Search

Background Periploca sepium is traditionally used in Chinese medicine to treat particularly rheumatic disorders and as a tonic. Periplocin was found as the most cytotoxic compound of its root bark and induced death receptor mediated apoptosis in liposarcoma cells. Sarcomas are a rare type of cancer with only a few treatment options. The five-year survival rate of advanced tumors is low. Purpose In this study, we investigated the effects of periplocin in two myxofibrosarcoma (MFS)cell lines, MUG-Myx2a and MUG-Myx2b, which are subclones of the same tumor and reflect the tumor´s heterogeneity, and in T60 primary myxofibrosarcoma cells. Methods The xCELLigence system and the CellTiter 96® AQueous assay were used for studying cell viability. FACS and Western blot experiments were used to investigate the effects of periplocin on apoptosis induction, cell cycle distribution, and the expression of cleaved PARP, caspase 3, p53, phospho-histone γH2AX, ERK/phospho ERK, p38/phospho p38, and, finally, JNK/phospho JNK. Additionally, the expression of the apoptotic markers Bim, NOXA, Bak, Bcl-2, Bcl-xl, and the death receptors IGFR, FADD, TRADD, TNFR1A, TRAIL-R1, and TRAIL-R2 were evaluated using reversed real-time PCR. Results Periplocin decreased dose-dependently the viability of all MFS cell lines and was more effective than the standard chemotherapeutic doxorubicin. It arrested the cells in the G2/M phase and led to caspase activation. Moreover, periplocin increased the mRNA expression of NOXA, Bak, Bcl-2, and death receptors such as TRAIL-R1 and TRAIL-R2 and the protein expression of ERK/phospho ERK, p38/phospho p38, and JNK/phospho JNK. In all cases, differences in the effects in the different subclones were observed. Conclusion Periplocin showed promising effects in MFS cells. The higher effectiveness compared to doxorubicin is an important aspect for further research with regard as a treatment option. The different effects of periplocin in the two subclones showed the great importance of intratumoral heterogeneity in MFS therapy.

Background Myeloperoxidase (MPO) activity increases in myocardial infarction due to neutrophil infiltration. MPO together with H2O2 and Cl- leads to the formation of HOCl, which induces plasmalogen modification. HOCl attacks the vinylether bond of plasmalogens inducing formation of alpha-chlorofatty aldehydes, like 2-chlorohexadecanal (2-ClHDA). 2-ClHDA impacts protein function by covalent modification, triggering cytotoxic responses. Methods To study 2-ClHDA-mediated protein modification in cardiomyocytes, click-chemistry with proteomic approaches was used. Therefore, an alkyne containing bioortholog of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA), was synthesized. Protein lysates of 2-ClHDyA-modified cardiomyocytes were clicked with tetramethylrhodamine (TAMRA) azide and separated by 2-DE. TAMRA-positive spots were trypsinized and identified by LC-MS/MS. Results During these analyses 51 2-ClHDyA-modified proteins were identified. Those proteins were mainly attributed to cellular processes involved in stress response, the cytoskeleton, and enzymes responsible for energy metabolism. These results suggest that cellular damage following myocardial infarction could be caused by impaired protein function due to modification by 2-ClHDA.

2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase–hydrogen peroxide–chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood–brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting., Highlights ► We studied the effects of 2-chlorohexadecanal (2-ClHDA) on brain endothelial cells. ► Brain endothelial cells take up and metabolize 2-ClHDA. ► 2-ClHDA impairs endothelial barrier integrity. ► 2-ClHDA induces mitochondrial dysfunction and activates caspase 3. ► Phloretin provides protection against 2-ClHDA-induced barrier dysfunction.

Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCl), formed by MPO from H(2)O(2) and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCl, (ii) to define the impact of exogenously added SM and HOCl-modified SM (HOCl-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCl-SM. MALDI-TOF-MS analyses revealed that HOCl, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCl-SM but not SM induced the formation of reactive oxygen species. HOCl-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCl-SM. Our results demonstrate that HOCl modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO-H(2)O(2)-chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis.

Drug delivery to the brain is severely restricted by formation of tight junctions between adjacent brain capillary endothelial cells (BCEC). In the present study we have evaluated the effects of protamine-oligonucleotide nanoparticles (proticles) on the functional properties of primary porcine BCEC and characterized uptake and transcytosis of proticles by these cells. Proticles had no adverse effects on BCEC properties relevant to blood-brain barrier (BBB) function. Transcytosis of (125)I-labeled proticles across polarized BCEC cultures occurred in a time- and concentration-dependent manner. As apolipoproteins were suggested to enhance cellular proticle uptake, proticle coating was performed with apoA-I, the major apolipoprotein component of high density lipoproteins. Adsorption of apoA-I on the surface of proticles resulted in significantly improved uptake and transcytosis properties as compared to uncoated proticles. ApoA-I coating enhanced proticle delivery to astrocytes in an in vitro model of the BBB almost twofold. Blocking of scavenger receptor class B, type I (the prime receptor for high density lipoprotein/apoA-I that is expressed on BCEC) reduced transcytosis of apoA-I-coated proticles to levels observed for uncoated proticles. Our data indicate that apoA-I-coating of proticles could be a feasible targeting technology to improve delivery across the BBB.

Corrigendum to “Mouse brain plasmalogens are targets for hypochlorous acid-mediated modification in vitro and in vivo” [Free Radic. Biol. Med. 49 (2010) 1655-1665] Andreas Ullen , Gunter Fauler , Harald Kofeler , Sabine Waltl , Christoph Nusshold , Eva Bernhart , Helga Reicher , Hans-Jorg Leis , Andrea Wintersperger , Ernst Malle , Wolfgang Sattler a,⁎ a Institute of Molecular Biology and Biochemistry, Center for Molecular Medicine, Medical University of Graz, 8010 Graz, Austria b Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, 8010 Graz, Austria c Center of Medical Research, Medical University of Graz, 8010 Graz, Austria d Research Unit of Osteology and Analytical Mass Spectrometry, University Children's Hospital, Medical University of Graz, 8010 Graz, Austria