apoptosis. The aim of this study was to explore the possible mechanism of arsenic trioxide effects on multiple myeloma lines.Materials and methodsThe human MM cell lines, KM3 and RPMI8226, were provided by Hematology Institution of Zhejiang University. The concentration of As203 used in cell culture system was from 0.5 urnol/ L to 20umol/ L 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.Both of the MM lines were treated with As2O3 in various concentration. The restrained proliferation was observed by drawing growth curve. MTT bioassay was used to examine the effect of As203 on cell growth and the concentration of 50% growth inhibition (IC50) was calculated respectively. Apoptosis andchanges of kB protein was observed by flow cytomerty. Changes and subcellular localization of lKB-a protein were observed by fluorescence microscopy. Western blot was also used to analyze the expression of NF-B in nuclear.ResultsAs2O3 inhibited the proliferation of both myeloma lines, and IC5o of KM3 and RPMI8226 was 9.61 umol/l and 8.01 umol/l respectively. After exposure to As2O3, both of the lines were induced to apoptosis. With As2O3 concentration varied from 0.5 umol/l to 20 umol/l the apoptosis rate of KM3 and RPMI8226 was concentration-dependent measured by FCM. After 24 hours treated with 20 umol/l As2O3 , the apoptosis rate of KM3 cells was 41.18% while that of RPMI8226 was 65.25%. It was seemed that RPMI8226 cell was more sensitive to As203. Flow cytometric analysis showed a significant increase in the percentage of G2/M phase cells(from 13.74% to 41.72%) and a decrease percentage of S phase cella(from 35.11% to 23.96%) in KM3 cells.The level of kB-a in cytoplasm was downregulated after As2O3 stimulation in both cell lines. The downregulation of kB-a from 79.68% to 51.57% was confirmed by FCM in KM3 treated with 10umol/L As2O3 (p<0.01) and the effect was concentration-dependent, while that in RPMI8226 changed from 79.72% to 34.08% (p<0.01) after the same treatment.Immunolfluorescence assay showed that I K. B- a localized in cytoplasm mostly. The level of I K B- a protein was downregulated after As203 stimulation. The results shown that both KM3 and RPMI8226 cell lines expressed constitutive NF-KB activity and As2O3 down-regulated the constituti...