br Single crystals of pale yellow
Single crystals of 3 (pale yellow irregular fragment with approximate dimensions 0.18 0.40 0.45 mm) were mounted on a glass fiber in a random orientation. The X-ray intensity data were measured on a Bruker Smart Breeze CCD system equipped with a graphite monochromated Mo-Ka radiation (l ¼ 0.71073 Å) at 100
(2) K, cooled by an Oxford Cryosystems 700 Series Cryostream. Space group assignments were based on systematic absences, E statistics and successful refinement of the structures. The struc-tures were solved by direct methods with the aid of successive difference Fourier maps and were refined using the Bruker SHELXTL Software Package. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were assigned to ideal positions and refined using a riding model. These data can be obtained free of charge from The Cambridge Crystallographic Data Center via www. ccdc.cam.ac.uk/data_request/cif (CCDC 1579386) or in the Supple-mentary Information.
4.2. Biological assays
Human renal cell carcinoma line Caki-1 was newly obtained for these studies from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA) and cultured in Roswell Park Memorial Institute (RPMI-1640) (Mediatech Inc., Manassas, VA) media con-taining 10% Fetal Bovine Serum, certified, heat inactivated, US origin (FBS) (Gibco, Life Technologies, US), 1% Minimum Essential Media (MEM) nonessential PCI32765 (NEAA, Mediatech) and 1% penicillinestreptomycin (PenStrep, Mediatech). IMR90 (human fetal lung fibroblast) cells were purchased from ATCC (Manassas, Virginia, USA) and maintained in Dulbecco's modified Eagle's me-dium (DMEM) (Mediatech) supplemented with 10% FBS, 1% NEAA and 1% Penicillin Streptomycin. HUVEC (human umbilical vein endothelial) cells were obtained from ATCC and cultured in Me-dium 200PRF (Gibco, Life Technologies, US).
4.2.2. Cell viability analysis
The cytotoxic profile (IC50) of bimetallic TieAu Titanocref (2), Titanofin (4) and monometallic Au cref (2), and fin (3) was deter-mined by assessing the viability of Caki-1 and IMR90 control cells treated with the appropriate cultured medium containing 0.1 mM, 1 mM, 10 mM and 100 mM of compounds for 72 h using the colori-metric cell viability assay PrestoBlue (Invitrogen). The cytotoxic profile of Auranofin and Titanocene dichloride (for comparative purposes) was also determined. All compounds were dissolved in DMSO except Cisplatin that was dissolved in H2O with a final DMSO concentration of 1%. In the supplementary information section there is a more complete cytotoxic profile that includes IC50, IC20, and IC10 values at 72 h and IC50 and IC20 values at 24 h (Table S3).
The cell death profile in Caki-1 cancerous cells cultured in the appropriate medium containing IC50 concentrations of bimetallic TieAu Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin for 24 h was analyzed by flow cytometry stained with Annexin V (FITC) dye (Invitrogen). Staurosporine and Ion-omycin treated cells were also stained with Annexin V dye and served as positive controls for apoptosis and cell death. FITC fluo-rescence intensity was detected with a flow cytometry analysis was conducted using a BD LSR II flow cytometer. 10*105 events per sample were recorded. The flow cytometer was calibrated prior to use.
The cell cycle profile in Caki-1 cancerous cells cultured in the complete RPMI medium containing IC20 concentrations of bime-tallic TieAu Titanocref (2), Titanofin (4) and Auranofin for 24 h was analyzed by flow cytometry wherein total DNA was stained with FxCycle Violet (FCV; DAPI) dye (Invitrogen). DAPI fluorescence in-tensity was detected with a BD LSR II flow cytometer and flow cytometry analysis was conducted using BD FACSDiva 8.0.2 10*105 events per sample were recorded. The flow cytometer was cali-brated prior to use.
4.2.5. Cell migration and invasion analysis
The anti-migratory profile of bimetallic TieAu Titanocref (2), Titanofin (4) and monometallic Au cref (2), and fin (3) Auranofin was assessed by in vitro scratch assay using Caki-1 cells treated with the appropriate cultured medium containing IC20 concentration of the compounds. The diluting agent (0.1% DMSO) served as positive control. Briefly, using 6-well collagen-coated plate cells were allowed to seed for 24 h. After which, cells were serum starved for 4 h, scratched by 200 mL tips. 24 h after injury, cells were photo-graphed using a Moticam camera mounted on a Zeiss microscope inverted microscope at 20X. The area invaded was measured in 5 randomly selected segments from each photo then averaged. Data were collected from two independent experiments performed.
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 cells 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.