Thioredoxin-1 is regulated via reactive oxygen species : low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

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.The ubiquitous redoxregulator thioredoxin-1 (trx-1) is essential for cellular redox status, cell growth and apoptosis. Reactive oxygen species (ROS) are involved in the pathogenesis of cardivascular diseases like atherosclerosis. In addition, ROS have recently been characterised as intra- and extracellular signalling molecules. Severe imbalance in cellular redox state leads to oxidative protein damage and cell death. Therefore, this thesis was focused on how trx-1 is regulated by ROS in endothelial cells, whether endothelial cell apoptosis is effected and whether antioxidants, nitric oxide and shear stress modulate trx-1 regulation. In this thesis it was shown that H2O2 modulates expression of trx-1 in a concentration dependent manner. Low doses of H2O2 like 10 and 50 µM induced trx-1 mRNA after 3 hours. On the protein level, a transient upregulation of trx-1 became evident after 6 hours incubation with low doses of ROS. These low doses of H2O2 protected endothelial cells from apoptosis. The anti-apoptotic effect was dependent on trx-1 protein expression. In contrast, high doses of H2O2 lead to degradation of trx-1. The antioxidant NAC and nitric oxide prevented degradation of trx-1 under conditions of high doses of H2O2. Investigations of the degradation mechanism revealed that trx-1 is degraded by the aspartatic protease Cathepsin D. Additionally, the protective effect of nitric oxide on trx-1 protein expression was shown in the tissue of eNOS-deficient mice as these had a lower trx-1 protein level in the kidney compared with wildtype control mice. In the progression of atherosclerosis and the stability of atherosclerotic plaques, endothelial cell apoptosis may play a key role. Trx-1 protects endothelial cells from apoptosis but is degraded under conditions of oxidative stress. Factors that stabilise trx-1 under conditions of oxidative stress like NAC and NO are of great importance for endothelial cell homeostasis

Significance of a new fluorodeoxyglucose-positive lesion on restaging positron emission tomography/computed tomography after induction therapy for non-small-cell lung cancer †

OBJECTIVES Restaging of patients with locally advanced non-small-cell lung cancer (NSCLC) is of paramount importance, since only patients with down-staging after induction therapy will benefit from surgery. In this study, we assessed the aetiology of new 18fluoro-2-deoxy-d-glucose (FDG)-positive focal abnormalities on restaging positron emission tomography/computed tomography (PET/CT) in patients with a good response after induction chemotherapy in the primary tumour and lymph nodes. METHODS Between 2004 and 2008, 31 patients with histological proven stage III NSCLC had a PET/CT prior and after induction chemotherapy. Their medical charts were retrospectively reviewed. RESULTS Restaging PET/CT revealed a new FDG-positive lesion in 6 of 31 (20%) patients. The initial clinical stage of the disease was IIIA N2 in four and IIIB T4 in two patients. The maximal standard uptake value in the primary tumour (P=0.043) and in the initially involved mediastinal nodes (P=0.068) decreased after induction treatment in all patients. The new PET/CT findings were located in an ipsilateral cervical lymph node in two patients, a contralateral mediastinal in one patient and an ipsilateral mammary internal lymph node in one patient. Two other patients had a lesion on the contralateral lung. Malignant lymph node infiltrations were excluded following fine-needle puncture, intraoperative biopsy or follow-up PET/CT. Contralateral pulmonary lesions were diagnosed as benign following mini thoracotomy and pulmonary wedge resection. CONCLUSIONS New solitary FDG-positive lesions on restaging PET/CT after induction chemotherapy for NSCLC are not rare in good responders to chemotherapy. In our experience, all these lesions were not associated with malignanc

Delayed granulomatous reaction to hyaluronic acid gel injection

Injectable hyaluronic acid (HA) derivatives are the most used resorbable dermal fillers used for soft tissue augmentation. While their use is considered safe, there have been reports of cutaneous granulomatous reactions. We describe the clinical, radiological, and cytological findings in a patient who presented a full year after cosmetic treatment with HA injections and discuss the various treatment options. Level of Evidence: Level V, therapeutic study

L1CAM protein expression is associated with poor prognosis in non-small cell lung cancer

Background: The L1 cell adhesion molecule (L1CAM) is potentially involved in epithelial-mesenchymal transition (EMT). EMT marker expression is of prognostic significance in non-small cell lung cancer (NSCLC). The relevance of L1CAM for NSCLC is unclear. We investigated the protein expression of L1CAM in a cohort of NSCLC patients. L1CAM protein expression was correlated with clinico-pathological parameters including survival and markers of epithelial-mesenchymal transition. Results: L1CAM protein expression was found in 25% of squamous cell carcinomas and 24% of adenocarcinomas and correlated with blood vessel invasion and metastasis (p < 0.05). L1CAM was an independent predictor of survival in a multivariate analysis including pT, pN, and pM category, and tumor differentiation grade. L1CAM expression positively correlated with vimentin, beta-catenin, and slug, but inversely with E-cadherin (all p-values < 0.05). E-cadherin expression was higher in the tumor center than in the tumor periphery, whereas L1CAM and vimentin were expressed at the tumor-stroma interface. In L1CAM-negative A549 cells the L1CAM expression was upregulated and matrigel invasion was increased after stimulation with TGF-beta1. In L1CAM-positive SK-LU-1 and SK-LC-LL cells matrigel invasion was decreased after L1CAM siRNA knockdown. Conclusions: A subset of NSCLCs with vessel tropism and increased metastasis aberrantly expresses L1CAM. L1CAM is a novel prognostic marker for NSCLCs that is upregulated by EMT induction and appears to be instrumental for enhanced cell invasion

LACTB is a tumour suppressor that modulates lipid metabolism and cell state

Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression

LACTB is a tumour suppressor that modulates lipid metabolism and cell state

Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression

Mechanistic insight into RET kinase inhibitors targeting the DFG-out conformation in RET-rearranged cancer

Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. Here, we provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors such as AD80 or ponatinib that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells we identify the CCDC6-RETI788N mutation and drug-induced MAPK pathway reactivation as possible mechanisms, by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors

Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors

<p>Abstract</p> <p>Background</p> <p>Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-β signaling and activation of the β-catenin pathway can induce EMT in intratumoral cancer cells.</p> <p>Methods</p> <p>We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized <it>in vitro </it>in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC).</p> <p>Results</p> <p>Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. <it>In vitro</it>, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating β-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-β was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-β levels and tumor grade.</p> <p>Conclusions</p> <p>Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.</p