Antiangiogenic and Anticancer Properties of Bifunctional Ruthenium(II

Article pubs.acs.org/molecularpharmaceutics

Antiangiogenic and Anticancer Properties of Bifunctional Ruthenium(II)−p‑Cymene Complexes: Influence of Pendant Perfluorous Chains Patrycja Nowak-Sliwinska,*,†,‡ Catherine M. Clavel,‡ Emilia Păunescu,‡ Marije T. te Winkel,†,‡ Arjan W. Griffioen,† and Paul J. Dyson*,‡ †

Angiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland

‡

ABSTRACT: Two bifunctional ruthenium(II)−p-cymene complexes with perfluorinated side chains, attached via pyridine ligands, have been evaluated in a series of in vitro and in vivo assays. Their effects on human endothelial (ECRF24 and HUVEC) cells, noncancerous human embryonic kidney (HEK-293) cells, and various human tumor cells were investigated. The complex with the shorter chain, 1, inhibits the proliferation of the tumor cell lines and ECRF24, whereas 2 selectively inhibits ECRF24 and HUVEC proliferation. Neither inhibits the migration of ECRF24 cells whereas both compounds inhibit sprout formation in HUVEC cells. Using three preclinical models, i.e., vasculature formation in the chorioallantoic membrane (CAM) of the chicken embryo, human A2780 ovarian carcinoma tumors xenografted on the CAM, and human LS174T colorectal adenocarcinoma tumors grown in athymic mice, the angiostatic and anticancer activities of these two complexes were studied. Overall, 1 inhibited tumor growth predominantly through an anticancer effect whereas 2 inhibited tumor growth predominately via an antiangiogenic mechanism. KEYWORDS: antiangiogenesis, bioorganometallic chemistry, CAM model, colorectal adenocarcinoma, ovarian carcinoma, ruthenium(II)−arene complexes, fluorine chemistry

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INTRODUCTION

tends to be low and part of their activity may be attributed to interactions with extracellular proteins.20,21 The development of new antiangiogenic drugs, which actively target the tumor microenvironment, represents an attractive alternative to cytotoxic agents for cancer therapy, and such compounds have been applied as monotherapies22,23 or in combination with other treatment strategies.23−26 The identification of the antiangiogenic properties of some ruthenium complexes12,17,27 encouraged us to pursue more detailed analysis of other compounds that present relevant antiproliferative activities. Recently, we reported the synthesis, characterization, and in vitro and in vivo evaluation of the antiproliferative properties of a series of ruthenium(II)−arene complexes designed to have thermoactive properties by virtue of a pendant perfluorinated chain.28,29 These derivatives are structurally related to the RAPTA class of compounds presenting the same “piano-stool” motif, with the PTA ligand being replaced by a pyridine ligand modified with a perfluorinated chain (Figure 1). We have previously shown that 1 (Figure 1), when evaluated at 37 °C,

Metal-based anticancer drugs used in the clinic are based on platinum, and, while there is continued interest in the development of new platinum-based drugs,1−3 there is growing interest in the antitumor properties of ruthenium-based compounds.4−6 The latter research area has been stimulated by the development of two ruthenium(III) complexes, namely, KP1019 (indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) 7 and NAMI-A (imidazolium trans[tetrachloro(dimethyl sulfoxide)(1H-imidazole)ruthenate(III)]).8,9 Both compounds are currently being developed in clinical trials.10,11 In addition, a growing number of ruthenium(II) complexes show promising biological properties.12−14 For example, organometallic ruthenium(II) complexes with the general formula [Ru(η6-arene)Cl2(PTA)] (arene = toluene and p-cymene, PTA = 1,3,5-triaza-7-phosphaadamantane), named RAPTA compounds, exhibit antimetastatic15,16 and antiangiogenic17 properties, as well as some activity on primary human tumor growth.18 Although the mechanism of action of these compounds remains to be fully elucidated, recent studies indicate that RAPTA compounds bind preferably to proteins.19,20 Such protein targeting of the RAPTA family is markedly different from the mechanism of action of platinum compounds. Moreover, cellular uptake of RAPTA compounds © XXXX American Chemical Society

