Antitumor activity of gold(III)-dithiocarbamato derivatives on prostate cancer cells and xenografts

Authors


Abstract

Among the nonplatinum antitumor drugs, gold(III)-dithiocarbamato derivatives have recently attracted considerable attention due to their strong in vitro and in vivo antiproliferative activity and reduced renal toxicity. Some of them, namely [AuCl2(DMDT)] (compound 1) and [AuBr2(ESDT)] (compound 2), have shown to be highly active against the androgen-resistant prostate cancer cell lines PC3 and DU145, both inhibiting cell proliferation in a dose-dependent way, and are more active than the reference drug cisplatin (cis-[PtCl2(NH3)2]). In particular, [AuCl2(DMDT)] was proved cytotoxic against cisplatin-resistant R-PC3 cells, with activity levels comparable to those induced on the parent cisplatin-sensitive PC3 cells, ruling out the occurrence of cross-resistance phenomena. Moreover, it causes early cell damage, slightly affecting the cell cycle, thus suggesting a different mechanism of action from clinically established platinum-based drugs. In fact, the investigated gold(III) complex alters mitochondrial functions, promoting mitochondrial membrane permeabilization and Cyt-c release, stimulating ROS generation, and strongly inhibiting the activity of the selenoenzyme TrxR, which is overexpressed in prostate cancer and associated with the onset of drug resistance. In addition, it induces apoptosis, caspase activation, Bcl-2 downregulation and Bax upregulation, reduces the expression of the phosphorylated form of the EGFR, and it inhibits PC3 cell migration. Finally, the treatment of PC3 prostate tumor-bearing nude mice with [AuCl2(DMDT)] significantly inhibited tumor growth in vivo, causing minimal systemic toxicity. Altogether, our results confirm that these gold(III)-dithiocarbamato derivatives have potential for the treatment of prostate cancer.

Prostate cancer is a major cause of cancer morbidity and mortality in men.1 Most prostate tumors initially respond to androgen depletion therapy, but ultimately progress to androgen-independent disease with few therapeutic options,2 so the development of new treatment strategies are currently under investigation.

Cisplatin was one of the first chemotherapeutic agents to exhibit a broad activity toward solid tumors, and still remains among the most widely used anticancer agents. Anyway, all drugs have drawbacks, and cisplatin is no exception. Thus, much effort in designing alternative both platinum and nonplatinum anticancer agents was aimed at making metal-based therapy safer to patients, in particular by lessening or removing unpredictable and severe side-effects, providing oral bioavailability, and overcoming both intrinsic and acquired resistance.3 In this regard, the idea of combining the well-known antitumor properties of some metal ions with the promising chemoprotective function of dithiocarbamates was proved to be a somewhat winning strategy.4–8 Dithiocarbamates are bidentate chelating ligands and, upon coordination to a metal ion, the resulting complexes are expected to be quite stable due to the so-called “chelate effect,” and possible decomposition with subsequent loss of the dithiocarbamato ligand is unlikely to occur. Moreover, as concerns square-planar complexes, the presence of a chelating dithiocarbamate should make the coordination of additional S-donor ligands (e.g., methionine and cysteine residues) trans to the -NCSS moiety less favorable because of the rather strong trans-influencing effect of the dithiocarbamato sulfur atoms, thus potentially preventing further interactions of the metal center with other thiol-containing biomolecules whose inhibition is generally associated with severe side-effects, such as kidney toxicity (nephrotoxicity).

Starting from some platinum(II)-dithiocarbamato complexes, we have been designing a number of other metals (i.e., gold(III), ruthenium(III), copper(II) and zinc(II)) derivatives that have been tested, at least preliminarily, for their in vitro cytotoxic activity toward a panel of human tumor cell lines. Among all, gold(III) complexes turned out to be the most promising in terms of greater in vitro and in vivo antitumor activity and reduced nephrotoxic side-effect compared to cisplatin as extensively demonstrated in animal models.9–11 The capability to overcome both acquired and intrinsic resistance showed by some types of tumors toward cisplatin confirm that they exert their antitumor activity by activating different apoptotic and nonapoptotic pathways compared to the clinically established platinum-based drugs. Although their mechanism of action has not been completely elucidated yet, some potential biological targets have been considered. Previous results indicated that DNA is not the main biological target of these gold complexes, whereas proteasome has been identified as a major in vitro and in vivo target.5 Moreover, these gold(III) compounds are able to activate the mitochondrial death pathways,12 by affecting the selenoenzyme thioredoxin reductase, which has been recently recognized as a potential target for the development of anticancer drugs for prostate cancer.13 Specific studies carried out on some model gold(III)-dithiocarbamato derivatives showed that they are able to exert a strong antiproliferative and proapoptotic effect on a panel of AML cell lines representing different FAB subtypes, and on the Ph+ (K562) cells.14

On account of these extremely positive results, we here report on the in vitro and in vivo antiproliferative activity and proapoptotic action of the gold(III)-dithiocarbamato derivatives [AuCl2(DMDT)] and [AuBr2(ESDT)] against androgen-resistant prostate cancer, to further investigate and elucidate their mechanism of action.

Abbreviations

AML: acute myelogenous leukemia; Bax: Bcl-2-associated x protein; Bcl-2: B-cell lymphoma/leukemia-2; BrdU: bromodeoxyuridine; Cyt-c: cytochrome-c; DMDT: N,N-dimethyldithiocarbamate; EGFR: epithelial growth factor receptor; ELISA: enzyme-linked immunoadsorbent assay; ESDT: ethylsarcosinedithiocarbamate; FAB: French-American-British; FACS: fluorescence-activated cell sorting; FITC: fluorescein isothiocyanate; FLICA: fluorochrome-labeled inhibitors of caspases; IgG: immunoglobulin G; L-NAC (or NAC): N-acetyl-L-cysteine; mAb: monoclonal antibody; Ph+: Philadelphia-positive; PI: propidium iodide; ROS: reactive oxygen species; Trx: thioredoxin; TrxR: thioredoxin reductase

Material and Methods

Chemicals

Methyl sulfoxide (DMSO), N-acetyl-L-cysteine (L-NAC), propidium iodide (PI), trypan blue (Sigma-Aldrich), cisplatin (Pharmacia and Upjohn), Iscove's modified Dulbecco's medium (IMDM), RPMI medium, penicillin, streptomycin, L-glutamine (Cambrex Bio Science) and fetal bovine serum (FBS) (Gibco) were used as received. All other reagents and solvents were of high purity and were used as purchased without any further purification. Gold(III) complexes 1 and 2 were synthesized as previously described.8 Before use, gold(III) complexes and cisplatin were dissolved in DMSO, aliquoted and stored at −80°C. Just before the experiments, calculated amounts of drug solutions (0.2 μM) were then added to IMDM and filter sterilized, to a final organic solvent concentration lower than 0.5% (v/v). All the tested gold(III) complexes were proved by 1H NMR studies to be stable in DMSO over 48 h.8

Cell lines and culture conditions

Human prostate cancer PC3 and DU145 cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cisplatin-resistant cell line R-PC3 was obtained by weekly treatment with 1 μM cisplatin. All cells were cultured at 37°C in 5% CO2 and moisture-enriched atmosphere in RPMI medium supplemented with 10% heat-inactivated FBS, 0.1% (w/v) L-glutamine and antibiotics (0.2 mg mL−1 penicillin and streptomycin).

Cell proliferation assay

2.5 × 103 cells mL−1 were seeded in 96-well flat-bottomed microplates in RPMI medium (100 μL) and incubated at 37°C in a 5% CO2 atmosphere for 24 h to allow cell adhesion, before drug testing. The medium was then removed and replaced with a fresh one containing the compounds to be studied (previously dissolved in DMSO, see above) at increasing concentrations (0.3, 0.6, 1.25, 2.5, 5, 10 μM), at 37°C for 72 h. Alternatively, cells were exposed to increasing concentrations (0.5–10 μM) of the investigated compounds at 37°C for 24 h, then washed and incubated for additional 72 h with fresh medium. Each treatment was performed in triplicate in three independent experiments. Cell proliferation was assayed using the BrdU Cell Proliferation ELISA kit (Roche Diagnostics) according to the manufacturer's instructions.

Evaluation of cell cycle progression and apoptosis

5 × 105 PC3 cells mL−1 were incubated in Petri dishes with complete medium containing 5 μM of either compound 1 or cisplatin at 37°C. After 24, 48 and 72 h, DNA fragmentation and cell cycle were evaluated by PI staining, as described elsewhere.14

Evaluation of APO2.7 expression, Annexin-V binding, changes in mitochondrial membrane potential, and production of mitochondrial ROS

5 × 105 PC3 cells mL−1 were incubated in Petri dishes with RPMI medium supplemented with 10% FBS containing 5 μM of either compound 1 or cisplatin at 37°C for 12 h. APO2.7 expression and Annexin-V binding were detected by flow cytometry, as described elsewhere.14 To evaluate the dissipation of the mitochondrial membrane potential, 200 nM CMXros (Molecular Probes, Eugene) were added to the cell culture for 30 min, then the cells were washed twice and analyzed by flow cytometry, as described elsewhere.15 For Bcl-2 and Bax analysis, fixed and permeabilized PC3 cells were incubated with FITC-conjugated mouse anti-human Bcl-2 (clone 124) (DAKO Citomation), or with 1 μg mL−1 of mouse anti-Bax generated from Bax-alpha (Becton-Dickinson-Pharmingen), followed by PE-conjugated goat anti-mouse IgG (Becton-Dickinson).14 Cyt-c release was assessed, with minor modifications, as explained elsewhere,16 Briefly, PC3 cells were permeabilized with 100 μg mL−1 digitonin (Sigma) and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) solution for 20 min at room temperature. After washing twice with PBS, cells were incubated with labeling medium (2% FBS, 0.2% sodium azide, 0.5% TritonX-100 in PBS) for 15 min, then with 1 μg mL−1 of the mouse anti-Cytochrome-c antibody (Becton-Dickinson-Pharmingen), followed by PE-conjugated goat anti-mouse IgG (Becton-Dickinson). For intracellular ROS evaluation, cells were incubated with 5 μM of MitoSox reagent working solution (Molecular Probes, Invitrogen) for 15 min at 37°C. Red fluorescence was immediately analyzed by flow cytometry. Viable antibody-labeled cells were identified according to their forward and right-angle scattering, electronically gated and analyzed on a FACScalibur flow cytometer (Becton-Dickinson), using CellQuest software (Becton-Dickinson). In another series of experiments, 25 × 103 PC3 cells mL−1 were seeded in 24-well plates and exposed to compound 1 (5 μM) in the presence or absence of the antioxidant L-NAC (5 nM) at 37°C for 24 h. Cells were then washed and medium replaced with fresh one, and viable cells were counted after 72 h using the trypan blue dye exclusion assay. Caspase activity was evaluated using the fluorochrome inhibitors of caspases (FLICA) CaspaTagTM caspase-3/7 (FAM-DEVD-FMK), caspase-9 (FAM-LETD) (Chemicon International, Milan, Italy). PC3 cells were treated with compound 1 (5 μM) at 37°C for 3, 6 and 12 h, then harvested, washed and resuspended in warmed complete medium supplemented with FLICA for 1 h at 37°C in a 5% CO2 atmosphere, and then analyzed immediately by flow cytometry.

TrxR enzyme activity assay

TrxR activity in cultured cells was assessed using the Thioredoxin Reductase Assay Kit (Sigma-Aldrich), according to the manufacturer's instructions. 2.5 × 105 cells mL−1 were treated with 5 μM of either compound 1 or cisplatin and, after 12 h, cells were lysed with lysis buffer (50 mM Tris-HCl, 0.1% Triton-X 100, 0.9% NaCl, pH 7.6) and frozen at −20°C for 30 min. Total protein content was analyzed with the protein assay dye reagent (Bio-Rad Laboratories). The cell lysate was then incubated in 100 mM of potassium phosphate with 10 mM EDTA and 0.24 mM NADPH (nicotinamide adenine dinucleotide phosphate) with and without a TrxR inhibitor. The reaction was started by adding DNTB (dinitrothiocyanobenzene) and monitored spectrophotometrically at 412 nm.

