br The anti invasion profile of bimetallic TieAu Titanocref
The anti-invasion profile of bimetallic TieAu Titanocref (2), Titanofin (4) and Auranofin was assessed by transwell assay by determining the invasion of Caki-1 cancerous Relebactam treated with the appropriate cultured medium containing IC20 concentration of the compounds. The diluting agent (0.1% DMSO) served as positive control. Briefly, Geltrex® a Reduced Growth Factor Basement Membrane Matrix (Invitrogen) is thawed and added to a 24-well transwell insert and solidified in a 37 C incubator for 30 min to form a thin gel layer. Cell solution of 5*105 cells in serum-free media is added on top of the Geltrex® coating to simulate invasion through the extracellular matrix. The insert was mounted into a well containing complete media. Invasion of cells toward the chemotactic gradient through the membrane to the underside was assessed after 24 h when the membrane is fixed and stained with hematoxylin and eosin. Then the invaded cells were captured with a Moticam camera mounted on a Zeiss inverted microscope at 20X. Cell numbers were counted from 5 randomly selected field of view from each photo then averaged. Data were collected from at least two independent experiments performed.
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 cells (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).