The influences of the compounds on the NF- B pathway were studied using the Raw Blue Cells assay from the InvivoGen. Raw Blue cells, which are derived from the macrophages, stably express a

NF-B induced gene, secreting embryonic alkaline phosphatase (SEAP). The change in the NF- NF-B pathway was determined by measuring the concentration of secreted SEAP in the medium using Quanti-Blue. The measurements were performed following the provided protocols with some adjustments.

The cells were seeded in the 96 wells plate with ~100,000 cells per well in 200 µL Dulbecco's Modified Eagle Medium (DMEM) containing 0.1% (v/v) zeocin and the plate was incubate overnight at 37°C. For the blank, no cells were seeded. The medium was removed and replaced by 180 µL of fresh medium containing 3 µM of the modulator compounds 23a or 23d. After 6 hour incubation at 37°C, 20 µL of NF- B stimulant (IFN- ) was added. Empty medium was used as the control. The plate was incubated overnight at 37°C. 150 µL of the incubated medium were transferred into a flat-bottom 96 wells plate containing 50 µL Quati-blue suspension. The plate was incubated for another 6 hours at 37°C before the absorbance was measured using a ThermoMax microplate reader at 650 nm.

The experiments were performed in triplicate, and the presented results were the average of three independent experiments with the standard deviation.


1. Martel-Pelletier, J.; Lajeunesse, D.; Reboul, P.; Pelletier, J. Therapeutic role of dual inhibitors of 5-LOX and COX, selective and non-selective non-steroidal anti-inflammatory drugs. Ann. Rheum. Dis. 2003, 62, 501-509.

2. Gillmor, S. A.; Villasenor, A.; Fletterick, R.; Sigal, E.; Browner, M. F. The structure of mammalian 15-lipoxygenase reveals similarity to the lipases and the determinants of substrate specificity. Nat. Struct.

Biol. 1997, 4, 1003-1009.

3. Sigal, E. The molecular biology of mammalian arachidonic acid metabolism. Am. J. Physiol. 1991, 260, L13-L28.

4. Epp, N.; Fürstenberger, G.; Müller, K.; de Juanes, S.; Leitges, M.; Hausser, I.; Thieme, F.; Liebisch, G.;

Schmitz, G.; Krieg, P. 12R-lipoxygenase deficiency disrupts epidermal barrier function. J. Cell Biol. 2007, 177, 173-182.

5. Ikei, K. N.; Yeung, J.; Apopa, P. L.; Ceja, J.; Vesci, J.; Holman, T. R.; Holinstat, M. Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation. J. Lipid Res.

2012, 53, 2546-2559.

6. Hunter, J. A.; Finkbeiner, W. E.; Nadel, J. A.; Goetzl, E. J.; Holtzman, M. J. Predominant generation of 15-lipoxygenase metabolites of arachidonic acid by epithelial cells from human trachea. Proc. Natl. Acad.

Sci. 1985, 82, 4633-4637.

7. Nadel, J. A.; Conrad, D. J.; Ueki, I. F.; Schuster, A.; Sigal, E. Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells. J. Clin. Invest. 1991, 87, 1139-1145.

8. Brash, A. R.; Boeglin, W. E.; Chang, M. S. Discovery of a second 15S-lipoxygenase in humans. Proc.

Natl. Acad. Sci. 1997, 94, 6148-6152.

9. Brown, C. D.; Kilty, I.; Yeadon, M.; Jenkinson, S. Regulation of 15-lipoxygenase isozymes and mucin secretion by cytokines in cultured normal human bronchial epithelial cells. Inflamm. Res. 2001, 50, 321-326.

10. Chanez, P.; Bonnans, C.; Chavis, C.; Vachier, I. 15-Lipoxygenase: a Janus enzyme? Am. J. Respir. Cell Mol. Biol. 2002, 27, 655-658.

11. Chu, H. W.; Balzar, S.; Westcott, J. Y.; Trudeau, J. B.; Sun, Y.; Conrad, D. J.; Wenzel, S. E. Expression and activation of 15-lipoxygenase pathway in severe asthma: relationship to eosinophilic phenotype and collagen deposition. Clin. Exp. Allergy 2002, 32, 1558-1565.

