Identification of the anti-inflammatory targets of interactive constituents of Hypericum perforatum (Hp). BIRT DF and HAMMER KDP
Dept. of Food Sci. & Human Nutr., Iowa State Univ., Ames, IA.
Background: Hp is used as a botanical therapy for infective disorders. The level of constituents in Hp extracts were lower than the concentration of pure constituents needed to reduce lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production in RAW 264.7 macrophages; suggesting an interaction of compounds for the activity. The goal of this study was to identify key constituents and investigate the gene targets for the anti-inflammatory effect.
Methods: A flavonoid-rich bioactivity guided fractionation was used with screening in the LPS-induced RAW 264.7 macrophage system to identify active fractions from each round and liquid chromatography-mass spectrometry (LC-MS) identified constituents present. CellTiter96TM Aqueous solution revealed no significant cytotoxicity with fractions or constituents at the doses studied. Microarray analysis was performed with Hp fraction, the 4 constituents combined at levels detected in the Hp fraction, and solvent control with/without LPS.
Results: A third round fraction (3A) (10 µg/ml) significantly reduced PGE2. Combining four constituents at concentrations detected in the LC-MS analysis (0.17 µM chlorogenic acid, 0.08 µM amentoflavone, 0.08 µM quercetin, and 0.03 µM pseudohypericin (PHCN) explained the anti-inflammatory activity of the fraction in light-activated conditions. The amount of each pure constituent needed to observe a significant reduction in PGE2 was > 50 times more than was found in fraction 3A. Of the 4 interacting compounds, only PHCN was required. With LPS, the 4 component system affected 162 genes and the fraction affected 780 genes; 40 genes were differentially expressed under both treatments. Important pathways for both treatments were the Janus kinase-signal transducer and activator of transcription and eicosanoid metabolism pathways.
Conclusions: 1) Interactions of constituents explained the anti-inflammatory activity of fraction 3A. 2) PHCN was required for the anti-inflammatory activity in combination with one or more other constituents. 3) The gene targets identified for the 4 component system were consistent with the reduction of LPS induced PGE2. This work supports the role of interactions of compounds in Hp targeting genes that are important in inflammation. Supported by grants ES012020 from NIEHS/ODS and 9P50AT004155-06 from NCCAM/ODS, NIH.
LC-MS for Label-Free Biomarker Discovery HORVATOVICH P1, GOVORUKHINA N1, SOBCZAK-ELBOURNE I1, KEMPERMAN R1, 4, CHRISTIN C1, HOEKMAN B1, VAN DER ZEE A2, SUITS F3, MUSKIET F2, HOEFSLOOT H4, SMILDE A4, BISCHOFF R1 1 University of Groningen, Groningen, The Netherlands
2 University Medical Center Groningen, Groningen, The Netherlands
3 IBM TJ Watson Research Center, Yorktown Heights, NY, USA
4 University of Amsterdam, Amsterdam, The Netherlands
Background: The discovery and validation of biomarkers is an important goal in many areas of biomedical research and drug development. Changes in individual proteins or peptides as well as in protein and peptide profiles in bodily fluids or tissue biopsies have been implicated in diagnosis and prognosis of various diseased states. Recent developments in analytical chemistry allow to obtain complex, quantitative data from hundreds to thousands of compounds (proteins, peptides, metabolites) that require novel data processing and statistical approaches to arrive at biomarker candidates.
Methods: Serum was prepared for proteomics analysis by Liquid-Chromatography Mass Spectrometry (LC-MS) [1]. The generated data were processed using in-house developed algorithms [2,3] and analyzed by multi-variate statistics in combination with variable selection algorithms.
Results: LC-MS analysis of sera from cervical cancer patients were analyzed prior to and approximately 6 months after therapy. While differences in protein profiles were minor for early-stage disease differences became significant for more advanced stage tumors. Follow up of patients over multiple years showed that certain biomarker candidates changed in agreement with the recurrence of disease. Some of these candidates were related to the glycosylation of serum proteins.
