Refine
Year of publication
Document Type
- Article (21)
- Doctoral Thesis (4)
Has Fulltext
- yes (25)
Is part of the Bibliography
- no (25)
Keywords
- Apoptosis (25) (remove)
Institute
- Medizin (25) (remove)
Background: NH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA.
Methods: Colon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors.
Results: HMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death.
Conclusion: Our results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy.
Searching for new strategies to trigger apoptosis in rhabdomyosarcoma (RMS), we investigated the effect of two novel classes of apoptosis-targeting agents, i.e. monoclonal antibodies against TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (mapatumumab) and TRAIL receptor 2 (lexatumumab) and small-molecule inhibitors of inhibitor of apoptosis (IAP) proteins. Here, we report that IAP inhibitors synergized with lexatumumab, but not with mapatumumab, to reduce cell viability and to induce apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1). Cotreatment-induced apoptosis was accompanied by enhanced activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and caspase-dependent apoptosis. In addition, IAP inhibitor and lexatumumab cooperated to stimulate the assembly of a cytosolic complex containing RIP1, FADD, and caspase-8. Importantly, knockdown of RIP1 by RNA interference prevented the formation of the RIP1·FADD·caspase-8 complex and inhibited subsequent activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and apoptosis upon treatment with IAP inhibitor and lexatumumab. In addition, RIP1 silencing rescued clonogenic survival of cells treated with the combination of lexatumumab and IAP inhibitor, thus underscoring the critical role of RIP1 in cotreatment-induced apoptosis. By comparison, the TNFα-blocking antibody Enbrel had no effect on IAP inhibitor/lexatumumab-induced apoptosis, indicating that an autocrine TNFα loop is dispensable. By demonstrating that IAP inhibitors and lexatumumab synergistically trigger apoptosis in a RIP1-dependent but TNFα-independent manner in RMS cells, our findings substantially advance our understanding of IAP inhibitor-mediated regulation of TRAIL-induced cell death.
In der vorliegenden Arbeit wird das Wachstums- und Zelltodverhalten von Tumoren des zentralen Nervensystems untersucht. Des Weiteren wird die Expression verschiedener Apoptose-assoziierter Faktoren in den Präparaten analysiert und mit Normalkontrollen verglichen. Es zeigt sich, dass Apoptose von Tumorzellen aller untersuchter Hirntumore und Malignitätsgrade vollzogen werden kann. Die Rate apoptotischer Zellen ist jedoch sehr variabel und korreliert nicht mit dem Malignitätsgrad der Tumore. Auch besteht keine Korrelation zwischen der Apoptose- und der Proliferationsrate. Die Ergebnisse legen insgesamt nahe, dass die Apoptoserate nicht als Marker für die Malignität von Tumoren des zentralen Nervensystems verwendet werden kann. Auch unter Einbeziehung Apoptose-assoziierter Faktoren ist eine Gradifikation der Tumore hinsichtlich der Malignität nicht möglich. So unterscheiden sich z.B. atypische (WHO-II) und anaplastische (WHO-III) Meningiome quantitativ und qualitativ nicht signifikant voneinander. Es können ebenfalls keine signifikanten Unterschiede hinsichtlich der Expression der untersuchten Apoptose-assoziierten Faktoren, sowie der Apoptose- und Proliferationsraten zwischen Medulloblastomen und primitiven neuroektodermalen Tumoren (PNETs) festgestellt werden. Dies spricht dafür, dass sich diese Tumore lediglich bezüglich ihrer Lokalisation im zentralen Nervensystem unterscheiden. Die Analyse der Apoptose-assoziierten Faktoren zeigt, dass alle untersuchten Faktoren grundsätzlich in allen untersuchten Tumoren vorkommen, während die Normalkontrollen diese Faktoren nicht exprimieren. Der Vollzug der Apoptose findet jedoch nicht in diesem Maße statt, da die Apoptoserate der Tumore (markiert durch TUNEL) stets wesentlich geringer ist als die Expressionsraten der Apoptose-assoziierten Faktoren. Es ist davon auszugehen, dass entdifferenzierte Tumorzellen entweder nur begrenzt in der Lage sind, ihr apoptotisches „Selbstzerstörungsprogramm“ in Gang zu setzen und zu Ende zu führen, oder, dass apoptosehemmende Mechanismen greifen. Um so interessanter wäre es, durch therapeutische Intervention Apoptose zu initiieren. Die Analyse der einzelnen Apoptose-assoziierten Faktoren liefert Hinweise darauf, an welchen Stellen des apoptotischen Systems eine solche Intervention ansetzen könnte: Die hochmalignen WHO-IV-Tumore zeigen eine signifikante Hochregulation der Effektor-Caspasen-3 und -6. Die physiologischen Aktivierungsmechanismen dieser Caspasen z.B. durch Caspase-2 und TNFalpha scheinen in diesen hochmalignen Tumoren jedoch weniger eine Rolle zu spielen, da diese Faktoren hier nur in geringem Ausmaß exprimiert werden. Jedoch könnten modifizierte, per se aktive Caspase-3- und -6-Moleküle eine interessante therapeutische Option zur Behandlung maligner Tumore des zentralen Nervensystems darstellen. Zu beachten ist aber unter anderem, dass z.B. Glioblastome auch geringe Expressionsraten apoptotischer Faktoren im peritumoralen, mikroskopisch nicht infiltrierten Normalgewebe zeigen. Dies könnte für eine peritumorale Dysfunktion des Hirngewebes sprechen. Welche Rolle dies bei der Behandlung mit Apoptose-stimulierenden Agenzien spielt und wie spezifisch die Anwendung solcher Stimulanzien für Tumorgewebe wären, muss Gegenstand weiterer Studien sein. Die untersuchten WHO-II- und –III-Tumore zeigen eine Hochregulation vor allem von Faktoren des extrinsischen Apoptoseweges (z.B. TNFalpha). Die Expressionsraten von TNFalpha korrelieren signifikant mit dem WHO-Grad der untersuchten Tumore. Interessante therapeutische Optionen könnten hier zum einen die Aktivierung des extrinsischen Apoptoseweges über TNFalpha sein, zum anderen könnte man versuchen, eine direkte Aktivierung über modifizierte Effektor-Caspasen herbeizuführen. Insgesamt existieren verschiedene mögliche Angriffsorte innerhalb des apoptotischen Netzwerkes der Zelle für eine thepeutische Intervention bei Tumoren des zentralen Nervensystems. Die Komplexität des Kaskade-artigen Systems legt nahe, dass eine therapeutische Intervention möglichst an dessen Ende erfolgen sollte, um möglichst viele Stör- und Hemmfaktoren zu umgehen.
Background/Aims: Sphingosine 1-phosphate (S1P) is considered as a key molecule regulating various cell functions including cell growth and death. It is produced by two sphingosine kinases (SK) denoted as SK-1 and SK-2. Whereas SK-1 has been extensively studied and has been appointed a role in promoting cell growth, the function of SK-2 is controversial, and both pro-proliferative and pro-apoptotic functions have been suggested. In this study we investigated whether renal mesangial cells isolated from transgenic mice overexpressing the human Sphk2 gene (hSK2-tg) showed an altered cell response towards growth-inducing and apoptotic stimuli.
Methods: hSK2-tg mice were generated by using a Quick KnockinR strategy. Renal mesangial cells were isolated by a differential sieving method and further cultivated in vitro. Lipids were quantified by mass spectrometry. Protein expression was determined by Western blot analysis, cell proliferation was determined by 3H-thymidine incorporation, and apoptosis was determined by a DNA fragmentation ELISA.
Results: We show here that kidneys and mesangial cells from hSK2-tg mice express the hSK2 as well as the endogenous mouse mSK2. hSK2 and mSK2 predominantly resided in the cytosol of quiescent transgenic cells. However, S1P accumulated strongly in the nucleus and only minimally in the cytosol of transgenic cells. Functionally, hSK2-tg cells proliferated less than control cells under normal growth conditions and were also more sensitive towards stress-induced apoptosis. On the molecular level, this was reflected by reduced ERK and Akt/PKB activation, and upon staurosporine treatment, by a sensitized mitochondrial pathway as manifested by reduced anti-apoptotic Bcl-XL expression and increased cleavage of caspase-9, downstream caspase-3 and PARP-1.
Conclusion: Altogether, these data demonstrate that SK-2 exerts an antiproliferative and apoptosis-sensitizing effect in renal mesangial cells which suggests that selective inhibitors of SK-2 may promote proliferation and reduce apoptosis and this may have impact on the outcome of proliferation-associated diseases such as mesangioproliferative glomerulonephritis.
Background: Ischemia-reperfusion injury (IRI) is a major challenge in liver transplantation. The mitochondrial pathway plays a pivotal role in hepatic IRI. Levosimendan, a calcium channel sensitizer, was shown to attenuate apoptosis after IRI in animal livers. The aim of this study was to investigate the effect of levosimendan on apoptosis in human hepatocytes.
Methods: Primary human hepatocytes were either exposed to hypoxia or cultured under normoxic conditions. After the hypoxic phase, reoxygenation was implemented and cells were treated with different concentrations of levosimendan (10ng/ml, 100ng/ml, 1000ng/ml). The overall metabolic activity of the cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and aspartate aminotransferase (AST) levels were determined in order to quantify hepatic injury. Fluorescence-activated cell sorting (FACS) analysis was applied to measure necrosis and apoptosis. Finally, Western blotting was performed to analyze apoptotic pathway proteins.
Results: Administration of levosimendan during reperfusion increases the metabolic activity of human hepatocytes and decreases AST levels. Moreover, apoptosis after IRI is reduced in treated vs. untreated hepatocytes, and levosimendan prevents down-regulation of the anti-apoptotic protein Bcl-2 as well as up-regulation of the pro-apoptotic protein BAX.
