European Journal of Pharmaceutical Sciences 45 (2012) 600605

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European Journal of Pharmaceutical Sciences

Autophagy induced by FTY720 promotes apoptosis in U266 cells

a r t i c l e

i n f o

a b s t r a c t
Despite recent treatment advances, multiple myeloma (MM) remains incurable and patients develop a progressively relapsing disease with subsequent poor prognosis. Studies have shown FTY720 has activities against a number of hematological malignancies including MM, no reports about autophagy induced by FTY720 in MM. Therefore, we investigated the potential application of FTY720 on MM using U266 cell line. We observed that FTY720 could induce caspase-3 dependent apoptosis in a dose- and time-dependent manner in U266 cells. FTY720 caused apoptosis by down-regulating antiapoptotic proteins Mcl-1, bcl-2, survivin and cleavage of Bid. Interestingly, FTY720 induce autophagy which could promote the apoptosis in U266 cells. Furthermore, activation of reactive oxygen species (ROS) regulates FTY720 induced apoptosis and autophagy in U266 cells. The study implicated that FTY720 could be a good candidate for MM treatment. 2012 Published by Elsevier B.V.

Article history: Received 28 September 2011 Received in revised form 14 December 2011 Accepted 23 December 2011 Available online 20 January 2012 Keywords: FTY720 Multiple myeloma Apoptosis Autophagy Reactive oxygen species

1. Introduction Multiple myeloma (MM) is a hematologic malignancy characterized by clonal proliferation of immunoglobulin-secreting plasma cells. It accounts about 10% of all hematological malignancies (Kumar et al., 2004). Despite recent treatment improvements including bortezomib and thalidomide, MM patients remains incurable and they are prone to relapse (Satoh et al., 2011). New therapeutic strategies are therefore required for MM patients. FTY720 (Gilenya; Fingolimod), a synthetic compound based on the fungal secondary metabolite myriocin (ISP-I), is a potent immunosuppressant which was approved (September 2010) by FDA as a new treatment for multiple sclerosis. It has the potential to be used in the treatment of organ transplants and cancer (Strader et al., 2011). Studies have shown FTY720 have the activities against a number of hematological malignancies including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML) with c-kit mutations, and mouse models of chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) and MM (Neviani et al., 2007; Liu et al., 2008, 2010; Roberts et al., 2010; Yasui et al., 2005). Although there is a report that FTY720 induces apoptosis in several multiple myeloma cell lines (Yasui et al., 2005), there are no reports about autophagy induced by FTY720 in MM. In this study, we assessed the potential of FTY720 on inducing autophagy in MM cell line U266 and investigated the associated mechanisms of cell
Corresponding author. Tel.: +86 18940251010.
E-mail address: [email protected] (Z. Liu). 0928-0987/$ - see front matter 2012 Published by Elsevier B.V. doi:10.1016/j.ejps.2011.12.014

apoptosis and autophagy, which are two different but related cell death pathways. 2. Materials and methods 2.1. Cell lines and cell culture U266 cells were cultured in RPMI 1640 medium which was supplemented with 15% FBS, 100 U/ml penicillin and 100 lg/ml streptomycin. Cells were incubated at 37 C in a humidied atmosphere containing 5% CO2. 2.2. Reagents and antibodies RPMI 1640 medium was purchased from Invitrogen Gibco. FTY720 was purchased from Cayman chemical company. Balomycin A1, N-acetyl-L-cysteine (NAC) and Tiron were purchased from SigmaAldrich. Anti-microtubule-associated protein 1 light chain 3(LC3B), anti-Bcl-2, anti-Bid, anti-phospho-P44/42 MAPK(Erk1/2), anti-Bax, anti-Bak, anti-GAPDH and anti-survivin antibodies were purchased from Cell Signaling Technology Inc. Anti-Mcl-1 was purchased from Santa Cruz (USA). 2.3. CCK-8 cell proliferation assay Inoculate U266 cell suspension (104/well) in a 96-well plate and prepare wells that contain known number of viable cells (to create a calibration curve) at the same time. Pre-incubate the plate in a humidied incubator (at 37 C, 5% CO2). Add 10 ll of the CCK-8

