Antje Ludwig

Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovarian cancer cells.

Telomerase is a promising target for human cancer gene therapy. Its inhibition allows telomere shortening to occur in cancer cells, which in turn is thought to trigger delayed senescence and/or apoptosis. We tested whether telomerase inhibition might have additional, immediate effects on tumor cell growth. Ovarian cancer cell lines with widely differing telomere lengths were efficiently transduced with an adenovirus expressing a ribozyme directed against the T motif of the catalytic subunit of human telomerase, hTERT. Three days after transduction, telomerase activity was significantly reduced and massive cell loss was induced in mass cultures from all four ovarian cancer cell lines tested, whereas transduction of telomerase-negative human fibroblasts did not attenuate their growth. The kinetics of induction of cell death in cancer cells was not significantly dependent on telomere length, and telomeres did not shorten measurably before the onset of apoptosis. The data suggest the existence of a “fast-track” mechanism by which diminution of telomerase can interfere with cancer cell growth and induce cell death, presumably by apoptosis. This phenomenon might be a consequence of the telomere capping function provided by telomerase in tumor cells. Uncapping of telomeres by ribozyme-mediated inhibition of telomerase bears therapeutic potential for ovarian cancer. Cancer Gene Therapy (2001) 8, 827–834

Multiple cardiac proteasome subtypes differ in their susceptibility to proteasome inhibitors

AIMS The proteasome is the proteolytically active core of the ubiquitin-proteasome system, which regulates vital processes and which can cause various diseases when it malfunctions. Therefore, the proteasome has become an attractive target for pharmaceutical interventions. Inhibition of the cardiac proteasome by specific proteasome inhibitors has been shown to attenuate cardiac hypertrophy and ischaemia reperfusion injury of the heart. We have resolved the cardiac proteasome into its subtypes and have addressed the key question of how proteasome inhibitors affect single cardiac proteasomal subtypes. METHODS AND RESULTS The 20S proteasome from rat heart was dissected into three different subpopulations (groups I-III), each comprising 4-7 different subtypes. The major group (group II) comprises standard proteasome subtypes; the two minor subpopulations (groups I and III) contain intermediate proteasome subtypes. All subtypes exhibit chymotrypsin-, trypsin-, and caspase-like activity but to different degrees. We have tested the effect of two common proteasome inhibitors on the chymotrypsin-like activity of all subtypes: 20-30 nmol/L MG132 caused 50% inhibition of all subtypes from groups I and II, whereas 100 nmol/L was necessary to affect group III subtypes to the same extent. However, another inhibitor, bortezomib (VELCADE), already used clinically, inhibited 50% of the activity of group III proteasome subtypes even below 20 nmol/L, a concentration showing almost no effect on group I and II proteasome subtypes. The caspase-like activity of group II proteasome subtypes was not affected by MG132 and was inhibited by bortezomib only at concentrations above 100 nmol/L. CONCLUSION These data show that different inhibitors have differential inhibitory effects on the various cardiac proteasome subtypes. Different cardiac subtypes are inhibited by the same dose of proteasome inhibitor to a different extent.

Combined Effect of Proteasome and Calpain Inhibition on Cisplatin-Resistant Human Melanoma Cells

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.

Effects of Lycopene on the Initial State of Atherosclerosis in New Zealand White (NZW) Rabbits

Background Lycopene is the main carotenoid in tomatoes, where it is found in high concentrations. Strong epidemiological evidence suggests that lycopene may provide protection against cardiovascular diseases. We therefore studied the effects of lycopene on diet-induced increase in serum lipid levels and the initiation of atherosclerosis in New Zealand White (NZW) rabbits. Methodology/Principal Findings The animals, divided into four groups of 9 animals each, were fed either a standard diet, a high-cholesterol diet containing 0.5% cholesterol, a high-cholesterol diet containing placebo beadlets, or a high-cholesterol diet plus 5 mg/kg body weight/day of lycopene (in the form of lycopene beadlets), for a period of 4 weeks. We found significantly elevated lycopene plasma levels in the animal group treated with lycopene beadlets. Compared to the high-cholesterol and the placebo group, this was associated with a significant reduction of 50% in total cholesterol and LDL cholesterol serum levels in the lycopene group. The amount of cholesteryl ester in the aorta was significantly decreased by lycopene. However, we did not observe a significant decrease in the extent of aortic surface lipid accumulation in the lycopene group. In addition, no differences in the intima-media thickness among groups were observed. Endothelial-dependent and endothelial-independent vasodilation in isolated rabbit aortic and carotid rings did not differ among any of the animal groups. Conclusions Lycopene supplementation for 4 weeks increased lycopene plasma levels in the animals. Although we found strongly reduced total and LDL cholesterol serum levels as well as significantly lower amounts of cholesteryl ester in the aortae in the lycopene-treated group, no significant differences in initial lesions in the aortae were detected.

