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Purpose Although regular water is composed of two hydrogens and one oxygen, referred to as H2O, a small amount of water on this planet contains alternative forms of elements with different molecular weights because of the addition of neutrons. The present study was dedicated to studying the effect of heavy water (D2O), in which the two hydrogens become substituted by deuterium, on the cell physiology of different human cells with particular focus on malignant melanoma cells. Methods Cells were cultured in regular medium in which the content of H2O was gradually substituted by D2O or deuterium-depleted water (DDW). Following this, the changes of basic cellular parameters, such as morphology, migration, proliferation, cell cycle, apoptosis and microtubule integrity were examined. Results It was found that raising the D2O content above the standard levels led to a concentration-dependent decrease in proliferation. Lowering the D2O levels below this level had no effect. Likewise, elevated D2O levels hampered migration. Moreover, cell-cycle analysis showed an increase of sub-G1 cells. Corroboratively, markers for apoptosis were induced (histone-associated DNA fragments, Bax, and PARP). In regard to microtubule integrity, only very high levels of D2O (75%) caused partial filament condensation. Conclusion D2O, although chemically identical with H2O, shows proapoptotic and antiproliferative effects on melanoma cells. These findings give a closer look of this interesting compound.

Background Adipose-derived stem cells (ASC) are known to transdifferentiate into a wide range of different cell species in vitro including along the epidermal lineage. This property makes them a promising tool for regenerative medicine to restore the epidermal barrier. Objective This study is dedicated to identify in vitro conditions enabling transdifferentiation to a keratinocyte-like phenotype. In particular, the impact of different culture conditions (media compositions, 2D, 3D cultures) and extracellular matrix (ECM) molecules was evaluated. Methods Adipose-derived stem cells derived from subcutaneous abdominal fat were characterized by stemness-associated markers and subjected to different media. Epithelial differentiation in 2D cultures was monitored by pan-cytokeratin expression using flow cytometry and immunocytochemistry. To evaluate the impact of different ECM molecules on epidermal stratification, 3D cultures were produced, lifted to the air-liquid interface (ALI) and examined by histological analysis and quantitative real-time RT-PCR. Results We identified a medium composition containing retinoic acid, hydrocortisone, ascorbic acid and BMP-4 enabling maximum pan-cytokeratin expression in 2D cultures. Moreover, adhesion to type IV collagen further promotes the pan-cytokeratin expression. When cultures were lifted to the ALI, significant stratification was observed, particularly in supports coated with type IV collagen or fibronectin. Moreover, epidermal differentiation markers (involucrin, cytokeratin 1 and 14) become induced. Conclusion Conditions with hampered wound healing such as non-healing ulcers demand new treatment regimes. The here introduced optimized protocols for transdifferentiation of ASC into keratinocyte-like cells may help to establish more effective treatment procedures.

The association between angiogenesis and chronic inflammatory diseases, such as psoriasis, seems to be an important phenomenon implicated in the pathogenesis of these medical conditions. Recent studies provide evidence that dimethylfumarate (DMF) has a profound anti-inflammatory as well as anti-tumorigenic action, although the effect of DMF on angiogenesis is unknown. Signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses. Therefore, we explored whether the known anti-inflammatory and anti-tumorigenic properties of DMF might be mediated in part by anti-angiogenic effects through the reduction in VEGFR2 expression. In this study, DMF was found to inhibit endothelial VEGFR2 expression; time- and concentration-dependent inhibition was demonstrated both at the level of protein and mRNA expression. This blockade was coincident with the inhibition of the formation of capillary-like structures. The DMF-dependent inhibition of VEGFR2 transcription was found to be mediated by an element located between base pairs -60 and -37, which contains two adjacent, consensus Sp1 transcription factor-binding sites, and the constitutive formation of complexes containing Sp1 at this site is decreased by DMF treatment. Inhibition of VEGFR-2 is shown to be one critical aspect in DMF-mediated anti-angiogenic effects.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. This system controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. Recent evidence also established the importance of the proteasome in tumor development, showing antitumor and antiangiogenic actions by using selective inhibitors in vivo. As signaling via the vascular endothelial growth factor receptor 2 (VEGFR2) pathway is critical for angiogenic responses to occur, we explored whether antiangiogenic effects due to proteasome inhibition were partly mediated through decreased endothelial VEGFR2 expression. This study shows that different proteasome inhibitors blocked VEGFR2 expression in a time-dependent and concentration-dependent manner. This blockade was paralleled by the respective inhibition of the formation of capillary-like structures and endothelial cell migration. In contrast, neither tie-2 nor VEGFR1 expression was significantly affected by proteasome inhibitor treatment. The suppressive effects on VEGFR2 expression were not conveyed by increased shedding or a decrease in protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. In line with this conclusion, proteasome inhibition significantly suppressed VEGFR2 mRNA accumulation. In addition, inhibitor treatment considerably decreased the transcriptional activity of 5′ deletional VEGFR2 promoter gene constructs. Proteasome inhibition–mediated repression was controlled by a GC-rich region that harbored one consensus Sp1-binding site. Subsequent EMSA analyses showed decreased constitutive Sp1-dependent DNA binding in response to proteasome inhibition. In addition, we could show that proteasome inhibitors reduced VEGFR2 mRNA stability. Therefore, VEGFR2 expression may constitute a critical molecular target of proteasome inhibitors that may mediate their antiangiogenic effects in vivo. [Cancer Res 2009;69(5):1976–84]