Vitamin E succinate inhibits survivin and induces apoptosis in pancreatic cancer cells

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Identifying novel chemotherapeutic and chemopreventive approaches is critical in the prevention and treatment of cancers such as pancreatic cancer. Vitamin E succinate (VES) is a redox-silent analog of the fat-soluble vitamin alpha-tocopherol. In the present study, we explored the antiproliferative action of VES and its effects on inhibitor of apoptosis proteins in pancreatic cancer cells. We show that VES inhibits cell proliferation and induces apoptosis in pancreatic cancer cells. Further, we demonstrate that VES downregulates the expression of survivin and X-linked inhibitor of apoptosis proteins. The apoptosis induced by VES was augmented by siRNA-mediated inhibition of survivin in PANC-1 cells. In summary, our results suggest that VES targets survivin signaling and induces apoptosis in pancreatic cancer cells.     

Survivin contributes to the anti-apoptotic activities of transforming growth factor alpha in mouse blastocysts through phosphatidylinositol 3'-kinase pathway

Transforming growth factor alpha (TGFA) is produced by epithelial cells in the oviducts and uteri and has the potential to act as an anti-apoptotic factor on preimplantation embryos expressing its receptor. Previously, we demonstrated that survivin (also known as BIRC5), an anti-apoptotic gene expressed in mouse preimplantation embryos, protects embryos from apoptosis. In this study, we investigated the role of survivin on TGFA-mediated inhibition of apoptosis in mouse blastocysts. Under the suboptimal conditions produced by single embryo culture, blastocysts showed an increase of apoptosis that correlated with a decrease of survivin expression. TGFA treatment significantly decreased apoptosis and increased the levels of survivin mRNA in a dose-dependent manner in blastocyst, and conversely, these activities were neutralized by an anti-TGFA antibody. Antibody treatment alone exerted little effect on either the occurrence of apoptosis or the levels of survivin mRNA. Upregulation of survivin expression by TGFA treatment was insignificant before the blastocyst stage. Using an antisense approach, we examined whether upregulation of survivin is responsible for the anti-apoptotic effect of TGFA in blastocysts. Apoptosis was inhibited by TGFA treatment in blastocysts, but the effect was abrogated by cotreatment with antisense oligonucleotides directed against survivin. These data suggest that survivin contributes to the anti-apoptotic activities of TGFA in blastocysts. We also found that the upregulation of survivin expression was mediated by activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Thus, TGFA inhibits apoptosis in mouse blastocysts through upregulation of survivin expression via the PI3K pathway.     

ICOS通过Survivin维持T细胞分裂及存活

有关T细胞共刺激分子信号转导方面的研究远落后于其功能研究,为探讨T细胞活化后诱导表达的共刺激分子ICOS(induciblecostimulate)维持T细胞存活、抑制活化后T细胞凋亡的作用是否与survivin相关,利用survivin重组腺病毒感染活化但不提供共刺激信号的T淋巴细胞,或者在活化后提供ICOS信号的条件下人工给予优势抑制survivin突变基因,CCK-8及TUNEL法分别检测活化晚期上述T细胞存活及凋亡情况.结果显示,T细胞活化后2～6天,ICOS抗体刺激可以明显增强survivin表达,survivin可维持无ICOS信号的T细胞存活减少其凋亡,突变型survivin在ICOS信号存在下抑制T细胞存活使其凋亡增加.结果提示,活化后表达的共刺激分子ICOS通过survivin维持T细胞分裂和存活.     

Rapamycin decreases survivin expression to induce NSCLC cell apoptosis under hypoxia through inhibiting HIF-1α induction

Survivin is a member of the inhibitor of apoptosis protein family that is overexpressed in various tumors and is important in restricting apoptosis. Understanding the molecular events of apoptosis may provide information for developing novel therapeutic agents targeting non-small cell lung cancer (NSCLCs). This study used three human NSCLC cell lines, NCI-H1299, SK-MES-1, and NCI-H460. Changes in apoptosis, the mRNA and protein expression of survivin under normoxia and hypoxia, with or without rapamycin treatment were analyzed. In addition, siRNA and ChIP assay were further applied to demonstrate the role of hypoxia-inducible factor 1 (HIF-1)α in regulating survivin expression regulation under hypoxia during rapamycin induced NSCLC cell apoptosis. Treatment with rapamycin resulted in significantly increased NSCLC cells apoptosis under hypoxia. We demonstrated for the first time that rapamycin inhibited hypoxia-induced survivin expression in NSCLC cell lines. We further demonstrated that HIF-1α participated in hypoxia-induced survivin expression, and that rapamycin inhibited hypoxia-induced HIF-1α expression by enhancing its degradation. The results above collectively showed that rapamycin inhibits HIF-1α-induced survivin expression under hypoxia to induce NSCLC apoptosis.     

