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Rationale: Signal transduction mediated by receptor tyrosine kinases (RTKs) such as EGFR and Met is often altered in lung cancer. Targeting RTKs in such tumors offers a benefit, however it is followed by a fast resistance. Therefore, in-depth mechanistic understanding of how tumor cells process the information from RTKs is essential for developing novel intervention strategies. Aim: We aim to understand whether Met receptor clearance from the plasma membrane and both, degree and duration of receptor phosphorylation could be modulated to encode the information about the stimulus. We aim to characterize changes in the proteome upon different stimuli and using mathematical modelling gain insight into the mechanisms of pathway activation. Methods: Mass Spectrometry and microscopy are used to examine perturbations induced by antibodies selectively recognizing Met, or Archazolid A which accumulates receptors in intracellular vesicles. Results: Applied antibodies differentially induce the residue-specific patterns of Met phosphorylation. As a result we observe alterations in activation of downstream signalling pathways and changes in the invasive potential of lung cancer cells. HGF stimulation after treatment with Archazolid A leads to a faster induction and quicker attenuation of Met activation and phosphorylation of downstream signalling pathways is impaired. Conclusion: Met autophosphorylation degree in response to the extracellular cues depends on the character of a stimulus. By modulating the phosphorylation of different Met residues we are able to alter cell fate decisions. Applying systems biology approach we hope to descibe the mechanistic properties of different receptor activation patterns.

Metal-organic frameworks (MOFs) are promising platforms for the synthesis of nanoparticles for diverse medical applications. Their fundamental design principles allow for significant control of the framework architecture and pore chemistry, enabling directed functionalization for nanomedical applications. However, before applying novel nanomaterials to patients, it is imperative to understand their potential health risks. In this study, the nanosafety of different MOF nanoparticles is analyzed comprehensively for diverse medical applications. The authors first evaluate the effects of MOFs on human endothelial and mouse lung cells, which constitute a first line of defense upon systemic blood-mediated and local lung-specific applications of nanoparticles. Second, we validated these MOFs for multifunctional surface coatings of dental implants using human gingiva fibroblasts. Moreover, biocompatibility of MOFs is assessed for surface coating of nerve guidance tubes using human Schwann cells and rat dorsal root ganglion cultures. The main finding of this study is that the nanosafety and principal suitability of our MOF nanoparticles as novel agents for drug delivery and implant coatings strongly varies with the effector cell type. We conclude that it is therefore necessary to carefully evaluate the nanosafety of MOF nanomaterials with respect to their particular medical application and their interacting primary cell types, respectively.

Cyclin-dependent kinase 5 (CDK5) has recently emerged as an attractive target in several tumour entities. Inhibition of CDK5 has been shown to have anti-angiogenic effects in vitro and in vivo. However, potent inhibitors of CDK5, which can be applied in vivo, are still scarce. We have recently developed a new series of 5-substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines that show a preference for inhibiting CDK5 and tested them in vitro and in vivo in a murine model of hepatocellular carcinoma.All compounds were initially examined for effects on proliferation of HUVECs. The most potent compounds were then tested on migration, and one of them, LGR2674, was selected for assessing effects on nuclear fragmentation, cell cycle, cell viability and metabolic activity. Furthermore, LGR2674 was tested in a tube formation assay and in vivo in a murine model of hepatocellular carcinoma, induced by s.c. injection of HUH7 cells (measurement of in vivo toxicity, tumour vascularization, tumour cell proliferation and tumour size).LGR2674 showed an EC50 in the low nanomolar range in the proliferation and migration assays. Cytotoxic effects started at 50 nM, a concentration that did not influence the cell cycle. In vivo, LGR2674 was well tolerated and caused a clear reduction in vessel density in the tumours; also tumour cell proliferation was inhibited and tumour growth retarded.Pyrazolo[4,3-d]pyrimidine is a novel scaffold for the development of potent CDK inhibitors with in vivo potential. Such structures are good candidates for broadening our pharmacological arsenal against various tumours.

Metastatic invasion is the major cause of cancer-related deaths. In this study, we introduce two-pore channels (TPC), a recently described class of NAADP- and PI(3,5)P2–sensitive Ca2+-permeable cation channels in the endolysosomal system of cells, as candidate targets for the treatment of invasive cancers. Inhibition of the channel abrogated migration of metastatic cancer cells in vitro. Silencing or pharmacologic inhibition of the two-pore channel TPC2 reduced lung metastasis of mammary mouse cancer cells. Disrupting TPC function halted trafficking of β1-integrin, leading to its accumulation in EEA1-positive early endosomes. As a consequence, invasive cancer cells were no longer able to form leading edges, which are required for adequate migration. Our findings link TPC to cancer cell migration and provide a preclinical proof of concept for their candidacy as targets to treat metastatic cancers. Cancer Res; 77(6); 1427–38. ©2017 AACR.

BACKGROUND AND PURPOSE The use of tubulin-binding compounds, which act in part by inhibiting tumour angiogenesis, has become an integral strategy of tumour therapy. Recently, tubulysins were identified as a novel class of natural compounds of myxobacterial origin, which inhibit tubulin polymerization. As these compounds are structurally highly complex, the search for simplified precursors [e.g. pretubulysin (Prt)] and their derivatives is mandatory to overcome supply problems hampering clinical development. We tested the anti-angiogenic efficacy of Prt and seven of its derivatives in comparison to tubulysin A (TubA). EXPERIMENTAL APPROACH The compounds were tested in cellular angiogenesis assays (proliferation, cytotoxicity, cell cycle, migration, chemotaxis, tube formation) and in vitro (tubulin polymerization). The efficacy of Prt was also tested in vivo in a murine subcutaneous tumour model induced with HUH7 cells; tumour size and vascularization were measured. KEY RESULTS The anti-angiogenic potency of all the compounds tested ran parallel to their inhibition of tubulin polymerization in vitro. Prt showed nearly the same efficacy as TubA (EC50 in low nanomolar range in all cellular assays). Some modifications in the Prt molecule caused only a moderate drop in potency, while others resulted in a dramatic loss of action, providing initial insight into structure–activity relations. In vivo, Prt completely prevented tumour growth and reduced vascular density to 30%. CONCLUSIONS AND IMPLICATIONS Prt, a chemically accessible precursor of some tubulysins is a highly attractive anti-angiogenic compound both in vitro and in vivo. Even more simplified derivatives of this compound still retain high anti-angiogenic efficacy.

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442.

