Neuroblastoma is the most common extracranial solid tumor that occurs during infancy. Despite the great progress has been made in contemporary clinic medicine some forms of neuroblastoma disease are still found very difficult to treat . This work focuses on the effects of histone deacetylase inhibitors (HDAC) in the neuroblastoma cell lines. It is known that HDAC inhibitors may contribute to recurrence of the tumor cells by affecting the chromatin structure and thus increase the expression of critical tumor suppressor genes. These genes activate apoptotic pathways that may even be independent of caspases. We observed the efficiency of used HDAC inhibitors as under standard conditions an in hypoxia (1 % O2). Inadequate amount of oxygen supply is one of the characteristic features of tumors and it also may contribute to chemoresistance. With the hypoxia-induced chemoresistance of tumor cells, the influence of HIF-1α is expected. Some HDAC inhibitors reduce the amount of HIF-1α in hypoxia and thus HIF transcription factor activity. Thus, the first part of this study is concerned with the acquisition of suitable experimental arrangement for the monitoring of induction of cellular death in human neuroblastoma cell lines SK-N-AS and UKF-NB-3. Secondly, this paper provides the evaluation of the influence...

Als Kapazitation bezeichnet man den im weiblichen Reproduktionstrakt stattfindenden Reifungsschritt, der Spermien das volle Fertilisierungspotential verleiht. Die molekularbiologischen Grundlagen dieses für eine erfolgreiche natürliche oder auch artifizielle Befruchtung essenziellen Prozesses sind bis heute nur unvollständig verstanden. Im Rahmen der vorliegenden Dissertation wurden die mit der Kapazitation einhergehenden funktionellen und strukturellen spermalen Veränderungen untersucht. Die kapazitative Stimulation führte zu einer gesteigerten Motilität bis hin zur Hyperaktivierung, zu einer vermehrt induzierten Akrosomenreaktion und zu einer deutlich reduzierten Apoptoseaktivität. Anhand von Inhibitionsexperimenten wurde die Rolle der potentiellen Signaltransduktoren Caspase-1, Calpain und Calmodulin analysiert. Dabei wies die Calmodulinantagonisierung auf eine ausgeprägte Calciumabhängigkeit aller untersuchten kapazitationsassoziierten Prozesse hin. Die Hemmung von Caspase-1 und Calpain führte zu einer Beeinträchtigung der Motilität und der Akrosomenreaktion ohne das Ausmaß der Apoptoseinduktion zu beeinflussen. Die vorstehend genannten Erkenntnisse wurden zur Evaluation verschiedener Ejakulataufbereitungsprotokolle genutzt. Dabei konnte gezeigt werden, dass die Kombination des modernen Verfahrens der immunomagnetische Zellseparation mit der etablierten Methode der Dichtegradientenzentrifugation dem einfachen Standard in Bezug auf die Anreicherung hochmotiler Spermien mit minimaler Apoptoseaktivität aus frischen wie auch aus kryokonservierten Ejakulaten deutlich überlegen war. Bedeutsam im Hinblick auf eine mögliche pratische Anwendung der immunomagnetischen Zellseparation erscheint der Befund, dass die durch das kombinierte Anreicherungsverfahren erhaltene Spermatozoensubpopulation im Hamsteroozytenpenetrationstest ein signifikant höheres Fertilisierungspotential zeigte.

Hepatitis C virus (HCV) is remarkable for its ability to establish persistent infection. Studies suggest that HCV core protein modulates immune responses to viral infection and can bind Fas receptor in vitro. To further examine the role of HCV core protein in Fas signaling, full-length (aa 1-192) and truncated (aa 1-152) HCV core proteins were expressed in Jurkat lymphocytes and cells were assayed for apoptotic response, caspase activation, and Fas activation. Jurkat expressing full-length but not truncated core protein exhibited ligand-independent apoptosis. Cytoplasmic targeting of truncated core protein recapitulated its ability to induce apoptosis. Activation of caspases 8 and 3 was necessary and sufficient for full-length core to induce apoptosis. Jurkat cells expressing full-length but not truncated core protein induced Fas receptor aggregation. HCV core activates apoptotic pathways in Jurkat via Fas and requires cytoplasmic localization of core. Infection of host lymphocytes by HCV may alter apoptotic signaling and skew host responses to acute infection.

Our previous studies have shown that β-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which β-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either β-arrestin 1 or β-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. β-arrestin 2 deficient-induced apoptosis was inhibited by transfection with β-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on β-arrestin 2. Furthermore, in the absence of either β-arrestin 1 or β-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either β-arrestin 1 or β-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of β-arrestin 1/2. Our study thus demonstrates that β-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.

β-arrestins, a family of regulatory and scaffold proteins, are well-known negative regulators of G-protein-coupled receptors (GPCRS) including opioid receptors. Recent studies have shown that β-arrestin2 plays a potential role in inhibiting cell death. It has been reported that opioids such as morphine induce cell death at high concentrations (>500 μM for 24 hours), which is similar to morphine plasma concentrations in cancer patients receiving chronic morphine treatment for pain relievers. However, the role of β-arrestin2 in opioid-induced cell death remains to be elucidated. We report here that β-arrestin2 significantly blocks morphine-induced number of cell death in human breast cancer MCF-7 and MDA-MB231 cells. Suppression of endogenous β-arrestin2 by specific RNA interfering (RNAi) and morphine treatment significantly attenuates the levels of phosphorylated Akt compared with inhibition of β-arrestin2 or morphine treatment alone. However, blockade of morphine-induced cell death by β-arrestin2 seems to be dependent on the inhibition of caspase-8, as inhibition of β-arrestin2 and morphine treatment significantly enhanced the levels of cleaved caspase-8. These studies show for the first time that β-arrestin2 blocks morphine-induced cell death through anti-apoptotic Akt and pro-apoptotic caspase-8 pathways. Therefore, targeting β-arrestin2 may be useful for treating side effects of opioids as pain relievers for cancer patients.

A novel marine active polypeptide (PCF), isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether UVB-induced HaCaT cell apoptosis occurs via the mitochondrial pathways Apaf-1/caspase-9 and Smac/XIAP/caspase-3. We then investigated the molecular mechanisms controlling the anti-apoptotic effect of PCF. Pre-treatment with PCF and caspase-9 inhibitor significantly inhibited UVB-induced apoptosis in HaCaT cells based on a DNA fragmentation assay and Hoechst 33258 staining. The expression of Apaf-1 and the cleavage of procaspase-9 were dose-dependently reduced by 1.42-5.96 mmol/L PCF pretreatment in UVB-irradiated HaCaT cells. This was followed by inhibition of cleavage of procaspase-3, whose activation induced cell apoptosis. Meanwhile, PCF significantly and dose-dependently enhanced the activation of ATPase. Furthermore, we demonstrated that PCF strongly inhibited the release of Smac from the mitochondria to cytosol by reducing the degradation of XIAP dose-dependently. We conclude that the protective effect of PCF against UVB irradiation in HaCaT cells may be attributed to the inhibition of the Apaf-1/caspase-9 and Smac/XIAP/caspase-3 apoptotic signaling pathways.