Cell migration assay

Cell migration was assessed using the scratch assay, as described elsewhere.17 Cells were grown to confluence in tissue culture dishes, then either compound 1 (2 μM) or medium were added. After 12 h, cells were washed twice with PBS and scraped up using a sterile pipette tip, then washed again and cultured in IMDM with 2% FCS for further 48 h. The migration rate was assessed by measuring the distance between the edges of the wound after 12, 24 and 48 h.

Evaluation of EGFR phosphorylation

After fixing in 2% paraformaldehyde (for 15 min at 4°C), the surface expression of the EGF receptor was analyzed by flow cytometry using the anti-EGFR mAb 528 (Santa Cruz Biotechnology). To detect tyrosine phosphorylated EGFR (pEGFR), cells were fixed in 2% paraformaldehyde in PBS for 15 min at 4°C, then permeabilized with 1% Tween 20 for 30 min at 4°C, and, finally, incubated with mouse anti-pEGFR (PY1197) (DAKO Citomation). The PE-conjugated goat anti-mouse IgG (Becton-Dickinson) was used as a secondary antibody.

Human prostate tumor xenograft experiments

Six-week-old female athymic nu/nu (nude) mice were purchased from Charles River. 3 × 106 PC3 cells suspended in 0.1 mL of matrigel were inoculated subcutaneously into the right flank of each mouse. When tumors reached sizes of about 28 mm3, mice were randomly divided into two groups of eight mice each, and treated every other day with s.c injection of either drug-free medium or containing 1 mg kg−1 of compound 1. Tumor size was measured three times a week using a caliper, and volumes were calculated according to a standard formula: (width2 × length × 3.14)/6. Mouse weights were monitored every other day. Mice were sacrificed after 19 days of treatment when control tumors had reached about 630 mm3. Mice organs were then excised and fixed in formalin for tissue toxicity analyses.

Statistical analyses

All values are given as means ± SD of not less than three measurements (unless otherwise stated). Statistical comparisons were drawn using Student's t-test. Differences were considered significant where p < 0.05.

Results

Effects of gold compounds on prostate cancer cells proliferation

We first evaluated the in vitro cytotoxic effect of the gold(III)-dithiocarbamato complexes [AuCl2(DMDT)] (1) and [AuBr2(ESDT)] (2) on the androgen-resistant prostate cancer PC3 and DU145 cell lines. For comparison purposes, cisplatin (3) was also evaluated under the same experimental conditions (Fig. 1). As reported in Figure 2a, treatment of PC3 and DU145 cells with either 1 or 2 inhibited cell proliferation in a dose-dependent way, and both showed higher cytotoxicity against prostate cancer cells than cisplatin, with statistically different IC50 values (p < 0.05). Among all, compound 1 was proved more effective than compound 2 with IC50 values about 4 and 10 times lower than cisplatin on PC3 and DU145 cells, respectively. Since PC3 cells are more invasive and resistant to chemotherapy than DU145 cells, and 1 resulted to be the best performing complex, it was selected for further in-depth investigations on PC3 cells. In this regard, cell growth recovery experiments were carried out, in which, after incubation for 24 h with increasing concentrations of 1, treated PC3 cells were washed and the medium replaced with fresh drugless medium, to establish the capability of cancer cells to start proliferating again upon removal of the gold compound. Remarkably, the effects induced by 1 were not reversed, suggesting that it exerts cytotoxic rather than cytostatic effects (Fig. 2b). Additional in vitro experiments, were performed to evaluate the cytotoxic activity on the cisplatin-resistant cell line R-PC3 (Fig. 2c). Again, compound 1 inhibited cells proliferation with IC50 value comparable to that previously recorded on the parent cisplatin-sensitive cell line.

Figure 1.

Chemical drawing of the investigated compounds: [AuCl2(DMDT)] (1, DMDT = N,N-dimethyldithiocarbamate), [AuBr2(ESDT)] (2, ESDT = ethylsarcosinedithiocarbamate), cis-[PtCl2(NH3)2] (cisplatin, 3).

Figure 2.

Representative survival curves of the tested prostate cancer cell lines treated with either the gold(III)-dithiocarbamato derivatives or cisplatin at increasing concentrations. (a) Inhibition of proliferation in PC3 and DU145 cells after 72 h exposure at 37°C to 1 (□), 2 (○), or cisplatin (▪), at increasing concentrations (0.3–2.5 μM). (b) Inhibition of proliferation in PC3cells first incubated for 24 h at 37°C with increasing concentrations of 1 (□) or cisplatin (▪) (0.5–10 μM), then washed and incubated for further 72 h with fresh medium at 37°C. (c) Inhibition of proliferation in cisplatin-sensitive (PC3) and -resistant (R-PC3) cells after 72 h exposure at 37°C to 1 (□: PC3; ○: R-PC3) or cisplatin (▪: PC3; •: R-PC3) at increasing concentrations (0.5–10 μM). In all assays cell proliferation was evaluated using the BrdU Cell Proliferation ELISA kit. Results represent the mean ± standard deviation (SD) of three replicate wells from three independent experiments. Negative exponential dose-response survival curves were calculated by linear regression and IC50 values (i.e., the half maximal inhibitory concentration representing the concentration of a substance required for 50% inhibition in vitro) calculated. The differences emerging between the treatment with either cisplatin or the investigated gold(III)-dithiocarbamato derivatives are statistically significant (p < 0.05).

Compound 1 induces apoptosis in PC3 cells

The capability to induce cell cycle modifications and apoptosis was subsequently tested by incubating PC3 cells with a single cytotoxic dose (5 μM) of either the investigated gold(III)-dithiocarbamato complex or cisplatin for 12 h. Treatment with complex 1 resulted in a substantial phosphatidylserine exposure together with a higher percentage of cells permeable to PI staining whereas cisplatin had no effect under the same experimental conditions (Fig. 3a).18 Cell cycle progression and DNA fragmentation were measured by PI staining and flow cytometric analyses. After incubation for 24 h, complex 1 caused a blockade at G1 phase and a marked reduction in the S phase of the cell cycle (Fig. 3b). DNA fragmentation was apparent after 48-72 h. On the other hand, after 72 h, an equivalent amount of cisplatin caused a blockade in the G2M phase of the cell cycle, with no significant DNA fragmentation (Fig. 3b).

Figure 3.

Induction of apoptosis and DNA fragmentation by compound 1. (a) FACS analysis of PC3 cells in drug-free medium and after 12 h incubation at 37°C with either compound 1 or cisplatin (5 μM), and double stained with Annexin-V-FITC and PI, and (b) subsequent detection of DNA fragmentation and cell cycle progression after 24, 48 and 72 h. The percentage of cells in the G0/G1 phase, S phase, and G2M phase are reported, as well as the percentage of sub-G1 cells. In all assays, experiments were carried out in triplicate.

Compound 1 affects mitochondrial functions and activates the caspase pathway in PC3 cells

In our previous studies, we have reported that the antiproliferative activity of gold(III)-dithiocarbamato derivatives may arise from several different mechanisms, including the alteration of mitochondrial membrane potential and permeability.12 Compared to cisplatin, treatment of PC3 cells with compound 1 led to a substantial increase of the 76A antigen (APO2.7), a decrease of mitochondrial membrane potential, and to Cyt-c release from the mitochondria (Fig. 4a). Moreover, activation of caspase 9 was observed after just 3 h, whereas caspase 3 was activated after 6 h of incubation (Fig. 4b). Finally, since Bcl-2 and Bax play a crucial role in regulating the intrinsic apoptotic pathway, the effects on their expression by complex 1 was investigated. Remarkably, it induced an increase in the pro-apoptotic molecule Bax and a significant decrease in the anti-apoptotic molecule Bcl-2 (Fig. 4c). On the contrary, under the same experimental conditions, cisplatin only slightly reduced Bcl-2 expression and induced negligible effects on Bax (Fig. 4c).

Figure 4.

Mitochondrial-mediated apoptosis, Bax and Bcl-2 modulation induced by compound 1. (a) FACS analysis of mitochondrial membrane permeabilization after treating PC3 cells at 37°C with either compound 1 or cisplatin (5 μM). After 12 h, APO2.7 expression, mitochondrial membrane permeabilization (CMXros) and Cyt-c release were assessed by flow cytometry. (b) FACS analysis of caspases after incubating PC3 cells at 37°C with either compound 1 or cisplatin (5 μM) for 3, 6 and 12 h; cells were then harvested, washed and resuspended in warmed complete medium supplemented with FLICA for 1 h at 37°C under 5% CO2, then washed again and analyzed by flow cytometry. (c) FACS analysis of Bcl-2 and Bax after treating PC3 cells at 37°C with either compound 1 or cisplatin (5 μM). After 12 h, Bax and Bcl-2 expression were assessed by flow cytometry. In all assays, experiments were carried out in triplicate. Dotted lines indicate background fluorescence of cells, as determined by isotype-matched immunoglobulins or autofluorescence. X- and Y-axes indicate the logarithms of the relative intensity of fluorescence and relative cell number, respectively.

Compound 1 induces ROS accumulation in mitochondria and inhibits the activity of the enzyme TrxR in PC3 cells

In our previous studies carried out on human cervical carcinoma HeLa cells, gold(III)-dithiocarbamato compounds were proved to inhibit the activity of the enzyme thioredoxin reductase and to induce ROS production, suggesting that deregulation of the thioredoxin reductase/thioredoxin redox system is a major mechanism involved in their anticancer activity.12 Therefore, we broadened our investigations to assess whether or not the proposed mechanism might be generalized and extended to prostate cancer cells. With reference to Figure 5a, compound 1 was proved to induce on PC3 cells a dramatic increase in mitochondrial peroxides, as evaluated by MitoSox assay, even after a short incubation time (i.e., 12 h). To establish a possible connection between ROS overproduction and decreased cell viability further experiments were performed, including PC3 cells preincubation with both the ROS scavenger L-NAC and complex 1 itself. As clearly shown in Figure 5b, pretreatment with L-NAC neutralizes the proapoptotic effects of 1, suggesting that its anticancer activity is strictly related to the production of ROS.

Figure 5.

ROS accumulation and inhibition of TrxR activity induced by complex 1. (a) Evaluation of ROS production in PC3 cells treated with either compound 1 or cisplatin (5 μM) at 37°C for 12 h by flow cytometry after incubation for 10 min at 37°C with MitoSox reagent. (b) Effect of the cotreatment with the antioxidant L-NAC: PC3 cells were cultured at different combinations of compound 1 and L-NAC (none: −/−; only L-NAC at 5 μM: ±; only complex 1 at 5 μM: ±; both L-NAC and complex 1 at 5 μM: +/+). After 24 h incubation, the medium was replaced with fresh one and viable cells were counted after 72 additional h. (c) Inhibition of TrxR in PC3 cells treated at 37°C with either compound 1 or cisplatin (5 μM) for 12 h, evaluated by means of the Thioredoxin Reductase Assay Kit.

TrxR is a selenoenzyme overexpressed in prostate cancer and it is involved in ROS detoxification.19 A short (12 h) incubation of PC3 cells with complex 1 at 5 μM markedly (about 90%) inhibited TrxR enzyme activity (Fig. 5c), whereas, glutathione reductase was unaffected by the same treatment (data not shown). Intriguingly, under the same experimental conditions, cisplatin was much less active in either inducing ROS accumulation or inhibiting TrxR activity.

Compound 1 inhibits PC3 cell migration and reduces EGFR expression

According to literature reports, EGFR phosphorylation is believed to affect the migration and proliferation of prostate cancer cells.20, 21 Treatment of PC3 cells with increasing amounts of complex 1, resulted in a dose-dependent decrease in both EGFR and its phosphorylated form (p-EGFR), as assessed by flow cytometry (Fig. 6a), whereas incubation with cisplatin under the same experimental conditions affected neither EGFR expression nor its phosphorylation. A representative experiment supporting this result, obtained using a high concentration of 1 (5 μM), is shown in Figure 6b, in which the effect on cell migration was measured by means of the scratch test.17 PC3 cells were cultured in the presence of a lower amount of compound 1, at a low serum concentration. After 48 h of incubation, the cell migration rate was significantly reduced (Fig. 6b, right panel), and cells failed to form a confluent monolayer like the one formed by untreated PC3 cells. The migration rate is clearly reported in the histogram graph of Figure 6b, and is calculated as the percentage of the surface area covered by the cells. Again, treatment with cisplatin under the same experimental conditions did not affect PC3 cell migration (data not shown).