12. Shannon, V. R.; Chanez, P.; Bousquet, J.; Holtzman, M. J. Histochemical Evidence for Induction of Arachidonate 15-Lipoxygenase in Airway Disease. Am. Rev. Respir. Dis. 1993, 147, 1024-1028.

13. Piao, Y.; Du, Y.; Oshima, H.; Jin, J.; Nomura, M.; Yoshimoto, T.; Oshima, M. Platelet-type 12-lipoxygenase accelerates tumor promotion of mouse epidermal cells through enhancement of cloning efficiency. Carcinogenesis 2008, 29, 440-447.

14. Rásó, E.; Döme, B.; Somlai, B.; Zacharek, A.; Hagmann, W.; Honn, K.V.; Tímár, J.; Molecular

identification, localization and function of platelet-type 12-lipoxygenase in human melanoma progression, under experimental and clinical conditions. Melanoma Res. 2004, 14, 245-250.

15. Gupta, S.; Srivastava, M.; Ahmad, N.; Sakamoto, K.; Bostwick, D. G.; Mukhtar, H. Lipoxygenase-5 is overexpressed in prostate adenocarcinoma. Cancer 2001, 91, 737-743.

16. Melstrom, L. G.; Bentrem, D. J.; Salabat, M. R.; Kennedy, T. J.; Ding, X.; Strouch, M.; Rao, S. M.; Witt, R. C.; Ternent, C. A.; Talamonti, M. S.; Bell, R. H.; Adrian, T. A. Overexpression of 5-Lipoxygenase in Colon Polyps and Cancer and the Effect of 5-LOX Inhibitors In vitro and in a Murine Model. Clin. Cancer Res. 2008, 14, 6525-6530.

17. Nathoo, N.; Prayson, R.A.; Bondar, J.; Vargo, L.; Arrigain, S.; Mascha, E.J.; Suh J.H.; Barnett,

G.H.; Golubic, M. Increased Expression of 5-Lipoxygenase in High-Grade Astrocytomas. Neurosurgery 2006, 58, 347-354.

18. Wilborn, J.; Bailie, M.; Coffey, M.; Burdick, M.; Strieter, R.; Peters-Golden, M. Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. J. Clin. Invest. 1996, 97, 1827-1836.

19. Zou, L. Y. Tumor 5-Lipoxygenase Expression Correlates with Gastric Cancer Metastasis and Its Selective Inhibitor Induces Cancer Cell Apoptosis. J. Cancer Mol. 2006, 2, 227.

20. Serhan, C. N.; Chiang, N.; Van Dyke, T.E. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat. Rev. Immunol. 2008, 8, 349.

21. Edenius, C.; Haeggström, J.; Lindgren, J. Å. Transcellular conversion of endogenous arachidonic acid to lipoxins in mixed human platelet-granulocyte suspensions. Biochem. Biophys. Res. Commun. 1988, 157, 801-807.

22. Hashimoto, A.; Hayashi, I.; Murakami, Y.; Sato, Y.; Kitasato, H.; Matsushita, R.; Iizuka, N.; Urabe, K.;

Itoman, M.; Hirohata, S.; Endo, H. Antiinflammatory mediator lipoxin A4 and its receptor in synovitis of patients with rheumatoid arthritis. J. Rheumatol. 2007, 34, 2144-2153.

23. Sobrado, M.; Pereira, M.P.; Ballesteros, I.; Hurtado, O.; Fernández-López, D.; Pradillo, J.M.; Caso, J.R.; Vivancos, J.; Nombela, F.; Serena, J.; Lizasoain, I.; Moro, M.A. Synthesis of lipoxin A4 by 5-lipoxygenase mediates PPARγ-dependent, neuroprotective effects of rosiglitazone in experimental stroke.

J. Neurosci. 2009, 29, 3875.

24. Smith, W. L.; DeWitt, D.L., Garavito, R.M. Cyclooxygenases: structural, cellular, and molecular biology.

Annu. Rev. Biochem. 2000, 69, 145.

