Influence of selenium on pancreatic carcinogenesis and the role of the selenoproteins cytosolic and mitochondrial thioredoxin reductase in the pancreas

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive cancers in humans. It is the fourth leading cause of cancer related deaths in Germany and in the United States. Most PDA occurs sporadically, but there are also approximately 5-10% of patients with a family history of pancreatic cancer. The high mortality of PDA is attributed to a lack of early detection methods and poor efficacy in therapies for advanced disease. As an alternative, preventive strategies in individuals with familial pancreatic carcinoma should be considered. Several epidemiological studies showed an inverse correlation between selenium-intake and mortality of certain types of cancer and particularly in gastrointestinal cancers. To this end, in the first part of this study, the influence of selenium as a preventive nutritional additive was investigated in a genetically defined pancreatic cancer mouse model, the EL-TGFatg/+;p53+/- mouse strain. As a major finding, the differentiation grade of the pancreatic carcinomas was heavily influenced by the selenium status. In the selenium-deficient group there were more non-differentiated pancreatic carcinomas than in the selenium-adequate group, which highlighted the implication of selenium or selenoproteins in tumour differentiation. Unexpectedly, however, there was no protective effect of selenium on total or pancreatic tumour latency. Within the selenoproteins, the thioredoxin reductases are strong candidates which may influence cell death and differentiation in pancreatic carcinogenesis. Their function is generally associated with tumour proliferation and also linked to the activation of the tumour suppressor p53. Consequently, the role of the thioredoxin reductases in the pancreas was studied in the second part of this thesis. The enzymatic activity of cytosolic (TXNRD1) and mitochondrial (TXNRD2) thioredoxin reductase in the pancreas and other organs was determined in relation to the selenium-status. TXNRD1 activity in the pancreas was moderate and decreased under selenium deficiency. TXNRD2, instead, showed very high pancreatic activity in relation to other organs and its activity was even increased under selenium-deficiency emphasising its special role in this organ. To further investigate the function of Txnrd1 and Txnrd2 in the pancreas, tissue-specific knockout mice were created and characterized. The Txnrd1 knockout mice did not show an overt phenotype. Interestingly although, pancreatic acinus cells in one year old mice showed a disturbed rough endoplasmic reticulum and alterations in serum amylase and lipase. These mice also had an impaired glucose tolerance. The pancreas of Txnrd2 knockout mice showed severe chronic pancreatitis and pancreatic atrophy at the end of an observation period of one year. The progressive pathogenic process started with mild pancreatitis, developing spontaneously at an age of four weeks. The chronic stage was characterized by the formation of different types of acinar-to-ductal metaplastic lesions, which could be classified in part as early precursor lesions of pancreatic carcinomas. The endocrine pancreas was not affected. The pancreas-specific Txnrd2 knockout mouse strain is the first genetically modified mouse model spontaneously developing acute and chronic pancreatitis. This strain constitutes a unique and powerful tool to model pancreatic pathogenesis, especially the yet unresolved process of transformation from inflammatory to malignant disease.Das duktale Adenokarzinom des Pankreas gehört zu den aggressivsten Krebsarten des Menschen. In Deutschland wie auch in den USA bilden Krebserkrankungen des Pankreas die viert häufigste durch Krebs hervorgerufene Todesursache. Obwohl die molekularen