Objective— Inhibitor of apoptosis proteins (IAPs), such as X-linked or cellular IAP 1/2 (XIAP, cIAP1/2), are important regulators of apoptosis. IAP antagonists are currently under clinical investigation as anticancer agents. Interestingly, IAPs participate in the inflammation-associated TNF receptor signaling complex and regulate NFκB signaling. This raises the question about the role of IAPs in inflammation. Here, we investigated the anti-inflammatory potential of IAP inhibitors and the role of IAPs in inflammatory processes of endothelial cells. Methods and Results— In mice, the small molecule IAP antagonist A-4.10099.1 (ABT) suppressed antigen-induced arthritis, leukocyte infiltration in concanavalin A-evoked liver injury, and leukocyte transmigration in the TNFα-activated cremaster muscle. In vitro, we observed an attenuation of leukocyte–endothelial cell interaction by downregulation of the intercellular adhesion molecule-1. ABT did not impair NFκB signaling but decreased the TNFα-induced activation of the TGF-β–activated kinase 1, p38, and c-Jun N-terminal kinase. These effects are based on the proteasomal degradation of cIAP1/2 accompanied by an altered ratio of the levels of membrane-localized TNF receptor-associated factors 2 and 5. Conclusion— Our results reveal IAP antagonism as a profound anti-inflammatory principle in vivo and highlight IAPs as important regulators of inflammatory processes in endothelial cells.

Evasion of cell death by overexpression of anti-apoptotic proteins, such as Bcl-2, is commonly observed in cancer cells leading to a lack of response to chemotherapy. Hence, there is a need to find new chemotherapeutic agents that are able to overcome chemoresistance mediated by Bcl-2 and to understand their mechanisms of action. Helenalin, a sesquiterpene lactone (STL), induces cell death and abrogates clonal survival in a highly apoptosis-resistant Bcl-2 overexpressing Jurkat cell line as well as in two other Bcl-2 overexpressing solid tumor cell lines (mammary MCF-7; pancreatic L6.3pl). This effect is not achieved by directly affecting the mitochondria-protective function of Bcl-2 in the intrinsic pathway of apoptosis since Bcl-2 overexpressing Jurkat cells do not show cytochrome c release and dissipation of mitochondrial membrane potential upon helenalin treatment. Moreover, helenalin induces an atypical form of cell death with necrotic features in Bcl-2 overexpressing cells, neither activating classical mediators of apoptosis (caspases, AIF, Omi/HtrA2, Apaf/apoptosome) nor ER-stress mediators (BiP/GRP78 and CHOP/GADD153), nor autophagy pathways (LC3 conversion). In contrast, helenalin was found to inhibit NF-κB activation that was considerably increased in Bcl-2 overexpressing Jurkat cells and promotes cell survival. Moreover, we identified reactive oxygen species (ROS) and free intracellular iron as mediators of helenalin-induced cell death whereas activation of JNK and abrogation of Akt activity did not contribute to helenalin-elicited cell death. Our results highlight the NF-κB inhibitor helenalin as a promising chemotherapeutic agent to overcome Bcl-2-induced cell death resistance.

BACKGROUND AND PURPOSE Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation.

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X) 18 -E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528 R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Small molecular inhibitors of Cyclin dependent kinases (Cdks) are currently being developed as anticancer therapeutics due to their antiproliferative properties. The purine Cdk specific inhibitor (R)-roscovitine (seliciclib, CYC202) represents one of the most promising of these compounds. It is currently evaluated in clinical trials concerning cancer therapy. Recently, we have shown that roscovitine exerts potent antiangiogenic effects and elucidated Cdk5 as a new player in angiogenesis. These findings introduce Cdk5 as novel target for antiangiogenic therapy, and Cdk5 inhibitors as an attractive therapeutic approach. Here, we present the antiangiogenic profile of 15 derivatives of roscovitine in vitro and in vivo and provide structure activity relationships of the roscovitine analogs. The (S)-isomer LGR561 and the respective (R)- and (S)-isomers LGR848 and LGR849 strongly inhibited proliferation and cell cycle progression, induced cell death, and reduced migration of endothelial cells in vitro. In comparison to roscovitine, these compounds showed an increased potency to inhibit Cdk2, Cdk5, Cdk7, and Cdk9. By analyzing the effects of LGR561, LGR848, and LGR849 on endothelial cell tube formation, mouse aortic ring sprouting, angiogenesis in the chick chorioallantoic membrane, and neovessel formation in the mouse cornea, we elucidate the two (S)-isomers LGR561 and LGR849 as highly potent inhibitors of angiogenesis. This study provides first information on how to modify roscovitine to develop Cdk inhibitors with increased antiangiogenic activity and suggests the application of existing and the development of new Cdk inhibitors to inhibit both, cancer cell proliferation and angiogenesis.

Hyperproliferation of vascular smooth muscle cells (VSMCs) is critically involved in the onset of atherosclerosis and restenosis. Although caffeic acid phenethyl ester (CAPE, 1), one of the main constituents of honeybee propolis, has been shown to exert a beneficial effect in models of vascular injury in vivo, detailed mechanistic investigations in vascular cells are scarce. This study has examined the antiproliferative activity of 1 in platelet-derived growth factor (PDGF)-stimulated primary rat aortic VSMCs and aimed to shed light on underlying molecular mechanisms. Compound 1 inhibited the proliferation of VSMCs upon exposure to PDGF in a dose-dependent manner by interfering with cell cycle progression from the G0/1- to the S-phase. Enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) as well as stabilization of hypoxia-inducible factor (HIF)-1α and subsequent induction of heme oxygenase-1 (HO-1) could be identified as molecular events contributing to the observed growth arrest in PDGF-activated VSMCs upon exposure to 1.

Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-α-l-(2″,4″-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC₅₀ 10.4 μM). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques.