Figure 6.

Compound 1-induced inhibition of EGFR expression and cell migration. (a) FACS analysis of EGFR and the corresponding phosphorylated form p-EGFR after treating PC3 cells at 37°C with increasing amounts of compound 1 (1–5 μM). EGFR (•) and p-EGFR (○) were analyzed using the anti-EGFR mAb 528 and the anti-p-EGFR mAb PY1197, respectively. Data are expressed as percentage of fluorescence intensity as compared with controls. Fluorescence histograms are also provided. (b) Scratch test analysis of PC3 cells migration after incubating PC3 cells either with or without complex 1 at 2 μM at 37°C for 12 h. Cells were then washed and scraped up three times in the confluent monolayer, and eventually cultured in low serum medium for additional 48 h. The migration rate is represented by the distance between the edges of the wound (defined by the dotted lines), indicating the surface area occupied by the migrating cells after 12, 24 and 48 h. Data in the histogram graph (▪: medium; ▪: compound 1) are given as mean ± SD.

Anticancer activity of compound 1 in prostate cancer xenografts

As a natural continuation of our studies, the in vivo antitumor activity of compound 1 was evaluated. PC3 cells were implanted into the right flank of 6-week-old female athymic nude mice and, once tumors reached a volume of about 28 mm3, mice were treated every other day with s.c. injection of either drug-free medium or containing 1 mg kg−1 of the investigated gold(III) compound. A significant tumor growth inhibition induced by 1 was recorded since after seven days of treatment. After nineteen days, tumors grew to an average 660 ± 71 mm3 volume in control mice, whereas treatment with complex 1 had an overall 85% inhibitory effect, since the treated tumors reached an average size of 100 ± 12 mm3 (p < 0.05; Fig. 7). Mouse body weights were monitored every other day and no significant changes were detected upon treatment. Remarkably, no histologically detectable cytotoxicity involving animals' heart, lung, spleen or liver was detected (data not shown).

Figure 7.

In vivo anticancer activity of compound 1 (xenografts). Tumor volume was measured in female athymic nude mice after subcutaneous (s.c.) injection of either drug-free medium or containing 1 mg kg−1 of compound 1 every other day. Tumor volumes were measured three times a week using a caliper. Points (♦: medium; ⋄: compound 1) represent the mean ± SD (bars) of eight animals in each group.

Discussion

Prostate cancer is the most common malignancy and the second cause of cancer-related death in males. Despite the benefits of androgen ablation in the early stages of the disease, many tumors recur in an androgen-independent form, with a dramatic increase in the mortality rates.1 Therefore, the design of more active and less toxic antitumor agents is a major goal to develop new targeted therapeutic strategies.

In this regard, gold complexes have recently gained increasing attention due to their strong tumor cell growth inhibiting effects generally achieved by exploiting pharmacodynamic and pharmacokinetic properties and mechanisms of action different from the other clinically-established metal-based drugs.22–26 Following the extremely promising results concerning the use of some gold(III)-dithiocarbamato complexes as antitumor agents,9 in this study, we investigated the biological activity and molecular mechanisms of two of them, namely [AuCl2(DMDT)] (1) and [AuBr2(ESDT)] (2), toward prostate cancer cell lines. Our results show that, in particular, compound 1 induces apoptosis in androgen-resistant cell lines, inhibits TrxR enzyme activity, induces ROS production, reduces the expression and functional activity of EGFR; and inhibits cell migration capability. The investigated compounds strongly inhibit the proliferation of androgen-resistant prostate cancer cells and are significantly more active than the reference drug cisplatin. Compound 1 was proved cytotoxic in vitro also against the cisplatin-resistant cell line R-PC3, with activity levels comparable to those detected for the parent cisplatin-sensitive cell line PC3, ruling out the occurrence of any cross-resistance phenomena. Finally, it actively inhibits prostate cancer tumor growth in vivo, showing outstanding antitumor activity in a xenograft model.

