A novel immunosuppressive agent FTY720 induced Akt dephosphorylation in leukemia cells

1. Our previous studies revealed that the immunosuppressive agent, FTY720, mainly induces mitochondria-involved apoptosis in some types of cancer cells, since Bcl-2 overexpression prevents the FTY720-induction of apoptotic stimuli. Furthermore, FTY720 induces G0/G1 cell cycle arrest. The present study further examines the correlation between intracellular signaling kinases with FTY720-induced mitochondria-involved apoptosis. 2. Human T cell leukemia Jurkat was exposed to FTY720. Dephosphorylation of Akt occurred in a time- and concentration-dependent manner. FTY720 also induced Bad (Ser(136)) and ribosomal p70S6 kinase (p70(S6k)) (Thr(389)) dephosphorylation. 3. FTY720-induced Akt dephosphorylation was not because of Akt upstream phosphatidylinositol 3'-kinase (PI 3-kinase) pathway inhibition. 4. FTY720 also induced Akt dephosphorylation in human B cell leukemia BALL-1. BALL-1 cells were resistant to FTY720-induced apoptosis. 5. Okadaic acid (OA) inhibited the FTY720-induced dephosphorylation of Akt and p70(S6k), suggesting that FTY720 promotes Ser/Thr protein phosphatase (PP) activity. 6. OA partially inhibited FTY720-induced caspase-3 activation. 7. PP2A or PP2A-like phosphatase was temporarily activated in cells exposed to FTY720. In addition, FTY720 activated purified PP2A (ABC). 8. Overall, the results suggest that FTY720 activated PP2A or PP2A-like phosphatase and dephosphorylated Akt pathway factors resulting in the enhancement of apoptosis via mitochondria.     

Activation of caspases and mitochondria in FTY720-mediated apoptosis in human T cell line Jurkat

FTY720, a novel immunosuppressive drug originally derived from a metabolite from Isaria sinclairii, is known to induce apoptosis in lymphocytes. In this study, we investigated the involvement of caspases and mitochondria in FTY720-mediated apoptosis using Jurkat cells, a human T cell line. Our results indicated that FTY720-induced activation of caspases 2, 3, 6, 8, 9 and 10, whereas caspases 1 and 5 were not activated. We also observed in the FTY720-treated cells a loss of mitochondrial membrane potential, a release of cytochrome c into cytosol and an exposed phosphatidylserine (PS) at the outer surface of the cell membrane. Pretreatment with a peptide inhibitor, benzyloxycarbonyl-Asp-CH2COC-2, 6-dichlorobenzene (Z-Asp-CH2-DCB), prevented apoptosis and externalization of phosphatidylserine, whereas the inhibitor did not prevent the mitochondrial events. This suggests that caspases may play a role downstream of the mitochondrial pathway. Therefore, caspase cascade in FTY720-treated cells may be initiated by activation of mitochondria.     

Evidence that FTY720 induces T cell apoptosis in vivo

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5-10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyer's patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly/aly mice, which lack Peyer's patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.     

小檗碱通过破坏线粒体功能选择性诱导淋巴瘤细胞的凋亡

目的研究小檗碱(BBR)对T淋巴瘤细胞凋亡的影响及机制。方法正常T淋巴细胞、T淋巴瘤细胞、Jurkat细胞用BBR 0,10,20和40μmol·L^-1处理24 h,多柔比星(Dox)40μmol·L^-1为阳性对照;Jurkat p0细胞用BBR 0和40μmol·L^-1处理24 h。流式细胞术检测细胞凋亡率,Seahorse XF24细胞代谢分析仪和H2DCFDA活性氧探针检测细胞氧消耗率(OCR)和活性氧(ROS)水平。CellTiter-Glo发光法细胞活力检测试剂盒检测细胞ATP水平,试剂盒测线粒体呼吸链复合物Ⅰ,Ⅱ,Ⅳ和Ⅴ的活性。Western印迹法检测细胞内胱天蛋白酶3、Bcl-2、Bax、NF-κB抑制蛋白激酶α/β(IKKα/β)、磷酸化IKKα/β(p-IKKα/β)、NF-κB抑制因子α(IκBα)、p-IκBα和P65蛋白表达水平。结果BBR 40 mmol·L^-1明显增加T淋巴瘤细胞和Jurkat细胞凋亡率(P<0.01),胱天蛋白酶3和Bax蛋白表达水平显著上调(P<0.01),Bcl-2表达显著下调(P<0.01),但对正常T淋巴细胞的凋亡并无影响;BBR 40 mmol·L^-1明显降低Jurkat细胞的OCR及线粒体呼吸链复合物Ⅰ活性(P<0.01),增加ROS水平(P<0.01),降低ATP水平(P<0.01),但不影响线粒体缺陷型淋巴瘤Jurkat p0细胞呼吸链和凋亡;BBR 40μmol·L-1明显下调Jurkat细胞中NF-κB通路相关蛋白p-IKKα/β/IKKα/β、p-IκBα/IκBβ和核内P65表达(P<0.01)。结论BBR通过破坏线粒体功能选择性诱导淋巴瘤T细胞的凋亡,可能与抑制NF-κB通路有关。     

Analysis of the mode of action of a novel immunosuppressant FTY720 in mice.

