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Apoptosis represents one of the most important forms of cell death in higher organisms and is typically dysregulated in human cancers, including pediatric tumors. This implies that ineffective engagement of cell death programs can contribute to tumor formation as well as tumor progression. In addition, the majority of cytotoxic therapeutic principles rely on the activation of cell death signaling pathways in cancer cells. Blockade of signaling networks that lead to cell death can therefore confer treatment resistance. A variety of genetic and epigenetic events as well as dysfunctional regulation of signaling networks have been identified as underlying causes of cell death resistance in childhood malignancies. Apoptosis pathways can be therapeutically exploited by enhancing proapoptotic signals or by neutralizing antiapoptotic programs. The challenge in the coming years will be to successfully transfer this knowledge into the development of innovative treatment approaches for children with cancer.
Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications
(2012)
Conjugation of ubiquitin to proteins (ubiquitylation) has emerged to be one of the most crucial post-translational modifications controlling virtually all cellular processes. What was once regarded as a mere signal for protein degradation has turned out to be a major regulator of molecular signalling networks. Deregulation of ubiquitin signalling is closely associated with various human pathologies. Here, we summarize the current knowledge of ubiquitin signalling in immune deficiencies and cancer as well as the available therapeutic strategies targeting the ubiquitin system in combating these pathogenic conditions.
Apigenin (4′,5,7-trihydroxyflavone) (Api) is an important component of the human diet, being distributed in a wide number of fruits, vegetables and herbs with the most important sources being represented by chamomile, celery, celeriac and parsley. This study was designed for a comprehensive evaluation of Api as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound. In the set experimental conditions, Api presents antiproliferative activity against the A375 human melanoma cell line, a G2/M arrest of the cell cycle and cytotoxic events as revealed by the lactate dehydrogenase release. Caspase 3 activity was inversely proportional to the Api tested doses, namely 30 μM and 60 μM. Phenomena of early apoptosis, late apoptosis and necrosis following incubation with Api were detected by Annexin V-PI double staining. The flavone interfered with the mitochondrial respiration by modulating both glycolytic and mitochondrial pathways for ATP production. The metabolic activity of human dendritic cells (DCs) under LPS-activation was clearly attenuated by stimulation with high concentrations of Api. Il-6 and IL-10 secretion was almost completely blocked while TNF alpha secretion was reduced by about 60%. Api elicited antiangiogenic properties in a dose-dependent manner. Both concentrations of Api influenced tumour cell growth and migration, inducing a limited tumour area inside the application ring, associated with a low number of capillaries.
We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.
Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.
Carcinogenesis is a multistep process. Besides somatic mutations in tumor cells, stroma-associated immunity is a major regulator of tumor growth. Tumor cells produce and secrete diverse mediators to create a local microenvironment that supports their own survival and growth. It is becoming apparent that iron acquisition, storage, and release in tumor cells is different from healthy counterparts. It is also appreciated that macrophages in the tumor microenvironment acquire a tumor-supportive, anti-inflammatory phenotype that promotes tumor cell proliferation, angiogenesis, and metastasis. Apparently, this behavior is attributed, at least in part, to the ability of macrophages to support tumor cells with iron. Polarization of macrophages by apoptotic tumor cells shifts the profile of genes involved in iron metabolism from an iron sequestering to an iron-release phenotype. Iron release from macrophages is supposed to be facilitated by ferroportin. However, lipid mediators such as sphingosine-1-phosphate, released form apoptotic tumor cells, upregulate lipocalin-2 (Lcn-2) in macrophages. This protein is known to bind siderophore-complexed iron and thus, may participate in iron transport in the tumor microenvironment. We describe how macrophages handle iron in the tumor microenvironment, discuss the relevance of an iron-release macrophage phenotype for tumor progression, and propose a new role for Lcn-2 in tumor-associated macrophages.
