Mechanism of Arsenic Trioxide-Induced Apoptosis on Multiple Myeloma Cells.

Multiple myeloma (MM) is an incurable B-cell malignancy. High-dose chemotherapy with stem cell support has achieved higher response rates than conventional therapy, but few patients remain in long-term remission. Arsenic trioxide (As₂O₃) is an effective treatment for acute promyelocytic leukemia. Current clinical studies are investigating arsenic as a therapeutic agent for malignancies. The mechanism of cytotoxicity inducted by As₂O₃ has not been known thoroughly. Constitutively activated NF-κB is a characteristic of different tumor entities including MM clones and malignant plasma cells from MM patients. It plays an important role in maintaining tumor cell proliferation and protecting cell from apoptosis. The study was to explore the possible mechanism of As₂O₃ effects on MM lines. Materials and methods: The human MM cell lines of KM3 and RPMI8226 were provided by Hematology Institution of Zhejiang University. The concentration of As₂O₃ in cell culture system was from 0.5μmol/L to 20μmol/L, 24 hours was selected as the termination point of cell culture. Cell culture without As₂O₃ and first-antibody-dismissed test were enrolled as controls. Both of the MM lines were treated with As₂O₃ in various concentrations. The restrained proliferation was observed by drawing growth curve. MTT bioassay was used to examine the effect of As₂O₃ on cell growth and the concentration of 50% growth inhibition( IC₅₀) calculated respectively. Apoptosis and changes of IκB-α protein was observed by flow cytometry(FCM). Changes and subcellular localization of IκB-α protein were observed by fluorescence microscopy. Western blot was also used to analyze the expression of NF-κB in nuclear. Results: As₂O₃ inhibited the proliferation of both MM lines, and IC₅₀ of KM3 and RPMI8226 was 9.61μmol/L and 8.01μmol/L respectively. After exposure to As₂O_3, both of the lines were induced to apoptosis. With As₂O₃ concentration varied from 0.5μmol/L to 20μmol/L the apoptosis rate of KM3 and RPMI8226 was concentration-dependent measured by FCM. After 24 hours treated with 20μmol/L As₂O_3, the apoptosis rate of KM3 cells was 41.18% while that of RPMI8226 was 65.25%. It seemed that RPMI8226 cell was more sensitive to As₂O₃. FCM 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 cells from 35.11% to 23.96%. The level of IκB-α in cytoplasm was down-regulated after As₂O₃ stimulation in both cell lines. The down-regulation of IκB-α from 79.68% to 51.57% was confirmed by FCM in KM3 treated with 10μmol/L As₂O₃ (p<0.01), the effect was concentration-dependent. While the IκB-α in RPMI8226 changed from 79.72% to 34.08% (p<0.01). Immunofluorescence assay showed that IκB-α localized in cytoplasm mostly. The level of IκB-α protein was down-regulated after As₂O₃ stimulation. The results showed that KM3 and RPMI8226 lines expressed constitutively activity NF-κB and As₂O₃ down-regulated the constitutive expression of NF-κB in both cell lines but with different kinetics. Conclusions: As₂O₃ could not only inhibit the proliferation but also induce apoptosis of KM3 and RPMI8226 cells. The mechanisms of its effect on MM cells may include decreasing the constitutive activity of NF-κB, subsequently down-regulating the expression of various gene products controlled by NF-κB, thus inducing apoptosis in MM cells. As₂O₃ could inhibit the proliferation of MM cells by changing MM cell cycle and inducing G2/M phase arrest.