Accelerated lymphocyte homing and apoptosis have been suggested to contribute to potent immunosuppressive effects of FTY720, however, its main mechanism of action remains to be fully elucidated. Here, we examined the mode of action of FTY720 in mice. FTY720, when given at a single dose of 1 mg/kg, markedly decreased the number of peripheral blood lymphocytes (PBL) but moderately increased the lymphocyte numbers in lymph nodes (LN) and Peyer's patches (PP) in normal mice, as previously observed in rats. However, the sharp decrease in PBL numbers was also observed in aly/aly mice lacking LN and PP, indicating that this phenomenon is not explained by accelerated lymphocyte homing to LN and PP. In addition, the finding that a single administration of FTY720 did not suppress proliferative responses of T cells suggested that the PBL reduction could occur without inhibiting lymphocyte functions. However, when administered at the same dose for 2 weeks, FTY720 induced severe systemic lymphopenia, as well as marked suppression of lymphocyte proliferative responses in normal mice. The same treatment also prolonged skin allograft survival in aly/aly mice. Our results suggest that FTY720 suppresses in vivo immune functions mainly by inducing systemic lymphopenia and also by inhibiting T cell functions.     

Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27-dependent G1/S cell cycle arrest in conjunction with NF-κB activation

2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21^Cip1/Waf1 and up-regulating p16^INK4A. Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27^Kip1. Bcl-2 and p27^Kip1 were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-κB activity and p50 levels were increased by 2-ME2 and suppression of NF-κB signaling reduced p27^Kip1 expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27^Kip1 in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27^Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.     

Goniothalamin-induced oxidative stress,DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells

Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4 h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1 h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2.     

Depletion of T lymphocytes ameliorates cardiac fibrosis in streptozotocin-induced diabetic cardiomyopathy

T cell infiltration has been associated with increased coronary heart disease risk in patients with diabetes mellitus. Effect of modulation of T cell trafficking on diabetes-induced cardiac fibrosis has yet to be determined. Therefore, our aim was to investigate the circulatory T cell depletion-mediated cardioprotection in streptozotocin-induced diabetic cardiomyopathy. Fingolimod (FTY720), an immunomodulatory drug, was tested in wild-type (WT) C57BL/6 and recombination activating gene 1 (Rag1) knockout (KO) mice without mature lymphocytes in streptozotocin-induced type 1 diabetic model. FTY720 (0.3 mg/kg/day) was administered intraperitoneally daily for the first 4 weeks with interim 3 weeks then resumed for another 4 weeks in 11 weeks study period. T lymphocyte counts, cardiac histology, function, and fibrosis were examined in diabetic both WT and KO mice. FTY720 reduced both CD4⁺ and CD8⁺ T cells in diabetic WT mice. FTY720-treated diabetic WT mouse myocardium showed reduction in CD3 T cell infiltration and decreased expression of S1P₁ and TGF-β1 in cardiac tissue. Fibrosis was reduced after FTY720 treatment in diabetic WT mice. Rag1 KO mice exhibited no CD4⁺ and CD8⁺ T cells in the blood and CD3 T cells in the heart. Diabetic Rag1 KO mouse hearts appeared no fibrosis and exhibited preserved myocardial contractility. FTY720-induced antifibrosis was abolished in diabetic Rag1 KO mice. These findings demonstrate that chronic administration with FTY720 induces lymphopenia and protects diabetic hearts in WT mice whereas FTY720 increases cardiac fibrosis and myocardial dysfunction in diabetic Rag1 KO mice without mature lymphocytes.     

FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis

FTY720 (fingolimod; 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, Novartis) is the prototype of a new generation of immunomodulators. The drug is the result of extensive chemical derivatisation based on the natural product myriocin, isolated from the ascomycete Isaria sinclairii. FTY720 bears structural similarity to sphingosine, a naturally occurring sphingolipid. As with sphingosine, FTY720 is effectively phosphorylated by sphingosine kinases in vivo and the phosphorylated drug targets G-protein-coupled receptors for sphingosine-1-phosphate (S1P). Gene deletion and reverse pharmacology studies have shown that FTY720 acts at S1P1 receptors on lymphocytes and the endothelium, thereby inhibiting the egress of T- and B cells from secondary lymphoid organs into the blood and their recirculation to inflamed tissues. Animal studies suggest that this novel mechanism translates into effective treatments for several autoimmune diseases and a recently completed Phase II clinical trial highlighted FTY720 as a potential therapy for relapsing-remitting multiple sclerosis.     

17-beta-estradiol alters Jurkat lymphocyte cell cycling and induces apoptosis through suppression of Bcl-2 and cyclin A

In this investigation, the effects and potential mechanisms of female sex steroid action on proliferation, cell cycling, and apoptosis in Jurkat CD4 + T lymphocytes were examined. 17-beta-Estradiol (estrogen) inhibited Jurkat T cell proliferation, stimulated accumulation of cells in S and G2/M phases of the cell cycle, and induced apoptosis over 72 h in a dose-dependent manner. 4-Pregnene-3,20-dione (progesterone) did not induce redistribution of the cells in the cell cycle but did induce cytostasis and slightly increased apoptosis. Simultaneous staining with anti-BrDU and propidium iodide indicated that estrogen-treated Jurkat T cells proceeded through S phase prior to apoptosis. Progesterone halted cell cycle progression; cells did not progress through S phase or incorporate BrDU. Both hormones decreased the percentage of cells in S or G2/M expressing cyclin A protein, but did not affect cyclin D protein expression. Cyclin A mRNA was markedly decreased by estrogen. Bcl-2 protein and mRNA were also reduced in estrogen but not progesterone-treated Jurkat T lymphocytes. This data shows that high concentrations of estrogen or progesterone significantly suppress lymphoproliferation in association with suppression of cyclin A. Additionally, bcl-2 protein levels were suppressed in association with estrogen-induced apoptosis. These findings demonstrate direct, hormone-specific effects on lymphocytes that may provide insight into their role in immunomodulation or the development of autoimmunity.     

Non-specific anti-proliferative effect of FTY720, a derivative of fungal metabolite from Iscaria sinclarii

FTY720 is a derivative of ISP-1 (myriocin), a fungal metabolite of the Chinese herb Iscaria sinclarii, with agonistic effect for sphingosine-1-phosphate receptor. In this study, we examined the potential adverse effect of FTY720 in terms of cell cytotoxicity and the relevance to its pharmacological action, using cell proliferation assay and functional aspect of activated macrophages producing NO and TNF-α. FTY720 potently blocked the proliferation of allogeneic T cells. However, this compound did not strongly suppress Con A-induced T cell blastogenesis and IL-2 induced CTLL-2 cell proliferation. Furthermore, FTY720 also non-specifically blocked the proliferation or viability of cancerous or primary cells tested, indicating its potential adverse effects. Interestingly, however, the non-specific inhibitory feature of FTY720 seems not to alter its pharmacological action, according to regulatory role of FTY720 on the production of TNF-α and NO from LPS-activated RAW264.7 cells, regardless of serum level. Therefore, our results suggest that FTY720 might have additional non-specific anti-proliferative activity, distinct from its pharmacological activity.     

Bcl-2 mediated inhibition of erucylphosphocholine-induced apoptosis depends on its subcellular localisation

The synthetic phospholipid derivative erucylphosphocholine (ErPC) is a potent inducer of apoptosis in human tumor cell lines. This membrane-targeted drug induces apoptosis independently from death receptor signaling through a mitochondrial pathway that is inhibited by over-expression of Bcl-2.Within the cell, Bcl-2 resides in membranes of mitochondria, endoplasmic reticulum (ER) and the nucleus. However, the importance of its subcellular localisation in distinct organelles for protection against apoptosis is not completely understood.To investigate the impact of Bcl-2 localised at defined subcellular compartments on its protective effects against ErPC-induced apoptosis, Bcl-2 expression was directed to the outer membrane of the mitochondria or the ER of Jurkat T Lymphoma cells, using Bcl-2 mutants with modified membrane anchors. The mitochondrial insertion sequence of ActA directed Bcl-2 to the mitochondria (Bcl-2/MT), the ER-specific sequence of cytochrome b5 to the ER (Bcl-2/ER). Additionally, Jurkat cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/ΔTM) were employed.While restricted expression of Bcl-2 either at membranes of the mitochondria or the ER strongly interfered with ErPC-induced mitochondrial damage and apoptosis, cytosolic Bcl-2/ΔTM exhibited only reduced protection. Thus, membrane localisation of Bcl-2 is a prerequisite for substantial protection against ErPC-induced apoptosis. For efficient long-term inhibition of ErPC-induced apoptosis Bcl-2 had to be present in the membranes of both compartments, the ER and the mitochondria.The finding that ER-targeted Bcl-2 interferes with ErPC-induced mitochondrial damage points to an involvement of the ER in apoptosis signaling upstream of the mitochondria and to a crosstalk between both compartments.     