Received: May 27, 2015 Revised: June 28, 2015 Accepted: July 9, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00417 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Proliferation Assay. Endothelial cells (5 × 103 cells/well) were seeded in gelatin-coated 96-well cell culture plates as described previously.50 Briefly, 24 h after seeding, culture medium containing or without the compounds was added and cells were grown for a further 72 h. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Migration (Scratch) Assay. The migration capability of endothelial cells was determined using the wound assay.51 In brief, ECRF24 were grown to confluence in gelatin-coated wells and “scratch wounds” (with an approximate width of 350 μm) were made in the monolayer by removing cells with a sterile scratch tool (Peira Scientific Instruments, Beerse, Belgium). Cells were washed with PBS, and the medium was replaced by fresh medium containing 10 ng/mL bFGF (Tebu-Bio) and incubated with or without tyrosine kinase inhibitors. Automated image acquisition was performed using a Leica DMI3000B microscope in combination with UGR grabbing software (DCI Laboratories, Peira Scientific Instruments). Bright field images taken at 5× magnification were processed for computational analysis of scratch sizes using UGR Scratch Assay 6.2 software (DCI Laboratories, Peira Scientific Instruments). Sprouting Assay. HUVEC spheroids were created using the hanging drop method.17 HUVEC were suspended in medium containing 20% methocel (Sigma-Aldrich, St. Louis, USA) at a concentration of 4 × 104 cells/mL, and 25 μL drops (containing 1000 cells) were aliquoted on the inside of the lid of a Petri dish. The lid was subsequently inverted to create the hanging drops and placed over the PBS containing Petri dish. After 24 h the spheroids were harvested and embedded in a collagen gel (2 mg/mL) at 20 spheroids per well of a 96-well plate. After solidification of the gel, medium containing the test compounds was added and cells were allowed to sprout into the collagen for 16 h. Sprouting spheroids (8 to 10 per condition) were pictured directly under a microscope (Leica Microsystems GmbH, Wetzlar, Germany) under ×10 magnification. Quantification of sprouting was performed using a semiautomatic macro based on the ImageJ platform.17 Flow Cytometry. Apoptosis was estimated by flow cytometric determination of subdiploid cells after DNA extraction and subsequent staining with PI as described previously.13 Briefly, cells were harvested and subsequently fixed in 70% ethanol at −20 °C. After 2 h the cells were resuspended in DNA extraction buffer (45 mM Na2HPO4, 2.5 mM citric acid and 1% Triton X-100, pH 7.4) for 20 min at 37 °C. PI was added to a final concentration of 20 μg/mL, and red log-scale fluorescence was analyzed on a FACSCalibur (BD Biosciences, NJ, USA). Apoptosis was quantified as the percentage of subdiploid cells. Cellular Ruthenium Uptake. Ruthenium cellular uptake was performed as described previously.28 Briefly, ECRF24 cells were grown to ca. 70% confluency and incubated with 1 or 2 (50 μM) for 5 h. At the end of the incubation period, cells were rinsed twice with 2.0 mL of PBS, detached by adding 0.5 mL of enzyme free cell dissociation solution (Millipore, Switzerland), and collected by centrifugation. The samples were digested in ICP-MS-grade concentrated hydrochloric acid (Sigma-Aldrich) for 3 h at room temperature and filled to a total volume of 8.0 mL with ultrapure water. Indium was added as an internal standard at a concentration of 0.5 ppb. Determinations of total metal contents were achieved on an Elan DRC II ICP-MS instrument (PerkinElmer, Switzerland) equipped with a

Figure 1. Structures of 1 and 2 and RAPTA-C, [Ru(η6-p-cymene)Cl2(PTA)] (PTA = 1,3,5-triaza-7-phosphaadamantane), used herein as a reference compound.