Mechanismen des duktalen Adenokarzinoms immer besser verstanden werden, nimmt diese Krebserkrankung meist einen tödlichen Verlauf. Die hohe Sterblichkeitsrate wird vor allem durch fehlende Möglichkeiten der Früherkennung und mangelnde Effektivität der Behandlungsmethoden bei fortgeschrittener Krankheit begründet. Zumeist tritt das Pankreaskarzinom spontan auf, jedoch bei 5-10% der Patienten lässt sich ein familiärer Hintergrund nachweisen. Für diese Patientengruppe sollten präventive Maßnahmen angestrebt werden. In epidemiologischen Studien konnten Hinweise zu einer inversen Korrelation von Selenaufnahme und altersabhängiger Sterblichkeit bei verschiedenen Krebsarten und vor allem bei gastrointestinalen Krebserkrankungen erarbeitet werden. Im ersten Teil der hier vorliegenden Studie wurde daher der Einfluss von Selen auf das Pankreaskarzinom des genetisch definierten EL-TGFatg/+;p53+/- Mausmodelles beforscht. Selen-defiziente Mäuse wurden mit Selen-adäquat ernährten Mäusen verglichen. Interessanter Weise wurde der Differenzierungsgrad der entstandenen Pankreas-karzinome hoch signifikant durch den Selenstatus der Mäuse beeinflusst. Selen-defizient ernährte Mäuse entwickelten hauptsächlich anaplastische Pankreaskarzinome, wohingegen Selen-adäquat ernährte Mäuse mehr differenzierte Tumoren aufwiesen. Unerwarteter Weise konnte jedoch kein protektiver Einfluss von Selen weder auf die Latenzzeit aller auftretender Tumoren, noch im einzelnen auf Pankreaskarzinome festgestellt werden. Innerhalb der Gruppe der Selenoproteine sind die Thioredoxinreduktasen potentielle Kandidaten welche den Zelltod und die Differenzierung von Pankreaskarzinomen beeinflussen. Ihre Funktion wird im Allgemeinen mit der Proliferation von Tumoren und der Aktivierung des Tumorsupressors p53 in Verbindung gebracht. Folglich wurde die Rolle der Thioredoxinreduktasen im Pankreas im zweiten Teil dieser Studie bearbeitet. Die enzymatische Aktivität der cytosolischen (TXNRD1) und mitochondrialen (TXNRD2) Thioredoxinreduktase im Pankreas und anderen Organen wurde im Allgemeinen und in Bezug auf den Selenstatus der Tiere bestimmt. Die enzymatische Aktivität von TXNRD1 im Pankreas war eher mäßig und sank unter Selen-defizienten Bedingungen noch weiter ab. TXNRD2 hingegen zeigte eine sehr starke enzymatische Aktivität im Pankreas und in Selen-defizienten Tieren erhöhte sich die enzymatische Aktivität von TXNRD2 im Pankreas sogar noch, was auf eine wichtige Rolle dieses Enzyms in diesem Organ schließen lässt. Um die Rolle von Txnrd1 und Txnrd2 im Pankreas aufzuklären wurden Gewebe-spezifische Knockout-Mäuse gezüchtet und charakterisiert. Txnrd1 Knockout-Mäuse zeigten zuerst keinen offensichtlichen Phänotyp. Interessanter Weise jedoch, konnte in Azinus-Zellen des Pankreas von ein Jahr alten Mäusen ein dilatatives bis hin zu völlig zerstörtem rauen Endoplasmatischem Retikulum beobachtet werden. Des Weiteren wurden Veränderungen der Amylase und Lipase Werte im Blutserum gemessen. Die Tiere hatten auch eine veränderte Glucose Toleranz. Das Pankreas der Txnrd2 Knockout-Mäuse wies eine schwerwiegende chronische Pankreatitis und voranschreitende Atrophie des Pankreasgewebes gegen Ende des Beobachtungszeitraums von einem Jahr auf. Die Mäuse entwickelten spontan eine akute Pankreatitis im Alter von vier Wochen. In der chronischen Pankreatitis wurden verschiedene Arten von azinären-duktalen Metaplasien gefunden, die zum Teil als frühe Vorläuferstadien von Pankreaskarzinomen klassifiziert werden konnten. Das endokrine Pankreas wies keine Veränderungen auf. Dieser Pankreas-spezifische Txnrd2 Knockout-Mausstamm ist das erste genetische Model welches spontan akute und chronische Pankreatitis entwickelt und bietet daher enorme Möglichkeiten für die Erforschung dieser inflamatorischen Erkrankung und ihrer Verbindung zu Krebserkrankungen des Pankreas