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solution to each well of the plate. Incubate the plate for 1 h in the incubator. Measure the absorbance at 450 nm using a microplate reader. 2.4. Apoptosis assay Apoptotic cells were evaluated by ow cytometry and DAPI staining. For ow cytometry, the 5 105 cells in 500 ll PBS were stained with 5 ll Annexin V-FITC and 5 ll propidium iodide at room temperature for 10 min. The cells were then analysed by owcytometry using 488 nm for excitation and red uorescence measured >600 nm using the PACS can owcytometer. Apoptosis cells were counted as Annexin V positive and both PI positive and negative cells. As for DAPI staining, U266 cells were treated with or without FTY720 20 lM for 24 h and then stained with DAPI for 5 min. The cells were dropped on the glass slide and covered with coverslip, then observed by orescent microscope (100). 2.5. Western blot assay Cells were harvested for various times after treatment. Cell debris was removed by centrifugation at 1200 rpm for 1 h at 4 C. The resulting supernatants were resolved on a 12% SDSPAGE under denatured reducing conditions and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk at room temperature for 30 min and incubated with different primary antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The signal was visualized using an enhanced chemiluminescence. 2.6. Statistical analysis Differences between two treatment groups were analyzed using a two-tailed Students t test. A signicant statistically difference was reported with p indicated where applicable. Data were reported as the mean SE from at least three independent experiments. 3. Results 3.1. FTY720 inhibits U266 cell survival and induces apoptosis FTY720 has been reported to induce cell death in a number of hematological malignancies. To understand FTY720 whether or not induced cell death in MM cell line, U266 cells, we rst treated U266 cells with different dosages of FTY720 and then measured cell viability after 24 h by CCK-8 assay. As shown in Fig. 1A, cell viability started to reduce at 5 lM and reach to 15% at 20 lM, suggesting FTY720 affects cell survival in MM cell line. We then measured cell viability at 2, 6, 24 h at a xed concentration of 20.0 lM. Fifty percent cells were killed even 2 h after treatment as compared to control group (Fig. 1B). Cell apoptosis was also measured at the same time by ow cytometry (FCM) using Annexin V and PI staining. Apoptosis rate (%) increased with the increased dose of FTY720 (Fig. 1C) and this was inconsistent with cell viability as measured by CCK-8 assay. The apoptotic cells in the control group (control group cells were treated with 1% DMSO in all the experiments) remained constant at 5% while samples treated with drug are at 1530% from 6 to 24 h in the time course experiment (Fig. 1D). This validates that FTY720 induced apoptosis of U266 cells. The cell viability and apoptosis induced by FTY720 showed a dose- and time-dependant manner. PI positive cells are also increased in a dose- and time-dependent manner (Fig. 1E and F). We conrmed the apoptosis induced by FTY720 in U266 cells by staining cells with DAPI. As shown in Fig. 1G some of the cells with FTY720 treatment have the chromatin condensation and chromatinorrhexis as compared to the cells without treatment.

