Charakterisierung zweier im Mammakarzinom überexprimierter Gene: LIV-1 und Stromelysin-3

Um neue Zielmoleküle für die rationale Medikamentenentwicklung gegen Tumorerkrankungen der Brust identifizieren zu können, wurden anhand von Genchip Hybridisierungsdaten die im Mammakarzinom überexprimierten Gene LIV-1 und Stromelysin-3 (SL-3) für eine weitere Analyse des Proteinexpressionsmusters bzw. der Tumorrelevanz ausgewählt. Die Überexpression auf mRNA Ebene wurde zuvor mittels quantitativer RT-PCR bestätigt. Für die Transkripte des putativen Zinktransporters LIV-1 wurde durch in situ Hybridisierung an 111 Tumor- und dazugehörigen Normalgeweben der Brust die Assoziation mit einem positiven Östrogenrezeptor-? Status sowie deren Überexpression in ca. 50% der Brusttumorproben bestätigt. Da gezeigt werden konnte, dass die LIV-1 Expressionshöhe die Aktivität des Östrogenrezeptor-? besser reflektiert als der derzeit in der klinischen Diagnostik verwendete Progesteronrezeptorstatus, könnten diese für die Entscheidung über eine anti-hormonale Therapie verwendet werden. Durch die Herstellung und Validierung eines LIV-1 spezifischen polyklonalen Antikörpers war es möglich, die Lokalisation des Proteins im endoplasmatischen Retikulum nachzuweisen. Anhand von immunhistochemischen Färbungen wurde festgestellt, da LIV-1 mRNA- und Proteinexpressionshöhen weder in Normal- noch Tumorgeweben der Brust miteinander korrelieren. Dies deutet auf das Vorhandensein posttranskriptionaler Regulationsmechanismen hin. Allerdings wurde nachgewiesen, dass die LIV-1 Proteinmengen für die Prognose der Länge des krankheitsfreien Intervalls geeignet sind. Im Gegensatz zur mRNA- war die Proteinexpression in aggressiven Tumoren im Vergleich zum Brustnormalgewebe signifikant erniedrigt, was auf eine Funktion als neues Tumorsuppressorprotein hindeutet. Da außerdem eine hohe Expression des Proteins in Normalgeweben lebenswichtiger Organe gezeigt wurde, scheint LIV-1 als potentielles Zielmolekül für die Entwicklung therapeutischer Inhibitoren ungeeignet zu sein. Für die sekretierte Matrix-Metalloprotease SL-3, deren prognostische Signifikanz bereits bekannt war, wurde gezeigt, dass eine Überexpression in humanen Brusttumorzelllinien deren Zellzahl in 3D Zellkulturmodellen erhöht. Dieser Effekt war abhängig von der IGF-1 Signalkaskade. Außerdem steigerte SL-3 sowohl in in vitro Softagartests als auch in in vivo Xenograft Tumormodellen in Nacktmäusen die Tumorgenität der Zelllinien. In vivo führte die SL-3 Überexpression zu einer höheren Anwachsrate sowie einer Volumenzunahme der Tumore. Dieser Effekt war ebenfalls abhängig von der Anwesenheit von Wachstumsfaktoren. Die gewonnenen Daten zeigen somit, dass LIV-1 als prognostischer Marker sowohl auf mRNA- als auch auf Proteinebene klinische Relevanz beweisen könnte. Sl-3 hingegen stellt aufgrund seiner hohen Überexpression im Mammakarzinom und der gezeigten Tumorrelevanz in vitro und in vivo einen viel versprechenden Kandidaten für die Entwicklung eines spezifischen Therapieansatzes dar

Osteogenic Predifferentiation of Human Bone Marrow-Derived Stem Cells by Short-Term Mechanical Stimulation

It is commonly accepted that bone marrow-derived stem cells (BMSCs) have to be expanded in vitro, but a prolonged time in culture decreases their multilineage potential. Mechanical and biological stimuli have been used to improve their osteogenic potential. While long-term stimulation has been shown to improve osteogenic differentiation, it remains to be seen whether short-term stimulation is also sufficient

Oxidative functionalization mechanisms in organic synthesis using fenton reaction systems

We argued an oxywater-oxene concept for hydrogen peroxide transformation in Fenton reaction systems and used this interpretation for mechanisms explanation of hydroperoxide monooxygen oxidative functionalization processes (alkane and arene hydroxylation, alkene epoxidation, Baeyer-Villiger ketone oxidation to ester, organonitrogen compounds N-oxidation and organosulfur compounds S-oxidation) and dioxygen alkene and alkadiene functionalization processes (synthesis of hydroperoxides and cyclic peroxides)

Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

BACKGROUND: Stromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized. METHODS: The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed. RESULTS: Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts. CONCLUSION: These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis

Cyclic strain disrupts endothelial network formation on Matrigel

Most forms of tissue healing depend critically on revascularisation. In soft tissues and in vitro, mechanical stimuli have been shown to promote vessel-forming activity. However, in bone defects, increased interfragmentary motion impairs vascular regeneration. Because these effects seem contradictory, we aimed to determine whether a range of mechanical stimuli exists in which angiogenesis is favoured. A series of cyclic strain magnitudes were applied to a Matrigel-based “tube formation” assay and the total lengths of networks formed by human microvascular endothelial cells measured at 24 h. Network lengths were reduced at all strain levels, compared to unstretched controls. However, the levels of pro-angiogenic matrix metalloproteases-2 and -9 in the corresponding conditioned media were unchanged by strain, and vascular endothelial growth factor was uniformly elevated in stretched conditions. By repeating the assay with the addition of conditioned media from mesenchymal stem cells cultivated in similar conditions, paracrine stimuli were shown to increase network lengths, but not to alter the negative effect of cyclic stretching. Together, these results demonstrate that directly applied periodic strains can inhibit endothelial organisation in vitro, and suggest that this may be due to physical disruption rather than biochemical modulation. Most importantly, the results indicate that the straining of endothelial cells and their assembly into vascular-like structures must be studied simultaneously to adequately characterise the mechanical influence on vessel formation

Mechanical influences on endothelial cell network formation in vitro

While both the restoration of the blood supply and an appropriate local mechanical environment are critical for uneventful bone healing, their influence on each other remains unclear. Human bone fracture haematomas (<72h post-trauma) were cultivated for 3 days in fibrin matrices, with or without cyclic compression. Conditioned medium from these cultures enhanced the formation of vessel-like networks by HMEC-1 cells, and mechanical loading further elevated it, without affecting the cells’ metabolic activity. While haematomas released the angiogenesis-regulators, VEGF and TGF-β1, their concentrations were not affected by mechanical loading. However, direct cyclic stretching of the HMEC-1 cells decreased network formation. The appearance of the networks and a trend towards elevated VEGF under strain suggested physical disruption rather than biochemical modulation as the responsible mechanism. Thus, early fracture haematomas and their mechanical loading increase the paracrine stimulation of endothelial organisation in vitro, but direct periodic strains may disrupt or impair vessel assembly in otherwise favourable conditions

Validation of β-actin used as endogenous control for gene expression analysis in mechanobiology studies : amendments

We write in response to the letter by Liu et al. [1] commenting\ud on our article, ‘‘Mesenchymal Stem Cells Regulate Angiogenesis\ud According to Their Mechanical Environment’’ [2]. The\ud study by Liu et al. demonstrates that the commonly used endogeneous\ud reference gene (ERG), b-actin, is upregulated by mechanical\ud loading, indicating a potential bias in the determined\ud target gene expression when normalizing to b-actin, such as in\ud our report on unchanged vascular endothelial growth factor\ud (VEGF) and hypoxia-inducible factors (HIF)-1a mRNA levels\ud in mechanically loaded mesenchymal stem cells (MSCs)

Toward biomimetic materials in bone regeneration : functional behavior of mesenchymal stem cells on a broad spectrum of extracellular matrix components

Bone defect treatments can be augmented by mesenchymal stem cell (MSC) based therapies. MSC interaction with the extracellular matrix (ECM) of the surrounding tissue regulates their functional behavior. Understanding of these specific regulatory mechanisms is essential for the therapeutic stimulation of MSC in vivo. However, these interactions are presently only partially understood. This study examined in parallel, for the first time, the effects on the functional behavior of MSCs of 13 ECM components from bone, cartilage and hematoma compared to a control protein, and hence draws conclusions for rational biomaterial design. ECM components specifically modulated MSC adhesion, migration, proliferation, and osteogenic differentiation, for example, fibronectin facilitated migration, adhesion, and proliferation, but not osteogenic differentiation, whereas fibrinogen enhanced adhesion and proliferation, but not migration. Subsequently, the integrin expression pattern of MSCs was determined and related to the cell behavior on specific ECM components. Finally, on this basis, peptide sequences are reported for the potential stimulation of MSC functions. Based on the results of this study, ECM component coatings could be designed to specifically guide cell functions