i.e., in the absence of a hyperthermia signal, displays a remarkable cancer cell selectivity.30 Compound 1 was also found to inhibit A2780 tumor growth in a CAM preclinical model.28 Moreover, 2, which displays little activity at 37 °C toward all tumor cell types tested, showed thermoactive properties in vitro, i.e., inhibiting cell viability when combined with mild hyperthermia (41.5 °C).30 Based on these observations further studies of the biological properties of 1 and 2 were undertaken, and, herein, we show that, at nontoxic doses, 1 has direct anticancer properties whereas 2 exerts predominately an antiangiogenic effect that leads to effective tumor growth inhibition.

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EXPERIMENTAL SECTION Compounds. RAPTA-C, 1, and 2 were prepared as previously described.30,47 The compounds were dissolved in DMSO immediately prior each experiment. Log P Determination. Stock solutions of 1 and 2 was prepared in octanol saturated with PBS. A 1:1 ratio of octanol and PBS was used for the shake-flask method while introducing the stock solution volume. The biphasic solution was shaken for 24 h and centrifuged to separate the 2 phases. A colorimetric calibration curve was prepared for each complex in octanol saturated with PBS. Absorbance spectra were recorded in a 1 cm quartz cuvette in a SpectraMax M5e reader (Molecular Devices). Fluorescence Measurements. HSA and apotransferrin were purchased from Sigma and used without further purification. The stock solutions of proteins (0.4 mM) were prepared in PBS, stored at 0−4 °C in the dark for a week, and diluted to 2.4 μM when used. Stock solutions of RAPTA-C, 1, or 2 (1 mM) were prepared in DMSO. Fluorescence measurements were performed using black 96-well plates on a SpectraMax M5e (Molecular Devices). Fluorescence quenching was measured after each successive addition of the complex stock solution in each measured well containing the protein at 280 nm for the excitation wavelength and the emission spectrum recorded in the range of 300−450 nm. Cell Culture. The immortalized endothelial ECRF24 cell line48 was maintained in DMEM/RPMI-1640 (1:1) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS, 1% penicillin, and streptomycin (BioConcept Ltd., Allschwil, Switzerland). Human umbilical vein endothelial cells (HUVEC) were isolated from normal human umbilical cords by perfusion with 0.125% trypsin/EDTA and cultured in RPMI culture medium supplemented as above and with 10% human serum.49 HUVEC and ECRF24 cells were cultured as previously described17 in 0.2% gelatin-coated tissue flasks. Human colorectal adenocarcinoma LS174T cells (Cell Line Service GmbH, Eppelheim, Germany) were cultured in DMEM medium supplemented with 10% fetal calf serum and 1% antibiotics (as above). All cells were cultured in a highly humidified atmosphere with 5% CO2 at 37 °C. B


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analyses were done using the Student’s t test, or ANOVA. *pvalues 500b 25.0 ± 2.0b >500b b 37.9 ± 1.7 >500b b 36.4 ± 1.9 >500b 43.1 ± 0.7b >500b 41.4 ± 2.6 >500 >200 >200 >500b >500b 53.4 ± 1.0 55.6 ± 4.2 >100 43.0 ± 5.0 Intracellular Uptake (pmol/106 cells) 148 694 Log P −0.03 0.07

RAPTA-C 230c 270c >50031 >500 >500d 170d >500e >500c 250e 300 ± 8.0e 107

a

Errors represent the standard error of the mean. Intracellular uptake in ECRF24 cells (pM in 106 cells, 5 h incubation). bValues taken from ref 30. cValues taken from ref 32. dValues taken from ref 33. eValues taken from ref 34.

