br In this assay the endothelial tube formation
In this assay, the endothelial tube formation of Human umbilical vein endothelial BCI-121 (HUVECs) on an ECM-like matrix was deter-mined as a function of length of the uninterrupted tubes (TL), and number of branching point or nodes in the tubes (TN) (Fig. 4). The number of tubes and nodes was counted using Image-J with the Angiogenesis plugin. The greater the inhibition of tube formation, that is the lower length of tube (TL) and the lower the number of nodes (TN) the higher the anti-angiogenic properties of a compound.
All three compounds, bimetallic Titanocref (2) and Titanofin (4) as well as control Auranofin induce similar disruption in tube integrity with an average of 50% in disruption tube length (TL) and 45% disruption in tube node (TN) formation. Disruption in vascular formation is a key attribute of many anti-angiogenic compounds and can prevent tumor growth and hinder metastasis. Limiting a tumor's access to viable vasculature serves to deprive the tumor of nutrients for growth and an exit avenue through which it can escape for metastasis.
Fig. 4. Effects on vascular endothelial cell reorganization into 3D structures. Human umbilical vein endothelial cells (HUVEC) were seeded with the appropriate media in plates coated with Geltrex® matrix and incubated at 37 C and 5% CO2. Thereafter, the 72 h IC10 of bimetallic Ti-Au Titanocref (2), Titanofin (4) and Auranofin or 0.1% DMSO was added. (A) Representative phase-contrast images captured 4 h then 24 h post dosing. Scale bar ¼ 100 mm. (B) Quantitation of tube formation was performed using Image-J with the Angio-genesis plugin. 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. r> 2.5. Inhibition of targets associated to cancer cisplatin resistance, metastasis and angiogenesis
2.5.1. Inhibition of thioredoxin reductase
Changes in intracellular anti-oxidant states are a distinctive feature of many chemo-resistant cancers. Overexpression of thio-redoxin reductase (TrxR), an enzyme that controls intracellular
redox state, is a critical condition for the survival of cisplatin-resistant cancer cells. Furthermore, TrxR overexpression has been causally linked to increased angiogenesis and therefore TrxR has become a salient therapeutic target [9,30,44,53e56]. We previously reported on the significant inhibition of TrxR in Caki-1 cells by Auranofin  and heterometallic titanocene-Au [26,27] and RueAu complexes [29,30]. We here measured the activity of (TrxR)
Fig. 5. Inhibition of proangiogenic factors TrxR and VEGF in Caki-1 cells by bimetallic Ti-Au Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin occurs in a time and dose dependent manner. A. inhibition of proangiogenic anti-apoptotic mitochondrial protein TrxR following treatment with IC20 concentrations of each compound for 24 h and 72 h. The values indicate the percentage of TrxR activity relative to DMSO treated cells. B. Inhibition of proangiogenic protein VEGF, following treatment with IC20 concentrations of each compound for 24 h and 72 h. The values indicate the percentage of VEGF expression relative to DMSO treated cells. Analysis of 150 ng of protein extracted from cell lysate. The data shown, and standard deviation of the sample mean, result from two independent trials.
in Caki-1 cells, following incubation with bimetallic TieAu Tita-nocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin as a positive control (Fig. 5). After 72 h of incubation Caki-1 TrxR activity is significantly reduced by Auranofin (86%), after 24 h of incubation there was not a significant change [26,30]. After 72 h of incubation the inhibition of TrxR by the bimetallic TieAu Titanocref (2) and Titanofin (4) is very similar (Titanocref, 87%; Titanofin 79%). The inhibition is larger than the also strong inhibition displayed by monometallic gold compounds cref (2) and fin (3) of 54% and 57% respectively.
Vascular endothelial growth factor (VEGF) is the key mediator of tumor angiogenesis through its up-regulated by oncogene and growth factors expression [57e59]. For solid tumors to grow beyond 0.5 cm in diameter, denovo angiogenesis is required for the delivery of nutrients and oxygen to the tumor site [49,57,58]. Therefore, VEGF along with other key growth factors produced by tumors in orchestrate an 'angiogenic switch', wherein vasculature is formed through and around the tumor, facilitating hyper-proliferation and exponential growth . Thus, given the signifi-cant tumor promoting capacities of VEGF we sought to assess whether or not its expression is affected by TieAu Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin on VEGF. We found that VEGF secretion is inhibited by both bimetallic TieAu Titanocref (2), Titanofin (4) and Auranofin after 72 h incu-bation with IC20 of each compound (41% (2), 59% (4) and 55% (AF) reduction respectively), while for monometallic Au cref (2) and fin (3) there was no significant inhibition of VEGF secretion with it being 22% and 17% respectively, no significant change was observed after 24 h incubation with IC20 of each compound. In this case the strong inhibition of VEGF could be correlated to the anti-angiogenic effects we found. VEGF is known to be a key regulator of angio-genesis and its downregulation is often correlated with reduced angiogenesis.