br Next we evaluated the
Next we evaluated the effects of bimetallic Titanocref (2), Tita-nofin (4) and monometallic Au Auranofin on 3847-29-8 arrest. Titanocref (2) and Titanofin (4) decreased G1/G0 by 32% and 33% respectively and increased the G2/M population by 38% and 36% respectively - compared to DMSO control. Auranofin increased G1/ G0 by 27% and induced an arrest of 76% of the cell population at G1/ G0 (as observed recently by us ), while also generating a peak at
SubG1 in 20% of the cells arrest consistent with apoptosis. This suggests that Auranofin induced complete G1/G0 arrest, as has already been reported for other cells lines treated with AF [44e48], or other anti-inflammatory drugs . The cell cycle shift of Tita-nocref (2) and Titanofin (4) are strikingly similar to each other and unlike that of Auranofin suggesting that perhaps the Ti moieties account in part for the distribution in G2/M and the prevent the complete switch to G1/G0 observed with Auranofin.
In view of all results obtained and the very high concentration needed of TDC to observe cytotoxic effects, we decided not to explore the effects of titanocene dichloride in Caki-1 cells any further.
2.3. Inhibition of migration and invasion
The increased local cell migration and later the distal invasion
Fig. 3. Cell Migration and Invasion Inhibition Assays for bimetallic Ti-Au Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin. A. Inhibition of migration (2D wound-healing scratch assay). Scratch assay showing that Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin interfere with Caki-1 migration. Panels show representative images of untreated cells at time points T0 (top row) when the compound is added to the assay up to 24 h (bottom row). B. Inhibition of invasion (3D transwell assay Geltrex) showing that Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin interfere with Caki-1 invasion. The values graphed are normalized to DMSO control. The invasion assay was performed using an ECM-like Reduced Growth Factor Basement Membrane Matrix (Geltrex) 3:1. Panels show representative images of cells 24 h after treatment with the 72 h IC20 concentration at which we observe less than 8% cell death after 24 h of incubation. The data reported in the graph, and standard deviation of the sample mean, result from two independent trials averaging quantitation from five fields of view per trial.
seen in advanced tumors are hallmarks of metastasis [50,51]. We therefore evaluated the anti-migration and anti-invasive properties of bimetallic Titanocref (2), Titanofin (4) and monometallic gold cref (1), fin (3) and Auranofin. The effect of (IC20) the compounds on migration was determined using a wound-healing 2D scratch assay on a collagen-coated plate (Fig. 3A). We chose the IC20 because it was determined that at that concentration about 80% of the cells (78% ± 2.31 depending on the compound) are alive. Migration is quantified by measuring the space in the wound gap that is occu-pied by cells 24 h after treatment. We observed that heterometallic Titanocref (2) and Titanofin (3) as well as Auranofin significantly reduced migration by 89%, 83% and 81% respectively. Gold mono-metallic cref (1) and fin (3) inhibit migration to a lesser degree 40% and 33% respectively. While the cytotoxicity and apoptotic prop-erties of Auranofin on different cancer cell lines are well known , the efficacy of Auranofin as an anti-migratory and perhaps anti-metastatic compound was unexpected and has only been recently reported by us . All the compounds studied interfere with invasion and follow a similar trend as that of migration with Auranofin and TieAu compounds being most effective (Fig. 3B). There is a clear trend that the monometallic gold compound fin (3) is a better inhibitor of invasion than cref (1), while their inhibition of migration is similar. This may be because proteolysis is necessary for the cells to infiltrate though the 3D matrix, but is not needed for 2D migration. Indeed fin (3) which is a better inhibitor of invasion is also a stronger inhibitor or MMP-9 and MMP-2 (see next sections) which are proteolytic agents known to be key players in migration and metastasis. As explained above, the most efficient compounds in these experiments were bimetallic Titanocref (2) and Titanofin
2.4. Inhibition of angiogenesis
Neovascularization plays an essential role in tumor malignancy. We chose to examine the formation of tube-like structures by HUVEC cells in an extracellular matrix as an in vitro model  to assess the potential perturbation of angiogenesis is the most effective and potent bimetallic TieAu Titanocref (2), Titanofin (4) and Auranofin.