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  • br Angioge http www glpbio com simage GC

    2020-08-18


    4.2.6. Angiogenesis analysis
    The antiangiogenic profile of bimetallic TieAu Titanocref (2), Titanofin (4) and Auranofin were determined by assessing the
    endothelial tube formation of Human umbilical vein endothelial Ibrutinib (HUVECs) treated with the appropriate cultured medium containing the above-mentioned compounds. Briefly, 96 well plates were coated with Geltrex®, Reduced Growth Factor Basement Membrane Matrix (Invitrogen) (50ml/well) and incubated at 37 C for 30 min to allow gelation to occur. HUVECs were added to the top of the gel at a density of 6000 cells/well in the presence or absence of Titanocref (2), Titanofin (4) or their monometallic controls (1 mM). The diluting agent (1% DMSO) served as positive control. Cells were incubated at 37 C with 5% CO2 for 24 h and pictures were captured with a Moticam camera mounted on a Zeiss mi-croscope inverted microscope at 10X. Quantification of tube for-mation was assisted by SCORE, a web-based image analysis system (S$CO BioLifescience). Tube formation quantified by Number of branching points (tube nodes, TN) and total length skeleton (tube length, TL). The data was obtained from the average of three wells per treatment condition.
    4.2.7. Thioredoxin reductase activity assay
    Caki-1 cells treated in vitro with IC50 concentrations of either Titanofin (2), Titanocref (4) the monometallic compounds cref (1), fin (3), and Auranofin or 0.1% DMSO were lysed after 24 h of treatment. The lysate was then mixed with Thioredoxin Reductase Assay buffer, after 20 min incubation DTNB (5, 50-dithiobis (2-nitrobenzoic) acid) was added TrxR levels were detected accord-ing to the manufacturer's instructions (Abcam kit, ab83463) using the BioTek Microplate Reader (BioTek U.S., Winooski, VT) at l ¼ 412 nm. Tests were done in duplicate. TrxR activity was calcu-lated based on the linear amount of TNB produced per mg of total protein and adjusted for background activity from enzymes other than TrxR in the lysates.
    Caki-1 cells treated in vitro with either IC20 concentrations of bimetallic TieAu Titanocref (2), Titanofin (4) and monometallic Au cref (2), and fin (3) and Auranofin or 0.1% DMSO were lysed after 72 h of treatment, cell culture supernatant was collected and VEGF expression was sassessed by a VEGF human ELISA kit (100663 Abcam).
    Caki-1 cells treated in vitro with either IC20 concentrations of bimetallic TieAu Titanocref (2), Titanofin (4) and monometallic Au cref (2), and fin (3) and Auranofin or 0.1% DMSO. After 72 h of treatment, cell culture supernatant was collected, and interleukin expression was determined by Multi-Target ELISA array kit (PathScan Cytokine Antibody Array Kit, Cell Signaling). The relative expression levels of the proteases were determined according to the manufacturer's protocol, and signal intensities were compared using HLImageþþ software (R&D).
    Caki-1 cells treated in vitro with either IC20 concentration of bimetallic TieAu Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin or 0.1% DMSO were lysed after 72 h of treatment. Before application to the array, protein concentration was determined by BCA. Then 150 mg of lysate was incubated for 24 h with the Proteome Profiler Human Protease Array Kit (ARY025, R&D Systems). The relative expression levels of the proteases were determined according to the manufacturer's protocol, and signal intensities were compared using HLImageþþ software (R&D).
    Abbreviations
    ATCC: American Type Culture Collection; ADAM: a disintegrin 
    and metalloproteinase; Auranofin: Gold (1þ); (2S,3R,4S,5R,6R)-3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate; triethyl-phosphane: Caki-1: human clear cell renal cell carcinoma: DPPF: 1,10-Ferrocenediyl-bis(diphenylphosphine); DTNB (5, 50-dithiobis (2-nitrobenzoic) acid: HCT 116: human colorectal carcinoma: HUVEC: human umbilical vein endothelial cells: ILs: Interleukins; IMes: 1,3-bis(2,4,6-trimethylphenyl)imidazole-2-ylidene; IRM90: human foetal lung fibroblast: mba: MMPs: matrix metal-loproteinase: NHC: N-heterocyclic carbene: TNB2 : 5-thio-2-nitrobenzoic acid: TrRx: thioredoxin reductase: VEGF: vascular endothelial growth factor.
    Conflicts of interest
    The authors declare no conflict of interest for this article.
    Acknowledgments
    We thank the National Cancer Institute (NC1) and National Institute of General Medical Sciences (NIGMS) for grant 1SC1CA182844 (M.C.). N.C. thanks Fundacion Alfonso Martín Escudero (Spain) for a postdoctoral fellowship. We are very grateful to Dr. Karen Hubbard (City College of New York, CUNY) for providing infrastructure and advice and Ciara Bagnall (City College of New York, CUNY) for her technical expertise in imaging and immunocytochemistry. We thank Dr. William H. Hersh (Queens College, CUNY) for crystallogrpahic studies of compound 3.