Survivin monomer plays an essential role in apoptosis regulation

Survivin was initially described as an inhibitor of apoptosis and attracted growing attention as one of the most tumor-specific genes in the human genome and a promising target for cancer therapy. Lately, it has been shown that survivin is a multifunctional protein that takes part in several crucial cell processes. At first, it was supposed that survivin functions only as a homodimer, but now data indicate that many processes require monomeric survivin. Moreover, recent studies reveal a special mechanism regulating the balance between monomeric and dimeric forms of the protein. In this paper we studied the mutant form of survivin that was unable to dimerize and investigated its role in apoptosis. We showed that survivin monomer interacts with Smac/DIABLO and X-linked inhibitor of apoptosis protein (XIAP) both in vitro and in vivo. Due to this feature, it protects cells from caspase-dependent apoptosis even more efficiently than the wild-type survivin. We also identified that mutant monomeric survivin prevents apoptosis-inducing factor release from the mitochondrial intermembrane space, protecting human fibrosarcoma HT1080 cells from caspase-independent apoptosis. On the other hand, our results indicate that only wild-type survivin, but not the monomer mutant form, enhances tubulin stability in cells. These findings suggest that survivin partly performs its functions as a monomer and partly as a dimer. The mechanism of dimer-monomer balance regulation may also work as a "switcher" between survivin functions and thereby explain remarkable functional diversities of this protein.     

Novel synthetic oleanane triterpenoid AMR-MeOAc inhibits K-Ras through ERK,Akt and survivin in pancreatic cancer cells

K-Ras activating mutations are a major problem that drives aggressive tumor growth and metastasis in pancreatic cancer. Currently, there are no effective targeted therapies for this genetically defined subset of cancers harboring oncogenic K-Ras mutations that confer drug resistance, aggressive tumor growth, metastasis and poor clinical outcome. We identified a novel synthetic oleanane triterpenoid compound designated AMR-MeOAc that effectively kills K-Ras mutant pancreatic cancer HPAF-II cells. The cytotoxic effects correlated with apoptosis induction, as was evidenced by increase of apoptosis cells upon the treatment of AMR-MeOAc in HPAF-II cells. Our studies revealed that AMR-MeOAc treatment inhibits cancer associated survival gene survivin. Moreover, AMR-MeOAc also led to down regulation of Akt, ERK1/2 and survivin protein levels. Our results indicate that AMR-MeOAc or its active analogs could be a novel class of anticancer agents against K-Ras driven human pancreatic cancer.     

Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84--> Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via beta1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-alpha-induced apoptosis. Evidence is provided that expression of beta1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-alpha-induced apoptosis. In contrast, the beta1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-alpha in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis.     

Compartmentalized phosphorylation of IAP by protein kinase A regulates cytoprotection

Cell death pathways are likely regulated in specialized subcellular microdomains, but how this occurs is not understood. Here, we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates the inhibitor of apoptosis (IAP) protein survivin on Ser20 in the cytosol, but not in mitochondria. This phosphorylation event disrupts the binding interface between survivin and its antiapoptotic cofactor, XIAP. Conversely, mitochondrial survivin or a non-PKA phosphorylatable survivin mutant binds XIAP avidly, enhances XIAP stability, synergistically inhibits apoptosis, and accelerates tumor growth, in vivo. Therefore, differential phosphorylation of survivin by PKA in subcellular microdomains regulates tumor cell apoptosis via its interaction with XIAP.     

Threonine 48 in the BIR domain of survivin is critical to its mitotic and anti-apoptotic activities and can be phosphorylated by CK2 in vitro

In this study we report that the protein kinase CK2 phosphorylates survivin specifically on threonine 48 (T48) within its BIR domain, and that T48 is critical to both the mitotic and anti-apoptotic roles of survivin. Interestingly, during mitosis T48 mutants localise normally, but are unable to support cell growth when endogenous survivin is removed by siRNA. In addition, while overexpression of survivin normally confers inhibition of TRAIL-mediated apoptosis, this protection is abolished by mutation of T48. Furthermore in interphase cells depletion of endogenous survivin causes redistribution of T48 mutants from the cytoplasm to the nucleus and treatment of cells expressing survivin-GFP with the CK2 inhibitor TBB phenocopies this nuclear redistribution. Finally, we show T48 mutants have increased affinity for borealin, and that this association and cell proliferation can be restored by introduction of a second mutation at T97. To our knowledge these data are the first to identify T48 as a key regulatory site on survivin, and CK2 as a mediator of its mitotic and anti-apoptotic functions.Key words: survivin, chromosomal passenger protein, CK2, mitosis, apoptosis, phosphorylation     

Inhibition of fibroblast growth factor 2-induced apoptosis involves survivin expression, protein kinase C alpha activation and subcellular translocation of Smac in human small cell lung cancer cells

To investigate the mechanism by which fibroblast growth factor 2 (FGF-2) inhibits apoptosis in the human small cell lung cancer cell line H446 subjected to serum starvation, apoptosis was evaluated by flow cytometry, Hoechst 33258 staining, caspase-3 activity, and DNA fragmentation. Survivin expression induced by FGF-2 and protein kinase Cα (PKCα) translocation was detected by subcellular frac-tionation and Western blot analysis. In addition, FGF-2-in-duced release of Smac from mitochondria to the cytoplasm was analyzed by Western blotting and immunofluorescence. FGF-2 reduced apoptosis induced by serum starvation and up-regulated survivin expression in H446 cells in a dose-dependent andtime-dependentmanner, andinhibitedcaspase-3 activity. FGF-2 also inhibited the release of Smac from mitochondria to the cytoplasm induced by serum starvation and increased PKCα translocation from the cytoplasm to the cell membrane. In addition, PKC inhibitor inhibited the expression of survivin. FGF-2 up-regulates the expression of survivin protein in H446 cells and blocks the release of Smac from mitochondria to the cytoplasm. PKCα regulated FGF-2-induced survivin expression. Thus, survivin, Smac, and PKCα might play important roles in the inhibition of apoptosis by FGF-2 in human small cell lung cancer cells.     

An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7

Survivin, an apoptosis inhibitor/cell-cycle regulator, is critically required for suppression of apoptosis and ensuring normal cell division in the G2/M phase of the cell cycle. It is highly expressed in a cell cycle-regulated manner and localizes together with caspase-3 on microtubules within centrosomes. Whether survivin is a physiologically relevant caspase inhibitor has been unclear due to the difficulties with obtaining correctly folded survivin and finding the right conditions for inhibition assay. In this study, recombinant, active human survivin was expressed in Escherichia coli and purified to homogeneity. The protein, existing as a homodimer in solution, binds caspase-3 and -7 tightly with dissociation constants of 20.9 and 11.5 nM, respectively, when evaluated by surface plasmon resonance spectroscopy. Consistently, survivin potently inhibits the cleavage of a physiological substrate poly(ADP-ribose) polymerase and an artificial tetrapeptide by caspase-3 and -7 in vitro with apparent inhibition constants of 36.0 and 16.5 nM, respectively. The data suggest that sequestering caspase-3 and -7 in inhibited states on microtubules is at least one mechanism of survivin in the suppression of default apoptosis in the G2/M phase. The localization of survivin on microtubules, which is essential for its function, should increase the protective activity at the action site.     

Threonine 48 in the BIR domain of survivin is critical to its mitotic and anti-apoptotic activities and can be phosphorylated by CK2 in vitro

In this study we report that the protein kinase CK2 phosphorylates survivin specifically on threonine 48 (T48) within its BIR domain, and that T48 is critical to both the mitotic and anti-apoptotic roles of survivin. Interestingly, during mitosis T48 mutants localise normally, but are unable to support cell growth when endogenous survivin is removed by siRNA. In addition, while overexpression of survivin normally confers inhibition of TRAIL-mediated apoptosis, this protection is abolished by mutation of T48. Furthermore in interphase cells depletion of endogenous survivin causes redistribution of T48 mutants from the cytoplasm to the nucleus and treatment of cells expressing survivin-GFP with the CK2 inhibitor TBB phenocopies this nuclear redistribution. Finally, we show T48 mutants have increased affinity for borealin, and that this association and cell proliferation can be restored by introduction of a second mutation at T97. To our knowledge these data are the first to identify T48 as a key regulatory site on survivin, and CK2 as a mediator of its mitotic and anti-apoptotic functions.     

Survivin as a Potential Mediator to Support Autoreactive Cell Survival in Myasthenia Gravis: A Human and Animal Model Study

The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular junction, associated with marked reduction of survivin-expressing circulating CD20+ cells. These data strongly suggest that survivin expression in cells with lymphocyte and plasma cell markers occurs in patients with myasthenia gravis and in two animal models of myasthenia gravis. Survivin expression may be part of a mechanism that inhibits the apoptosis of autoreactive B cells in myasthenia gravis and other autoimmune disorders.