Introduction Chemoresistance represents a major problem for the treatment of leukemia patients, which frequently manifests in the multidrug resistance (MDR) phenotype1. The drug refractoriness to structurally related and unrelated drugs can be caused by the P-glycoprotein (P-gp), an efflux pump belonging to the superfamily of ATP-binding cassette (ABC) proteins1, 2. Multiple large clinical studies have evaluated the effectiveness of potential P-gp inhibitors, but all of them failed clinically, except for the alkaloid tetrandrine (TET)3. Given the high structural complexity and the pulmonary toxicity of the plant-derived TET in animal studies4, we are seeking to develop new bisbenzylisoquinoline derivatives (BBIQDs) with similar potency and reduced toxicity to overcome P-gp-mediated chemoresistance in leukemia. Methods Calcein-AM, Cell-TiterBlue® cell viability and propidium iodide cell death were performed as described by the manufacturers. TUNEL assays were conducted as described previously5. For the assessment of in vivo toxicity, female C57BL/6-Tyr mice (Envigo) were treated with 57 or 90 nmol/kg of the corresponding P-gp inhibitor on three consecutive days. Results BBIQDs were synthesized and screened for their potential to inhibit P-gp-mediated drug efflux using calcein-AM as model substrate in vincristine-resistant leukemia cells (VCR-R CEM). The in vitro characterization revealed small molecule hits with a similar potency compared with the reference molecule TET. Consequently, the lead compounds were able to overcome chemoresistance to vincristine (VCR) when applied as a combination therapy, as assessed by cell viability and cell death assays. More specifically, the usage of P-gp inhibitors as add-on to VCR enabled a decrease of the IC50 value from 0.73 µM to up to 0.03 µM, corresponding to a maximum dose modifying factor of 24. Additionally, the ability of BBIQDs to reverse chemoresistance to imatinib in MDA cells overexpressing the breast cancer resistance protein (BCRP), another major efflux transporter responsible for MDR in leukemia6, 7, was tested. As no relevant chemosensitization was observed here, we concluded that our BBIQD hits are specific inhibitors of P-gp. Furthermore, in vivo toxicity was evaluated in a mouse model, showing that the BBIQDs were well-tolerated at the highest tested dose of 90 nmol/kg. Additionally, TUNEL assays on organ slides indicated lack of toxicity in lungs, heart and brain. Finally, the P-gp inhibitors will be tested for their efficacy to overcome chemoresistance in vivo using a zebra fish leukemia model. Conclusion Taken together, we have identified derivatives of the alkaloid TET with a similar potency to inhibit P-gp mediated drug efflux. Remarkably, the application of the BBIQDs as add-on to VCR was able to reverse chemoresistance in P-gp-mediated, VCR-resistant leukemia cells, while no toxicity issues were observed in a mouse model. References Marin, J.J., Briz, O., Rodriguez-Macias, G., Diez-Martin, J.L. & Macias, R.I. Role of drug transport and metabolism in the chemoresistance of acute myeloid leukemia. Blood Rev30, 55-64 (2016). Joshi, P., Vishwakarma, R.A. & Bharate, S.B. Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer. Eur J Med Chem138, 273-292 (2017). Kelly, R.J. et al. A pharmacodynamic study of the P-glycoprotein antagonist CBT-1(R) in combination with paclitaxel in solid tumors. Oncologist17, 512 (2012). Jin, H. et al. Pulmonary toxicity and metabolic activation of tetrandrine in CD-1 mice. Chem Res Toxicol24, 2142-2152 (2011). Leanza, L. et al. Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo. Cancer Cell31, 516-531 e510 (2017). Burger, H. et al. Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump. Blood104, 2940-2942 (2004). Mao, Q. & Unadkat, J.D. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update. AAPS J17, 65-82 (2015). Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Objective Effective systemic drugs against restenosis upon percutaneous transluminal coronary angioplasty (PTCA) are largely lacking. Polyphenols have been suggested to ameliorate post-angioplasty restenosis. Hawthorn ( Crataegus spp.) extracts, which are among the most frequently used herbal medicinal products against mild forms of congestive heart failure, contain polyphenols, but have not been investigated in this context. We aimed to assess the potential of the hawthorn extract WS ® 1442 to prevent balloon catheter-induced intimal hyperplasia and to elucidate the underlying mechanisms. Methods We analyzed the effects of WS ® 1442 on serum-induced vascular smooth muscle cell (VSMC) and endothelial cell (EC) growth and migration, growth factor-induced proliferation, growth factor receptor activity, and neointima formation in the rat carotid artery model. Results WS ® 1442 (100μg/ml) decreased VSMC migration by 38% and proliferation by 44%, whereas EC migration and proliferation were unaltered. The extract inhibited VSMC DNA synthesis induced by platelet-derived growth factor (PDGF) (IC 50 : 47μg/ml), but not that of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Along this line, WS ® 1442 blocked recombinant human PDGF receptor (PDGFR)-β kinase activity (IC 50 : 1.4μg/ml) and decreased PDGFR-β activation and extracellular signal-regulated kinase (ERK) activation in VSMCs. In rats, orally administered WS ® 1442 significantly reduced neointima formation after balloon catheter dilatation of the carotid artery. Conclusion WS ® 1442 inhibits migration and proliferation of VSMCs, but not of ECs, and reduces balloon catheter-evoked neointima formation probably through inhibition of PDGFR-β. Thus, the present study suggests a novel adjunct pharmacological strategy to prevent angioplasty-related restenosis.

Treatment of pancreatic cancer remains a major challenge and new anticancer drugs are urgently required. Our study presents the marine natural compound spongistatin 1 as a promising experimental drug. Spongistatin 1 was applied in an orthotopic in vivo model of human pancreatic cancer. Spongistatin 1 significantly reduced tumor growth, which correlates with a strong apoptosis induction (DNA-fragmentation) and long-term effects on clonogenic survival of pancreatic tumor cells (L3.6pl) in vitro. In addition, the formation of metastasis was reduced in spongistatin 1-treated mice, which is in line with a diminished MMP-9 activity in tumor tissue determined by zymography. Based on the pronounced efficacy of spongistatin 1, the underlying mechanisms were studied in more detail. In vitro adhesion, as well as migration, and invasion assays showed spongistatin 1 to influence these critical steps in the metastatic cascade. Furthermore, spongistatin 1 induced anoikis in L3.6pl cells. Exposure to spongistatin 1 leads to phosphorylation, and thus inactivation of the antiapoptotic protein Bcl-2 in pancreatic tumor cells. siRNA experiments silencing Bcl-2 suggest a role of Bcl-2 in anoikis and cell migration. Taken together, spongistatin 1 not only proved to be a potent experimental drug but also served as a chemical tool to examine the role of the antiapoptotic protein Bcl-2 in pancreas carcinoma, thereby supporting the hypothesis of a link between apoptosis signaling and metastasis.

Aims Despite the lower patency of venous compared with arterial coronary artery bypass grafts, ∼50% of grafts used are saphenous vein conduits because of their easier accessibility. In a search for ways to increase venous graft patency, we applied the results of a previous pharmacological study screening for non-toxic compounds that inhibit intimal hyperplasia of saphenous vein conduits in organ cultures. Here we analyse the effects and mechanism of action of leoligin [(2 S ,3 R ,4 R )-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl (2 Z )-2-methylbut-2-enoat, the major lignan from Edelweiss ( Leontopodium alpinum Cass.). Methods and results We found that leoligin potently inhibits vascular smooth muscle cell (SMC) proliferation by inducing cell cycle arrest in the G1-phase. Leoligin induced cell death neither in SMCs nor, more importantly, in endothelial cells. In a human saphenous vein organ culture model for graft disease, leoligin potently inhibited intimal hyperplasia, and even reversed graft disease in pre-damaged vessels. Furthermore, in an in vivo mouse model for venous bypass graft disease, leoligin potently inhibited intimal hyperplasia. Conclusion Our data suggest that leoligin might represent a novel non-toxic, non-thrombogenic, endothelial integrity preserving candidate drug for the treatment of vein graft disease.

