Anna Mazur

Publications of Anna Mazur

• Fibroblast activation protein-α promotes tumor growth and invasion of breast cancer cells through non-enzymatic functions.

  Authors: Yan Huang, Avis E Simms, Anna Mazur, Sophie Wang, Noel R León, Barry Jones, Nazneen Aziz, Thomas Kelly

  Clinical & experimental metastasis. 05/2011; 28(6):567-79.

  Fibroblast activation protein-α (FAP) is a cell surface, serine protease of the post-prolyl peptidase family that is expressed in human breast cancer but not in normal tissues. Previously, we showedFibroblast activation protein-α (FAP) is a cell surface, serine protease of the post-prolyl peptidase family that is expressed in human breast cancer but not in normal tissues. Previously, we showed that FAP expression increased tumor growth rates in a mouse model of human breast cancer. Here the role of the proteolytic activities of FAP in promoting tumor growth, matrix degradation and invasion was investigated. Mammary fat pads of female SCID mice were inoculated with breast cancer cells that express FAP and the mice treated with normal saline or Val-boroPro (talabostat); Glu-boroPro (PT-630); or 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine (LAF-237) that inhibit prolyl peptidases. Other mice were injected with breast cancer cells expressing a catalytically inactive mutant of FAP and did not receive inhibitor treatment. PT-630 and LAF-237 did not slow growth of tumors produced by any of the three cell lines expressing FAP. Talabostat slightly decreased the growth rates of the FAP-expressing tumors but because PT-630 and LAF-237 did not, the growth retardation was likely not related to the inhibition of FAP or the related post-prolyl peptidase dipeptidyl peptidase IV. Breast cancer cells expressing a catalytically inactive mutant of FAP (FAP(S624A)) also produced tumors that grew rapidly. In vitro studies revealed that cells expressing wild type FAP or FAP(S624A) degrade extracellular matrix (ECM) more extensively, accumulate higher levels of matrix metalloproteinase-9 (MMP-9) in conditioned medium, are more invasive in type I collagen gels, and have altered signaling compared to control transfectants that do not express FAP and form slow growing tumors. We conclude that the proteolytic activity of FAP participates in matrix degradation, but other functions of the protein stimulate increased tumor growth.

• [The influence of oxidised cholesterol derivatives on morphology of hepatocytes in rat's liver].

  Authors: Anna Mazur, Andrzej Wiczkowski, Robert Kubina

  Wiadomości lekarskie (Warsaw, Poland : 1960). 01/2011; 64(2):79-83.

  Products of oxidizing cholesterol-called oxysterols, are having different physicochemical properties and various from not-modified sterols, biological activity. Amongst characteristic effects ofProducts of oxidizing cholesterol-called oxysterols, are having different physicochemical properties and various from not-modified sterols, biological activity. Amongst characteristic effects of these connections are: stopping the activity of HMG-CoA reductase, antiproliferative action, apoptosis induction, changes in the structure and functioning of the cell membrane and influence on remodeling of the immunological system. Mechanisms of oxysterols influence on the cellular level include a broad spectrum of complicated biological activities and they haven't been examined to the end yet. In order to experiment execution the rat livers have been collected and the microscope sections have been made by standard technics, staining by haematoxylin and eosin (H&E). In each preparation 222 hepatic cells surrounding 10 central veins have been analysed and 222 hepatic cells occuring around 10 triads. The total surface ofcell, nucleus and cytoplasm have been taken for analysis. In all researched cases the analysed parameters have been reduced in area liver traid and central vein. Decreasing of area value of cell, nucleus and cytoplasm have been observed. On the basis of our examinations we determine, that oxysterols derived from the diet have influence on parenchymatous liver cells morphology in regions of hepatic triad and the central vein. We have concluded, that cytotoxic effect of oxysterols depend mainly on stopping the growth of these hepatic cells and lead to many disorders connected with the influence ofoxysterols, not only to cells but also to the all organism.

• Characterization of human hepatic and extrahepatic UDP-glucuronosyltransferase (UGTs) enzymes involved in the metabolism of classical cannabinoids.

  Authors: Anna Mazur, Cheryl F Lichti, Paul L Prather, Agnieszka K Zielinska, Stacie M Bratton, Anna Gallus-Zawada, Moshe Finel, Grover Paul Miller, Anna Radominska-Pandya, Jeffery H Moran

  Drug metabolism and disposition: the biological fate of chemicals. 05/2009;

  Tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT) dependentTetrahydrocannabinol (Delta(9)-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT) dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450 dependent oxidations, but little work has been done characterizing phase II metabolic pathways. This study tests the hypothesis that there are specific human UGTs responsible for classical cannabinoid metabolism. The activities of 12 human recombinant UGTs towards classical cannabinoids [cannabinol (CBN), cannabidiol (CBD), (-)-Delta(8)-THC, (-)-Delta(9)-THC, (+/-)-11-hydroxy-Delta(9)-THC (11-OH-THC), and (-)-11-nor-9-carboxy-Delta(9)-THC (COOH-THC)] were evaluated using HPLC-MS/MS and labeling assays. Despite activity by UGT1A1, -1A3, -1A8, -1A9, -1A10, and -2B7 toward CBN, CBD, 11-OH-THC, and COOH-THC, only selected UGTs demonstrate sufficient activity for further charaterization of steady-state kinetics. CBN was the most recognized substrate as evidenced by activities from hepatic UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect the the introduction of an aromatic ring to Delta(9)-THC, leading to favorable pi stacking with phenylalanines in the UGT active site. Similarly, oxidation of Delta(9)-THC to 11-OH-THC results in UGT1A9 and UGT1A10 activity toward the cannabinoid. Further oxidation to COOH-THC surprisingly leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of COOH-THC is the main metabolite found in urine, and thus these hepatic enzymes play a critical role in the metabolic clearance of cannabinoids. Taken together, glucuronidation of cannabinoids depends on upstream processing including enzymes such as CYP2C9 and CYP3A4.

• Assessing Cytochrome P450 and UDP-Glucuronosyltransferase Contributions to Warfarin Metabolism in Humans.

  Authors: Grover P Miller, Drew R Jones, Shane Z Sullivan, Anna Mazur, Suzanne N Owen, Neil C Mitchell, Anna Radominska-Pandya, Jeffery H Moran

  Chemical research in toxicology. 05/2009;

  As a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable ofAs a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying specific enantiomeric (R and S) contributions of warfarin (WAR) and the corresponding hydroxywarfarins (OH-WAR) and glucuronides (-GLUC) generated by cytochrome P450s (CYP) and UDP-glucuronosyltransferases (UGTs), respectively. Evaluation of quality control samples and three commercially available human samples showed that our analytical approach has the ability to measure 24 unique WAR metabolites in human urine. Evaluation of the human data also provides new insights for evaluating WAR toxicity and begins characterizing important UGT metabolic pathways responsible for WAR detoxification. Data revealed the significance of specific metabolites among patients and the corresponding enzymatic capacity to generate these compounds, including the first report of direct WAR glucuronidation. On the basis of total OH-WAR levels, (S)-7-OH-WAR was the predominant metabolite indicating the significance of CYP2C9 in WAR metabolism, although other CYP2C enzymes also contributed to clearance of this isomer. (R)-WAR hydroxylation to OH-WARs was more diverse among the patients as reflected in varying contributions of CYP1A2 and multiple CYP2C enzymes. There was wide variation in the glucuronidation of WAR and the OH-WARs with respect to the compounds and patients. 6- and 7-OH-WAR were primarily (>70%) excreted as glucuronides unlike 4'-OH-WAR and 8-OH-WAR. For all patients, UGT1A1 is likely responsible for 6-O-GLUC production, although UGT1A10 may also contribute in one patient. 7-O-GLUC levels reflected contributions from potentially five different UGT1A enzymes. In all cases, WAR, 4'-OH-WAR, 8-OH-WAR, and the corresponding glucuronides were minor metabolites with respect to the others. Taken together, these data suggest that both P450 and UGT reactions contribute to the generation of excretable products in human urine, thereby generating complex metabolic networks.

