The Role Of FTY720 In Glioblastoma And Its Mechanisms

Glioblastoma (GB) is the most common and most aggressive malignant primary brain tumor in humans, with short survival time, poor feedback to surgery, vulnerable to relapse, and put variety of tolerates to radio and chemotherapy. Therefore, new therapeutic strategies should be developed to overcome cancer cells, inhibit the recurrence of cancer and improve the prognosis of patients. FTY720 is a potent immunosuppressant which has been approved by the US Food and Drug Administration (FDA) to treat relapsed multiple sclerosis. In addition. FTY720 was recently shown to be therapeutically active against several solid tumors. It has been reported that FTY720 could suppress invasion and migration, induce apoptosis and inhibit the growth of solid tumor in nude mice in hepatocellular carcinoma. In addition, in prostate cancer. FTY720 not only induces mitochondrial apoptosis but also increases the sensitivity of cancer cells to radiotherapy. Furthermore, in gastric cancer, lung cancer, breast cancer and ovarian cancer. FTY720 also shows anti-cancer effects, however the specific mechanism is unclear. Phosphoinositide-3-kinase (PI3K) family is involved in multiple signaling pathways. PI3K-protein kinase B (AKT) signaling pathway includes PI3K, AKT, mammalian target of rapamycin (mTOR), p70S6K and so on. PI3K/AKT/mTOR/ p70S6K signaling pathway is closely related the differentiation of normal cell. The abnormal activation of this pathway plays a vital role in cancer development. Researches have showed this pathway is inextricably linked with the biological behavior of cancer cell such as apoptosis. autophagy, invasion and metastasis. Therefore, we hypothesized that FTY70 exhibits aiti-cancer effects in glioblastoma through the PI3K/AKT/mTOR/p70S6K signaling pathway.In our study, we examined the effects of FTY720 on apoptosis. autophagy. necrosis, invasion and metastasis in glioblastoma both in vitro and in vivo. In addition, we studied the role of PI3K/AKT/mTOR/p70S6K signaling pathway in FTY720-induced biological behavior changes in glioblastoma.This thesis is divided into five parts:Part ⅠThe effects of FTY720 on apoptosis and necrosis in glioblastoma cellsObjective:To establish multiple glioblastoma cell line models. To study the effects of FTY720 on apoptosis and necrosis in glioblastoma cells and distinguish the FTY720-induced apoptosis is endogenous apoptosis (Markers:caspase-8) or exogenous apoptosis (Markers:Bcl-2， Bcl-xL, Bax， cytochrome c). To find a new target for glioblastoma cells.Methods:Human glioblastoma cell lines U251MG and U87MG and mouse primary astrocytes were treated with FTY720 at the indicated concentrations for different time points, and the cell viability was analyzed by CCK-8 assay. Cells were treated with DMSO (control) or 15 μM FTY720 for 12 h, and cell death was determined by flow cytometry followed by Annexin V/PI staining. Cells were exposed to FTY720 for various periods at a concentration of 15 μM or for 12 h at various concentrations. After treatment, the expression levels of caspase 3. PARP. RIP1 and RIP3 were detected by Western blot analysis. Cells were treated with FTY720 for 12 h at increasing concentrations and proteins were then extracted followed by immunoblotting analysis for measurement of caspase-8, Bcl-xL, Bcl-2, Bax and cytochrome c expression levels.Results:FTY720 could induce cell death in glioblastoma cell lines. FTY720 could induce the cleavage of caspase-3 and PARP and increase the expression of RIP1 and RIP3. In addition, after treatment with FTY720. the endogenous apoptosis markers such as cleaved caspase-8 increase significantly. However, the exogenous apoptosis markers such as Bcl-2, Bcl-xL, Bax and cytochrome c remain unchanged, indicating that FTY720-induced apoptosis is endogenous apoptosis instead of exogenous apoptosis.Conclusions:FTY720 induces endogenous apoptosis and necroptosis in glioblastoma cell lines.Part ⅡThe effects of FTY720 on autophagy and the role of autophagy in FTY720-induced apoptosis and necroptosis in glioblastoma cells.Objective:To determine whether FTY720 could induce autophagyand study the relationship among FTY720-induced autophagy, apoptosis and necroptosis in glioblastoma cells.Methods:Cells were treated with FTY720 for various periods at a concentration of 15μM or exposed to various concentrations of FTY720 for 12 h, then Western blot analysis was performed to detect the expression of LC3、Beclin1、Atg3、Atg5、Atg7 and Atg12. Cells were treated with DMSO (control) or 15 μM FTY720 for 12 h, stained with LC3 antibody and appropriate secondary antibody and observed under a fluorescence microscope (During autophagy, LC3 is relocated to the autophagosomal membranes, monitoring LC3 from a diffuse form to the accumulation of puncta is an effective method to detect autophagosomes). Cells were treated as described above. After treatment, cells were harvested and subjected to transmission electron microscopy (TEM) to detect the formation of autophagic vacuoles. Cells were treated as described above. After treatment, cells were stained with AO and then subjected to fluorescence microscope to detect the formation of acidic vesicular organelles (AVOs). Cells were treated with FTY720 for various concentrations in 12 h, the expression of p62 was detected by western blotting. Cells were pr-treated with101 nM BafA1 for 1 h and then co-treated with 15 μM FTY720 for 12 h. Protein levels of p62 and LC3 were detected. Cells were exposed to either 15 μM FTY720 or 3 mM 3-MA. or a combined treatment of FTY720 and 3-MA for 12 h. After treatment. the cell viability and the protein levels of LC3, caspase-8. caspase-3 and PARP were analyzed by CCK-8 and Western blot analysis respectively. Cells were transfected with either Beclin 1 shRNA or a non-target control shRNA for 72 h and then co-treated with 15 μM FTY720 for 12 h, and the cell viability and protein levels of PARP were examined by CCK-8 and Western blot respectively. Cells were treated as described above. Then apoptosis and necrosis were analyzed by flow cytometry following Annexin V/PI staning.Results:Western blot analysis showed that FTY720 increased the expression of LC3-Ⅱ、Beclin 1、Atg3、Atg5、Atg7、Atg12 in U251MG and U87MG cells. Results of the immunofluorescence revealed that FTY720 induced the formation of LC3 puncta in cells, indicating the formation of autophagosome. Upon treatment of FTY720, autophagic vacuoles containing cellular material or organelle were observed in the cytoplasm by TEM. Furthermore, treatment with FTY720 led to the formation of red fluorescent AVOs and the expression of p62 exhibited a concentration-dependent decreased in respond to FTY720. In addition. BafAl significantly increased the expression of p62 and LC3-Ⅱ. Moreover, The BafAl-induced p62 and LC3-Ⅱ expression was obviously augmented when combined with FTY720. Moreover, cell death induced by FTY720 was significant decreased when FTY720 was co-treated with 3-MA, indicating that 3-MA rescued FTY720-induced cell death. And consistent with the reduction of cell death. FTY720-induced cleavage of caspase-8, caspase-3 and PARP was decreased in the presence of 3-MA. Moreover, the cell death and cleavage of PARP induced by FTY720 were significantly decreased in Beclin 1 shRNA-treated cells. Flow cytometry analysis showed that not only apoptosis but also necrosis were decreased when FTY720 was co-treated with 3-MA or shRNA which knocked down Beclin 1.Conclusions:FTY720 induces autophagy in glioblastoma cell lines and FTY720-induced autophagy severed as an upstream of extrinsic apoptosis and necrosis.Part ⅢThe effects of FTY720 on invasion and migration in glioblastoma cell lines.Objective:To explore whether FTY720 could inhibit the invasion and migration of glioblastoma cell lines.Methods:Cells were treated with FTY720 at the indicated concentrations for 24 h. and the cell proliferation was analyzed by CCK-8 assay. Cells were wounded and then treated with FTY720 (0.1.5,3 and 6 μM) for 24 h in DMEM. At 0.12 and 24 h, phase-contrast pictures of the wounds at three different locations were taken. Transwell invasion and migration assay were used to detect the effects of FTY720 on migration and invasion of U251MG and U87MG cells. The protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 were analyzed in U251MG and U87MG cells treated with FTY720 (0,1.5,3 and 6 μM) for 24 h using Western blot. Cells were exposed to various concentrations of FTY720 for 24 h, the expression of ROCK1, ROBO1, E-cadherin, N-cadherin and Vimentin was determined.Results:At concentrations above 7 μM, FTY720 significantly inhibited the proliferation of U251MG and U87MG cells, however, at concentrations below 7 μM, the inhibition rate was not obvious; therefore we chose a concentration range of FTY720 lower than 7 μM for our subsequent studies. Wound healing assay and transwell assay showed that FTY720 significantly decreased the invasion and migration ability of glioblastoma cells in concentration - and time- dependent manners. FTY720 significantly decreased the expression of MMP-2 and MMP-9 while increased the expression of TIMP-1 and TIMP-2 in a concentration-dependent manner, demonstrated that FTY720 inhibited the migration and invasion of glioblastoma cells partially via the down-regulation of MMPs and up-regulation of TIMPs. FTY720 significantly decreased the expression of N-cadherin, Vimentin, ROBO1 and ROCK1 while increased the expression of E-cadherin.Conclusion:FTY720 could reduce glioblastoma cells migration and invasion. Part Ⅳ The role of PI3K/AKT/mTOR signaling pathway in FTY720-induced changes of glioblastoma biological behavior. Objective:To explore whether FTY720 induced autophagy and reduced invasion and migration of glioblastoma cell lines through the PI3K/AKT/mTOR signaling pathway.Methods:Cells were exposed to 15 μM FTY720 for different durations or different concentrations of FTY720 for 12 h, and the expression levels of phospho-AKT, AKT, phospho-mTOR, mTOR, phospho-p70S6K and p70S6K were detected by Western blot. Cells were pretreated with 10 μM LY294002 for 1 h followed by the treatment of FTY720. The expression of p-AKT, AKT, LC3, MMP-2, MMP-9, TIMP-1 and TIMP-2 was determined by Western blot.Results:FTY720 treatment obviously decreased phosphorylation of AKT, mTOR and p70S6K in concentration- and time-dependent manners. However, the expression levels of total AKT, mTOR and p70S6K remained unchanged. Pretreatment of LY294002 further attenuated the expression of MMP-2 and MMP-9 and increased the expression of LC3-Ⅱ, TIMP-1 and TIMP-2.Conclusion:FTY720-induced induction of autophagy and inhibition of migration and invasion in glioblastoma cells were partly through the PI3K/AKT/mTOR signaling pathway.Part ⅤThe effects of FTY720 on autophagy, apoptosis, necroptosis, migration and invasion in vivo.Objective:To determine whether FTY720 could show anti-cancer effects in vivo.Methods:U251MG and U87MG cells (5.0x106) were suspended in 100μl PBS and then injected subcutaneously into either side of the posterior flank of the male BALB/c athymic nude mice. When the tumors reached 70 to 100 mm3 in volume, intraperitoneal injections of saline or FTY720 (5 or 10 mg/kg) were administered every 24 h for 10 days and tumor volume was measured for another 10 days after the cessation of treatment. Tumor growth was measured every 2 days and tumor volumes were determined by external measurements and calculated according to V= [L x W2] x 0.52, where V is the volume, L is the length and W is the width. After the treatment, the tumors were harvested and the markers of autophagy, apoptosis, necroptosis, invasion and migration and PI3K/AKT signaling pathway were analyzed by Western blot.Results:FTY720 treatment led to a notable inhibition of tumor growth without affecting body weight obviously. Moreover, compared to the low-dose group, the high-dose group resulted in smaller tumors, suggesting a dose-dependent effect. Compared with saline-treated group. FTY720-treated groups showed increased expression of LC3-Ⅱ, PARP, RIP1, RIP3 and decreased expression of p-AKT, MMP-2, MMP-9 in a dose-dependent manner.Conclusion:FTY720 could induce autophagy, apoptosis and necroptosis, reduce invasion and migration and inhibit the PI3K/AKT signaling pathway in vivo.SummaryOur results provided evidence that FTY720 induced autophagy, apoptosis. necroptosis and reduced invasion, migration in glioblastoma via the PI3K/AKT/mTOR/p70S6K signaling pathway both in vitro and in vivo. These results make FTY720 an attractive therapeutic agent for developing alternative treatment protocols, and possibly, for combining with other anticancer agents to overcome drug resistance and achieve better outcomes.