25. Anderson, G. D.; Hauser, S. D.; McGarity, K. L.; Bremer, M. E.; Isakson, P. C.; Gregory, S. A. Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J. Clin. Invest. 1996, 97, 2672-2679.

26. Nielsen, O. H.; Ahnfelt-Ronne, I.; Elmgreen, J. Abnormal metabolism of arachidonic acid in chronic inflammatory bowel disease: enhanced release of leucotriene B4 from activated neutrophils. Gut 1987, 28, 181-185.

27. Reuter, B. K.; Asfaha, S.; Buret, A.; Sharkey, K. A.; Wallace, J. L. Exacerbation of inflammation-associated colonic injury in rat through inhibition of cyclooxygenase-2. J. Clin. Invest. 1996, 98, 2076-2085.

28. Hendel, J.; Nielsen, O. H. Expression of cyclooxygenase-2 mRNA in active inflammatory bowel disease.

Am. J. Gastroenterol. 1997, 92, 1170-1173.

29. Kargman, S. L.; O'Neill, G. P.; Vickers, P. J.; Evans, J. F.; Mancini, J. A.; Jothy, S. Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res. 1995, 55, 2556-2559.

30. Cianchi, F.; Cortesini, C.; Bechi, P.; Fantappiè, O.; Messerini, L.; Vannacci, A.; Sardi, I.; Baroni, G.;

Boddi, V.; Mazzanti, R.; Masini, E. Up-regulation of cyclooxygenase 2 gene expression correlates with tumor angiogenesis in human colorectal cancer. Gastroenterology 2001, 121, 1339-1347.

31. Wisastra, R.; Ghizzoni, M.; Boltjes, A.; Haisma, H. J.; Dekker, F. J. Anacardic acid derived salicylates are inhibitors or activators of lipoxygenases. Bioorg. Med. Chem. 2012, 20, 5027-5032.

32. Brash, A. R. Lipoxygenases: Occurrence, Functions, Catalysis, and Acquisition of Substrate. J. Biol.

Chem. 1999, 274, 23679-23682.

33. Ghizzoni, M.; Boltjes, A.; Graaf, C. d.; Haisma, H. J.; Dekker, F. J. Improved inhibition of the histone acetyltransferase PCAF by an anacardic acid derivative. Bioorg. Med. Chem. 2010, 18, 5826-5834.

34. Ghizzoni, M.; Wu, J.; Gao, T.; Haisma, H. J.; Dekker, F. J.; George Zheng, Y. 6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site. Eur. J. Med. Chem. 2012, 47, 337-344.

35. Uchiyama, M.; Ozawa, H.; Takuma, K.; Matsumoto, Y.; Yonehara, M.; Hiroya, K.; Sakamoto, T.

Regiocontrolled Intramolecular Cyclizations of Carboxylic Acids to Carbon-Carbon Triple Bonds Promoted by Acid or Base Catalyst. Org. Lett. 2006, 8, 5517-5520.

36. Harrowven, D. C.; Woodcock, T.; Howes, P. D. Total Synthesis of Cavicularin and Riccardin C:

Addressing the Synthesis of an Arene That Adopts a Boat Configuration. Angew. Chem. Int. Ed. Engl.

2005, 44, 3899-3901.

37. Sista, P.; Nguyen, H.; Murphy, J. W.; Hao, J.; Dei, D. K.; Palaniappan, K.; Servello, J.; Kularatne, R. S.;

Gnade, B. E.; Xue, B.; Dastoor, P. C.; Biewer, M. C.; Stefan, M. C. Synthesis and Electronic Properties of Semiconducting Polymers Containing Benzodithiophene with Alkyl Phenylethynyl Substituents.

Macromolecules 2010, 43, 8063-8070.

38. Tranchimand, S.; Tron, T.; Gaudin, C.; Iacazio, G. First Chemical Synthesis of Three Natural Depsides Involved in Flavonol Catabolism and Related to Quercetinase Catalysis. Synth. Comm. 2006, 36, 587-597.