Loss of Npn1 from motor neurons causes postnatal deficits independent from Sema3A signaling

AbstractThe correct wiring of neuronal circuits is of crucial importance for the function of the vertebrate nervous system. Guidance cues like the neuropilin receptors (Npn) and their ligands, the semaphorins (Sema) provide a tight spatiotemporal control of sensory and motor axon growth and guidance. Among this family of guidance partners the Sema3A-Npn1 interaction has been shown to be of great importance, since defective signaling leads to wiring deficits and defasciculation. For the embryonic stage these defects have been well described, however, also after birth the organism can adapt to new challenges by compensational mechanisms. Therefore, we used the mouse lines Olig2-Cre;Npn1cond and Npn1Sema− to investigate how postnatal organisms cope with the loss of Npn1 selectively from motor neurons or a systemic dysfunctional Sema3A-Npn1 signaling in the entire organism, respectively. While in Olig2-Cre+;Npn1cond−/− mice clear anatomical deficits in paw posturing, bone structure, as well as muscle and nerve composition became evident, Npn1Sema− mutants appeared anatomically normal. Furthermore, Olig2-Cre+;Npn1cond mutants revealed a dysfunctional extensor muscle innervation after single-train stimulation of the N.radial. Interestingly, these mice did not show obvious deficits in voluntary locomotion, however, skilled motor function was affected. In contrast, Npn1Sema− mutants were less affected in all behavioral tests and able to improve their performance over time. Our data suggest that loss of Sema3A-Npn1 signaling is not the only cause for the observed deficits in Olig2-Cre+;Npn1cond−/− mice and that additional, yet unknown binding partners for Npn1 may be involved that allow Npn1Sema− mutants to compensate for their developmental deficits

Croconaine-based nanoparticles enable efficient optoacoustic imaging of murine brain tumors

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application

Levels of the Autophagy-Related 5 Protein Affect Progression and Metastasis of Pancreatic Tumors in Mice

[Background and Aims]: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. [Methods]: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/–;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. [Results]: A5+/–;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/–;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/–;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/–;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. [Conclusions]: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.This study was supported in part by the Mildred-Scheel-Professur der Deutschen Krebshilfe 111464, DFG AL 1174/6-1 to H.A., DFG DI 2299/1-1 to K.N.D., DFG SFB1321 (S01) to K.S. and W.W., and the German Federal Ministry of Education and Research to the German Center for Diabetes Research (DZD e.V.) to J.A

Positron emission tomographic monitoring of dual phosphatidylinositol-3-kinase and mTOR inhibition in anaplastic large cell lymphoma

Background: Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[F-18] fluoro-D-glucose (FDG) and the thymidine analog, 3'-deoxy-3'-[F-18] fluorothymidine (FLT). Methods: The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects. Results: SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue. Conclusion: Dual PI3K/mTOR inhibition using BGT226 is effective in ALK-positive anaplastic large cell lymphoma and can be monitored with both FDG-PET and FLT-PET early on in the course of therapy

Sensory reanimation of the hand by transfer of the superficial branch of the radial nerve to the median and ulnar nerve

BACKGROUND: It remains a surgical challenge to treat high-grade nerve injuries of the upper extremity. Extra-anatomic reconstructions through the transfer of peripheral nerves have gained clinical importance over the past decades. This contribution outlines the anatomic and histomorphometric basis for the transfer of the superficial branch of the radial nerve (SBRN) to the median nerve (MN) and the superficial branch of the ulnar nerve (SBUN). METHODS: The SBRN, MN, and SBUN were identified in 15 specimens and the nerve transfer performed. A favorable site for coaptation was chosen and its location described using relevant anatomical landmarks. Histomorphometric characteristics of donor and target were compared to evaluate the chances of a clinical success. RESULTS: A suitable location for dissecting the SBRN was identified prior to its first bifurcation. Coaptations were possible near the pronator quadratus muscle, approximately 22 cm distal to the lateral epicondyle of the humerus. The MN and SBUN had to be dissected interfasciculary over 82 ± 5.7 mm and 49 ± 5.5 mm, respectively. Histomorphometric analysis revealed sufficient donor-to-recipient axon ratios for both transfers and identified the SBRN as a suitable donor with high axon density. CONCLUSION: Our anatomic and histomorphometric results indicate that the SBRN is a suitable donor for the MN and SBUN at wrist level. The measurements show feasibility of this procedure and shall help in planning this sensory nerve transfer. High axon density in the SBRN identifies it or its branches an ideal candidate for sensory reanimation of fingers and thumbs

Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging

Bacterial outer membrane vesicles (OMVs) are increasingly used as carriers for drug delivery. Here the authors encapsulate biopolymer melanin into OMVs, extending their use to optoacoustic imaging both in vitro and in vivo, and demonstrate the potential of this tool for photothermal therapy applications

Characterization of Magnetic Viral Complexes for Targeted Delivery in Oncology

Abstract Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring. In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r 2 and r 2 * relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 -97 % reduction of r 2 and 35 -82 % increase of r 2 * compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model. In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy