Background Ethyl pyruvate (EP) exerts anti-inflammatory and anti-oxidative properties. The aim of our study was to investigate whether EP is capable of inhibiting the oxidation of LDL, a crucial step in atherogenesis. Additionally, we examined whether EP attenuates the cytotoxic effects of highly oxidized LDL in the human vascular endothelial cell line EA. hy926. Methods Native LDL (nLDL) was oxidized using Cu2+ ions in the presence of increasing amounts of EP. The degree of LDL oxidation was quantified by measuring lipid hydroperoxide (LPO) and malondialdehyde (MDA) concentrations, relative electrophoretic mobilities (REMs), and oxidation-specific immune epitopes. The cytotoxicity of these oxLDLs on EA. hy926 cells was assessed by measuring cell viability and superoxide levels. Furthermore, the cytotoxicity of highly oxidized LDL on EA. hy926 cells under increasing concentrations of EP in the media was assessed including measurements of high energy phosphates (ATP). Results Oxidation of nLDL using Cu2+ ions was remarkably inhibited by EP in a concentration-dependent manner, reflected by decreased levels of LPO, MDA, REM, oxidation-specific epitopes, and diminished cytotoxicity of the obtained oxLDLs in EA. hy926 cells. Furthermore, the cytotoxicity of highly oxidized LDL on EA. hy926 cells was remarkably attenuated by EP added to the media in a concentration-dependent manner reflected by a decrease in superoxide and an increase in viability and ATP levels. Conclusions EP has the potential for an anti-atherosclerotic drug by attenuating both, the oxidation of LDL and the cytotoxic effect of (already formed) oxLDL in EA. hy926 cells. Chronic administration of EP might be beneficial to impede the development of atherosclerotic lesions.

Merkel cell carcinoma (MCC) is an aggressive metastatic neuroendocrine skin cancer associated with high recurrence rates as well as regional and distant metastasis resulting in a high mortality. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid playing a decisive role in promoting tumor cell survival, proliferation, migration and metastasis via intracellular and extracellular targets. SPHK1 and 2 are the enzymes that phosphorylate pro-apoptotic sphingosine to form pro-survival S1P and are upregulated in different cancer entities. The objective of this study was to determine the importance of intracellular S1P in MCC cell survival and proliferation. The SPHK1 and 2 expressions were analyzed by qRT-PCR in 16 MCC cell lines. The well characterized MCC cell lines MKL1 and WaGa were used for functional studies in vitro. Since the cell of origin of MCC is still controversial, normal human dermal fibroblasts (NHDF) were used as non-transformed control cells. Treatment with SKI-II, an inhibitor of SPHK1 and 2, on MKL1 and WaGa resulted in dose dependent decrease of cell viability and increased apoptosis. In contrast, we did not see any effect of SKI-II inhibitor on NHDF cell viability as determined by CellTiter-Glo 3D cell viability assay and FITC Annexin V FACS analysis. Further, S1P-ELISA showed a decrease in intracellular S1P concentration and LC/MS analysis revealed an increase in ceramide and sphingomyelin species upon treatment with SKI-II in both MKL1 and WaGa cell lines. In addition, immunoblotting revealed decrease in pAKT levels and increased Caspase3 and PARP cleavage in SKI-II treated MKL1 and WaGa. In conclusion our in vitro data suggest that targeting the S1P axis represents a potential therapeutic strategy in MCC.

Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1–6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed “LPA” throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways.

Increased Lipoprotein associated phospholipase A(2) (LpPLA(2)) has been associated with inflammatory pathologies, including Type 2 Diabetes. Studies on LpPLA(2) and Gestational Diabetes Mellitus (GDM) are rare, and have focused mostly on maternal outcome. In the present study, we investigated whether LpPLA(2) activity on foetal lipoproteins is altered by maternal GDM and/or obesity (a major risk factor for GDM), thereby contributing to changes in lipoprotein functionality. We identified HDL as the major carrier of LpPLA(2) activity in the foetus, which is in contrast to adults. We observed marked expression of LpPLA(2) in placental macrophages (Hofbauer cells; HBCs) and found that LpPLA(2) activity in these cells was increased by insulin, leptin, and pro-inflammatory cytokines. These regulators were also increased in plasma of children born from GDM pregnancies. Our results suggest that insulin, leptin, and pro-inflammatory cytokines are positive regulators of LpPLA(2) activity in the foeto-placental unit. Of particular interest, functional assays using a specific LpPLA(2) inhibitor suggest that high-density lipoprotein (HDL)-associated LpPLA(2) exerts anti-oxidative, athero-protective functions on placental endothelium and foetus. Our results therefore raise the possibility that foetal HDL-associated LpPLA(2) might act as an anti-inflammatory enzyme improving vascular barrier function.

Merkel cell carcinoma (MCC) is an aggressive metastatic neuroendocrine skin cancer associated with high recurrence and mortality rates. Sphingolipids (SL) represent a major class of bioactive lipids potently regulating cell proliferation, migration, and apoptosis. In particular pro-survival sphingosine 1-phosphate (S1P) plays a prominent role in tumor biology. De novo synthesis of S1P and its precursor ceramide begins from condensation of serine and palmitoyl-CoA catalysed by serine palmitoyltransferases (SPTLC) 1-3. The objective of this study was to elucidate whether pharmacological interference with S1P synthesis would impact MCC cell proliferation, survival, and apoptosis. MKL1 and WaGa cell lines (reflecting phenotypic properties of primary MCC tumors) were used as in vitro MCC models. Since the cell of origin of MCC is still controversial, normal human dermal fibroblasts (NHDF) were used as non-transformed control cells. qPCR and Western blotting revealed the expression of SPTLC 1-3. Myriocin, a pharmacological inhibitor of SPTLC1-3, was used to inhibit the first step of SL synthesis. Using CellTiter-Glo assay and flow cytometry, we observed dose dependent decrease in cell viability and induction of apoptosis in MCC cells. Of note, NHDF viability was unaffected by myriocin. LC/MS analysis and S1P-ELISA showed significant reduction in intracellular ceramide and S1P concentrations in myriocin treated MCC cells. This was accompanied by decreased phosphorylation of AKT and increased Caspase3 and PARP cleavage. In conclusion our in vitro data suggest that targeting the S1P axis in MCC could represent a potential anti-proliferative strategy in MCC.

Soft tissue sarcomas represent a rare group of malignant tumors frequently exhibiting increased metastatic potential and chemotherapeutic resistance. Treatment is often unsuccessful or the efficacy limited. Therefore, there is an urgent need for the discovery of new lead compounds. In this study, we investigated the effects of 25-O-acetyl-23,24-dihydro-cucurbitacin F (ADCF) on cell viability, cell cycle distribution and apoptosis induction in three different sarcoma cell lines. The compound was previously isolated as most active constituent from Quisqualis indica L. (Combretaceae) which gained interest as a result of a systematic bioactivity-based screening of plants used in traditional Chinese medicine [1]. In soft tissue sarcoma cells, ADCF reduced cell viability concentration dependently (IC50 values after 48h: SW-872: 16.2 µM; SW-982: 4.3 µM; TE-671: 1.2 µM). In SW-872 and TE-671 cells, ADCF additionally arrested the cells at the G2/M interphase and led to a significant reduction of cyclin B1, cyclin A, CDK1, CDK2 and survivin. Moreover, it induced apoptosis caspase-3 dependently [2]. However, in SW-982 cells, the cell cycle was only slightly changed and caspase-3 was not activated when treated with the IC50.

Objective: It was our aim to investigate effects of human LDL, copper-, or AAPH-oxidized over different periods of time to different degrees (ox-LDL), on viability and electrophysiological parameters of isolated ventricular myocytes of guinea pigs.Methods: Guinea pig ventricular myocytes were incubated with ox-LDL or native LDL (at 0.5 mg/ml) for 12 h, and afterwards myocyte damage, action potentials, and transmembrane ion currents were studied (at 37 degrees C).Results: Ox-LDL was found to induce severe myocyte damage, whereas native LDL had no effect. Myocyte damage was dependent on the content of total lipid hydroperoxides in both copper-oxidized and AAPH-oxidized LDL. Incubation with ox-LDL led to intense contractile and electrophysiological effects including prolongation of action potential duration, depolarization of resting membrane potential, spontaneous activity, generation of afterdepolarizations, and modification of transmembrane ion currents (e.g. inward rectifier, calcium, and background currents).Conclusions: Ox-LDL induced cell damage and irregular electrical activity in ventricular myocytes. These effects were dependent on the lipid hydroperoxide content of ox-LDL and were similar to oxidative stress (OS) induced by various OS-generating systems. The observed effects may play a role for functional cardiac abnormalities in patients with increased ox-LDL levels. (c) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

The aim of this investigation was to study L-type and T-type Ca(2+) current (I(CaL) and I(CaT)) in short-term cultured adult guinea pig ventricular myocytes. The isolated myocytes were suspended in serum-supplemented medium up to 5 days. Using whole-cell patch clamp techniques ICaL and ICaT were studied by applying voltage protocols from different holding potentials (-40 and -90 mV). After 5 days in culture the myocytes still showed their typical rod shaped morphology but a decline in cell membrane capacitance (26 %). The peak density of ICaT was reduced significantly between day 0 (-1.6+/-0.37 pA/pF, n=9) and day 5 (-0.4+/-0.13 pA/pF, n=11), whereas peak ICaL density revealed no significant differences during culturing. The I(CaT)/I(CaL) ratio dropped from 0.13 at day 0 to 0.05 at day 5. Compared with day 0 I(CaL) the steady state inactivation curve of day 1, day 3 and day 5 myocytes was slightly shifted to more negative potentials. Our data indicate that guinea pig ventricular L-type and T-type Ca(2+) channels are differently regulated in culture.

It was the aim of our study to investigate the effects of the sulphonylurea glibenclamide on voltage dependent potassium currents in human atrial myocytes. The drug blocked a fraction of the quasi steady state current (ramp response) which was activated positive to −20 mV, was sensitive to 4‐aminopyridine (500 μM) and was different from the ATP dependent potassium current IK(ATP). Glibenclamide dose dependently inhibited both, the peak as well as the late current elicited by step depolarization positive to −20 mV. The IC50 for reduction in charge area of total outward current was 76 μM. The double‐exponential inactivation time‐course of the total outward current was accelerated in the presence of glibenclamide with a τfast of 12.7±1.5 ms and a τslow of 213±25 ms in control and 5.8±1.9 ms (P<0.001) and 101±20 ms (P<0.05) under glibenclamide (100 μM). Our data suggest, that both repolarizing currents in human atrial myocytes, the transient outward current (Ito1) and the ultrarapid delayed rectifier current (IKur) were inhibited by glibenclamide. In human ventricular myocytes glibenclamide inhibited Ito1 without affecting the late current. Our data suggest that glibenclamide inhibits human voltage dependent cardiac potassium currents at concentrations above 10 μM. British Journal of Pharmacology (1999) 128, 1175–1180; doi:10.1038/sj.bjp.0702904