The aims of the study were to meet the demand of new tubulin antagonists with fewer side effects by characterizing the antiangiogenic properties of the experimental compound spongistatin 1, and to elucidate nonmitotic mechanisms by which tubulin antagonists inhibit angiogenesis. Although tubulin-inhibiting drugs and their antiangiogenic properties have been investigated for a long time, surprisingly little is known about their underlying mechanisms of action. Antiangiogenic effects of spongistatin 1 were investigated in endothelial cells in vitro, including functional cell-based assays, live-cell imaging, and a kinome array, and in the mouse cornea pocket assay in vivo. Spongistatin 1 inhibited angiogenesis at nanomolar concentrations (IC50: cytotoxicity>50 nM, proliferation 100 pM, migration 1.0 nM, tube formation 1.0 nM, chemotaxis 1.0 nM, aortic ring sprouting 500 pM, neovascularization in vivo 10 μg/kg). Further, a kinome array and validating data showed that spongistatin 1 inhibits the phosphorylation activity of protein kinase Cα (PKCα), an essential kinase in angiogenesis, and its translocation to the membrane. Thus, we conclude that PKCα might be an important target for the antiangiogenic effects of tubulin antagonism. In addition, the data from the kinase array suggest that different tubulin antagonists might have individual intracellular actions.—Rothmeier, A. S., Ischenko, I., Joore, J., Garczarczyk, D., Fu¨rst, R., Bruns, C. J., Vollmar, A. M., Zahler, S. Investigation of the marine compound spongistatin 1 links the inhibition of PKCα translocation to nonmitotic effects of tubulin antagonism in angiogenesis.

Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive stimulation of DC may lead to DC unresponsiveness and exhaustion. In this study, we investigated how short-term versus long-term DC activation with a Toll-like receptor 9 agonist influences DC phenotype and function. Murine DC were generated in the presence of the hematopoietic factor Flt3L (FL-DC) to obtain both myeloid and plasmacytoid DC subsets. Short activation of FL-DC for as little as 4 h induced fully functional DC that rapidly secreted IL-12p70 and IFN-alpha, expressed high levels of costimulatory and MHC molecules and efficiently presented antigen to CD4 and CD8 T cells. Furthermore, short-term activated FL-DC overcame immune suppression by regulatory T cells and acquired high migratory potential toward the chemokine CCL21 necessary for DC recruitment to lymph nodes. In addition, vaccination with short-term activated DC induced a strong cytotoxic T-cell response in vivo and led to the eradication of tumors. Thus, short-term activation of DC generates fully functional DC for tumor immunotherapy. These results may guide the design of new protocols for DC generation in order to develop more efficient DC-based tumor vaccines.

The standardised Ginkgo biloba extract EGb(®) 761 (Dr. Willmar Schwabe Pharmaceuticals, Karlsruhe, Germany) is one of the most widely used herbal remedies. Indications for this extract range from dementia to peripheral vascular disease, based on well‐documented vascular effects. Surprisingly, the actions of EGb(®) 761 on angiogenesis as a function of vascular cells have not been investigated to date. The anti‐cancer activity of EGb(®) 761 in vitro and epidemiological data showing reduced risk for ovarian cancer in regular users have prompted us to investigate this issue. We show an anti‐angiogenic profile of EGb(®) 761 in vitro (inhibited proliferation, migration and tube formation of endothelial cells) and in vivo in the chicken chorio‐allantoic membrane (CAM) assay. An analysis of the underlying mechanisms indicates inhibition of growth factor‐induced extracellular signal‐regulated kinase (ERK) phosphorylation by EGb(®) 761. Inhibitory effects of EGb(®) 761 on ERK as well as of the upstream kinases map‐erk‐kinase (MEK) and rapidly growing fibrosarcoma (Raf)‐1 could be completely reversed by pre‐treatment with sodium vanadate (inhibitor of tyrosine phosphatases). Sodium vanadate also reversed the EGb(®) 761‐induced inhibition of endothelial cell migration. Focusing on tyrosine phosphatases upstream of the Raf‐MEK‐ERK cascade, we identified the tyrosine phosphatase Src homology‐2 domain‐containing phosphatase 1 (SHP‐1) as one target of EGb(®) 761. SHP‐1 was rapidly activated by EGb(®) 761, and silencing SHP‐1 (siRNA) abrogated reduction of endothelial proliferation by EGb(®) 761. In summary, we identify EGb(®) 761 as a potent anti‐angiogenic drug. The underlying mechanism is the activation of protein tyrosine phosphatases, leading to inhibition of the Raf‐MEK‐ERK pathway. These findings provide a rational basis for using EGb(®) 761 for an additional therapeutic indication: anti‐angiogenesis‐based tumour prevention and adjuvant therapy.

The freeze-drying properties of gelatin nanoparticles were investigated with the goal of providing practicable nanoparticle formulations for in vitro applications or clinical studies. Various excipients and rehydration protocols were assessed, and gelatin nanoparticles loaded with oligonucleotides were successfully freeze-dried and rehydrated. An NF-kappaB decoy oligonucleotide-loaded gelatin nanoparticle formulation was developed and applied in a drug targeting approach in an animal model. The high concentrations of nanoparticles achieved after rehydration with reduced volumes proved to be critical for the in vivo effect. Finally, short term storage stability under accelerated conditions was assessed for dried gelatin nanoparticles formulated in sucrose, trehalose, mannitol, or a mannitol/sucrose mixture. Size, size distribution, and residual moisture content were investigated. Sucrose- and trehalose-containing formulations exhibited the greatest stability, but mannitol-containing formulations also showed notable stabilization despite their crystalline nature.

A pathological increase of the permeability of the mitochondrial membranes may culminate in the irreversible rupture of the mitochondrial outer membrane. Such a permeability transition is lethal because it results in the release of death-inducing molecules from mitochondria and/or metabolic failure. Current methods to assess this outer membrane damage are mostly indirect or scarcely representative of the overall mitochondrial population. Here we present an analytical and preparative approach using free flow electrophoresis to directly distinguish rat liver mitochondria that have undergone the permeability transition from unaffected organelles or from organelles that are damaged to a minor degree. Mitochondrial populations, which considerably differ in outer membrane integrity or cytochrome c content, were separated by this means. We further show that the relative abundance of each population depends on the dose of the permeability transition inducer and the duration of the treatment time. Finally, we have employed this approach to investigate the impairment of mitochondria that were isolated from livers subjected to ischemia/reperfusion damage.

We have recently identified the MAPK phosphatase (MKP)-1 as a novel mediator of the antiinflammatory properties of glucocorticoids (dexamethasone) in the human endothelium. However, nothing is as yet known about the signaling pathways responsible for the up-regulation of MKP-1 by dexamethasone in endothelial cells. Knowledge of the molecular basis of this new alternative way of glucocorticoid action could facilitate the identification of new antiinflammatory drug targets. Thus, the aim of our study was to elucidate the underlying molecular mechanisms. Using Western blot analysis, we found that dexamethasone rapidly activates ERK, c-jun N-terminal kinase (JNK), and p38 MAPK in human umbilical vein endothelial cells. By applying the kinase inhibitors PD98059 (MAPK kinase-1) and SP600125 (JNK), ERK and JNK were shown to be crucial for the induction of MKP-1. Using EMSA and a decoy oligonucleotide approach, the transcription factors activator protein-1 (activated by ERK and JNK) and cAMP response element-binding protein (activated by ERK) were found to be involved in the up-regulation of MKP-1 by dexamethasone. Interestingly, dexamethasone induces the generation of reactive oxygen species (measured by dihydrofluorescein assay), which participate in the signaling process by triggering JNK activation. Our work elucidates a novel alternative mechanism for transducing antiinflammatory effects of glucocorticoids in the human endothelium. Thus, our study adds valuable information to the efforts made to find new antiinflammatory principles utilized by glucocorticoids. This might help to gain new therapeutic options to limit glucocorticoid side effects and to overcome resistance.