Atherosklerotische Stenosen der Karotiden sind eine häufige Erkrankung mit variablem Verlauf und stellen durch ihre potentielle Emboligenität einen wichtigen Risikofaktor für zerebrale Ischämien dar. Klinische und paraklinische Parameter helfen, das individuelle Schlaganfallrisiko bei Patienten mit hochgradigen ACI-Stenosen einzuschätzen, das unmittelbar nach einem thrombembolischen Ereignis besonders hoch ist. Als histomorphologisches Korrelat dieser "Vulnerabilität" wird die Ruptur der fibrotischen Deckplatte der Plaque propagiert, die häufiger bei symptomatischen Patienten nachzuweisen ist. Sie korreliert mit der Infiltration der Gefäßwand durch aktivierte Leukozyten, die über molekulare und zelluläre Interaktionen die Zell- und Bindegewebskomposition der Plaque verändern können. Die strukturelle Integrität atherosklerotischer Läsionen beruht auf der extrazellulären Vernetzung von kollagenem Bindegewebe, das überwiegend von phänotypisch veränderten glatten Gefäßmuskelzellen produziert wird. Eine Hypothese besagt, dass die im Rahmen der Inflammation stattfindende Zunahme proapoptotischer Mediatoren über eine Ausdünnung der zellulären und bindegewebigen Strukturen zu einem Verlust an mechanischer Stabilität führt und somit eine symptomatische Ruptur begünstigt. Da der Nachweis einer Ruptur mit Exponierung des thrombogenen nekrotischen Kerns allerdings nur in einem Teil der symptomatischen Plaques und umgekehrt auch in einem Teil der asymptomatischen nachgewiesen werden kann, ist aber bislang unklar, ob o.g. Abläufe in der humanen Karotis-Atherosklerose tatsächlich mit einer klinischen Relevanz einhergehen. In der vorliegenden Arbeit wurde daher das Auftreten der Apoptose von glatten Gefäßmuskelzellen (mittels DNA in situ end labeling Technik, TUNEL-Färbung) in 38 prospektiv gesammelten Endarterektomie-Präparaten hochgradiger Karotisstenosen quantitativ erfasst und statistisch in Beziehung gesetzt zu Parametern der Plaque-Instabilität, klinisch definiert durch kürzliche (< 60 Tage zurückliegende) ischämische Ereignisse (n=19) und histopathologisch definiert über den Nachweis einer Plaque-Ruptur (n=14). Außerdem wurde eine morphometrische Analyse der einzelnen Plaque-Komponenten durchgeführt und deren Ergebnisse zu den zellulären und klinischen Parametern in Beziehung gesetzt. Die Morphometrie ergab keine signifikanten Unterschiede zwischen symptomatischen vs. asymptomatischen und rupturierten vs. unrupturierten Plaques was die Größe der fibrotischen Deckplatte, die durchschnittliche Dicke (Kern-Lumen-Distanz) und die dünnsten bzw. dicksten Stellen der Deckplatte anbelangt. Anzahl und Konzentration apoptotischer glatter Muskelzellen war deutlich (p<0,001) erhöht in symptomatischen, klinisch instabilen, Karotisplaques. Allerdings waren die Apoptose-Raten in Präparaten, die eine Plaque-Ruptur aufwiesen, nicht signifikant erhöht. Darüber hinaus fand sich kein Hinweis darauf, dass erhöhte Apoptose-Raten zu einem quantifizierbaren Verlust glatter Gefäßmuskelzellen in der fibrotischen Deckplatte führen. Auf dem Boden dieser Ergebnisse kann gefolgert werden, dass erhöhten Apoptose-Raten glatter Gefäßmuskelzellen in der humanen Atherosklerose offenbar eine tragende Bedeutung bei der Entwicklung thrombembolischer Ereignisse zukommt. Allerdings wird die Annahme, dass erhöhte Apoptose-Raten über einen Verlust an glatten Gefäßmuskelzellen Einfluss auf die morphometrischen Eigenschaften der fibrotischen Deckplatte atherosklerotischer Karotis-Läsionen nehmen und zu deren Ausdünnung führen durch die vorliegende Untersuchung nicht gestützt. Vielmehr scheint es plausibel, dass die Apoptose glatter Muskelzellen im Rahmen inflammatorischer Prozesse Einfluss auf die Komposition der Karotisplaque nimmt und so über eine Desintegration der zellulären und bindegewebigen Bestandteile zu reduzierter mechanischer Widerstandskraft und Rupturneigung führt.