Contrast-enhanced MR imaging of atherosclerosis using citrate-coated superparamagnetic iron oxide nanoparticles: calcifying microvesicles as imaging target for plaque characterization.

Objective To evaluate the suitability of citrate-coated very small superparamagnetic iron oxide particles (VSOP) as a contrast agent for identifying inflammation in atherosclerotic lesions using magnetic resonance imaging (MRI). Methods and results VSOP, which have already been evaluated as a blood pool contrast agent for MR angiography in human clinical trials, were investigated in Watanabe heritable hyper-lipidemic rabbits to determine to what extent their accumulation in atherosclerotic lesions is a function of macrophage density and other characteristics of progressive atherosclerotic plaques. In advanced atherosclerotic lesions, a significant MRI signal loss was found within 1 hour after intravenous administration of VSOP at the intended clinical dose of 0.05 mmol Fe/kg. Histological examinations confirmed correlations between the loss of MRI signal in the vessel wall and the presence of Prussian blue-stained iron colocalized with macrophages in the plaque cap, but surprisingly also with calcifying microvesicles at the intimomedial interface. Critical electrolyte magnesium chloride concentration in combination with Alcian blue stain indicates that highly sulfated glycosaminoglycans are a major constituent of these calcifying microvesicles, which may serve as the key molecules for binding VSOP due to their highly complexing properties. Conclusion Calcifying microvesicles and macrophages are the targets for intravenously injected VSOP in atherosclerotic plaques, suggesting that VSOP-enhanced MRI may render clinically relevant information on the composition and inflammatory activity of progressive atherosclerotic lesions at risk of destabilization.

Smoking decreases the level of circulating CD34+ progenitor cells in young healthy women - a pilot study

BackgroundDecreased levels of circulating bone marrow-derived progenitor cells have been associated with risk factors and cardiovascular diseases. Smoking is the most important modifiable risk factor for atherosclerosis in young women. The aim of this pilot study was to assess in healthy premenopausal women without other risk factors for cardiovascular disease the influence of nicotine abuse on the number of circulating progenitor cells in relation to endothelial function.MethodsThe number of endothelial progenitor cells, measured as colony-forming units in a cell-culture assay (EPC-CFU) and the number of circulating CD34 + and CD34 + /CD133 + cells, measured by flow cytometry, was estimated in 32 women at the menstrual phase of the menstrual cycle. In addition, flow-mediated dilation (FMD) was assessed as a marker for vascular function. In a subgroup of these women (n = 20), progenitor cells were also investigated at the mid-follicular and luteal phases of the menstrual cycle.ResultsCompared to non-smokers, the abundance of circulating CD34 + cells was significantly lower in smoking women in the menstrual, mid-luteal, and mid-follicular phases of the menstrual cycle. The number of CD34 + progenitor cells was revealed to have significant positive correlation with FMD in young healthy women, whereas CD34 + /CD133 + progenitor cells and EPC-CFU showed no significant correlation.ConclusionThe number of CD34 + progenitor cells positively correlates with FMD in young healthy women and is decreased by smoking.

Epigenetic Regulation of Cell Adhesion and Communication by Enhancer of Zeste Homolog 2 in Human Endothelial Cells

The histone methyltransferase enhancer of zeste homolog 2 (Ezh2) mediates trimethylation of lysine 27 in histone 3, which acts as a repressive epigenetic mark. Ezh2 is essential for maintaining pluripotency of stem cells, but information on its role in differentiated cells is sparse. Whole-genome mRNA expression arrays identified 964 genes that were regulated by >2-fold 72 hours after small interfering RNA-mediated silencing of Ezh2 in human umbilical vein endothelial cells. Among them, genes associated with the gene ontology terms cell communication and cell adhesion were significantly overrepresented, suggesting a functional role for Ezh2 in the regulation of angiogenesis. Indeed, adhesion, migration, and tube formation assays revealed significantly altered angiogenic properties of human umbilical vein endothelial cells after silencing of Ezh2. To identify direct target genes of Ezh2, we performed chromatin immunoprecipitation experiments followed by whole-genome promoter arrays (chromatin immunoprecipitation-on-chip) and identified 5585 genes associated with trimethylation of lysine 27 in histone 3. Comparative analysis with our mRNA expression data identified 276 genes that met our criteria for putative Ezh2 target genes, upregulation by >2-fold after Ezh2 silencing and association with trimethylation of lysine 27 in histone 3. Notably, we observed a striking overrepresentation of genes involved in wingless-type mouse mammary tumor virus integration site (WNT) signaling pathways. Epigenetic regulation of several of these genes by Ezh2 was specifically confirmed by polymerase chain reaction analysis of DNA enrichment after chromatin immunoprecipitation using an antibody specific for trimethylation of lysine 27 in histone 3. Combining mRNA expression arrays and chromatin immunoprecipitation-on-chip analysis, we identified 276 Ezh2 target genes in endothelial cells. Ezh2-dependent repression of genes involved in cell adhesion and communication contributes to the regulation of angiogenesis.