• Identification of hydroxywarfarin binding site in human UGT1A10: Phenylalanine90 is crucial for the glucuronidation of 6- and 7-hydroxy- but not 8- hydroxywarfarin.

  Authors: Grover P Miller, Cheryl F Lichti, Agnieszka K Zielinska, Anna Mazur, Stacie M Bratton, Anna E Gallus-Zawada, Moshe Finel, Jeffery H Moran, Anna Radominska-Pandya

  Drug metabolism and disposition: the biological fate of chemicals. 09/2008;

  Recent studies show that the extrahepatic human UDP-glucuronosyltransferase (UGT)1A10 is capable of Phase II glucuronidation of several major cytochrome P450 metabolites of warfarin (Coumadin) (i.e.Recent studies show that the extrahepatic human UDP-glucuronosyltransferase (UGT)1A10 is capable of Phase II glucuronidation of several major cytochrome P450 metabolites of warfarin (Coumadin) (i.e. 6-, 7-, and 8-hydroxywarfarin). This study expands on this finding by testing the hypothesis that the UGT1A10 F(90)-M(91)-V(92)-F(93) amino acid motif is important for proper recognition and conjugation of hydroxywarfarin derivatives. Site-directed mutagenesis studies demonstrate that F(90) is critical for 6- and 7-hydroxywarfarin glucuronidation based on the complete loss of enzymatic activity toward these substrates. In contrast, V92A and F93A mutants lead to higher rates of substrate turnover, have minimum changes in Km values, and demonstrate substrate inhibition kinetics. A completely different activity profile is observed in the presence of 8-hydroxywarfarin. No change in either activity or affinity is observed with F90A when compared to wild type, while F93A and V92A mutants show increases in Vmax (3- and 10-fold, respectively) and minimum changes in Km. LC-tandem mass spectrometry studies show that enzymatic products produced by mutants are identical to wild-type products produced in the presence of 6-, 7-, and 8-hydroxywarfarin. Because F(90) is not critical for the glucuronidation of 8-hydroxywarfarin, there is likely another, different amino acid responsible for binding this compound. In addition, an inhibitory binding site may be formed in the presence of 6- and 7-hydroxywarfarin. This new knowledge and continued characterization of the hydroxywarfarin binding site(s) for UGT1A10 will help to elucidate the molecular mechanism of hydroxywarfarin glucuronidation and potentially result in more effective anticoagulant therapies.

• Biopanning of single-chain antibodies expressing phages reveals distinct expression patterns of angiogenic and arteriogenic vessels.

  Authors: Anna Mazur, Andrea Deylig, Wolfgang Schaper, Thomas Meinertz, Wulf D Ito

  Endothelium : journal of endothelial cell research. 02/2003; 10(4-5):277-84.

  "Therapeutic angiogenesis" requires targeted delivery of growth factors for maximal benefit and limitation of potential hazards such as enhancement of tumor or plaque angiogenesis. Physiological"Therapeutic angiogenesis" requires targeted delivery of growth factors for maximal benefit and limitation of potential hazards such as enhancement of tumor or plaque angiogenesis. Physiological distinctions between angiogenesis and collateral growth suggest the possibility of targeting selectively collateral endothelium. This article describes the generation of collateral-targeting single-chain antibodies (scFv). Membrane preparations of growing collateral arteries from rats were used to produce collateral-targeting antibodies (CTAs) via immunization of mice. ScFv were generated from CTA-producing hybridoma and cloned into the pV gene of M13 phages. Phages expressing collateral-targeting scFv (CT scFv) were selected via repeated exposure to activated collateral arteries followed by reamplification. CT scFv could specifically be amplified, selected, and sequenced. Phages expressing CT scFv bound selectively to proliferating collateral vessels as identified by positive Polycyclic Nuclear Antigen (PCNA) staining and homed specifically to collateral endothelium after in vivo injection but bound neither to control vessels nor to tumor vessels. This study reveals major differences between angiogenic and collateral endothelium and delivers a tool that will allow the stimulation of collateral growth without promoting tumor or plaque angiogenesis.