Regulation of the antiapoptotic protein cFLIP by the glucocorticoid Dexamethasone in ALL cells
(2018)
We recently reported that the Smac mimetic BV6 and glucocorticoids, e.g. Dexamethasone (Dexa), synergize to induce cell death in acute lymphoblastic leukemia (ALL) in vitro and in vivo. Here, we discover that this synergism involves Dexa-stimulated downregulation of cellular FLICE-like inhibitory protein (cFLIP) in ALL cells. Dexa rapidly decreases cFLIPL protein levels, which is further enhanced by addition of BV6. While attenuating the activation of non-canonical nuclear factor-kappaB (NF-κB) signaling by BV6, Dexa suppresses cFLIPL protein but not mRNA levels pointing to a transcription-independent downregulation of cFLIPL by Dexa. Analysis of protein degradation pathways indicates that Dexa causes cFLIPL depletion independently of proteasomal, lysosomal or caspase pathways, as inhibitors of the proteasome, lysosomal enzymes or caspases all failed to protect from Dexa-mediated loss of cFLIPL protein. Also, Dexa alone or in combination with BV6 does not affect overall activity of the proteasome. Importantly, overexpression of cFLIPL to an extent that is no longer subject to Dexa-imposed downregulation rescues Dexa/BV6-mediated cell death. Vice versa, knockdown of cFLIP increases BV6-mediated cell death, thus mimicking the effect of Dexa. Altogether, these data demonstrate that Dexa-mediated downregulation of cFLIPL protein promotes Dexa/BV6-mediated cell death, thereby providing novel insights into the synergistic antitumor activity of this combination treatment.
The deregulation of Polo-like kinase 1 is inversely linked to the prognosis of patients with diverse human tumors. Targeting Polo-like kinase 1 has been widely considered as one of the most promising strategies for molecular anticancer therapy. While the preclinical results are encouraging, the clinical outcomes are rather less inspiring by showing limited anticancer activity. It is thus of importance to identify molecules and mechanisms responsible for the sensitivity of Polo-like kinase 1 inhibition. We have recently shown that p21Cip1/CDKN1A is involved in the regulation of mitosis and its loss prolongs the mitotic duration accompanied by defects in chromosome segregation and cytokinesis in various tumor cells. In the present study, we demonstrate that p21 affects the efficacy of Polo-like kinase 1 inhibitors, especially Poloxin, a specific inhibitor of the unique Polo-box domain. Intriguingly, upon treatment with Polo-like kinase 1 inhibitors, p21 is increased in the cytoplasm, associated with anti-apoptosis, DNA repair and cell survival. By contrast, deficiency of p21 renders tumor cells more susceptible to Polo-like kinase 1 inhibition by showing a pronounced mitotic arrest, DNA damage and apoptosis. Furthermore, long-term treatment with Plk1 inhibitors induced fiercely the senescent state of tumor cells with functional p21. We suggest that the p21 status may be a useful biomarker for predicting the efficacy of Plk1 inhibition.
Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.
BAG3, a multifunctional HSP70 co-chaperone and anti-apoptotic protein that interacts with the ATPase domain of HSP70 through its C-terminal BAG domain plays a key physiological role in cellular proteostasis. The HSP70/BAG3 complex determines the levels of a large number of selective client proteins by regulating their turnover via the two major protein degradation pathways, i.e. proteasomal degradation and macroautophagy. On the one hand, BAG3 competes with BAG1 for binding to HSP70, thereby preventing the proteasomal degradation of its client proteins. By functionally interacting with HSP70 and LC3, BAG3 also delivers polyubiquitinated proteins to the autophagy pathway. BAG3 exerts a number of key physiological functions, including an involvement in cellular stress responses, proteostasis, cell death regulation, development, and cytoskeletal dynamics. Conversely, aberrant BAG3 function/expression has pathophysiological relevance correlated to cardiomyopathies, neurodegeneration, and cancer. Evidence obtained in recent years underscores the fact that BAG3 drives several key hallmarks of cancer, including cell adhesion, metastasis, angiogenesis, enhanced autophagic activity, and apoptosis inhibition. This review provides a state-of-the-art overview on the role of BAG3 in stress and therapy resistance of cancer, with a particular focus on BAG3-dependent modulation of apoptotic signaling and autophagic/lysosomal activity.