3.2. FTY720 could induce caspase-3 dependent cell death in U266 cells Apoptosis is characterized by activation of executioner caspase and induced caspase-dependent cell death. FTY720 has also been reported to induce caspase-independent cell death in chronic lymphocytic leukemia and mantle cell lymphoma cells (Liu et al., 2008; Roberts et al., 2010). When U266 cells were treated with FTY720, we observed that the cell viability was only about 20% at the dose of 20 lM. In the presence of Z-VAD-fmk, a pancaspase inhibitor, we found that cell viability rate increased to 4060%, suggesting that Z-VAD-fmk could rescue cell death caused by FTY720 in a dosedependent manner (Fig. 2A). Exposure of U266 cells to different concentrations of FTY720 induced cleavage of caspase-3 (Fig. 2B). We conclude FTY720 can induce a caspase-dependent cell death in U266 cells. 3.3. FTY720 induced apoptosis of U266 via apoptosis-related proteins Many apoptosis-related proteins play important roles in apoptosis. Mcl-1, survivin, bcl-2 and Bid are antiapoptotic proteins and degradations of these proteins are required for the induction of apoptosis (Krishna et al., 2011; Zhao et al., 2010). To conrm that the observed cell death is mediated by these apoptosis-related proteins, we examined the protein levels of Mcl-1, survivin, bcl-2, P-ERK, Bak, Bax and cleaved Bid in U266 cells treated with FTY720. As shown in Fig. 3, the expression of Mcl-1, survivin, bcl-2 were reduced after the treatment and cleavage of Bid was increased, conrmed FTY720 induced apoptosis by regulating antiapoptotic proteins expression. 3.4. FTY720 induces autophagy in U266 cells Autophagy is a process by which cells undergo partial autodigestion that prolongs survival for a short time under starvation conditions. It provides nutrients that are necessary to maintain cell viability. However, constitutive autophagy also leads to cell death (Tsujimoto and Shimizu, 2005). Conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II is a marker of autophagosome degradation. To investigate whether FTY720 could induce autophagy in U266 cells, we examined LC3-I to LC3-II conversion in U266 cells by immunoblot with anti-LC3 antibody. We observed increased amount of conversion from LC3-I to LC3-II after the treatment and this conversion is dose-dependent (Fig. 4A). Interestingly, LC3-II accumulated in the presence of Balomycin A1, an inhibitor of autophagosomelysosome fusion and LC3-II degradation, in cells treated with FTY720 (Fig. 4B), conrmed that autophagic ux was induced by FTY720. 3.5. FTY720 induced autophagy promotes apoptosis in U266 cells Since autophagy can either promote cell survival or cause cell death, we seek to understand the role of autophagy played in the FTY720 induced cell death. We examined cell viability and apoptosis after FTY720 treatment in the presence or absence of autophagy inhibitor, Balomycin A1. Cell viability was higher in the presence of Balomycin A1 with FTY720 treatment (Fig. 5A), indicating that Balomycin A1 could rescue cell death caused by FTY720. This suggested that autophagy induced by FTY720 contributed to MM cell death. Then we further examined whether or not autophagy had any effect on apoptosis. We measured cell apoptosis in U266 cells treated with FTY720 in the presence or absence of Balomycin A1. The apoptosis rate is lower in the group treated with FTY720 and Balomycin A1 together than that in the group treated with FTY720 alone (Fig. 5B). This strongly suggested that autophagy induced by FTY720 promoted apoptosis. In consistent with our observation, expression of anti-apoptotic protein Mcl-1 and survivin was

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Fig. 1. FTY720 could induce cell death in U266 cells. (A) U266 cells were treated with 0, 2.5, 5, 10, 20 lM FTY720 for 24 h, then cell viability was evaluated by CCK-8 assay. (B) 266 cells were treated with 20 lM FTY720 for 2, 6 and 24 h, then CCK-8 assay was performed. (C) U266 cells were treated with 0, 2.5, 5, 10, 20 lM FTY720 for 24 h, then cells were assayed for apoptosis by ow cytometry. (D) U266 cells were treated with 20 lM FTY720 for 6, 16 and 24 h, then cells were assayed for apoptosis by ow cytometry. (E) U266 cells were treated with 0, 2.5, 5, 10, 20 lM FTY720 for 24 h, then cells were stained with PI and assayed by ow cytometry. (F) U266 cells were treated with 20 lM FTY720 for 2, 6 and 24 h, then cells were stained with PI and assayed by ow cytometry. (G) U266 cells were treated with 20 lM FTY720 for 24 h, then the cells were stained with DAPI and observed by uorescent microscopy (100). The apoptotic cells were pointed by arrows. p < 0.0005, p < 0.005, p < 0.05, indicates signicant difference versus control group (Students t test).

accumulated in the presence of Balomycin A1 (Fig. 5C). It is possible that autophagy could help to degrade the antiapoptotic proteins in the lysosome and synergize the cell death induced by FTY720. 3.6. FTY720 induced apoptosis and autophagy via the activation of ROS in U266 cells To dene the mediator between apoptosis and autophagy induced by FTY720 in U266 cells, we examined reactive oxygen species (ROS), which plays an important role in regulating both apoptosis and autophagy. Using two ROS scavengers NAC and Tiron, we observed that they all can rescue FTY720 induced apoptosis as measured by ow cytometry (Fig. 6A and B), indicating that

FTY720 induced apoptosis via the activation of ROS. Furthermore, both NAC and Tiron blocked LC3-I to LC3-II conversion based on protein expression (Fig. 6C and D), suggesting that ROS also contributes to autophagy induced by FTY720. All together, these results indicated that ROS is one of the mediators between autophagy and apoptosis. 4. Discussion There are about one out of 100,000 people in the world suffering from MM. Even with new therapies, MM patients are still incurable (Satoh et al., 2011). FTY720 has initially been used as an immunosuppressant and is promising in treating relapsing-remit-

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Fig. 2. FTY720 could induce caspase-3 dependent apoptosis in U266 cells. (A) U266 cells were treated with 5 lM FTY720 in the presence or absence of Z-VAD-fmk for 24 h, then CCK-8 assay was performed. p < 0.0005, p < 0.05, indicates signicant difference versus the group treated with FTY720 (Students t test). (B) U266 cells were treated with FTY720 0, 2.5, 5, 10, 20 lM for 24 h, then Western blot analysis was performed for cleaved caspase-3. The equal loading of protein was conrmed by probing with GAPDH.

Fig. 4. FTY720 could induce autophagy in U266 cells. (A) U266 cells were treated with FTY720 0, 2.5, 5, 10, 20 lM for 24 h, then Western blot analysis was performed for LC3B. (B) U266 cells were treated with FTY720 in the presence or absence of Balomycin A1 for 24 h, then Western blot analysis was performed for LC3B. The equal loading of protein was conrmed by probing with GAPDH.

Fig. 3. FTY720 induced apoptosis of U266 via the apoptosis-related proteins. U266 cells were treated with FTY720 0, 5, 10, 20 lM for 24 h, then Western blot analysis was performed for Mcl-1,survivin, Bcl-2, Bid, Bax, Bak, P-ERK. The equal loading of protein was conrmed by probing with GAPDH.

ting multiple sclerosis patients (Del Santo et al., 2011). In the past ten years, FTY720 has been studied in several hematological malignancies (Neviani et al., 2007; Liu et al., 2008, 2010; Roberts et al., 2010). Very limited studies were investigated for its application in MM (Yasui et al., 2005). In our study, for the rst time, we showed that FTY720 induced both apoptosis and autophagy in MM cell line U266 cells. We further demonstrated that both apoptosis and autophagy contributed to cell death. Interestingly, activation of ROS is one of the mechanisms underlying apoptosis and

autophagy induced by FTY720. All of these provided evidences for its clinical applications in the future. FTY720 likely has multiple effects on inducing cell death including apoptosis (caspase-dependent and -independent), autophagy and necrosis. In our study we focused on the function of caspase 3-dependent apoptosis and autophagy in FTY720 induced cell death. Our results showed that FTY720 could induce caspase-3 dependent apoptosis in U266 cells, which is consistent with previous study (Yasui et al., 2005), in a time- and dose-dependent manner. Autophagy is a highly regulated and evolutionarily conserved process that leads to the lysosomal degradation of damaged proteins, organelles and other macromolecules, with subsequent recycling of bioenergetic intermediates (Levine and Kroemer, 2008). During autophagy, cytoplasmic constituents are sequestered into double-membraned autophagosomes, which are then delivered to lysosomes and degraded. Upon the induction of autophagy, phosphatidyl ethanol amine is covalently linked to the cytosolic protein LC3-I to yield LC3-II, which then associates with the autophagosome (Mizushima, 2004; Kabeya et al., 2004). This conversion is commonly used as a marker for autophagy (Mizushima, 2004). We found exposure of U266 cells to FTY720 caused the increase of LC3-II in a dose-dependent manner. The increased level

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Fig. 5. Autophagy caused by FTY720 could promote apoptosis in U266 cells. (A) U266 cells were treated with 20 lM FTY720 in the presence or absence of 10 nM Balomycin A1 for 24 h, then CCK-8 assay was performed. (B) U266 cells were treated with 20 lM FTY720 in the presence or absence of 10 nM Balomycin A1 for 6, 16 and 24 h, then cells were assayed for apoptosis by ow cytometry. p < 0.05, indicates signicant difference versus the group treated with FTY720 (Students t test). (C) U266 cells were treated with FTY720 0, 2.5, 5, 10 and 20 lM in the presence or absence of 10 nM Balomycin A1 for 24 h, then Western blot assay was performed for Mcl-1 and survivin, the equal loading of protein was conrmed by probing GAPDH.

Fig. 6. FTY720 induced apoptosis and autophagy via the activation of ROS in U266 cells. (A) U266 cells were treated with 15 lM FTY720 in the presence or absence of NAC 1, 5 mM for 24 h, then cells were assayed for apoptosis by ow cytometry. (B) U266 cells were treated with 15 lM FTY720 in the presence or absence of Tiron 1, 5 mM for 24 h, then cells were assayed for apoptosis by ow cytometry. p < 0.05, indicates signicant difference versus the group treated with FTY720(Students t test). (C) U266 cells were treated with 20 lM FTY720 in the presence or absence of NAC 1, 5 and 10 mM for 24 h, then Western blot analysis was performed for LC3B. (D) U266 cells were treated with FTY720 15 and 20 lM in the presence or absence of 5 mM Tiron for 24 h, then Western blot analysis was performed for LC3B. The equal loading of protein was conrmed by probing with GAPDH.

of LC3-II is due to either the stimulation of the LC3 conversion or the inhibition of the fusion of autophagosome with lysosome that block lysosomal turnover of LC3. We further showed that LC3-II accumulated in the presence of Balomycin A1, an inhibitor of autophagosomelysosome fusion, in cells treated with FTY720, conrmed that autophagic ux was enhanced by FTY720 in U266 cells. Autophagy is a double-edged sword in tumorigenesis. In general, autophagy is utilized to keep the cells to be alive. It is induced to keep the cells survive not only in the normal cells that during nutrient starvation but also in rapidly growing cancer cells that have outgrown their vascular supply and are exposed to metabolic

stress. Although autophagy can serve as protective function to the cells, constitutive activation of autophagy may induce cell death (type II programmed cell death) (Tsujimoto and Shimizu, 2005). We found that the cell viability was increased when the cells were treated with FTY720 and Balomycin A1 together compared with the cells treated with FTY720 only (Fig. 5A), suggesting the autophagy induced by FTY720 is responsible for MM cell death. It has been reported that FTY720 can induce autophagy in ovary cancer cells and acute lymphoblastic leukemia cells (Zhang et al., 2010; Wallington-Beddoe et al., 2011). Previous studies have also shown that the autophagy induced by FTY720 worked as a protective function in ovary cancer cells and acute lymphoblastic leukemia

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cells (Zhang et al., 2010; Wallington-Beddoe et al., 2011). The discrepancies may be due to the complex and diverse interplays between autophagy and cell death. Depending on cell types, environment and stimulus, autophagy and cell death can have inhibitory, additive or even synergistic effects (Wirawan et al., 2012). So we think the autophagy induced by the same drug in different cell types may lead to either cell survival or cell death. The crosstalk between autophagy and apoptosis is controversial. Sandrine Eimer et al. showed that inhibition of autophagy induced a marked increase in the death-inducing activity of erlotinib (Eimer et al., 2011). While Qiao Cui et al. showed that inhibition of autophagy induced lowered apoptotic level (Cui et al., 2007). In some cases, autophagy and apoptosis may interconvert (Yanagisawa et al., 2003). We found there is functional association between autophagy and apoptosis in the cells subjected to FTY720. Balilomycin A1, an inhibitor of autophagy, could rescue drug induced apoptosis, suggesting that autophagy induced by FTY720 could sensitize apoptosis in U266 cells. It is possible that MM cells may be particularly prone to undergo autophagy and apoptosis by caspase-dependent mechanisms. This result also suggested that the function of FTY720 can benet from autophagy enhancement. We also found that Balomycin A1 could rescue the down-regulation of Mcl-1 and survivin caused by FTY720. This suggested that the inhibition of autophagy could reduce the degradation of antiapoptotic proteins in lysosome. Many signals including sphingolipids, death-receptor signaling molecules have long been known to activate both apoptosis and autophagy. Conversely, signaling pathways including PI3K/Akt and NF-jB signaling pathway can inhibit both apoptosis and autophagy (Levine, 2007). ROS, a critical regulator of both apoptosis and autophagy (Azad et al., 2009), can be generated in apoptotic and non-apoptotic modes of cell death. Low levels of ROS are important for cellular function and survival signaling, excessive ROS can lead to cell death through several mechanisms, including apoptosis, necrosis and autophagy. When U266 cells were treated with FTY720 and ROS scavenger NAC or Tiron, both NAC and Tiron could rescue apoptosis induced by FTY720, suggesting that FTY720 induced apoptosis via the activation of ROS in U266 cells. In addition, the ROS scavengers decreased the level of LC3-II, which means FTY720 even induce autophagy through ROS signaling pathway. Our results suggested that ROS is one of the mediators between apoptosis and autophagy caused by FTY720. Our results showed apoptosis and autophagy induced by FTY720 in U266 share the same stimuli and signal pathway, and interactions between them enhance the death rate of U266 cells. Although FTY720 was initially studied for its immunosuppressive actions, its potential roles has now increased to a number of malignancies include MM. MM is still incurable and more effective chemotherapeutic protocols are urgently needed. To this regard, the ability of FTY720 to induce cell death in U266 cells, if

conrmed in vivo, may represent a relatively selective therapy for the treatment of MM. References
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