br Conclusions br This study expands on
This study expands on the discovery of the high efficacy in vitro in a human clear-cell renal carcinoma cell line displayed by bime-tallic titanocene-gold derivatives. One of the compounds (Tita-nocref) had been described as extremely efficacious in vitro and
Fig. 7. Heat map visualization of protein expression data. Each row represents a protein of oncological interest and each column represents a specific compound incubated for 72 h at IC20. Markers features whose levels vary significantly (p < 0.05) between at least two treatment groups are projected on the heat map and used for drug response clustering. Analysis of 150 ng of protein extracted from cell lysate collected from bimetallic Titanocref 2 and Titanofin 4, monometallic cref 1, fin 3 and Auranofin treated PCI-32765 is IC20. Relative expression value of each protein interrogated is plotted in red-green color scale. The red color of the tile indicates high abundance and green indicates low abundance. The color gradients indicate the degree of expression relative to the DMSO control. The data shown result from two independent trials. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
in vivo previously. In this study we include a more complete mechanistic view on Titanocref and on a second related compound (Titanofin) that contains the same gold-phosphane fragment as Auranofin. Comparisons were made with Auranofin as control and with the monometallic gold precursors necessary to prepare these bimetallic titanocene-gold derivatives. While we have used tita-nocene dichloride (TDC) as titanocene monometallic control, in some cases the high amounts (well over 100 mM) needed to observe effects have precluded us to use it for relevant comparisons in most assays. The bimetallic compounds Titanocref and Titanofin strongly inhibit migration, invasion, and angiogenic assembly along with molecular markers associated with these processes such as prom-etastatic IL(s), MMP(s), TNF-a and VEGF. Bimetallic titanocene-gold compounds have thus emerged as potential chemotherapeutics for renal cancer as they hinder three of the most harmful behaviors of the tumor microenvironment that is, local migration of tumor cells, their invasion into the vasculature for metastasis and the formation of de novo blood vessels through which cancerous cells can
metastasize. We evaluated Auranofin and observed great similar-ities in efficacies to the bimetallic compounds in Caki-1 cells as an inhibitor of proliferation and hindering metastatic phenotypes. Auranofin also curbed expression of VEGF and IL(s). Points of dissimilarity are that Auranofin induced cell arrest at G1/G0, while Titanocref and Titanofin induced arrest at G2/M. These results along with those published for Titanocref in vivo warrant further efficacy, pharmacokinetic and histopathological studies and mechanistic exploration in vivo for bimetallic titanocene-gold derivatives. In addition, the relevant results found for Auranofin also warrant further exploration of this FDA approved agent for rheumatoid arthritis agent in renal cancer treatment.
4.1.1. General and instrumentation for the characterization and stability studies of the new compounds
NMR spectra were recorded in a Bruker AV400 (1H NMR at 400 MHz, 13C 1H NMR at 100.6 MHz and 31P 1H NMR at 161.9 MHz. Chemical shifts (d) are given in ppm using CDCl3 as the solvent, unless otherwise stated. 1H and 13C NMR resonances were measured relative to solvent peaks considering tetramethylsi-lane ¼ ppm, and 31P 1H NMR was externally referenced to H3PO4 (85%). Coupling constants J are given in hertz. IR spectra (4000e250 cm 1) were recorded on a Nicolet 6700 Fourier trans-form infrared spectrophotometer on solid state (ATR accessory). Elemental analyses were performed on a Perkin-Elmer 2400 CHNS/ O series II analyzer. Mass (MS) spectra (electrospray ionization, ESI) were performed on a Waters Q-Tof Ultima. Stability studies were performed in a Cary 100 Bio UVevisible spectrophotometer. The pH was measured in an OAKTON pH conductivity meter in 1:99 DMSO/ H2O solutions.
All compounds involving titanium centers were prepared and handled with rigorous exclusion of air and moisture under a ni-trogen atmosphere by using standard nitrogen/vacuum manifold and Schlenk techniques. Solvents were purified by use of a PureSolv purification unit from Innovative Technology, Inc. Titanocene dichloride and [AuCl(PEt3)] were purchased from Aldrich, Aur-anofin was purchased from Strem and 4-mercaptobenzoic acid from TCI America Inc. and used without further purification. [Au(Hmba)(PPh3)] , (1), [(h-C5H5)2TiMe2] , and [(h-C5H5)2TiMe(m-mba)Au(PPh3)] [25,26], (2) Titanocref were prepared as previously reported.
4.1.3. Crystal structure determination