Curcumin—a rhizomal phytochemical from the plant Curcuma longa—is well known to inhibit cell proliferation and to induce apoptosis in a broad range of cell lines. In previous studies we showed that combining low curcumin concentrations and subsequent ultraviolet A radiation (UVA) or VIS irradiation induced anti-proliferative and pro-apoptotic effects. There is still debate whether curcumin induces apoptosis via the extrinsic or the intrinsic pathway. To address this question, we investigated in three epithelial cell lines (HaCaT, A431, A549) whether the death receptors CD95, tumor necrosis factor (TNF)-receptor I and II are involved in apoptosis induced by light and curcumin. Cells were incubated with 0.25–0.5 µg/mL curcumin followed by irradiation with 1 J/cm2 UVA. This treatment was combined with inhibitors specific for distinct membrane-bound death receptors. After 24 h apoptosis induction was monitored by quantitative determination of cytoplasmic histone-associated-DNA-fragments. Validation of our test system showed that apoptosis induced by CH11 and TNF-α could be completely inhibited by their respective antagonists. Interestingly, apoptosis induced by curcumin/light treatment was reversed by none of the herein examined death receptor antagonists. These results indicate a mechanism of action independent from classical death receptors speaking for intrinsic activation of apoptosis. It could be speculated that a shift in cellular redox balance might prompt the pro-apoptotic processes
Apoptosis is deregulated in most, if not all, cancers, including hematological malignancies. Smac mimetics that antagonize Inhibitor of Apoptosis (IAP) proteins have so far largely been investigated in acute myeloid leukemia (AML) cell lines; however, little is yet known on the therapeutic potential of Smac mimetics in primary AML samples. In this study, we therefore investigated the antileukemic activity of the Smac mimetic BV6 in diagnostic samples of 67 adult AML patients and correlated the response to clinical, cytogenetic and molecular markers and gene expression profiles. Treatment with cytarabine (ara-C) was used as a standard chemotherapeutic agent. Interestingly, about half (51%) of primary AML samples are sensitive to BV6 and 21% intermediate responsive, while 28% are resistant. Notably, 69% of ara-C-resistant samples show a good to fair response to BV6. Furthermore, combination treatment with ara-C and BV6 exerts additive effects in most samples. Whole-genome gene expression profiling identifies cell death, TNFR1 and NF-κB signaling among the top pathways that are activated by BV6 in BV6-sensitive, but not in BV6-resistant cases. Furthermore, sensitivity of primary AML blasts to BV6 correlates with significantly elevated expression levels of TNF and lower levels of XIAP in diagnostic samples, as well as with NPM1 mutation. In a large set of primary AML samples, these data provide novel insights into factors regulating Smac mimetic response in AML and have important implications for the development of Smac mimetic-based therapies and related diagnostics in AML.
Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells
(2016)
Polo-like kinase 1 (Plk1) has been established as one of the most promising targets for molecular anticancer intervention. In fact, various Plk1 inhibitors have been identified and characterized. While the data derived from the bench are prospective, the clinical outcomes are less encouraging by showing modest efficacy. One of the explanations for this discrepancy could be unintendedly targeting of non-malignant cells by Plk1 inhibitors. In this work, we have addressed the effect of Plk1 inhibition in adipose tissue-derived mesenchymal stem cells (ASCs). We show that both visceral and subcutaneous ASCs display monopolar spindles, reduced viability and strong apoptosis induction upon treatment with BI 2536 and BI 6727, the Plk1 kinase domain inhibitors, and with Poloxin, the regulatory Polo-box domain inhibitor. While Poloxin triggers quickly apoptosis, BI 2536 and BI 6727 result in mitotic arrest in ASCs. Importantly, survived ASCs exhibit DNA damage and a pronounced senescent phenotype. In addition, Plk1 inhibition impairs ASCs’ motility and homing ability. These results show that Plk1 inhibitors target slowly proliferating ASCs, an important population of anti-inflammation and immune modulation. The toxic effects on primary cells like ASCs could be partially responsible for the reported moderate antitumor activity in patients treated with Plk1 inhibitors.
Smac mimetics antagonize IAP proteins, which are highly expressed in several cancers. Recent reports indicate that Smac mimetics trigger a broad cytokine response and synergize with immune modulators to induce cell death. Here, we identify a differential requirement of TRAIL or TNFα as mediators of IFNα/Smac mimetic-induced cell death depending on the cellular context. Subtoxic concentrations of Smac mimetics cooperate with IFNα to induce cell death in various solid tumor cell lines in a highly synergistic manner as determined by combination index. Mechanistic studies show that IFNα/BV6 cotreatment promotes the formation of a caspase-8-activating complex together with the adaptor protein FADD and RIP1. Assembly of this RIP1/FADD/caspase-8 complex represents a critical event, since RIP1 silencing inhibits IFNα/BV6-induced cell death. Strikingly, pharmacological inhibition of paracrine/autocrine TNFα signaling by the TNFα scavenger Enbrel rescues HT-29 colon carcinoma cells, but not A172 glioblastoma cells from IFNα/BV6-induced cell death. By comparison, A172 cells are significantly protected against IFNα/BV6 treatment by blockage of TRAIL signaling through genetic silencing of TRAIL or its cognate receptor TRAIL receptor 2 (DR5). Despite this differential requirement of TNFα and TRAIL signaling, mRNA and protein expression is increased by IFNα/BV6 cotreatment in both cell lines. Interestingly, A172 cells turn out to be resistant to exogenously added recombinant TNFα even in the presence of BV6, whereas they display a high sensitivity towards TRAIL/BV6. In contrast, BV6 efficiently sensitizes HT-29 cells to TNFα while TRAIL only had limited efficacy. This demonstrates that a differential sensitivity towards TRAIL or TNFα determines the dependency on either death receptor ligand for IFNα/Smac mimetic-induced cell death. Thus, by concomitant stimulation of both death receptor systems IFNα/Smac mimetic combination treatment is an effective strategy to induce cell death in TNFα- or TRAIL-responsive cancers.