previously reported.30 Here, we extended the study of 1 and 2 to SW480 (human colorectal adenocarcinoma) and LS174T (colorectal adenocarcinoma) cells, and also investigated their antiproliferative activity against immortalized human endothelial cells (ECRF24), and freshly isolated human umbilical cord endothelial cells (HUVEC) in order to assess potential antiangiogenic properties, see Table 1. The IC50 values of 1 range from 25 μM in A2780cisR cells to 44.2 μM in A2780 cells, and it is slightly less cytotoxic to ECRF24 cells (IC50 53.4 μM) and inactive on HEK-293 and HUVEC cells. Complex 2 exhibits a markedly different behavior, being moderately active only on endothelial cells, with IC50 values of 55.6 μM and 43 μM for ECRF24 and HUVEC cells, respectively. In addition, intracellular ruthenium uptake of 1 and 2 was determined in ECRF24 cells (Table 1), since 1 and 2 are equally cytotoxic to this cell line (53.4 and 55.6 μM, respectively). Low internalization of ruthenium was determined for 1 (148 pM), slightly higher than that of RAPTA-C (107 pM), whereas an approximately 5-fold higher C


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Figure 2. Migration inhibition of ECRF24 (A) and MDA-MB-231 (B) cells after exposure to 1 or 2. Wound closure in ECRF24 and MDA-MB-231 cultures after incubation of 1 (6−25 μM) and 2 (6−25 μM). DMSO 0.1% in DMEM culture medium and sunitinib (sut, 20 μM) were used as controls. **p < 0.01 (for sunitinib). Error bars represent standard error of the mean. n = 6.

Figure 3. Effect of 1 and 2 on HUVEC sprout formation. (A) Representative image of a sprouting spheroid after incubation with 2 (50 μM) for 24 h. Quantification of sprout number (B) and total sprout length/spheroid (C) following exposure to 1 and 2 expressed as a percentage of the control. Errors represent the standard error of the mean, **p < 0.01.

level of ruthenium internalization was determined for the ECRF24 cells treated with 2 (694 pM), i.e., the compound with the longer hydrophobic chain. For a comparison, uptake of 1 (pmol/106 cells) in A2780 and HEK293 cells after a 24 h incubation (at a dose of 250 μM) is almost an order of magnitude higher.14 In a 2D migration assay on ECRF24 or MDA-MB-231 cells, 1 and 2 did not inhibit the mobility of cells up to concentrations of 25 μM (Figures 2A and 2B). The activity of 1 and 2 on endothelial cell sprout formation was assessed using a bFGF-driven sprouting assay with HUVEC.17 Interestingly, incubation of HUVEC spheroids with 1 or 2 at 50 μM resulted in significant inhibition of sprout formation (p < 0.01, Figure 3). A representative image of a sprouting HUVEC spheroid is provided in Figure 3A, together with image-based quantification of sprouting for the control cells and cells treated with 1 and 2. An inhibition of sprouting by 45% for 1 and 48% for 2 in the number of sprouts (**p < 0.01, Figure 3B) was determined, and the mean sum of the sprout lengths/spheroid was inhibited by 60% and 61% (**p < 0.01, Figure 3C) for 1 and 2, respectively. Since neither compound inhibits migration, the reduction of sprouting in EC is presumably a result of the effect of 1 and 2 to inhibit proliferation. Complexes 1 and 2 induce apoptosis in ECRF24 cells in a dose-dependent manner (Table 2), determined by FACS analysis of subdiploid cells after exposure to a 50 μM dose. A 5.3-fold and 3.4-fold increase in apoptotic cells compared to the control was observed for 1 and 2, respectively. As reported

Table 2. Cell-Cycle Changes in ECRF24 Cells Treated with 1 or 2a compound (μM) CTRL 1 (6) 1 (12) 1 (25) 1 (50) 2 (6) 2 (12) 2 (25) 2 (50)

G1/G0 (±SEM) 63.0 66.3 68.0 61.5 51.8 67.0 64.0 62.5 67.0

± ± ± ± ± ± ± ± ±

0.2 3.9 0.0 0.7 0.3 2.8 2.8 2.8 3.5

G2/M (±SEM) 31.0 26.0 22.0 27.0 23.5 27.0 29.5 28.5 29.5

± ± ± ± ± ± ± ± ±

0.6 5.4 0.0 0.0 3.5 3.5 2.1 0.7 3.5

apoptosis (±SEM) 3.0 4.6 6.1 8.0 15.9 3.6 2.4 5.4 6.9

± ± ± ± ± ± ± ± ±

0.1 1.1 0.3 1.3 3.0 0.1 1.3 0.6 1.2

a

Changes to the cell-cycle were evaluated by FACS analysis of propidium iodide stained cells after 24 h incubation with 1 or 2 at concentrations ranging from 6 to 50 μM, n = 4. Errors represent the standard error of the mean.

previously, 1 induces apoptosis in MDA-MB-231 cells,28 with a 3.3-fold increase (at 50 μM) in apoptosis versus the control. Interactions of 1 and 2 with Serum Proteins. Following intravenous administration ruthenium compounds have been shown to bind to serum proteins, in particular albumin and transferrin, which are believed to play a crucial role in drug transport and metabolism.35−42 The binding properties of 1, 2, and RAPTA-C with human serum albumin (HSA) and apotransferrin were assessed using a fluorescence spectroscopy quenching assay (Table 3). HSA fluorescence results from tryptophan residues, one in the case of HSA (Trp214) and six in the case of apotransferrin. All complexes led to a significant fluorescence quenching on HSA and transferrin after 30 h. The D


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Molecular Pharmaceutics Table 3. Ka and KSV values for 1, 2, and RAPTA-C Binding to Human Serum Albumin (HSA) and Apotransferrin Determined by Fluorescence Quenching and Calculated Using the Stern−Volmer Equation

(CAM) of the chicken embryo following intravenous administration (between development days 11 and 14) (Figure 4). Morphological changes in the CAM organization recorded on development day 15 were imaged via epifluorescence microscopy and quantified using an automated imageprocessing method with a descriptor of branching points/ mm2 (Figure 4).43 Complex 1 administered at 50 μM (0.5 mg/kg) was previously shown to induce an antivascular effect represented by a 26.3% reduction in the number of branching points/ mm2.28 The presence of avascular zones (black areas in fluorescence angiographies indicated by arrows, Figure 4) was observed for 2 administered at 42 μM (0.47 mg/kg), with a 38% reduction of the number branching points/mm2 versus the control (***p = 2.8 × 10−5), mainly along the larger vessels. The avascular zones observed are most likely due to antiangiogenic activity of 2. For comparison, NAMI-A in the CAM-sponge model was shown to reduce the number of vessels in the dose range between 120 μM and 240 μM.44 A similar antivascular effect in the CAM model was observed for RAPTA-C and RAPTA-T administered topically in the developmental CAM (EDD 7−9) at a dose of 154 μg/ embryo.17 Antitumor Effect of 1 and 2 in Human Ovarian Carcinoma and Human Colorectal Adenocarcinoma. The antitumor activity of 1 was previously studied in vivo using the preclinical model of human ovarian A2780 carcinoma grown on the chicken chorioallantoic membrane CAM (Figure 5A).28 For comparison, 2 and a combination treatment of 1 + 2 were evaluated using the same model and protocol. Embryos were randomized and treated within the following groups via intravenous administration of 0.1% DMSO (CTRL), 2 (50 μM; 0.56 mg/kg, 80 μL), or the mixture of 1 + 2 (premixed at ratio 1:1; 25 μM each; 0.5 mg/kg, 80 μL). To investigate the

compound (μM) parameters

1

2

RAPTA-C

−1

Binding Constant Ka (M ) HSA 0.67 × 103 3.24 × 104 1.00 × 103 3 6 apotransferrin 3.32 × 10 1.84 × 10 1.00 × 106 4 −1 Stern−Volmer Quenching Constant KSV (10 M ) HSA 0.5 2.4 0.8 apotransferrin 0.4 0.9 1.0

most efficient fluorescence quenching was observed for apotransferrin in the presence of 1, with a fluorescence reduction of 63%, followed by 2 and RAPTA-C (36 and 32% reduction, respectively). HSA, in contrast, is essentially equally quenched by the three complexes, with a total fluorescence decrease between 49 and 54%. The binding constants of the complexes to HSA and apotransferrin were determined (Table 3), with the highest binding constants (Ka) estimated for 2 (Ka = 3.24 × 104 M−1), i.e., the more lipophilic complex (note that these values are lower than the binding constant of KP1019 to HSA).39 The calculated Stern−Volmer quenching constant (KSV) values for NAMI-A with apotransferrin and halotransferrin are, respectively, 1.28 × 104 M−1 and 1.28 × 104 M−1.40,42 The KSV values in apotransferrin were lower for 1 (0.4 × 104 M−1), 2 (0.9 × 104 M−1), and RAPTA-C (1.0 × 104 M−1), see Table 3. Interestingly, the KSV for HSA with 2 was almost 10fold higher than for 1 and 4-fold higher than for RAPTA-C. Influence of 1 and 2 on Vasculature Formation in the CAM Model. As previously reported for 1,28 the antiangiogenic activity of 2 was investigated in the chorioallantoic membrane

Figure 4. In vivo activity of 2 on the CAM model. Complex 2 at 8 μM induces only a mild vascular effect and at 42 μM 2 significantly (***p = 2.8 × 10−5) changes the vasculature architecture compared to the CTRL. Quantification was performed by measurement of the branching points/mm2. Error bars represent the standard error of the mean, n = 5−7. E


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Treatment with 1 induced significant tumor growth inhibition, by 88% versus the CTRL group, on the last day of the experiment (***p = 1.3 × 10−5).28 Administration of 2 led to inhibition of tumor growth by 63% (*p = 0.03). In cell proliferation inhibition assays at 37 °C 2 was active exclusively on ECRF24 cells, and consequently tumor growth inhibition may be attributed to an antiangiogenic mechanism. The specificity of 2 toward endothelial cells contrasts with other ruthenium-based compounds that possess less specific antiangiogenic characteristics, i.e., RAPTA-C, RAPTA-T, and DAPTA-C,17 triruthenium clusters,45 and NAMI-A.46 The premixed combination of 1 + 2 administrated iv at doses corresponding to half of the monotherapy doses of both compounds inhibited tumor growth efficiently, by 86% (*p = 0.047), the reduction being similar to that of 1 administrated at a 2-fold higher dose. Note that RAPTA-C administered in the same tumor model at 0.2 mg/kg (administered for 5 days) inhibited A2780 tumor growth by 75%.18 Embryo body weight loss was not observed in any of the treatment groups, indicative of a lack of general toxicity. The ability of 2 to reduce tumor growth was also studied on human adenocarcinoma LS174T carcinoma implanted subcutaneously on the hind leg of athymic mice. Compound 1 was evaluated on the same in vivo model injected intraperitoneally (ip) every 4 days.29 The median tumor volumes treated with 1 or 2 versus the control groups are shown in Figure 6. Significant tumor inhibition was observed for 1 (by 61% versus control, **p < 0.01) and for 2 (by 66% vs control, **p < 0.01). Tissue sections of treated tumors were subject to immunohistochemical analysis of CD31 to delineate the number of endothelial cells/blood vessels (Figure 6C). The number of stained vessels was significantly lower following treatment with 2. A significant reduction of 32% (**p = 0.006) was observed. No inhibition of vessel density was observed after treatment with 1. The diameter of remaining vessels in tumors treated with 1 was, however, smaller than those present in the control tumors. Combined, these data indicate that treatment with 1 leads to the inhibition of tumor growth via a direct anticancer effect,

Figure 5. In vivo anticancer activity of complexes 1, 2, and 1 + 2 in human ovarian carcinoma tumors (A2780) grown on the CAM using experimental protocol shown in (A). (B) Tumor growth curves of the following conditions: CTRL (100 μL), 1 (50 μM, 80 μL),28 2 (50 μM, 80 μL), or 1 + 2 (premixed ratio 1:1; 25 μM each, 80 μL). Inset shows a representative tumor in the CTRL group. In all the treatment groups significant tumor growth inhibition compared to the CTRL was observed (***p = 1.3 × 10−5 for 1, *p = 0.03 for 2 and *p = 0.047 for 1 + 2). Error bars represent standard error of the mean.

effect of 2 on tumor growth, A2780 ovarian carcinoma cells were inoculated at EDD 7 of the CAM and monitored for 11 days. Established and vascularized tumors were detected 3 days postimplantation (EDD 10). Treatment was performed by iv injections on four consecutive days (Figure 5). Tumors grew to an average size of approximately 150 mm3 by EDD 17 when left untreated. Tumor growth curves are shown in Figure 5B with differences in tumor volume between the treatment groups noticeable from the fourth day of treatment.

Figure 6. LS174T adenocarcinoma growth inhibition by 1 (A) and 2 (B) in athymic mice. Mice were treated with 1 (75 mg/kg, 300 μL ip) or 2 (12.5 mg/kg, 300 μL ip) given every 4 days (indicated by arrows). The vehicle control was treated with three doses of 10% DMSO in sterile saline (0.9% NaCl) also given every 4 days. Values plotted are medians ± the standard error of the mean, SEM (ANOVA, **p < 0.01, ***p < 0.001). (C). Histochemical analysis of tumors stained for the presence of endothelial cells (CD31) and its quantification. Values plotted are medians ± the standard error of the mean. F


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Results of a Clinical Phase I Study in Tumor Patients. Chem. Biodiversity 2008, 5, 2140−2155. (8) Sava, G.; Gagliardi, R.; Bergamo, A.; Alessio, E.; Mestroni, G. Treatment of metastases of solid mouse tumours by NAMI-A: comparison with cisplatin, cyclophosphamide and dacarbazine. Anticancer Res. 1999, 19, 969−972. (9) Rademaker-Lakhai, J. M.; van den Bongard, D.; Pluim, D.; Beijnen, J. H.; Schellens, J. H. A Phase I and pharmacological study with imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a novel ruthenium anticancer agent. Clin. Cancer Res. 2004, 10, 3717− 3727. (10) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.; Zorbas, H.; Keppler, B. K. From bench to bedside–preclinical and early clinical development of the anticancer agent indazolium trans[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J. Inorg. Biochem. 2006, 100, 891−904. (11) Leijen, S.; Burgers, S. A.; Baas, P.; Pluim, D.; Tibben, M.; van Werkhoven, E.; Alessio, E.; Sava, G.; Beijnen, J. H.; Schellens, J. H. M. Phase I/II study with ruthenium compound NAMI-A and gemcitabine in patients with non-small cell lung cancer after first line therapy. Invest. New Drugs 2015, 33, 201−214. (12) Nazarov, A. A.; Baquie, M.; Nowak-Sliwinska, P.; Zava, O.; van Beijnum, J. R.; Groessl, M.; Chisholm, D. M.; Ahmadi, Z.; McIndoe, J. S.; Griffioen, A. W.; van den Bergh, H.; Dyson, P. J. Synthesis and characterization of a new class of anti-angiogenic agents based on ruthenium clusters. Sci. Rep. 2013, 3, 1485. (13) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, W.; Keppler, B. K. NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chem. Sci. 2014, 5, 2925− 2932. (14) Clavel, C. M.; Paunescu, E.; Nowak-Sliwinska, P.; Griffioen, A. W.; Scopelliti, R.; Dyson, P. J. Discovery of a Highly Tumor-Selective Organometallic Ruthenium(II)-Arene Complex. J. Med. Chem. 2014, 57, 3546−3558. (15) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto, M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. In vitro and in vivo evaluation of ruthenium(II)-arene PTA complexes. J. Med. Chem. 2005, 48, 4161−4171. (16) Bergamo, A.; Masi, A.; Dyson, P. J.; Sava, G. Modulation of the metastatic progression of breast cancer with an organometallic ruthenium compound. Int. J. Oncol. 2008, 33, 1281−1289. (17) Nowak-Sliwinska, P.; van Beijnum, J. R.; Casini, A.; Nazarov, A. A.; Wagnières, G.; van den Bergh, H.; Dyson, P. J.; Griffioen, A. W. Organometallic Ruthenium(II) Arene Compounds with Antiangiogenic Activity. J. Med. Chem. 2011, 54, 3895−3902. (18) Weiss, A.; Berndsen, B. H.; Dubois, M.; Müller, M.; Schibli, R.; Griffioen, A. W.; Dyson, P. J.; Nowak-Sliwinska, P. In vivo anti-tumor activity of the organometallic ruthenium(II)-arene complex [Ru(η6-pcymene)Cl2(pta)] (RAPTA-C) in human ovarian and colorectal carcinomas. Chem. Sci. 2014, 5, 4742−4748. (19) Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.; Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, H. F.; Ang, W. H.; Droge, P.; Rothlisberger, U.; Dyson, P. J.; Davey, C. A. Ligand substitutions between ruthenium−cymene compounds can control protein versus DNA targeting and anticancer activity. Nat. Commun. 2014, 5, 3462. (20) Babak, M. V.; Meier, S. M.; Huber, K. V. M; Reynisson, J.; Legin, A. A.; Jakupec, M. A.; Berger, W.; Dyson, P. J.; Superti-Furga, G.; Keppler, B. H.; Hartinger, C. G. Target profiling of an antimetastatic RAPTA agent by chemical proteomics: relevance to the mode of action. Chem. Sci. 2015, 6, 2449−2456. (21) Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc. Jpn. Acad., Ser. B 2010, 86, 410−425. (22) Carmeliet, P.; Jain, R. K. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011, 473, 298−307. (23) Griffioen, A. W. Therapeutic approaches of angiogenesis inhibition: are we tackling the problem at the right level? Trends Cardiovasc. Med. 2007, 17, 171−176.