Conclusions: 1) Biomarker candidates have been discovered that correlate with the response to therapy for cervical cancer. 2) Dedicated data processing and statistical analysis reduced the number of variables to a number that is in-line with the number of analyzed samples.
References:
Govorukhina, N. I.; Reijmers, T. H.; Nyangoma, S. O.; van der Zee, A. G. J.; Jansen, R. C.; Bischoff, R. J. Chromatogr. A2006, 1120, 142-150.
Christin, C.; Smilde, A. K.; Hoefsloot, H. C.; Suits, F.; Bischoff, R.; Horvatovich, P. Anal Chem. 2008, in press.
Suits, F.; Lepre, J.; Du, P.; Bischoff, R.; Horvatovich, P. Anal. Chem.2008, 80, 3095-3104.
Reactive Oxygen Species Have a Causal role in the Development of Insulin Resistance in Diabetic, Hypercortisolemic and Chronic Inflammatory States: Amelioration with the Antioxidants Alpha-lipoic acid BITAR SM1, AL-ALI W2, AL-MULLA F2 1Kuwait University. Faculty of Medicine, Department of Pharmacology, Safat, Kuwait; 2 Kuwait University. Faculty of Medicine, Department of Pathology, Safat, Kuwait.
Background:Insulin resistance, featured by an inexorable decline in skeletal muscle glucose utilization and/or an excessive hepatic glucose production, represents a major pathogenic importance in a cluster of clinical disorders including diabetes mellitus, hypercortisolemia, inflammation, and coronary artery disease. A novel concept suggests that excessive generation of reactive oxygen species (ROS) contribute to the development of insulin resistance in most if not all the aforementioned disease states.
Methods:Euglycemic-hyperinsulinemic clamp studies with an insulin infusion index of 5 mU/kg bw/min were used to measure endogenous glucose production (EGP), glucose infusion rate (GIR), glucose disposal rate (GDR) and skeletal muscle glucose utilization index (GUI). Moreover, the status of oxidative stress as reflected by urinary levels of isoprostane and tissue contents of protein-bound carbonyls and thiobarbituric acid reactive substrates (TBARS) were also assessed as a function of diabetes, hypercortisolemia and chronic low grade inflammation.
Results: Post-absorptive basal EGP and circulating levels of insulin, glucose and free fatty acid were elevated in GK and to a lesser extent in Dex and TNF alpha-treated rats,compared to their corresponding control values. In contrast, steady state GIR and GDR of the hyperglycemic/hyperinsulinemic animals were reduced,concomitantly with impaired insulin‘s ability to suppress EGP. The suppression of skeletal muscle glucose utilization in these animals was associated with a decrease in insulin‘s ability to promote the phosphorylation of tyrosine residues of insulin receptor substrate-1. Similarly, the translocation of glucose transporter-4 from intracellular compartment to plasma membrane in response to insulin was also reduced in these animals. Oxidative stress-based markers (e.g. urinary isoprostane, carbonyl-bound proteins, TBARS) were elevated in response to diabetes, hypercotisolemia and chronic low grade inflammation. Nullification of the heightened state of oxidative stress in the aforementioned animal models with antioxidants such as alpha lipoic acid ameliorated hepatic and skeletal muscle insulin resistance.
Conclusions:Collectively, the above data suggest that excessive generation of ROS in connection with their detrimental effects on lipid and protein molecules have a causal role in multiple forms of insulin resistance.