39. Back, D. F.; Manzoni de Oliveira, G.; Ballin, M. A.; Corbellini, V. A. Complexes of vanadyl and uranyl ions with a benzoxazole derivative: Synthesis, structural features and remarks on luminescence properties.

Inorg. Chim. Acta 2010, 363, 807-812.

40. Gibian, M. J.; Galaway, R. A. Steady-state kinetics of lipoxygenase oxygenation of unsaturated fatty acids.

Biochemistry. 1976, 15, 4209-4214.

41. Ha, T. J.; Kubo, I. Lipoxygenase Inhibitory Activity of Anacardic Acids. J. Agric. Food Chem. 2005, 53, 4350-4354.

42. Copeland, R. A.; Williams, J. M.; Giannaras, J.; Nurnberg, S.; Covington, M.; Pinto, D.; Pick, S.;

Trzaskos, J. M. Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase.

Proc. Natl. Acad. Sci. 1994, 91, 11202-11206.

43. Verhagen, J.; Vliegenthart, J. F.; Boldingh, J. Micelle and acid-soap formation of linoleic acid and 13-L-hydroperoxylinoleic acid being substrates of lipoxygenase-1. Chem. Phys. Lipids 1978, 22, 255-259.

44. Leskovac, V. Comprehensive Enzyme Kinetics. 2004; pp 111-116.

45. Gilbert, N. C.; Bartlett, S. G.; Waight, M. T.; Neau, D. B.; Boeglin, W. E.; Brash, A. R.; Newcomer, M. E.

The Structure of Human 5-Lipoxygenase. Science 2011, 331, 217-219.

46. Gilbert, N. C.; Rui, Z.; Neau, D. B.; Waight, M. T.; Bartlett, S. G.; Boeglin, W. E.; Brash, A. R.;

Newcomer, M. E. Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663. FASEB J. 2012, 26, 3222-3229.

47. PyMOL The PyMOL Molecular Graphics System. PyMOL 2010, 1.3, .

48. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E.

UCSF Chimera? A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605-1612.

49. Lee, K. S.; Kim, S. R.; Park, H. S.; Park, S. J.; Min, K. H.; Lee, K. Y.; Jin, S. M.; Lee, Y. C. Cysteinyl leukotriene upregulates IL-11 expression in allergic airway disease of mice. J. Allergy Clin. Immunol.

2007, 119, 141-149.

50. Okamoto, F.; Saeki, K.; Sumimoto, H.; Yamasaki, S.; Yokomizo, T. Leukotriene B4 Augments and Restores FcγRs-dependent Phagocytosis in Macrophages. J. Biol. Chem. 2010, 285, 41113-41121.

51. Thompson, C.; Cloutier, A.; Bossé, Y.; Poisson, C.; Larivée, P.; McDonald, P. P.; Stankova, J.; Rola-Pleszczynski, M. Signaling by the Cysteinyl-Leukotriene Receptor 2: Involvement in Chemokine Gene Transcription. J. Biol. Chem. 2008, 283, 1974-1984.

52. Deb, A.; Haque, S. J.; Mogensen, T.; Silverman, R. H.; Williams, B. R. G. RNA-Dependent Protein Kinase PKR Is Required for Activation of NF-κB by IFN-γ in a STAT1-Independent Pathway. J. Immunol. 2001, 166, 6170-6180.

53. Boström, J.; Greenwood, J. R.; Gottfries, J. Assessing the performance of OMEGA with respect to retrieving bioactive conformations. J. Mol. Graph. Model. 2003, 21, 449-462.

54. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J.

AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput.

Chem. 2009, 30, 2785-2791.

55. Koes, D. R.; Baumgartner, M. P.; Camacho, C. J. Lessons Learned in Empirical Scoring with smina from the CSAR 2011 Benchmarking Exercise. J. Chem. Inf. Model. 2013 .

In document University of Groningen Modulation of lipoxygenase activity and chemistry-based detection of protein nitration in inflamation Wisastra, Rosaline (Page 36-39)

Related documents