Conclusion: The present study suggests a protective effect of levosimendan on human hepatocytes. Our findings suggest that treatment with levosimendan during reperfusion attenuates apoptosis of human hepatocytes by influencing BAX and Bcl-2 levels.
Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNFα-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNFα/TNFR1, as the TNFα-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-κB (NF-κB) activation is critically required for apoptosis, as inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in IκBα-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-κB-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.
BH3 mimetics are promising novel anticancer therapeutics. By selectively inhibiting BCL-2, BCL-xL, or MCL-1 (i.e. ABT-199, A-1331852, S63845) they shift the balance of pro- and anti-apoptotic proteins in favor of apoptosis. As Bromodomain and Extra Terminal (BET) protein inhibitors promote pro-apoptotic rebalancing, we evaluated the potential of the BET inhibitor JQ1 in combination with ABT-199, A-1331852 or S63845 in rhabdomyosarcoma (RMS) cells. The strongest synergistic interaction was identified for JQ1/A-1331852 and JQ1/S63845 co-treatment, which reduced cell viability and long-term clonogenic survival. Mechanistic studies revealed that JQ1 upregulated BIM and NOXA accompanied by downregulation of BCL-xL, promoting pro-apoptotic rebalancing of BCL-2 proteins. JQ1/A-1331852 and JQ1/S63845 co-treatment enhanced this pro-apoptotic rebalancing and triggered BAK- and BAX-dependent apoptosis since a) genetic silencing of BIM, BAK or BAX, b) inhibition of caspase activity with zVAD.fmk and c) overexpression of BCL-2 all rescued JQ1/A-1331852- and JQ1/S63845-induced cell death. Interestingly, NOXA played a different role in both treatments, as genetic silencing of NOXA significantly rescued from JQ1/A-1331852-mediated apoptosis but not from JQ1/S63845-mediated apoptosis. In summary, JQ1/A-1331852 and JQ1/S63845 co-treatment represent new promising therapeutic strategies to synergistically trigger mitochondrial apoptosis in RMS.
ADAM15 protein amplifies focal adhesion kinase phosphorylation under genotoxic stress conditions
(2012)
ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.
Secondary plant metabolites reveal numerous biological activities making them attractive as resource for drug development of human diseases. As the majority of cancer drugs clinically established during the past half century is derived from nature, cancer researchers worldwide try to identify novel natural products as lead compounds for cancer therapy. Natural products are considered as promising cancer therapeutics, either as single agents or in combination protocols, to enhance the antitumor activity of additional therapeutic modalities. Most natural compounds exert pleotrophic effects and modulate various signal transduction pathways. A better understanding of the complex mechanisms of action of natural products is expected to open new perspectives in coming years for their use alone or in combination therapies in oncology. Two major strategies to identify novel drug candidates from nature are the bioactivity-guided fractionation of medicinal plant extracts to isolate cytotoxic chemicals and the identification of small molecules inhibiting specific targets in cancer cells. In the present review, we report on our own efforts to unravel the molecular modes of action of phytochemicals in cancer cells and focus on resveratrol, betulinic acid, artesunate, dicentrine and camptothecin derivatives.
Background: Alcohol drinking is associated with a serious risk of developing health problems as well as with a large number of traumatic injuries. Although chronic alcohol misuse is known to contribute to severe inflammatory complications, the effects of an acute alcohol misuse are still unclear. Here, the impact of acute alcohol drinking on leukocyte counts and their cellular functions were studied.
Methods: Twenty-two healthy volunteers (12 female, 10 male) received a predefined amount of a whiskey-cola mixed drink (40% v/v), at intervals of 20 min, over 4 h to achieve a blood alcohol concentration of 1‰. Blood samples were taken before drinking T0, 2 h (T2), 4 h (T4), 6 h (T6), 24 h (T24) and 48 h (T48) after starting drinking alcohol. Leukocytes, monocytes and granulocyte counts and their functions regarding the production of reactive oxidative species (ROS), phagocytosis and apoptosis were analyzed by flow cytometry.
Results: Total leukocyte counts significantly increased at T2 and T4, while granulocyte and monocyte counts decreased at T4 and T6 vs. T0. Monocytes increased significantly at T24 and T48 vs. T0. While the total number of ROS-producing leukocytes and notably granulocytes significantly increased, in parallel, the intracellular ROS intensity decreased at T2 and T6. The numbers of ROS-positive monocytes have shown a delayed modulation of ROS, with a significant reduction in the total number of ROS-producing cells at T48 and a significantly reduced intracellular ROS-intensity at T24. Phagocyting capacity of leukocytes significantly decreased at T4 and T6. In general leukocytes, and notably granulocytes demonstrated significantly increased early (T2), while monocyte exerted significantly increased late apoptosis (T24 and T48).
Conclusions: Alcohol drinking immediately impacts leukocyte functions, while the impact on monocytes occurs at even later time points. Thus, even in young healthy subjects, alcohol drinking induces immunological changes that are associated with diminished functions of innate immune cells that persist for days.