br timepoint because no significant effect was observed
timepoint because no significant effect was observed after 24 h 72 h at IC20 prior similar assay. From this screen we identified that our compounds had significant effect on nine markers of onco-logical interest in addition to the fourteen reported earlier in the paper. A salient point arose from our analysis, there seems to be some stratification of markers that are affected by the bimetallic and not the monometallic compounds and vice versa. This strati-fication represented in a heat map (Fig. 7) warrants further exploration. Heat maps are two-dimensional representations of a data set from which one seeks to extract patterns between features such as treatment type for example. The protein expression level values can be arranged using stratification allowing for an infor-mative visual summary.
In an attempt to determine if there is any clear pattern in the inhibition of protein of oncological for bimetallic compounds we made a heat map (Fig. 7) including bimetallic TieAu (Titanocref 2 and Titanofin 4) and monometallic Au (cref 1 and fin 3) as well as Auranofin (used as control in all studies described here).
Fig. 6. Bimetallic Ti-Au Titanocref (2), Titanofin (4) and monometallic Au cref (2), fin (3) and Auranofin induced changes in the expression levels of prometastatic cytokines (TNF-a, and interleukins) and matrix proteases (MMMPs -matrix metalloproteinases) in Caki-1 cells. Analysis of 150 ng of protein extracted from cell lysate collected from cell treated with IC20 of the compounds for 72 h. The data shown, and standard deviation of the sample mean, result from two independent trials.
Markers whose levels vary significantly (p < 0.05) between at least two treatment groups (Titanocref 2, Titanofin 4, cref 1, fin 3 and Auranofin) are projected on the heat map and used for clus-tering. The data plotted is reported in bar graph form in Figs. 5 and 6 and S20 (In SI). In the heat map (Fig. 7) there are seven additional markers of interest in cancer progression that were inhibited by either bimetallic, monometallic or both PCI32765 of compounds that were not discussed earlier in the text. The degree of inhibition can be appreciated in the heat-map where DMSO serves as the 100% expression baseline.
From a protein array screen, we observed that the compounds interfered with the protein expression levels of CapG, ErbB2, FGF-basic, FOXC2, HIF-1a, Tenascin C and CD31/PECAM-1 all of which are involved in several aspects of tumor malignancy and their in-dividual or collective overexpression correlate to poor prognosis. The capping proteins CapG and ErbB2 play a key role in breast and lung cancer invasion and is a predictor of poor outcome [74e76]. Basic fibroblast growth factor (FGF-basic) is known to stimulate angiogenesis directly driving up the proliferation, migration and overall survival of tumors [77e79]. The inhibition of Forkhead box protein C2 (FOXC2) is reported to restore drug sensitivity in some cancer cells, and reinstate epithelial phenotype to metastatic cells [80,81]. While the cell-adhesion and signaling molecule PECAM-1 is known to modulate resistance to genotoxic chemotherapy such as cisplatin treatment .
High expression of Hypoxia-inducible factor 1-alpha (HIF-1a) also correlates to poor prognosis in several cancer types [83e85]. The extracellular matrix protein tenascin C is overexpressed in the invasive front of several cancer types during local invasion, and it is also associated with poor clinical outcome [86e88].
Along with the fifteen markers discussed earlier, the broad hitting range of inhibition of pro-tumorigenic signaling molecules by monometallic Au cref (2), fin (3) and more importantly by bimetallic Titanocref (2), Titanofin (4) is a testament to their great potential in inhibiting tumor progression on several fronts. Aur-anofin has also emerged as a significant inhibitor of a number of factors critical of tumor malignant progression and metastasis in human clear-cell renal carcinoma Caki-1 as exhibited throughout this body of work and that recently published by our group . These findings are informative not only in guiding further rational design, but also on the merit that it adds mechanistic insights to an FDA approved drug that is actively being explored as a potential anticancer agent. Broadening our understanding of the mechanism by which Auranofin archive efficacy is a valuable contribution to the study of its potential as safe, readily available in the clinic and efficient anticancer agent.
What has become evident from our evaluations is that however effective the monometallic compounds might be, overall the bimetallic compounds are more potent and affect a broader spec-trum of molecular targets and cellular behaviors than any single isolate monometallic as can be observed in the compounds inhi-bition of migration, invasion, angiogenesis as well as their inhibi-tion of VEGF, MMP(s) and IL(s).