A novel small molecule, 1-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (EOD), was synthesized in our laboratory. Previously, we reported pharmacological properties of EOD, triggering apoptosis in Human umbilical vein endothelial cells (HUVECs). Here, we further investigated the effects of EOD on the growth of A549 human lung cancer cells. EOD treatment induced apoptosis in A549 cells via up-regulating the expression of P53 protein, blocking cell cycle partly at G1 phase, and ultimately activating caspase-3. In contrast, caspase-8 might be irrelevant to EOD-triggered apoptosis. This study indicated that EOD might be a potential chemopreventive agent for lung cancer. The work would encourage us to add more novel compounds to our 'library' of small molecules derived through modern synthetic organic chemistry, and would drive us to determine the proteins that the compounds target.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20782号 / 生博第388号 / 新制||生||51(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Chronic kidney disease (CKD) affects 13.4% of adults in America; and 38% in people aged 65 years or older[1]. In addition, cardiovascular disease (CVD) is the leading cause of death in CKD patients with end-stage kidney disease. CKD is associated with chronic inflammation, which contributes to the progression of CVD[2]. Furthermore, CKD alter apolipoprotein profile and elevate plasma lipid levels. It has been reported that 68.8% of CKD patients are associated with hyperlipidemia[3]. Therefore, hyperlipidemia is the critical risk factor for cardiovascular morbidity and mortality in CKD patients [4, 5]. In addition, trained immunity has been shown to play a critical role in chronic inflammatory diseases[6]. However, whether trained immunity promotes the inflammation in hyperlipidemia-CKD remains unclear. Circulating lipopolysaccharide (LPS) is significantly increased in atherosclerotic and CKD patients[7]. Clinical data indicates that circulating LPS is positively associated with the progression of CKD, and its levels even higher in patients with hemodialysis or dialysis[8]. Studies found that circulating LPS is delivered into cytosol for caspase-4/11 activation[9]. The Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) involving 10,061 patients found that targeting interleukin-1β (IL-1β) innate immunity pathway is significantly lowered the rate of recurrent cardiovascular events independent of lipid-level lowering[10]. Therefore, inhibiting the secretion of proinflammatory cytokine IL-1β has high potential to future development of novel therapeutics for hyperlipidemia-CKD accelerated CVD. Gasdermin D (GSDMD) is cleaved by inflammatory caspase-1 and caspase-4. N-terminal GSDMD binds to plasma membrane forming protein channel [11] and mediates the secretion of IL-1β[12, 13]. We found that caspase-1 activation was significantly decreased in caspase-4/11 deficient high-fat diet (HFD)-CKD mice, indicating that caspase-4 could regulate caspase-1 activation in HFD-CKD. Whether increased cytosolic LPS contribute to the increased vascular inflammation via caspase-4/11-GSDMD-IL-1β pathway remains unknown. In this study, we used HFD fed 5/6 nephrectomy CKD mice in vivo and cytosolic LPS stimulation in human aortic endothelial cell (HAECs) in vitro. We made the following results: 1) Inflammatory pathways are significantly increased in the aorta of HFD-CKD compared to HFD-Sham, normal diet (ND)-CKD, and ND-Sham. 2) Expression levels of endothelial cell activation markers (ICAM1 and VCAM1) are significantly increased in the aorta of HFD-CKD mice compared to HFD-Sham, ND-CKD, and ND-Sham. 3) Caspase-4 activation and N-GSDMD cleavage are significantly increased in the aorta of HFD-CKD mice compared to HFD-Sham, ND-CKD, and ND-Sham and in cytosolic LPS stimulated HAECs. 4) The increased inflammatory pathways and increased expression of adhesion molecules are decreased in the deficiency of caspase-4 in vivo and in the presence of caspase-4 inhibitor and N-terminal GSDMD cleavage inhibitor in vitro. 5) The increased mitochondrial ROS promote endothelial cell activation via caspase-4-GSDMD axis. Taken together, the caspase-4/11-GSDMD axis mediates endothelial cell activation and vascular inflammation in the aorta of HFD-CKD mice compared to controls. Furthermore, the increased endothelial cell activation and vascular inflammation are restored by caspase-4/11 deficiency in the aorta of HFD-CKD mice. These evidence indicate that inhibiting caspase-4/11-GSDMD axis could be a potential therapeutic target for inhibiting vascular inflammation associated with hyperlipidemia-CKD. / Biomedical Sciences

Indiana University-Purdue University Indianapolis (IUPUI) / The anti-apoptotic protein Survivin and the cyclin-dependent kinase p34Cdc2 are involved in cell cycle progression and apoptosis. Activation of Cdc2 is required for its pro-apoptotic activity, which can be inhibited by phosphorylation at Tyrosine-15 (Tyr15). In transduced IL-3-dependent murine BaF3 hematopoietic cells, over-expression of wild-type-(wt)-Survivin increased Cdc2-Tyr15 phosphorylation, while over-expression of a dominant-negative-(dn)-T34A-Survivin construct decreased its phosphorylation. The increased phospho-Tyr15 levels associated with ectopic Survivin directly correlated with enhanced BaF3 cell survival in the absence of growth factors, and low phospho-Tyr15 levels observed in cells expressing ectopic dn-Survivin correlated with decreased survival. BaF3 cells transduced with Internal Tandem Duplication (ITD) mutations of the Flt3 receptor that results in increased Survivin levels, also contained increased levels of Tyr15 phosphorylated Cdc2. In BaF3 cells over-expressing wt-Survivin, 2-fold higher levels of Wee1 protein were observed compared to cells expressing control vector alone. Treatment of control BaF3 cells with the caspase-3 inhibitor Ac-DEVD-CHO increased both Cdc2-Tyr15 phosphorylation and Wee1 protein levels. In a similar fashion over-expression of wt-Survivin in these cells maintained high levels of Tyr15 phosphorylated Cdc2 and Wee1 protein. In MCF7 human breast cancer cells that lack caspase-3, increase of Tyr15 phosphorylated Cdc2 and Wee1 kinase protein by caspase-3, -7 or a pan-caspase inhibitor was absent, linking Survivin and caspase-3 to the increase of Wee1 and Tyr15 phosphorylation of Cdc2. To further link Survivin and Cdc2, we treated cells with AICAR and 17-AAG that inhibit Hsp90, which is known to be required for Survivin stability. Treatment of BaF3 cells expressing wt-Survivin with AICAR and 17-AAG decreased Cdc2-Tyr15 phosphorylation compared to vehicle-treated control cells. Taken together, these results indicate that Survivin protects the Cdc2-Tyr15-targeting kinase Wee1 from degradation by caspase-3 which leads to increased inhibitory Cdc2-Tyr15 phosphorylation resulting in reduced apoptosis and enhanced survival.