Potent In Vitro and In Vivo Anticancer Activity of New Bipyridine and Bipyrimidine Gold (III) Dithiocarbamate Derivatives

We synthesized eight new bipyridine and bipyrimidine gold (III) dithiocarbamate-containing complexes (C1–C8) and tested them in a panel of human cancer cell lines. We used osteosarcoma (MG-63), lung (A549), prostate (PC3 and DU145), breast (MCF-7), ovarian (A2780 and A2780cis, cisplatin- and doxorubicin-resistant), and cervical (ME-180 and R-ME-180, cisplatin resistant) cancer cell lines. We found that C2, C3, C6, and C7 were more cytotoxic than cisplatin in all cell lines tested and overcame cisplatin and doxorubicin resistance in A2780cis and R-ME-180 cells. In the PC3 prostate cancer cell line, the gold (III) complex C6 ([Au2(BPM)(DMDTC)2]Cl4) induced apoptosis and double-stranded DNA breaks, modified cell cycle phases, increased Reactive Oxigen Species (ROS) generation, and reduced thioredoxin reductase and proteasome activities. It inhibited PC3 cell migration and was more cytotoxic against PC3 cells than normal human adipose-derived stromal cells. In mice bearing PC3 tumor xenografts, C6 reduced tumor growth by more than 70% without causing weight loss. Altogether, our results demonstrate the anticancer activity of these new gold (III) complexes and support the potential of C6 as a new agent for prostate cancer treatment.

dimethyldithiocarbamate hydrate (143.2 mg in 20 mL distilled water) was slowly added, and the reaction mixture was stirred for 1 h. The product appeared as a pale yellow precipitate. The precipitate was collected by filtration, washed with distilled water (3 × 10 mL) and dried under vacuum.

Chemical characterization of gold(III) compounds
Due to the poor solubility of the gold(III) compounds in water, they were dissolved in 99.8% ethanol. The pH of buffers was monitored on a Accumet XL50 pH meter. A GR-2000 electrical balance was used to weigh the various chemicals. Electrochemical measurements for cyclic voltammetry and square wave voltammetry were performed using Autolab instruments (Metrohm; Netherlands). The electrochemical workstation had three electrodes (from CH Instruments): a glassy carbon electrode (GCE) as the working electrode, platinum as the counter electrode, and Ag/AgCl as the reference electrode (in saturated KCl). The GCE was polished as a mirror-like surface with alumina slurry on a synthetic cloth before every electrochemical analysis. Square wave voltammetry and cyclic voltammetry were scanned from 0 to 1.3 V for the various analyses. Elemental analyses of gold(III) compounds (C1-C8) were performed on PerkinElmer Series 11 (CHNS/O), Analyzer 2400.
The solid state FTIR spectra of sodium dimethyldithiocarbamate hydrate, sodium diethyldithiocarbamate trihydrate, and sodium dibenzyldithiocarbamate hydrate (free ligands) and their corresponding gold(III) complexes were recorded on a PerkinElmer FTIR 180 spectrophotometer or NICOLET 6700 FTIR using potassium bromide (KBr) pellets over the range 4000-400 cm −1 . 1 H and 13 C NMR spectra were recorded on a LAMBDA 500 spectrophotometer operating at 500.01 and 125.65 MHz respectively, corresponding to a magnetic field of 11.74 T. Tetramethylsilane was used as an internal standard for 1 H and 13 C. The 13 C NMR spectra were obtained with 1 H broadband decoupling, and the spectral conditions were: 32 k data points, 0.967 s acquisition time, 1.00 s pulse delay and 45 g pulse angle.

Gold (III) compound interactions with lysozyme, tryptophan and guanine
The electrochemical investigation of the interactions between the gold(III) compounds (C1-C8) and lysozyme, tryptophan and guanine was performed using the Autolab instrument described above, with a three-electrode system (CH Instruments): platinum wire counter electrode (CHI115), Ag/AgCl reference electrode (in 3 M KCl, CHI111) and glassy carbon working electrode (CHI112) inserted into a 5.0 ml glass cell. Solutions of 1 mM lysozyme, 5 mM tryptophan and 5 mM guanine were prepared in double distilled water, and the experiment was performed in 0.1 M phosphate buffer at pH 6.8.

Cellular uptake of gold (III) compounds
PC3 cells (1 × 10 6 cells seeded in 100 × 20 culture dishes) were treated for 2 h in duplicate with 3 µM C4, C5, C6 or C7 in complete culture medium. After treatment, monolayers were washed with ice-cold PBS four times, and the cells were detached with trypsin-EDTA and washed three times with ice-cold PBS by centrifugation. The cell pellet was solubilized in 700 µL of HNO3-HCl solution (1:3 molar ratio) for 2 h at 100 °C, and then diluted with 4 mL water. Samples were analyzed for gold on an Agilent 7500 inductively coupled plasma mass spectrometer (ICP-MS). Results were expressed as ng gold/10 6 cells. The experiment was performed a total of two times and the results were expressed as mean and SD.

Growth inhibition curves for C6 in adipose-derived stromal cells
Human adipose-derived stromal cells (ADSCs) were from Lonza (Verviers, Belgium). ADSCs were maintained in MSGM bullet kit (Lonza) and experiments were performed in DMEM (Cambrex Bio Science, Milan, Italy) supplemented with 10 % FBS. To evaluate effects of C6, ADSCs were seeded in 96-well flatbottomed microplates (5.0 × 10 3 cells in 100 µL per well) and incubated for 24 h (to allow cell adhesion) before drug testing. The medium was removed and replaced with fresh medium containing C6 at increasing concentrations (from 0.1 to 1 µM). Cells were incubated at 37 °C for 72 h. Each treatment was performed in triplicate. Cell growth was measured using the MTT assay.