FTY720 Induces Apoptosis and Autophagy in Multiple Myeloma Cells

Abstract 5103

Despite recent treatment advances, multiple myeloma(MM) remains incurable. FTY720 has initially been used as an immunosuppressant and is promising in treating relapsing/remitting multiple sclerosis. FTY720 has also been studied in several hematological malignancies including MM, but there are no reports about autophagy induced by FTY720 in MM. Therefore, we assessed the efficacy of FTY720 on MM cell line U266 and investigated the associated mechanisms of cell death. First we examined whether or not FTY720 induce cell death in U266 cells. We treated U266 cells with different dosage of FTY720 and then cell viability was measured at 24 hours by CCK-8 assay. The results showed cell viability started to reduce at 5uM and reach to 15% at 20uM, suggesting FTY720 affects cell survival in MM cell line. We then measured cell viability at 2□ A6□ A24 hours at fix concentration of 20.0 mM, 50% cells were killed even at 2 hours as compared with control group. Cell apoptosis was also measured by flow cytometry(FCM). Apoptosis rate(%) increased in a dose-dependent manner after FTY720 treatment. The percent Annexin+/7AAD- cells in the control group remained constant 5% and 15% to 30% in the drug treated time course experiments. The cell viability and apoptosis induced by FTY720 showed a dose- and time-dependant manner. Z-VAD-fmk, a pancaspase inhibitor, could rescue cell death caused by FTY720 and exposure of U266 cells to different concentration of FTY720 induced cleavage of caspase-3. We conclude FTY720 can induce a caspase-dependent cell death in U266 cells. Mcl-1, survivin, bcl-2 and Bid are antiapoptotic proteins and degradation of these proteins are required for the induction of apoptosis. In our study, the expression of Mcl-1, survivin and bcl-2 were reduced after the treatment which confirmed FTY720 induced apoptosis by down-regulating the expression of antiapoptotic proteins. Consistent with this, cleavage of Bid was also increased after the FTY720 treatment. Autophagy is another cell death pathway characterized by intracellular degradation system that delivers cytoplasmic constitutions to the lysosome. Conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II is a marker of autophagosome degradation. We observed increasing amount of conversion from LC3-I to LC3-II after FTY720 treatment, suggesting that FTY720 could induce autophagy in U266 cells. This conversion is dose-dependent. Interestingly, LC3-II accumulated in the presence of Bafilomycin A1, an inhibitor of autophagosome-lysosome fusion and LC3-II degradation, in cells treated with FTY720, confirmed that autophagic flux was induced by FTY720. To understand the role of apoptosis and autophagy played in the FTY720 induced cell death, we examined cell viability and apoptosis after FTY720 treatment in the presence or absence of Bafilomycin A1. Cell viability was higher and apoptosis was lower in the presence of Bafilomycin A1 after FTY720 treatment, indicating that Bafilomycin A1 could rescue cell death and apoptosis caused by FTY720. The results suggested that autophagy and apoptosis synergized to induce MM cell death after FTY720 treatment. We found Bafilomycin A1 rescued FTY720 induced cell death is dependent on the accumulation of anti-apoptotic protein Mcl-1 and survivin, it is possible that autophagy helps to degrade the anti-apoptotic proteins in the lysosome and synergize the cell death induced by FTY720. To investigate the mediators of FTY720-induced apoptosis and autophagy, we examined reactive oxygen species (ROS), which plays an important role in regulating both apoptosis and autophagy. Potential antioxidants N-acetyl-L-cysteine (NAC) and Tiron both could rescue apoptosis induced by FTY720, suggesting that FTY720 induced apoptosis via the activation of ROS. Furthermore, both NAC and Tiron blocked the conversion of LC3-I to LC3-II, indicating that FTY720 leads to ROS production, which results in autophagosome development and autolysosomal degradation. These studies indicate autophagy induced by FTY720 contributes to cell death in U266 cells and suggest that the function of FTY720 maybe can benefit from autophagy enhancement. To this regard, the ability of FTY720 to induce autophagy-dependent cell death in U266 cells, if confirmed in vivo, may represent a relatively selective therapy for the treatment of MM.