Proteasome inhibition prevents experimentally-induced endothelial dysfunction.

AIMS We recently demonstrated that non-toxic inhibition of the proteasome upregulates antioxidative enzymes and leads to an adaptive transcriptional pattern in endothelial cells. We therefore hypothesized that proteasome inhibition could prevent experimentally-induced endothelial dysfunction. As there are conflicting data about the effects of proteasome inhibition on endothelial function, we investigated whether proteasome inhibition could prevent experimentally-induced endothelial dysfunction. MAIN METHODS Endothelial dysfunction in isolated rat aortic rings was induced by incubation of rings with TNFalpha for 48 h. To study the effects of the inhibition of the proteasome, selected rings were co-treated with proteasome inhibitors. Vasorelaxation and expression of genes involved in endothelial function were evaluated. KEY FINDINGS Incubation of rat aortic rings with TNFalpha for 48 h led to significant dose-dependent reduction of acetylcholine-induced vasorelaxation. Co-incubation with TNFalpha and the proteasome inhibitor MG132 resulted in dose-dependent improvement of endothelium-dependent vasorelaxation in comparison to rings treated with TNFalpha alone. Levels of eNOS mRNA and protein were reduced despite improved vascular function after treatment with MG132. MG132 markedly suppressed mRNA levels of NADPH oxidase subunits and increased SOD1 expression. Superoxide production was reduced in rings incubated with MG132 in comparison to controls. TNFalpha-induced upregulation of the potent vasoconstrictor endothelin was abolished by MG132. SIGNIFICANCE Proteasome inhibition prevents TNFalpha-induced vascular dysfunction by reduction of superoxide production and suppression of endothelin levels. The balance between vasoconstriction and vasodilatation is shifted in favour of endothelium-dependent vasodilation.

Rapid binding of electrostatically stabilized iron oxide nanoparticles to THP-1 monocytic cells via interaction with glycosaminoglycans

Magnetic resonance imaging (MRI) with contrast agents that target specific inflammatory components of atherosclerotic lesions has the potential to emerge as promising diagnostic modality for detecting unstable plaques. Since a high content of macrophages and alterations of the extracellular matrix are hallmarks of plaque instability, these structures represent attractive targets for new imaging modalities. In this study, we compared in vitro uptake and binding of electrostatically stabilized citrate-coated very small superparamagnetic iron oxide particles (VSOP) to THP-1 cells with sterically stabilized carboxydextran-coated Resovist®. Uptake of VSOP in both THP-1 monocytic cells and THP-derived macrophages (THP-MΦ) was more efficient compared to Resovist® without inducing cytotoxicity or modifying normal cellular functions (no changes in levels of reactive oxygen species, caspase-3 activity, proliferation, cytokine production). Importantly, VSOP bound with high affinity to the cell surface and to apoptotic membrane vesicles. Inhibition of glycosaminoglycan (GAG) synthesis by glucose deprivation in THP-MΦ was associated with a significant reduction of VSOP attachment suggesting that the strong interaction of VSOP with the membranes of cells and apoptotic vesicles occurs via binding to negatively charged GAGs. These in vitro experiments show that VSOP-enhanced MRI may represent a new imaging approach for visualizing high-risk plaques on the basis of targeting pathologically increased GAGs or apoptotic membrane vesicles in atherosclerotic lesions. VSOP should be investigated further in appropriate in vivo experiments to characterize accumulation in unstable plaque.

Synthesis and biological activity of optimized belactosin C congeners

Successful biochemical studies of the natural products belactosin A and C as well as their more stable acylated derivatives have proved them to be powerful proteasome inhibitors and thereby potential candidates as pharmacologically relevant active compounds. In order to understand their structure-biological activity relations in detail and to find ways of improving their biological activity, four new modified belactosin congeners have been synthesized and tested. One of them (compound 6) turned out to be a more potent inhibitor against HeLa cells than the known proteasome inhibitor MG132.