whereas tumor growth inhibition by 2 corresponds predominantly to an antiangiogenic effect.

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CONCLUSIONS Herein, we show that two closely related ruthenium(II)−pcymene complexes, differing only in the length of a pendant fluorous chain attached to a pyridine ligand, inhibit tumor growth effectively. Interestingly, the mechanism of tumor growth inhibition of the two compounds is profoundly different. One compound, i.e., 1, with the shorter pendant fluorous chain, inhibits tumor growth via anticancer and antiangiogenic mechanisms whereas 2 involves predominantly an antiangiogenic mechanism. This research shows that finetuning of drug activities is possible through minor structural changes, an approach that may have an impact on future cancer therapy.

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AUTHOR INFORMATION

Corresponding Authors

*P.N.-S.: VU University Medical Center, De Boelelaan 1118, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Tel: + 31 20 4443374. Fax: +31 20 4443844. E-mail: [email protected]. *P.J.D.: Swiss Federal Institute of Technology (EPFL), SB ISIC LCOM, BCH 2402, Av. Forel 2, CH-1015 Lausanne, Switzerland. Tel: +41 21 693 98 54. Fax: +41 21 693 97 80. Email: paul.dyson@epfl.ch. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for technical assistance from Tse J. Wong (VUMC, Amsterdam). We thank the Swiss National Science Foundation and EPFL for financial support.

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ABBREVIATIONS USED CAM, chicken chorioallantoic membrane; ECRF24, immortalized endothelial cell line; HUVEC, human umbilical vein endothelial cells; EDD, embryo development day; HSA, human serum albumin; Ka, binding constant; bFGF, basic fibroblast growth factor

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REFERENCES

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Antiangiogenic and Anticancer Properties of Bifunctional Ruthenium(II

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