Evasion of apoptosis, for example, by inhibitor of apoptosis (IAP) proteins, contributes to treatment resistance and poor outcome in acute myeloid leukemia (AML). Here we identify a novel synergistic interaction between the small-molecule second mitochondria-derived activator of caspases (Smac) mimetic BV6, which antagonizes X-linked IAP, cellular IAP (cIAP)1 and cIAP2, and the demethylating agents 5-azacytidine or 5-aza-2′-deoxycytidine (DAC) to induce cell death in AML cells, including apoptosis-resistant cells. Calculation of combination index (CI) confirms that this drug combination is highly synergistic (CI 0.02–0.4). In contrast, BV6 and DAC at equimolar concentrations do not cause synergistic toxicity against normal peripheral blood lymphocytes, pointing to some tumor cell selectivity. Molecular studies reveal that BV6 and DAC cooperate to trigger the activation of caspases, mitochondrial perturbations and DNA fragmentation, consistent with apoptotic cell death. However, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to protect against BV6/DAC-induced cell death and even significantly increases the percentage of Annexin-V/propidium iodide double-positive cells. Importantly, BV6/DAC-induced cell death in the presence of zVAD.fmk is significantly reduced by pharmacological inhibition of key components of necroptosis signaling, that is, receptor-interacting protein (RIP) 1 using necrostatin-1 or mixed lineage kinase domain-like protein (MLKL) using necrosulfonamide. This indicates a switch from BV6/DAC-induced cell death from apoptosis to necroptosis upon caspase inhibition. Thus, BV6 cooperates with demethylating agents to induce cell death in AML cells and circumvents apoptosis resistance via a switch to necroptosis as an alternative mode of cell death. The identification of a novel synergism of BV6 and demethylating agents has important implications for the development of new treatment strategies for AML.
Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis. Keywords: leukotriene, apoptosis, cell proliferation, mitogenic effects, cytotoxicity
Der ubiquitäre Redoxregulator Thioredoxin-1 (Trx-1) hat wichtige Funktionen für den zellulären Redoxstatus, Zellwachstum und Apoptose. Reaktive Sauerstoffspezies (ROS) sind beteiligt an der Pathogenese kardiovaskulärer Erkrankungen wie der Atherosklerose und werden zunehmend in ihrer Rolle als intra- und extrazelluläre Signalmoleküle charakterisiert. Ein Ungleichgewicht zwischen der Entstehung von ROS und ihrem Abbau durch antioxidative Systeme führt zu oxidativem Stress, zur Oxidation von Proteinen und letztlich zum Zelltod. Daher wurde in dieser Doktorarbeit untersucht, wie reaktive Sauerstoffspezies Trx-1 in Endothelzellen regulieren, welchen Einfluss dies für die Endothelzellapoptose hat und welche Bedeutung Antioxidantien, Stickstoffmonoxid (NO) und Schubspannung haben. In dieser Arbeit wurde gezeigt, dass H2O2 konzentrationsabhängig die Expression von Trx-1 beeinflusst. Geringe Konzentrationen H2O2 wie 10 und 50 µM induzierten Trx-1-mRNA nach 3 Stunden. Auf Proteinebene fand sich dann nach 6 Stunden eine transiente Hochregulation von Trx-1. Diese geringen Konzentrationen von H2O2 wirkten antiapoptotisch. Dieser antiapoptotische Effekt war von der Trx-1 Proteinexpression abhängig. Im Gegensatz dazu kam es bei hohen Konzentrationen H2O2 zu einer Degradierung von Trx-1. Durch das Antioxidans NAC und NO konnte der Abbau von Trx-1 unter höheren H2O2-Konzentrationen verhindert werden. Untersuchungen zum Mechanismus des Degradierungsprozesses ergaben, dass Trx-1 durch die Aspartatprotease Cathepsin D abgebaut wird. Der protektive Effekt von NO auf die Trx-1 Expression konnte auch im Gewebe eNOS-defizienter Mäuse gezeigt werden, da bereits eNOS-defiziente Mäuse in den Nieren weniger Trx-1 Protein aufwiesen im Vergleich zu Wildtyp-Kontrollmäusen. Bei der Entstehung endothelialer Läsionen und der Stabilität atheromatöser Plaques spielt die Endothelzellapoptose vermutlich eine wichtige Rolle. Trx-1 schützt Endothelzellen vor Apoptose, wird jedoch unter oxidativem Stress abgebaut. Faktoren, die Trx-1 unter oxidativem Stress stabilisieren wie NAC und NO, kommt daher eine besondere Bedeutung für die Endothelzellhomöostase zu.