Synthesis of Unnatural Ceramide Analogs and Their Antiproliferative Properties Against a Panel of Cancer Cells BITTMAN R1 AND ARTHUR G2 1Department of Chemistry and Biochemistry, Queens College and The Graduate Center of CUNY, Flushing, NY 11367, USA
2Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, CANADA
Abstract: 2S,3R-Ceramide 1 occupies the “hub” of sphingolipid metabolism and serves as a coordinator of eukaryotic stress responses and other biological activities such as cell growth and differentiation. Ceramide 1 plays a key role in programmed cell death (apoptosis). However, since 1 is a naturally occurring lipid, it is recognized by endogenous enzymes and can be converted into anti-apoptotic lipids via phosphorylation and glycosylation. We sought to prepare unnatural ceramide analogs that may be longer lived in cells because they are unrecognized by enzymes. We found that some of the unnatural ceramide analogs have greater antiproliferative activity than 1 against human breast cancer cell lines in vitro. One of the unnatural ceramide analogs we synthesized has an exocyclic double bond in the sphingoid base (compound 2), whereas another has a disulfide linkage in the N-acyl chain (compound 3). Their antiproliferative activities against three human breast cancer cell lines (BT549, MDA-MB-231, MCF-7), a lung cancer cell line (A549), a prostate cancer cell line (DU145), and a cervical cell line (HeLa) were analyzed in vitro and compared with the activity of (2S,3R)-N-octanoylceramide (1). The sulfur-containing-ceramide analog 3 and the exo-ceramide analog 2 exhibited a higher antiproliferative activity than natural ceramide 1. Caspases in cells treated with compound 3were activated, indicating that the cells underwent apoptosis. A ceramide analog containing a tetrahydrofuranyl ring (compound 4) and a phytoceramide analog (compound 5) exhibited a much higher antiproliferative activity than natural N-palmitoyl-D-erythro-ceramide against T47D, MCF7, and MDA-MB-468 cells. The syntheses, antiproliferative activity, and mechanistic studies of the apoptotic properties of these ceramide analogs will be presented.
Peltophorum africanum Sond (Fabaceae) has a role in disease control BIZIMENYERA ES*A, OGUTTU JW A AND KOMA LM B a Department of Agriculture, Animal Health and Human Ecology, University of South Africa, Private Bag X6, Florida 1710, South Africa
b Department of Surgery and Theriogenology, Faculty of Veterinary Medicine, Makerere University, P O Box 7062, Kampala, Uganda Background. About 80% of people in the developing world, particularly those from rural communities where modern drugs are unaffordable, inaccessible or, unavailable, depend on phytomedicine for primary healthcare. However, most medical and veterinary professionals distrust herbal medicines due to lack of scientific evidence of efficacy and safety. Hence, there is need for their validation, before herbal medicines gain wider acceptance and use. Traditional healers, pastoralists and rural farmers use extracts of Peltophorum africanum (a medicinal plant widely spread in southern Africa and other tropical regions), to treat diarrhea, dysentery, pain, infertility, HIV-AIDS and to promote well-being and resistance to disease. The extracts of the plant inhibit HIV-type 1 reverse transcriptase and protease.
Methodology. Dried leaves bark and root from mature P. africanum trees were extracted with acetone. Chromatograms were made on silica gel plates. Minimum inhibitory concentrations (MIC) were determined for five bacteria (Gram positive and Gram negative), and five fungal pathogens. Qualitative screening for antioxidants was done by spraying chromatograms with 0.2% 2, 2-diphenyl-1-picryl hydrazyl (DPPH) , and quantification done in comparison with L-ascorbic acid and Trolox (6-hydroxy-2, 5, 7, 8-tetranethylchromane-2-carboxylic acid). Anthelmintic activity was evaluated by effects of extracts on the egg hatching and larval development of parasitic nematodes Haemonchus contortus and Trichostrongylus colubriformis.
Results. The extracts showed substantial activity against both Gram-positive and Gram-negative bacteria, with Minimum Inhibitory Concentration (MIC) values of 0.08 mg ml-1 for Staphylococcus aureus and 0.16 mg ml-1 for Pseudomonas aeruginosa. The extracts showed higher antifungal activity than amphoterin B. The acetone extracts of the bark, and root of P. africanum showed higher antioxidant activity than L-ascorbic acid (Vitamin-C) and Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchromane-2-carboxylic acid), a synthetic vitamin-E analogue, and much higher than Ginkgobiloba extract (EGb 761). The respective EC50 for the P. africanumroot and bark extracts, L-ascorbic acid, and EGb761 were 3.82µg/ml, 4.37µg/ml, 5.04µg/ml, and 40.72 µg/mL. The standardised extract of Ginkgobiloba (EGb 761) is widely employed for its significant benefit in neurological disorders. The extracts inhibited egg hatchability and larval development (from L1 to infective stage L3) of both Haemoncus contortus and Trichostrongylus colubriformis (both parasitic nematodes of ruminants) at concentrations of 0.1-1.0 mg ml-1. The plant extracts, at the concentration of 5-25 mg ml-1 completely lysed larval forms (L1) and eggs of the nematodes.