MK800-62F1, a new inhibitor of apoptotic cell death, from Streptomyces diastatochromogenes MK800-62F1. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity

A new compound, MK800-62F1, was isolated from a cultured broth of Streptomyces diastatochromogenes MK800-62F1. It inhibited H2O2-induced apoptosis in human small cell lung carcinoma Ms-1 cells as well as in human T-cell leukemia Jurkat cells. In addition, MK800-62F1 also inhibited camptothecin-induced apoptosis in Jurkat cells, which was mediated by intracellular H2O2 generation. MK800-62F1 did not exhibit antioxidative activity in vitro, suggesting that inhibition of apoptosis by MK800-62F1 was not due to the scavenging of H2O2, rather it was due to the modulation of the downstream event of H2O2 generation.     

FTY720 preferentially depletes naive T cells from peripheral and lymphoid organs

The sphingosine-1-phosphate receptor agonist FTY720 induces lymphopenia by inhibiting lymphocyte egress from thymus and lymph nodes. The immediate effect of the drug on T cells in blood and lymphoid tissues is well documented, however effects on peripheral T cell sub-populations have not been studied. We therefore analyzed the changes in T cell subset compositions in liver, lung, kidney, spleen, lymph nodes and blood induced by FTY720-treatment using 9-parameter flow cytometry. In untreated mice, naive T cells were present in all peripheral organs. Naive T cells were depleted from peripheral organs within 3 days by FTY720, and with slower kinetics from lymphoid organs. Antigen-experienced T cell subsets were less affected by FTY720-treatment and substantial numbers were retained in the periphery. The proportion of CD8(+)CD44(+)CD43(+) Gr-1(+) effector memory cells increased after FTY720-treatment, while that of CD8(+)CD44(+)CD62L(+) central memory cells was unchanged. Our data demonstrate that naive T cells pass peripheral tissues as part of their default recirculation pathway. FTY720 treatment primarily affects the recirculation of naive and central memory cells, both of which re-circulate through lymph nodes on a regular basis, but does not influence effector memory cells. This suggests that treatment with FTY720 may not interfere with immune functions mediated locally by tissue-resident peripheral effector/memory T cells.     

新型免疫抑制剂FTY720对大鼠胶原诱导性关节炎的抑制作用

目的观察新型免疫抑制剂FTY720对大鼠胶原诱导性关节炎发生和发展的影响。方法以胶原诱导大鼠关节炎模型,从第1次免疫造模开始,大鼠分别予以FTY720(0.1、0.6mg·kg-1)和雷公藤多苷(6、12mg·kg-1)灌胃治疗,连续4周。观察记录大鼠活动和关节炎指数、体重、足底厚度和足爪肿胀;血常规分析大鼠外周血淋巴细胞、单核细胞和中性粒细胞变化;流式分析外周血CD4+和CD8+T淋巴细胞变化;组织学及免疫组织化学分析炎性关节病理改变和滑膜组织中CD4+和CD8+T淋巴细胞的变化。结果 FTY720给药后能抑制大鼠关节炎的发生和发展,明显降低大鼠外周血淋巴细胞特别是CD4+T淋巴细胞数量和滑膜组织中CD4+T淋巴细胞数量。结论 FTY720可有效控制大鼠胶原诱导性关节炎的发生和发展,其作用机制可能是通过抑制外周血CD4+T淋巴细胞浸润到滑膜组织。     

Ciglitizone and 15d PGJ2 induce apoptosis in Jurkat and Raji cells

Several studies have shown that PPARgamma agonists play a role in the regulation of lymphocytes function and apoptosis. However, the molecular mechanism(s) underlying the immunomodulatory effects of PPARgamma agonists are not defined yet. In this study, the effects of PPARgamma (15d PGJ2 and ciglitizone) ligands on proliferation, cytokine production and apoptosis of Jurkat and Raji cells (human T and B lymphocytes, respectively) were examined. Ciglitizone and 15d PGJ2 presented antiproliferative and cytotoxic effects on Jurkat and Raji cells as shown by [14C]-thymidine incorporation and cell viability assay. In addition, 15d PGJ2 inhibited cytokine production (IL-2 in Jurkat cells and IL-10 in Raji cells). The mechanism whereby PPARgamma agonists induced cytotoxicity is via apoptosis as shown by DNA fragmentation, nuclear condensation and phosphatidylserine externalization. The induction of apoptosis by ciglitizone and 15d PGJ2 on Jurkat and Raji cells may explain the suppression of cytokine production and the decrease in proliferation observed in both cell types. The apoptotic process was associated with a decrease in mitochondrial membrane potential and a marked down-regulation of the c-myc expression. These findings might play a key role in the apoptosis of T and B lymphocytes induced by PPARgamma agonists.     

FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis

FTY720 (fingolimod; 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, Novartis) is the prototype of a new generation of immunomodulators. The drug is the result of extensive chemical derivatisation based on the natural product myriocin, isolated from the ascomycete Isaria sinclairii. FTY720 bears structural similarity to sphingosine, a naturally occurring sphingolipid. As with sphingosine, FTY720 is effectively phosphorylated by sphingosine kinases in vivo and the phosphorylated drug targets G-protein-coupled receptors for sphingosine-1-phosphate (S1P). Gene deletion and reverse pharmacology studies have shown that FTY720 acts at S1P₁ receptors on lymphocytes and the endothelium, thereby inhibiting the egress of T- and B cells from secondary lymphoid organs into the blood and their recirculation to inflamed tissues. Animal studies suggest that this novel mechanism translates into effective treatments for several autoimmune diseases and a recently completed Phase II clinical trial highlighted FTY720 as a potential therapy for relapsing-remitting multiple sclerosis.     

The New Isothiocyanate 4-(Methylthio)Butylisothiocyanate Selectively Affects Cell-Cycle Progression and Apoptosis Induction of Human Leukemia Cells

We investigated proliferation and apoptosis induction in Jurkat T-leukemia cells by the new isothiocyanate 4-(methylthio)butylisothiocyanate (MTBITC). To help elucidate whether the effects of MTBITC are specific for cancer cells, we tested MTBITC on freshly isolated, non-transformed human peripheral T lymphocytes. The effects of MTBITC are leukemic-cell-specific and consist of derangements in a critical point of cell-cycle control (G2/M transition). In fact, an increase in the proportion of G2 cells (from about 18% to 50%) was apparent following 24 h of treatment, associated with a decrease in the protein expression of cyclin B1. The expression of cyclin-dependent kinase (CDK) 1 was more mildly attenuated by MTBITC. Our results demonstrated that high concentrations of MTBITC can also induce apoptosis, through an increase of p53 and bax, but not bcl-2, protein expression. No effects of MTBITC were demonstrated on non-transformed T lymphocytes. Taking into account its in vitro antineoplastic activity and selectivity toward leukemia cells, MTBITC can be viewed as a conceptually promising agent in cancer therapy.     

γ-Tocotrienol induces apoptosis in human T cell lymphoma through activation of both intrinsic and extrinsic pathways

Tocotrienols are members of vitamin E family and possess broad biological activities including antioxidant, anti-inflammatory and antitumor effects. In the present study, we examine the potential of α-tocotrienol (AT) and γ-tocotrienol (GT) in inhibiting the proliferation of human T cell lymphoma Jurkat cells and elucidate the pathways involved in anti tumor effects of GT. GT but not AT inhibited proliferation and induced apoptosis in Jurkat cells in a dose dependent manner. GT treatment resulted in elevated mitochondrial ROS production, activation of JNK and suppression of ERK and p38 MAPK. GT also induced calcium release, loss of mitochondrial membrane potential and cytochrome c release from the mitochondria. These changes were accompanied by increase in Bax expression with a concomitant decrease in Bcl-xl expression suggesting activation of mitochondrial apoptotic pathway. GT induced increase in mitochondrial ROS was abrogated by catalase. Besides, GT also up-regulated surface expression of Fas and FasL on Jurkat cells. Further, caspase activation and PARP degradation were also seen in cells treated with GT. Inhibitors of caspase-8 and caspase-9 significantly abrogated GT mediated apoptosis. In contrast GT was not toxic to normal human peripheral blood mononuclear cells suggesting differential cytotoxicity towards normal lymphocytes and transformed lymphoma cells. Cellular uptake studies with tocotrienols showed higher intracellular accumulation of GT as compared to AT which may be responsible for its better antitumor activity. Our results show antitumor effects of GT in human lymphoma cells via increased mitochondrial ROS generation and activation of both intrinsic and extrinsic apoptotic pathways.     

Autophagy induced by FTY720 promotes apoptosis in U266 cells

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.