Impfungen simulieren eine Infektion mit einem bestimmten Pathogen und induzieren das Immunsystem. Wird das eingebrachte Antigen effizient uber MHC-Molekule auf antigenprasentierenden Zellen den T-Zellen prasentiert, wird die adaptive Komponente des Immunsystems stimuliert. Der Vorteil daraus ist, dass nun die Immunantwort sehr spezifisch fur das Antigen ist und dass ein immunologisches Gedachtnis gebildet wird, das beim Kontakt mit dem echten Pathogen sehr rasch reagieren kann. Verschiedene Impfstoffe und -strategien werden inzwischen angewendet, um der breiten Bevolkerung und auch den Spezialfallen der Neugeborenen und Alteren einen umfassenden Schutz vor zahlreichen Infektionskrankheiten zu bieten.

Beneficial effects of Ginkgo biloba on peripheral arterial occlusive disease have been repeatedly shown in clinical trials, especially after use of EGb® 761, a standardized special extract. Since the underlying mechanisms are widely unknown, we aimed to elucidate the molecular basis on which EGb® 761 protects against endothelial dysfunction in vitro and in vivo. Application of therapeutically feasible doses of EGb® 761 for 48 h caused endothelial nitric oxide (NO) production by increasing endothelial nitric oxide synthase (eNOS) promoter activity and eNOS expression in vitro. Phosphorylation of eNOS at a site typical for Akt (Ser 1177) was acutely enhanced by treatment with EGb® 761, as was Akt phosphorylation at Ser 478. Furthermore, the extract caused acute relaxation of isolated aortic rings and NO-dependent reduction of blood pressure in vivo in rats. These influences on eNOS represent a putative molecular basis for the protective cardiovascular properties of EGb® 761.

The alkaloid cryptolepine is thought to mediate the anti-inflammatory effects of the climbing shrub, Cryptolepis sanguinoleta. The underlying mechanism of action, however, is largely unknown. In the present study, we show that the synthetic cryptolepine-hydrochloride (2.5-10microM) dose-dependently inhibits lipopolysaccharide (LPS)-induced nitric oxide production in the murine macrophage cell line RAW 264.7. We furthermore demonstrate a strong inhibition of nuclear factor (NF)-kappaB, a transcription factor primarily involved in inflammatory and immune responses, by cryptolepine (2.5-10microM) using a luciferase reporter gene assay in human HEK 293 cells. Examining the individual steps of NF-kappaB activation in the presence of cryptolepine we could exclude an inhibitory effect on degradation of IkappaB or nuclear translocation of NF-kappaB by the alkaloid. However, EMSA of nuclear extracts from LPS-activated RAW cells revealed reduced DNA binding activity of NF-kappaB by cryptolepine in vivo and in vitro. This indicates that cryptolepine may exhibit its anti-inflammatory action by blocking DNA binding of activated NF-kappaB and thus transcription of NF-kappaB-regulated proinflammatory proteins.

Objective— Cell–matrix interactions are crucial for regulating cellular activities, such as migration. This is of special importance for morphogenic processes, such as angiogenesis (the development of new blood vessels). Most of our understanding of cell migration relies on 2-dimensional (2D) experiments. However, the awareness that 3D settings might elicit different results has increased. Knowledge about endothelial cell (EC) behavior in 3D environments and the influence of matrix composition on EC migration, in particular, is still limited. Approach and Results— We characterize the migration of single ECs through 2 structurally different hydrogels: spongy Matrigel and fibrillar collagen I. Our observations reveal an elongated migration phenotype in Matrigel and a rounded phenotype with pronounced cell blebs (blebs >2 µm) in collagen I, which have not previously been described in ECs. Directed migration seems to depend on Rac1 and Cdc42 in collagen, but not in Matrigel (shown using appropriate pharmacological inhibitors). By applying anti-integrin antibodies and supplementing laminin in collagen gels, we identify laminin as the main determinant of the elongated phenotype. Laminin seems to induce a morphological switch between modes of migration. As an in situ proof of principle, we performed live imaging of EC migration during vascular growth in a murine retina in the absence and presence of anti-integrin antibodies. Conclusions— We show that, surprisingly, ECs can evade the pharmacological inhibition of central signaling pathways involved in migration (contractility, small GTPases, and proteolysis) by shifting gears between modes of migration. This finding indicates an unexpected contextual plasticity of EC behavior.

Several natural compound interfere with microtubules or the actin cytoskeleton. Compounds interfering with the microtubules like Vinca-alkaloids or taxanes, are extensively used for cancer therapy. In contrast, knowledge about pharmacological properties of actin binding drugs is poor and drugs interfering with actin are far from clinical use. Rhizopodin is a natural compound that strongly affects the actin cytoskeleton at nanomolar concentrations. Initial work revealed interesting anti-bacterial and cytotoxic effects, but the cellular effects and pharmacological properties of rhizopodin have not been characterized. We hypothesized that rhizopodin might exert anti-cancer activity. Therefore, the aim of this study was to characterize the cellular and pharmacological effects of rhizopodin in cancer. Effects of rhizopodin demonstrated prominent effects on the actin cytoskeleton as shown in the actin-pyrene assay and by immunostaining of cancer cells. To investigate cellular effects of rhizopodin, we analyzed cell proliferation, cell death induction by propidium iodide exclusion and western blot, as well as migration by impedance measurement using the xCELLligence device in MDA-MB-231 breast cancer and T24 bladder cancer cell lines. Rhizopodin inhibited proliferation and induced cell death of MDA-MB-231 and T24 cells at nanomolar concentrations. PARP cleavage by rhizopodin suggests caspase-dependent cell death induction. Importantly, rhizopodin potently inhibited MDA-MB-231 and T24 cancer cell migration at subtoxic doses where no actin aggregation was observed, indicating a specific underlying signaling of rhizopodin. In summary, our study elucidates rhizopodin as actin-binding natural compound that exerts potent anti-cancer effects. Therefore, our work provides the basis for further in depth characterization of rhizopodin as an antitumoral agent.