Conclusion. P. africanum extracts have therefore, potential for treatment of inf7ction-related diseases by either directly inhibiting bacterial growth or by stimulating the immune system of the host. The traditional use of P. africanum concoctions against diarrhea, dysentery and unthriftness, may be also due to anthelmintic activity as these signs are consistent with parasitic gastroenteritis. Gastrointestinal nematodes exasperate diarrhea in HIV-AIDS patients, as well as disease-related production losses arising from stock mortality, severe weight loss and poor production in ruminants. Antioxidants are also important in boosting the immunity, critical in the management of helminthosis. There is ample scientific and empirical evidence supporting the use of plant-derived antioxidants in the control of neurological diseases, as antioxidants have neuro-protective (preventing apoptosis), as well as neuro-regenerative roles. Due to the high antioxidant activity of its extracts, P. africanum has prospects in the management or control of neurodegenerative diseases. Thus there is great potential of P. africanum extracts in disease control.
Brain cholesterol? Long secret life behind a barrier
BJÖRKHEM I Karolinska Institutet, Karolinska University Hospital Huddinge, Division of Clinical Chemistry, 14186 Huddinge, Sweden
The blood-brain barrier is almost completely impermeable for cholesterol in both directions. All cholesterol present in the brain is thus a product a local synthesis. Since there is a low but significant synthesis of cholesterol in the adult mammalian brain we hypothesized that there may be a compensatory flux of a cholesterol metabolite from the brain across the blood-brain barrier. About 14 years ago we identified this metabolite as 24S-hydroxycholesterol (24OHC) and showed that in contrast to cholesterol itself, this oxysterol can cross the blood-brain barrier. By measuring the concentration difference between the internal jugular vein and an artery in human volunteers, we demonstrated the production of 24OHC by the brain to be about 6 mg/24h. Since the uptake of 24OHC by the liver was fund to be about the same, it is evident that almost all 24OHC in the circulation originates from the brain.
In spite of the fact that cholesterol does not pass the blood-brain barrier, hypercholesterolemia is a risk factor for Alzheimer Disease (AD). We hypothesized that there may be a metabolite of cholesterol fluxing in the opposite direction from the circulation into the brain. This metabolite was identified as 27-hydroxycholesterol (27OHC), also using the catheterization approach. The uptake of this oxysterol by the human brain was found to be about 5 mg/24h. Since there is a good correlation between levels of cholesterol and 27OHC in the circulation, it seems likely that the uptake of 27OHC by the brain is related to the levels of cholesterol in the circulation.
In spite of the relatively high influx, levels of 27OHC in the brain are very low, indicating an efficient metabolism. The major metabolite was identified as 7a-hydroxy-3-oxo-4-cholestenoic acid. This acid very efficiently passed a model for the blood-brain barrier and we found a net flux of it from the human brain into the circulation. The conversion of 27OHC into the steroid acid can be regarded as a regulated detoxification. 27OHC is an efficient suppressor of cholesterol synthesis and we have shown that the compound is able to increase amyloid formation in neuroblastoma cells.
Different pathogenetic aspects of the oxysterol crosstalk over the blood-brain barrier will be discussed in the lecture and it is suggested that the flux of 27OHC from the circulation into the brain is the missing link between hypercholesterolemia and AD.
Therapeutic Differentiation of Insulinoma Cell Lines Treated with Streptozotocin BLOCH K, VARDI P.