According to the above reported experimental results, complex 1 exerts a powerful cytotoxic effect on PC3 cells by inducing apoptosis, as assessed by Annexin-V and APO2.7 staining assays, which confirmed a strong induction of APO2.7, that is a mitochondrial membrane protein exposed on the cell surface of cells undergoing apoptosis. Moreover, it induces mitochondrial membrane depolarization, cytochrome-c release and caspase 9 activation, indicating that its activity is exerted through the mitochondrial intrinsic apoptotic pathway. In fact, compound 1 significantly reduces the expression of the pro-survival protein Bcl-2 and, at the same time, increases the expression of the pro-apoptotic Bax protein, two regulators playing a major role in the mitochondrial apoptotic pathway.27, 28 The tested gold(III) complex causes early cell damage and slightly affects the cell cycle, thus suggesting a different mechanism of action compared to the clinically established platinum-based anticancer drugs.

The thioredoxin/thioredoxin reductase (Trx/TrxR) system is a major antioxidant system involved in maintaining the intracellular redox state and inhibiting the formation of pro-apoptotic molecules induced by ROS overproduction.29, 30 Higher levels of Trx and TrxR, compared to those observed in the corresponding counter-healthy cells from the same patient, are found in many different types of tumors and transformed cell lines,30, 31 including prostate cancer.19 Increased levels of Trx have been also associated with resistance to several anticancer drugs still in clinical use for the treatment of prostate cancer, including cisplatin32 and docetaxel.33 Thus, both the key role in regulating apoptosis and the high levels of expression detected in different cancer histotypes have prompted interest in the development of drugs that, either on their own or in combination with other chemotherapeutic agents, might target the Trx system.34 A number of metal-based compounds, including gold(III)-dithiocarbamato derivatives,12 were proved potent inhibitors of purified TrxR enzyme in vitro, but only a few studies have reported on its inhibition in cancer cell lines.34, 35 In this regard, compound 1 is an important representative case since treatment with the object gold(III) complex strongly inhibits TrxR enzyme activity in PC3 cells and induces the production of large amounts of ROS.

Androgen-independent prostate cancer is characterized by a greater invasive potential than its hormone-responsive counter-part. A successful therapeutic approach is aimed at suppressing not only cell proliferation, but also the metastatic potential of any cells escaping from the primary tumor site to colonize the bone marrow.36 As far as we are aware of, our study shows for the first time that an antitumor agent, that is, compound 1, is able to both inhibit PC3 cell migration and reduce the expression of the surface-expressed and phosphorylated forms of EGFR, whose ligands are paracrine and autocrine growth factors for prostate cancer and are involved in prostate cancer metastasis.37–39

Gold compounds whose anticancer activity has been already reported in the literature have often shown promising in vitro cytotoxicity that, unfortunately, was not confirmed by in vivo studies afterwards.25 On the contrary, the antitumor activity of compound 1in vivo is fully consistent with in vitro results. In fact, administration of 1 mg kg−1 every other day of 1 caused an overall 85% reduction of prostate tumor xenografts in nude mice after a 19-day treatment (compared to control untreated mice). Remarkably, chemotherapy was well tolerated by treated mice which suffered from minimal systemic toxicity and histology showed no detectable damage to main animals' organs. It is worth noting that this result is extremely important since the antitumor activity in vivo has been evaluated against a human prostate tumor (xenograft) implanted on immunodepressed nude mice and not just towards a murine tumor, thus providing a more reliable outcome.

All together, the results here reported provide both new insights into the possible mechanism of action of gold(III)-dithiocarbamato derivatives as potential anticancer agents and a solid starting point for their recognition as suitable candidates for further pharmacological testing, in particular for the treatment of prostate cancer either on their own or in combination with other chemotherapeutic drugs.

Acknowledgements

The authors thank EU (Marie Curie European Re-Integration Grant for L.R.) for financial support.

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