We recently introduced CDK5 as target in HCC, regulating DNA damage response. Based on this and on our previous knowledge about vascular effects of CDK5, we investigated the role of CDK5 in angiogenesis in HCC, one of the most vascularized tumors. We put a special focus on the transcription factor HIF-1α, a master regulator of tumor angiogenesis. The interaction of CDK5 with HIF-1α was tested by Western blot, PCR, reporter gene assay, immunohistochemistry, kinase assay, co-immunoprecipitation, mass spectrometry, and mutation studies. In vivo, different murine HCC models, were either induced by diethylnitrosamine or subcutaneous injection of HUH7 or HepG2 cells. The correlation of vascular density and CDK5 was assessed by immunostaining of a microarray of liver tissues from HCC patients. Inhibition of CDK5 in endothelial or HCC cells reduced HIF-1α levels in vitro and in vivo, and transcription of HIF-1α target genes (VEGFA, VEGFR1, EphrinA1). Mass spectrometry and site directed mutagenesis revealed a stabilizing phosphorylation of HIF-1α at Ser687 by CDK5. Vascular density was decreased in murine HCC models by CDK5 inhibition. In conclusion, inhibiting CDK5 is a multi-modal systemic approach to treat HCC, hitting angiogenesis, as well as the tumor cells themselves.

The invasive potential of cancer cells strongly depends on cellular stiffness, a physical quantity that is not only regulated by the mechanical impact of the cytoskeleton but also influenced by the membrane rigidity. To analyze the specific role of membrane rigidity in cancer progression, we treated cancer cells with the Acetyl-CoA carboxylase inhibitor Soraphen A and revealed an alteration of the phospholipidome via mass spectrometry. Migration, invasion, and cell death assays were employed to relate this alteration to functional consequences, and a decrease of migration and invasion without significant impact on cell death has been recorded. Fourier fluctuation analysis of giant plasma membrane vesicles showed that Soraphen A increases membrane rigidity of carcinoma cell membranes. Mechanical measurements of the creep deformation response of whole intact cells were performed using the optical stretcher. The increase in membrane rigidity was observed in one cell line without changing the creep deformation response indicating no restructuring of the cytoskeleton. These data indicate that the increase of membrane rigidity alone is sufficient to inhibit invasiveness of cancer cells, thus disclosing the eminent role of membrane rigidity in migratory processes.

Summary Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

Phamacological activities of a standard ethanol extract G1 from Brazilian green propolis, typified as BRP1, was evaluated in mouse models of pain and inflammation. Intraperitoneal injection ( I. P.) of G1 inhibited acetic acid-induced abdominal constrictions with an ID (50) = 0.75 +/- 0.05 mg/kg, and in the formalin test the ID (50) values were 0.85 +/- 0.07 mg/kg and 13.88 +/- 1.12 mg/kg, respectively, for the neurogenic and inflammatory phases. The extract was ineffective when assessed in the hot-plate assay. In serotonin-induced paw edema, G1 led to a maximal inhibition (MI) of 51.6 % after 120 min when administered I. P. and of 36 % after 15 min by the oral route ( O. R.). When the inflammatory agent was complete Freund's adjuvant, inhibition of paw edema was also observed after administration of the extract by both routes. In the capsaicin-induced ear edema the ID (50) values were 1.09 +/- 0.08 mg/kg ( I. P.) and 10.00 +/- 0.90 mg/kg ( O. R.). In the acute carrageenan-induced inflammatory reaction induced by carrageenan, G1 reduced the number of neutrophils in the peritoneal cavity with IC (50) values of 0.72 +/- 0.08 mg/kg and 4.17 +/- 0.50 mg/kg, by I. P. or O. R. administration, with a preferential migration of polymorphonuclear neutrophils. IN VITRO, G1 decreased nitric oxide production in LPS-stimulated RAW 264.7 cells (IC (50) = 41.60 microg/mL), and also the luciferase activity in TNF-alpha-stimulated HEK 293 cells transfected with NF-kappaB-luciferase reporter gene driven by the nuclear factor kappaB (NF-kappaB) (IC (50) = 200 microg/mL). This extract, which at low concentrations induces anti-inflammatory and analgesic effects in mouse models, presents a high content of flavonoids, known to inhibit inducible NOS (iNOS) activity. These data taken together led us to reinforce the hypothesis in the literature that the anti-inflammatory effect of propolis may be a due to inhibition of iNOS gene expression, through interference with NF-kappaB sites in the iNOS promoter.

In contrast to aspirin, salicylate, its active metabolite, possesses profound anti-inflammatory properties without blocking cyclooxygenase. Inhibition of the transcription factor nuclear factor-kappaB (NF-kappaB) has been discussed to play a role in the anti-inflammatory profile of salicylate. However, NF-kappaB-independent effects of salicylate have been assumed but have up to now been poorly investigated. Therefore, the aim of the present study was to investigate NF-kappaB-independent anti-inflammatory mechanisms of salicylate in human umbilical vein endothelial cells using interleukin-4 (IL-4) as NF-kappaB-independent proinflammatory stimulus and P-selectin as inflammatory read-out parameter. Using quantitative real-time reverse transcription-polymerase chain reaction, we found that salicylate decreases IL-4-induced P-selectin expression. As judged by Western blot analysis, salicylate increased endothelial heme oxygenase-1 (HO-1) protein levels. Using both the HO-1 inhibitor tin(II) protoporphyrin IX and HO-1 antisense oligonucleotides, we causally linked the induction of HO-1 to the decrease of P-selectin. Moreover, we were interested in the signaling mechanisms leading to the up-regulation of HO-1 by salicylate. c-Jun NH2-terminal kinase (JNK) was found to be activated by salicylate, and we could causally link this activation to the induction of HO-1 by using the JNK inhibitor 1,9-pyrazoloanthrone. By applying activator protein-1 (AP-1) decoys, it was shown that the transcription factor AP-1 is crucially involved in the up-regulation of HO-1 downstream of JNK. In summary, our study introduces HO-1 as novel NF-kappaB-independent anti-inflammatory target of salicylate in human endothelial cells. Moreover, we elucidated the JNK/AP-1 pathway as crucial for the induction of HO-1 by salicylate.

Rekombinante Immunsuppressiva bilden mittlerweile eine beachtliche, keineswegs homogene Gruppe. All diesen Wirkstoffen ist gemeinsam, dass sie extrazellulare Targets ansteuern, blockieren oder markieren. Bei diesen Targets handelt es sich um losliche Faktoren oder Membranstrukturen, die fast alle Komponenten eines uberaus komplexen Kommunikations- und Amplifikationssystems sind. Auffallig ist, dass im Bereich rekombinanter Immunsuppressiva in doch recht kurzer Zeit enorm viele neue Wirkstoffe entwickelt wurden. Das liegt daran, dass die eigentliche Molekulentwicklung der Natur uberlassen wird: Entweder handelt es sich – wie im Fall von Anakinra – um humane Proteine oder es handelt sich um Antikorper bzw. Antikorperderivate, die in aller Regeln das Immunsystem der Maus oder der Ratte “entwickelt”, nachdem ihm das Antigen prasentiert wurde. Die Herausforderung in diesem Bereich liegt in erster Linie darin, relevante Targets zu identifizieren. Sind diese definiert, ist es nicht mehr sehr schwer, ein Protein zu entwickeln, welches das Target blockiert. Die klinische Erfahrung muss allerdings erst noch zeigen, welche dieser Targets wirklich valide sind und welche Wirkstoffe in der Therapie reussieren.

Die Gewebetransplantation zum Ersatz erkrankter Organe ist heute eine wichtige Behandlungsmethode und gehort zum klinischen Alltag. Seit der ersten Nierentransplantation im Jahre 1954 in den USA sind allein in Deutschland bis 2003 uber 70.000 Organe ubertragen worden. Ein stetig steigender Bedarf an Organen bestimmt Diskussionen um wichtige ethische Fragen der Transplantationsmedizin.

trans-Resveratrol (RV), a polyphenolic stilbene derivative found in grape skin and other food products, has been proposed to exert beneficial effects in cardiovascular disease. Our group has shown previously that RV inhibits angiotensin II (Ang II)-induced Akt activation and, consequently, vascular smooth muscle cell (VSMC) hypertrophy. In this work, to identify the molecular target of RV, we investigated the impact of RV on early signaling cascades in rat aortic VSMCs triggered by Ang II and epidermal growth factor (EGF). We show that RV does not influence Ang II-mediated transactivation of EGF-receptor but potently inhibits EGF-induced phosphorylation of Akt kinase, suggesting that RV acts downstream of EGF-receptor transactivation in VSMCs. Recent evidence indicates that the adapter molecule Gab1, together with the protein tyrosine phosphatase Shp2, is critically involved in regulating the strength and duration of phosphatidylinositol-3-kinase (PI3K) and Akt activation upon EGF stimulation in fibroblasts. Our results show that stimulation of VSMCs with EGF as well as Ang II leads to a rapid tyrosine phosphorylation of Gab1 and its association with the p85 subunit of PI3K. RV attenuates these processes. Experiments performed in Shp2-deficient fibroblasts revealed that RV does not inhibit EGF-stimulated Akt activation in these cells, suggesting that Shp2 is necessary for the inhibitory effect of RV on the PI3K/Akt pathway. Furthermore, RV treatment activates Shp2. We therefore propose that RV blocks Akt activation in Ang II- and EGF-stimulated VSMCs by activating Shp2, thus preventing interaction between Gab1 and PI3K that is necessary for further signal transduction.

The consumption of garlic reduces the risk of cardiovascular disease and cancer, S-allylcysteine sulfoxide (alliin), allicin (DATi), diallyl disulfide (DADS), S-allylcysteine (SAC) and several storage dipeptides are the organo-sulphur compounds (OSC) involved in the protective mechanism of garlic against cardiovascular disorders and carcinogenesis. Thus it is very interesting to quantify simultaneously all these compounds in different garlic powders obtained in several cultural conditions. The quantification of OSC by a new ion-pair HPLC method allowed showing the general sulphur-dependence positive effect of garlic on cardiovascular disorder and carcinogenesis and the variable specific activity of each implicated OSC. The screening of 11 garlic tablets proposed on the market showed the variability and particularly the differential instability of each OSC. From these results, a new garlic tablet was realised and each step was controlled by this method. This analytical method proved to be a very powerful tool for the understanding of the garlic protective mechanism against cancer and cardiovascular diseases and the development and quality control of garlic tablets.

Cardiovascular diseases are known as the major causes of death or disability in western countries. Decreased bioavailability of endothelial derived nitric oxide (NO) is recognized as an important promoter in cardiovascular disease. In vivo studies suggest that phytoestrogens, especially isoflavones from soy, enhance endothelium-dependent vasoreactivity. We hypothesized that isoflavones may affect the expression of endothelial-type nitric oxide synthase (eNOS) and thereby NO formation in vitro. Human EA.hy926 endothelial cells were treated with the soybean isoflavones biochanin A and formononetin and with their metabolites genistein and daidzein. eNOS promoter activity was examined by a luciferase reporter gene assay (20 h). Active eNOS was detected by quantifying conversion of L-arginine to L-citrulline and by measuring NO released from endothelial cells using the fluorescent probe DAF-2 (20-96 h).eNOS promoter activity increased in response to isoflavone treatment (20 h). NO and L-citrulline production by EA.hy926 cells rose up to 1.7-fold of control levels after stimulation with genistein for 48-96 h. From these results, we conclude that the suggested positive effects of soy isoflavones on vascular reactivity may be indeed mediated via a long-term effect on the eNOS system.

The marine alkaloid ascididemin (ASC) was shown to exert cytotoxicity even against multidrug-resistant cancer cells. Here, we address the signaling pathways utilized by ASC to trigger apoptosis in Jurkat leukemia T cells. We show that ASC (0.5-20 microM) induces a mitochondrial pathway that requires the activation of the initiator caspase-2 upstream of mitochondria. ASC-triggered apoptosis occurred independent of CD95, but required mitochondrial dysfunction. The activation of caspase-2 was shown to precede the processing of caspase-8, -9 and -3. The specific caspase-2 inhibitor zVDVADfmk abrogated ASC-induced DNA fragmentation almost completely. Overexpression of Bcl-x(L) blocked caspase-8 but not caspase-2 processing. Conversely, caspase-2 inhibition strongly reduced caspase-9 activation. As a possible link between caspase-2 and mitochondrial dysfunction, Bid was found to be cleaved by ASC. In addition, JNK was activated by ASC upstream of mitochondria via reactive oxygen species. The specific JNK inhibitor SP600125 partially inhibited caspase-2 and -9 processing as well as cytochrome c release and DNA fragmentation indicating that JNK contributes to, but is not necessary for ASC-mediated apoptosis. Thus, ASC triggers a pathway in which early activation of caspase-2 provides a possible link between its DNA-damaging activity and the induction of mitochondrial dysfunction. The activation of JNK contributes to this signaling upstream of mitochondria.

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.

Background/Aims : Silibinin is the major pharmacologically active compound of the Silybum marianum fruit extract silymarin. Its well-known hepatoprotective activities are mostly explained by antioxidative properties, inhibition of phosphatidylcholine synthesis or stimulation of hepatic RNA and protein synthesis. Here, we characterized the hepatoprotective potential of silibinin as an immune-response modifier in T cell-dependent hepatitis in vivo. Methods : Silibinin was tested in the mouse model of concanavalin A (ConA)-induced, T cell-dependent hepatitis. Liver injury was assessed by quantification of plasma transaminase activities and intrahepatic DNA fragmentation. Plasma cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA), intrahepatic cytokine and inducible NO synthase (iNOS) mRNA levels by reverse transcriptase polymerase chain reaction, intrahepatic iNOS expression by immunofluorescent staining, and intrahepatic nuclear factor kappa B (NF-κB) activation by electrophoretic mobility shift assay. Results : Silibinin significantly inhibited ConA-induced liver disease. Silibinin proved to be an immune-response modifier in vivo, inhibiting intrahepatic expression of tumor necrosis factor, interferon-γ, interleukin (IL)-4, IL-2, and iNOS, and augmenting synthesis of IL-10. In addition, silibinin inhibited intrahepatic activation of NF-κB. Conclusions : Silibinin, suppressing T cell-dependent liver injury as an immune-response modifier, might be a valuable drug in therapeutic situations in which intrahepatic immunosuppression is required.

Garlic is proposed to have immunomodulatory and anti-inflammatory properties. This paper shows that garlic powder extracts (GPE) and single garlic metabolites modulate lipopolysaccharide (LPS)-induced cytokine levels in human whole blood. GPE-altered cytokine levels in human blood sample supernatants reduced nuclear factor (NF)-kappaB activity in human cells exposed to these samples. Pretreatment with GPE (100 mg/L) reduced LPS-induced production of proinflammatory cytokines interleukin (IL)-1beta from 15.7 +/- 5.1 to 6.2 +/- 1.2 micro g/L and tumor necrosis factor (TNF)-alpha from 8.8 +/- 2.4 to 3.9 +/- 0.8 micro g/L, respectively, whereas the expression of the anti-inflammatory cytokine IL-10 was unchanged. The garlic metabolite diallydisulfide (1-100 micro mol/L) also significantly reduced IL-1beta and TNF-alpha. Interestingly, exposure of human embryonic kidney cell line (HEK293) cells to GPE-treated blood sample supernatants (10 or 100 mg/L) reduced NF-kappaB activity compared with cells exposed to untreated blood supernatants as measured by a NF-kappaB-driven luciferase reporter gene assay. Blood samples treated with extract obtained from unfertilized garlic (100 mg/L) reduced NF-kappaB activity by 25%, whereas blood samples treated with sulfur-fertilized garlic extracts (100 mg/L) lowered NF-kappaB activity by 41%. In summary, garlic may indeed promote an anti-inflammatory environment by cytokine modulation in human blood that leads to an overall inhibition of NF-kappaB activity in the surrounding tissue.

Resveratrol (RV), a polyphenolic stilbene derivative, has been proposed to exert a plethora of beneficial cardiovascular effects. Of these, in particular, inhibition of vascular smooth muscle cell (VSMC) proliferation shows great promise for preventing cardiovascular disease. In the present study, we show that RV leads to a reversible arrest in early S phase of the VSMC cycle, accompanied by an accumulation of hyperphosphorylated retinoblastoma protein. In contrast to studies with other cell systems, RV decreases cellular levels of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). This is of particular interest because phosphorylated p53 protein (serine(15)) is strongly enhanced by this substance. We further found that RV only slightly inhibits phosphorylation of Erk 1/2, protein kinase B/Akt, and p70(S6) kinase upon serum stimulation. Thus, inhibition of these kinases is not likely to contribute to the cell cycle effect of RV. Importantly, the observed S phase arrest is not linked to an increase in apoptotic cell death: there was no detectable increase in apoptotic nuclei and in levels of the proapoptotic protein Bax. This is the first study elucidating the molecular pathways mediating the antiproliferative properties of RV in VSMCs.

TNF-α is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type → tnfr2° and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-κB activation and cytokine production in the livers of both wild-type and tnfr2° mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2°, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.

Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-x(L), and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards ajoene suggesting that the activation of JNK is not necessary for ajoene-induced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced ajoene-induced cell death. The survival kinase Akt, in contrast, did not participate in ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, ajoene-mediated activation of JNK and p38 has no impact on ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards ajoene.

Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells in vitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 µM) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikert et al., FEBS Lett 2001, 506:131-134) on aspects of validation procedures as well as limitations and pitfalls of this method.

Background: Atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory and cytoprotective potential. The aim of this study was to characterize induction of heme oxygenase (HO)-1 by ANP in human umbilical vein endothelial cells (HUVEC). Methods: HUVEC were treated with ANP, 8-bromo-cyclic GMP (cGMP), or cANF in the presence or absence of various inhibitors. HO-1 was determined by Western blot and RT-PCR, c-jun N-terminal kinase (JNK) and ERK by the use of phospho-specific antibodies. Activator protein (AP)-1 activation was assessed by gelshift assay. Reporter gene assays were performed using native or mutated AP-1 binding sites of the HO-1 promoter. TNF-α-induced cell death was investigated by Hoechst staining, fluorescence-activated cell sorting analysis, caspase-3-measurement, and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Results: ANP (10−9–10−6 mol/liter) induced the expression of HO-1 protein and mRNA. Induction was mediated via the guanylate-cyclase-coupled receptor because 8-Br-cGMP mimicked the effect of ANP, whereas the clearance receptor agonist cANF did not induce HO-1. Endogenously produced cGMP also induced HO-1 because phosphodiesterase inhibition markedly elevated HO-1. The lack of effect of the cGMP-dependent protein kinase inhibitor 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-pCT-cGMPS) suggested no involvement for this cGMP effector pathway in the signal transduction. ANP lead to activation of the transcription factor AP-1, and subsequently of JNK, as well as of ERK. Cotreatment of the cells with U0126 or SP600125, as well as reporter gene assays revealed the involvement of AP-1/JNK activation in HO-1 induction. Abrogation of HO-1 induction by PD-98059 showed also a role for ERK. Treatment of HUVEC with ANP did not protect from TNF-α-induced apoptosis. Conclusion: This work characterizes the induction of HO-1 by ANP in HUVEC, which is shown to be mediated via JNK/AP-1 and ERK pathways. ANP-induced HO-1 does not confer protection against TNF-α-induced apoptosis.

Atrial natriuretic peptide (ANP) has been shown to reduce tumor necrosis factor-α (TNF-α)-induced activation of endothelial cells via inhibition of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways. The aim of this study was to determine whether ANP is able to inhibit TNF-α-induced expression of monocyte chemoattractant protein-1 (MCP-1) in endothelial cells and to elucidate the mechanisms involved. Pretreatment of human umbilical vein endothelial cells (HUVEC) with ANP significantly reduced TNF-α-induced expression of MCP-1 protein and mRNA. The effects of ANP were shown to be mediated via the guanylyl-cyclase (GC)-coupled A receptor. Activation of the other GC-coupled receptor (natriuretic peptide receptor-B) by the C-type natriuretic peptide as well as activation of soluble GC with S-nitroso-L-glutathione (GSNO) exerted similar effects as ANP, supporting a role for cyclic guanosine monophosphate (cGMP) in the signal transduction. Antisense experiments showed a requirement of MAPK phosphatase-1 (MKP-1) induction and therefore, inhibition of p38 MAPK in the ANP-mediated inhibition of TNF-α-induced expression of MCP-1. To investigate a potential interplay between TNF-α-induced activation of p38 MAPK and NF-κB, the p38 MAPK inhibitor SB203580 and a dominant-negative p38 MAPK mutant were used. The results indicated that the blockade of p38 MAPK activity leads to an increased activation of NF-κB and therefore, suggest a counter-regulatory action of p38 MAPK and NF-κB. As antisense experiments revealed a pivotal role for MKP-1 induction and therefore, p38 MAPK inhibition in ANP-mediated attenuation of MCP-1 expression, this action seems to be rather independent of NF-κB inhibition.