Tel Aviv University, Felsenstein Medical Research Center, Petach Tikva, Israel
Background: Streptozotocin (STZ) is a member of a group of alkylating antineoplastic drugs, and is clinically active against insulinomas. STZ toxicity depends on glucose transporter protein-2 (GLUT-2) expression and generation of free radicals. As with many other chemotherapeutic drugs, repeated treatment with STZ, may induce a selection of resistant cell populations. Differentiation therapy has been proposed as a promising approach to selectively engage the process of tumor cell differentiation during chemotherapy. According to this approach, cytotoxic agents can induce drug resistance, but in certain conditions, can also lead to recovery of normal cell homeostasis. Aim: To estimate toxin resistance and differentiation of insulinoma cell survival following exposure to STZ.
Methods: A parental highly differentiated mouse insulinoma cell line (BTC-tet) and low differentiated rat insulinoma cell line (RINm) were repeatedly exposed to STZ. The cell populations (RIN-S and BTC-S) surviving such treatment were examined as to their resistance to STZ, hydrogen peroxide, nitric oxide and cytokines. Western blot analysis was applied to estimate GLUT-2 and bcl-2 expression in parental and STZ selected cells. Insulin content and secretion, cell proliferation, morphological and chromosomal characteristics were studied.
Results: Repeated STZ treatment of parental insulinoma cell lines resulted in the selection of cell sub-populations with multiple resistance to different toxins. The enhanced toxin tolerance may be explained by a low level of GLUT-2 and high expression of bcl-2 in selected cells. In addition, STZ selected cells displayed a lower rate of cell proliferation when compared to untreated cells. Moreover, BTC-S and RIN-S cells showed 2-5 times higher levels of intracellular insulin content and improved insulin response to glucose than their parental cells. STZ-based selection changed cell morphology and increased frequency of polyploidy in RIN-S (2.2% ) compared to RINm cells (0.7%).
Conclusions: Repeated STZ treatment of insulinoma cell lines results in the selection of cell subpopulations possessing multiple toxin resistance, a low rate of proliferation and enhanced functional activity. Further characterization of STZ selected beta cells could provide useful lessons for optimization of differentiation therapy of cancer.
Development of a new 3D-Human Airway Epithelium/ Whole-blood Co-culture Model Combined with Multi-Analyte Profile (MAP) Analyses for Assessing Drug Effects Blum M1, Stein GM1, Constant S2, Wiszniewski L2, Huang S2, Mapes J3, Spain M3, Schmolz M1 1EDI GmbH, Reutlingen, Germany, 2Epithelix Sàrl, Plan-Les-Ouates, Genève Suisse, 3Rules-Based Medicine Inc., Austin, TX, USA
Background: The dialogue between cells of the immune system and cells of various tissues controls immune reactions and is in part mediated by a variety of cytokines, chemokines etc. This network may be strongly influenced by the application of drugs. Aim of our investigations was to develop an innovative organo-typical human airway epithelial co-culture model for the analysis of immunopharmacological activities of drugs.
Methods: A differentiated airway epithelium, MucilAir, was combined with whole-blood cultures in a two-chamber system to study the effects of betamethasone applied onto the epithelium sitting on activated immune cells from a healthy donor mimicking an inflamed tissue environment. 92 mediators and other parameters were tested in the supernatants of the cell cultures by multiplexed bead assays (RBM MAP analysis).
Results: Betamethasone exhibited its typical, strong pharmacological effect profile on both, the immune and the epithelial cells: It dose-dependently inhibited a variety of pro-inflammatory mediators, being either T helper cell type 1- (Th1), Th2-, or macrophage-associated, such as interferon (IFN)- interleukin (IL)-5 and tumor necrosis factor (TNF)-, respectively. In contrast, IL-10 as a mediator of regulatory T cells was up-regulated after 24h of co-culture. Furthermore, epithelial cells were cultured for another 6 days showing a dose-dependent effect on e.g. the monocyte chemotactic protein-1 (MCP-1).
Conclusions: From the data we will present in this poster, it is evident that the highly complex, organo-typical co-culture model provides an excellent, in vivo-like tool to study in vitro not only the pharmacokinetics and pharmacodynamics of inhaled drugs, but also the harmful effects of toxicants that get access to the human lung.