Die Bedeutung der Apoptose und die zugrundeliegenden Mechanismen in verschiedenen pathophysiologischen Zuständen des Herzens sind noch weitgehend ungeklärt und es bleibt zu zeigen, daß die Apoptose-Signaltransduktion ähnlich reguliert wird, wie aus in vitro-Versuchen bekannt ist. Deshalb wurde die Apoptose in verschiedenen Tiermodellen kardialer Erkrankungen untersucht werden, um Hinweise auf die zugrundeliegende Signal-transduktion, durch Analyse der Proteine Bcl-2 und Bax, der finalen Exekutor-Caspase Caspase-3 oder p53 zu bekommen. Apoptose in der durch Hyperlipidämie induzierten Atherosklerose: In Aorten von 'Froxfield Heritable Hypercholesterolemic'-Kaninchen (genetische Hyperlipidämie) korrelierte die Apoptose von vaskulären glatten Muskelzellen und Makrophagen in fortgeschrittenen fibrösen Plaques mit einem 18-fachen Anstieg des proapoptotischen Bax. In Aorten Cholesterin gefütterter 'New Zealand White'-Kaninchen (0,25% Cholest., 12 Wochen) konnte eine erhöhte Baxexpression in Endothelzellen nachgewiesen werden, ohne daß morphologische Veränderungen zu beobachten waren. Die Apoptose in akut abgestoßenen allogenen Herztransplantaten (Rattenmodell) war von einer erhöhten Bax-Expression und einer totalen, posttranslationalen Degradation des antiapoptotischen Bcl-2 in ein spezifisches Degradationsprodukt durch eine Serinprotease gekennzeichnet. Die Rolle des wichtigen kardiovaskulären Mediators Stickstoffmonoxid (NO) auf die Apoptose wird kontrovers diskutiert. Da in der Zellkultur protektive Effekte von NO gezeigt werden konnten, wurde deren physiologische Relevanz in der durch Ischämie/Reperfusion induzierten Apoptose ex vivo im Langendorff-Rattenherzen untersucht. Es konnte gezeigt werden, daß Hemmung der endogenen NO-Synthese mit L-NG-Monomethyl-L-Arginin (LNMMA, 1mM) die Apoptose potenzierte und mit einer Aktivierung der Caspase-3 korrelierte. Bcl-2 und Bax wurden nicht reguliert. Untersuchung der Regulation der Proteinexpression der eNOS (endotheliale NO-Synthase) durch den proinflammatorischen/ proatherogenen Tumor-Nekrose-Faktor-[Alpha] (TNF[Alpha]) in der Endothelzellkultur (HUVEC) gaben Hinweise auf einen, die eNOS schützenden, Interaktionspartner. Zusammenfassend konnte in allen untersuchten Modellen für Herz(-Kreislauf)-Krankheiten Apoptose nachgewiesen werden, die jeweils spezifische Charakteristika zeigt, deren genauere Aufklärung interessante Ziele zukünftiger präventiver und therapeutischer Maßnahmen verspricht. Die Befunde weisen zudem auf antiapoptotische Effekte von NO - insbesondere durch die endotheliale NO-Synthaseaktivität - hin, deren genauere Charakterisierung dazu beitragen könnte, pathophysiologische Zustände der kardiovaskulären Biologie zu erklären.
Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1–100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa.