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Ruthenium(II) p-cymene complex bearing 2,2’-dipyridylamine targets caspase3 deficient MCF-7 breast cancer cells without disruption of antitumor immuneresponse

Goran N. Kaluerovic, Tamara Krajnovic, Miljana Momcilovic, StanislavaStosic-Grujicic, Sanja Mijatovic, Danijela Maksimovic-Ivanic, EvamarieHey-Hawkins

PII: S0162-0134(15)30081-7DOI: doi: 10.1016/j.jinorgbio.2015.09.006Reference: JIB 9807

To appear in: Journal of Inorganic Biochemistry

Received date: 24 July 2015Revised date: 6 September 2015Accepted date: 9 September 2015

Please cite this article as: Goran N. Kaluerovic, Tamara Krajnovic, Miljana Momcilovic,Stanislava Stosic-Grujicic, Sanja Mijatovic, Danijela Maksimovic-Ivanic, Evamarie Hey-Hawkins, Ruthenium(II) p-cymene complex bearing 2,2’-dipyridylamine targets caspase3 deficient MCF-7 breast cancer cells without disruption of antitumor immune response,Journal of Inorganic Biochemistry (2015), doi: 10.1016/j.jinorgbio.2015.09.006

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Ruthenium(II) p-cymene complex bearing 2,2’-dipyridylamine targets

caspase 3 deficient MCF-7 breast cancer cells without disruption of

antitumor immune response

Goran N. Kaluđerovića,b,*

, Tamara Krajnovićc, Miljana Momcilovic

c, Stanislava Stosic-

Grujicicc, Sanja Mijatović

c, Danijela Maksimović-Ivanić

c and Evamarie Hey-Hawkins

a

aInstitute of Inorganic Chemistry, Leipzig University, Johannisallee 29, D-04103 Leipzig,

Germany.

bDepartment of Bioorganic Chemistry, Leibniz-Institute of Plant Biochemistry,

Weinberg 3, D 06120 Halle (Saale) Germany.

cInstitute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar

despota Stefana 142, 11060 Belgrade, Serbia.

Dedicated to Prof. Giovanni Natile on the occasion of his 70th birthday.

Abstract

[Ru(6-p-cym)Cl{dpa(CH2)4COOEt}][PF6] (cym = cymene; dpa = 2,2’-dipyridylamine;

complex 2) was prepared and characterized by elemental analysis, IR and multinuclear NMR

spectroscopy, as well as ESI-MS and X-ray structural analysis. The structural analog without

a side chain [Ru(6-p-cym)Cl(dpa)][PF6] (1) as well as 2 were investigated in vitro against

518A2, SW480, 8505C, A253 and MCF-7 cell lines. Complex 1 is active against all

investigated tumor cell lines while the activity of compound 2 is limited only to caspase 3

deficient MCF-7 breast cancer cells, however, both are less active than cisplatin. As CD4+Th

cells are necessary to trigger all the immune effector mechanisms required to eliminate tumor

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cells, besides testing the in vitro antitumor activity of 1 and 2, the effect of ruthenium(II)

complexes on the cells of the adaptive immune system have also been evaluated. Importantly,

complex 1 applied in concentrations which were effective against tumor cells did not affect

immune cell viability, nor did exert a general immunosuppressive effect on cytokine

production. Thus, beneficial characteristics of 2 might contribute to the overall therapeutic

properties of the complex.

Keywords: ruthenium(II), cisplatin, anticancer drugs, immune cells, cytokines

1. Introduction

Over the last few decades many efforts have been made in the field of cancer therapy [1–4].

The treatment of many types of cancer has cisplatin and its analogues as mainstay drugs in

current clinical chemotherapy. However, the clinical drawbacks of cisplatin are apparent,

including the limited applicability, the intrinsic or acquired resistance, and the serious side

effects [5]. In recent years, ruthenium-based complexes have emerged as promising antitumor

and antimetastatic agents with equal or even greater antitumor activity and lower toxicity [6].

Superiority of ruthenium complexes from the classical platinum-based drugs reflect not only

in their cytotoxic activity, but also in the extremely low toxicity against normal cells [7–9].

Up to now, various ruthenium complexes were investigated as potential anticancer agents

[10–19]. Two families of ruthenium(II) complexes were the mostly investigated as potential

agents in treatment of cancer. Thus, octahedral ruthenium(III) complexes, such as [LH]trans-

[RuCl4(L--N)n(S-DMSO)2–n] (n = 1, L = imidazole; n = 2, L= indazole) which reached

clinical trials [20–25] as well as ruthenium(II) arene complexes, “piano stool” type, i.e.

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[Ru(6-arene)Cl(N

N)]X (N

N = chelating diamine ligand; X = Cl, PF6, BPh4), showed

promising anticancer activity in both in vitro and in vivo studies [26,27].

Inflammation and immunity affect all phases of tumor growth from initiation to progression

and dissemination [28]. T helper (Th) cells are fundamental for optimal induction of both

humoral and cellular effector mechanisms [29]. Both innate and adaptive immunity have been

shown to participate in this response. Adaptive immune responses to Ags released by dying

cells play a critical role in the spontaneous as well as therapy-induced tumor rejection [30].

Ample studies have identified proinflammatory cytokines as crucial mediators in cancer

treatments. Recently, the binuclear ruthenium(II) complex, [{RuCl2(η6-p-cym)}2-{(3-

py)COO(CH2CH2O)4CO(3-py)}] (py = pyridine), was reported, which modulates immune

system cell functions in vitro by inhibiting T cell differentiation towards pathogenic

Th1/Th17 phenotype and inducing a regulatory phenotype characterized by IL-10 and IL-4

production [31]. This ruthenium(II) complex was found ineffective against several tumor cell

lines [32].

This paper focuses on the synthesis and characterization of [Ru(6-p-

cym)Cl{dpa(CH2)4COOEt}][PF6] (cym = cymene; dpa = 2,2’-dipyridylamine; complex 2) as

well as its biological activity and of its structural analog [Ru(6-p-cym)Cl(dpa)][PF6] (1).

With the aim to contribute to the understanding of the antitumor action mechanism of

ruthenium(II) compounds the in vitro activity of 1 and 2 was investigated against tumor cells

along with normal cells of the adaptive immune system.

2. Materials and methods

2.1. Materials and measurements

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All reactions and manipulations were carried out under argon using standard Schlenk

techniques. NMR spectra (1H,

13C,

31P) were recorded at 27 °C on Varian Gemini VXR 400

spectrometers. Chemical shifts are relative to solvent signals (acetone-d6, 2.06, C 30.5,

206.8; CDCl3, 7.24, C 77.0) as internal references; (31

P) is relative to external H3PO4

(85%). Microanalyses (C, H) were performed in the Microanalytical Laboratory of the

University of Halle using a CHNS-932 (LECO) elemental analyzer. Mass spectra (ESI-MS)

were recorded on an FTICR MS Bruker-Daltonics ESI mass spectrometer (APEX II, 7 T).

Ethyl 5-bromovalerate was purchased from Sigma Aldrich (Germany). [{RuCl2(η6-p-cym)}2]

was prepared according to a literature procedure [33].

2.2. Preparation of dpa(CH2)4COOEt and complexes 1 and 2

2.2.1. Preparation of dpa(CH2)4COOEt

Compound dpa(CH2)4COOEt was prepared by an adapted literature procedure for N-

neopentyl-N-(pyridine-2-yl)pyridine-2-amine [34]. Solid NaH (198 mg, 8.25 mmol) was

added in small portions to a solution of 2,2’-dipyridylamine (1g, 5.85 mmol) in dry DMF (10

mL), stirred under N2 at 0 °C. After 1.5 h Br(CH2)4COOEt (1.5 mL, 9.25 mmol) was added

and the reaction mixture was heated at 75 °C for 2 days. Afterwards EtOH (5 mL) was used to

quench the reaction (caution!) and the solvent was removed under reduced pressure. The

obtained oil was treated with diethyl ether (10 mL), filtered and the diethyl was removed. The

remaining oil was purified by column chromatography on silica gel (n-hexane:ethylacetate =

8:2). Yield: 1.10 g (63%). ESI-MS (CHCl3/CH3OH), positive mode: Calcd for

[C17H21N3NaO2]+ 322.1, m/z 322.2 [M+Na]

+.

1H NMR (400 MHz, CDCl3): δ 1.25 (t, 3H,

C12

H3), 1.75 (m, 4H, C7H2+C

8H2), 2.35 (m, 2H, C

9H2), 4.10 (m, 2H, C

11H2), 4.21 (m, 2H,

C6H2), 6.83 (dd,

3J(H

4,H

3) = 6.9 Hz,

3J(H

4,H

5) = 6.1 Hz, 2H, H

4), 7.07 (d,

3J(H

2,H

3) = 8.0 Hz,

2H, H2), 7.49 (dd,

3J(H

3,H

2) = 8.0 Hz,

3J(H

3,H

4) = 6.9 Hz, 2H, H

3), 8.32 (d,

3J(H

5,H

4) =

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6.1 Hz, 2H, H5).

13C NMR (100 MHz, CDCl3): δ 15.2 (C

12), 22.4 (C

8), 26.8 (C

7), 33.3 (C

9),

47.7 (C6), 60.1 (C

11), 114.6 (C

2), 116.9 (C

4), 137.0 (C

3), 148.3 (C

5), 156.1 (C

1), 173.6 (C

10).

Numbering of carbon atoms is given in Scheme 1.

2.2.2. Preparation of [RuCl2(η6-p-cym)(dpa)][PF6] (1)

1 was prepared by a modified literature procedure [35]. The amine dpa (56 mg, 0.33 mmol)

was added to a dry methanol solution (25 mL) of [{RuCl2(η6-p-cym)}2] (100 mg, 0.16 mmol).

The solution was heated under reflux and stirred for 4 h. Afterwards the reaction mixture was

allowed to cool to r.t., and solid [NH4][PF6] (270 mg, 1.66 mmol) was added and the mixture

stirred for an additional 0.5 h. The obtained yellow precipitate was filtered off, washed with

diethyl ether and dried in vacuum. Yield: 160 mg (83%). The structure and purity were

confirmed by elemental analysis, ESI-MS and 1H and

13C NMR spectroscopy. Anal. Found:

C, 41.10; H, 3.84; N, 7.27. Calcd for C20H23N3ClF6PRu (586.90): C, 40.93; H, 3.95; N, 7.16.

ESI-MS (DMSO/CH3OH), positive mode: Calcd for [C20H23ClN3Ru]+ 442.1, m/z 442.1 [M–

PF6]+.

1H NMR (400 MHz, acetone-d6): δ 1.26 (d,

3J(H,H) = 7.0 Hz, 6H, C

gH3), 2.14 (3H,

CeH3), 2.75 (m, 1H, C

fH), 2.83 (s, 1H, NH), 5.70 (d,

3J(H,H) = 6.7 Hz, 2H, C

bH), 5.81 (d,

3J(H,H) = 6.7 Hz, 2H, C

cH), 7.23 (dd,

3J(H

4,H

3) = 6.9 Hz,

3J(H

4,H

5) = 5.8 Hz, 2H, H

4), 7.33

(d, 3J(H

2,H

3) = 7.9 Hz, 2H, H

2), 8.00 (dd,

3J(H

3,H

2) = 7.9 Hz

3J(H

3,H

4) = 6.9 Hz, 2H, H

3),

8.70 (d, 3J(H

5,H

4) = 5.8 Hz, 2H, H

5).

13C NMR (100 MHz, acetone-d6): δ 19.0 (C

e), 23.0 (C

g),

32.3 (Cf), 85.6 (C

c), 87.0 (C

b), 101.4 (C

a), 108.3 (C

d), 115.7 (C

2), 121.2 (C

4), 142.1 (C

3),

154.8 (C1), 156.3 (C

5). Numbering of carbon atoms is given in Scheme 1.

2.2.3. Preparation of [RuCl(η6-p-cym){dpa(CH2)4COOEt}][PF6] (2)

2 was prepared analogously to 1; instead of dpa, the derivative dpa(CH2)4COOEt (98 mg,

0.326 mmol) was used. Yield: 175 mg (75%). Anal. Found: C, 45.29; H, 4.44; N, 5.89. Calcd

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for C27H35N3O2ClF6PRu (715.05): C, 45.35; H, 4.93; N, 5.88. IR (KBr, cm–1

): 3015 (w), 2966

(w), 1730 (m), 1598 (w), 1574 (w), 1457 (m), 1440 (m), 1392 (w), 1351 (w), 1244 (w), 1181

(m), 1099 (w), 1080 (w), 1029 (w), 885 (w), 835 (vs), 788 (m), 774 (m), 759 (w), 742 (w),

556 (s), 453 (w), 297 (w). ESI-MS (CHCl3/CH3OH), positive mode: Calcd for

[C27H35ClN3O2Ru]+ 570.1, m/z 570.2 [M–PF6]

+.

1H NMR (400 MHz, CDCl3): δ 1.28 (9H,

CgH3+C

12H3), 1.85 (7H, C

eH3+C

7H2+C

8H2), 2.44 (br s, 2H, C

9H2), 2.74 (m, 1H, C

fH), 4.09

(m, 4H, C6H2+C

11H2), 5.48 (d,

3J(H,H) = 6.5 Hz, 2H, C

bH), 5.71 (d,

3J(H,H) = 6.5 Hz, 2H,

CcH), 7.20 (dd,

3J(H

4,H

3) = 7.0 Hz,

3J(H

4,H

5) = 6.0 Hz, 2H, H

4), 7.30 (d,

3J(H

2,H

3) = 8.1 Hz,

2H, H2), 7.90 (dd,

3J(H

3,H

2) = 8.1 Hz

3J(H

3,H

4) = 7.0 Hz, 2H, H

3), 8.66 (d,

3J(H

5,H

4) = 6.0

Hz, 2H, H5).

13C NMR (100 MHz, CDCl3): δ 14.2 (C

12), 17.9 (C

e), 22.1 (C

8), 22.3 (C

g), 26.7

(C7), 30.5 (C

f), 33.2 (C

9), 49.9 (C

6), 60.4 (C

11), 83.7 (C

c), 86.0 (C

b), 100.6 (C

a), 105.3 (C

d),

116.0 (C2), 121.2 (C

4), 141.0 (C

3), 154.0 (C

5), 157.9 (C

1), 173.4 (C

10).

31P NMR (162 MHz,

CDCl3): δ –144.4 (hep, P). Numbering of carbon atoms is given in Scheme 1.

2.3. Crystal structure determination

Crystals suitable for X-ray structural analysis were obtained mother liquor of 2 after filtration.

The data of 2 were collected with a CCD Oxford Xcalibur S and an Oxford Gemini S

diffractometer, (λ(Mo Kα) = 0.71073 Å) using the multiscan mode. The structure was solved

by direct methods and refined on F2 with SHELXL-2014 [36]. The hydrogen atoms were

placed in calculated positions with fixed displacement parameters (riding model), and were

refined isotropically. The Diamond program was used for the presentation of the structure

[37]. The crystallographic details are listed in Table 1. CCDC 1413063 contains the

supplementary crystallographic data for this paper. These data can be obtained free of charge

from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk.

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Table 1. Crystal data and structure refinement for 2

Compound 2

Chemical formula C27H35N3O2F6PClRu

Formula weight 715.07

Crystal system Monoclinic

Space group P21/c

a (Å) 10.3288(1)

b (Å) 18.8479(2)

c (Å) 15.8486(2)

α (°) 90

β (°) 108.705(1)

γ (°) 90

V (Å3) 2922.38(6)

Z 4

Dcalc (g cm-3

) 3.200

μ (mm-1

) 0.725

F(000) 752.0

Crystal size (mm3) 0.13 × 0.11 × 0.11

Data collection

Monochromator graphite

Radiation, Mo Kα (Å) 0.71073

Temperature (K) 200

θ Range (°) 2.9–27.5

Index range –13 ≤ h ≤ 13

–24 ≤ k ≤ 24

–20 ≤ l ≤ 20

Tmin/ Tmax 0.907/ 0.921

Number of measured reflections 48139

Number of independent reflections 6708

Refinement

Refinement on F2

Data/restraints/parameters 6708 / 0 / 370

R[F2 > 4σ(F

2)] 0.025

wR(F2)

a 0.054

Goodness-of-fit on F2 0.995

Δρmin/Δρmax (e Å−3

) −0.48/ 0.53 a w = 1/[σ

2(Fo

2) + (0.0178P)

2 + 3.5701P] where P = (Fo

2 + 2Fc

2)/3

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2.4. Reagents, cells and animals

RPMI-1640, fetal calf serum (FCS), dimethyl sulfoxide (DMSO), phosphate-buffered saline

(PBS), carboxyfluorescein diacetate succinimidyl ester (CFSE), crystal violet (CV), 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sulforhodamine B (SRB) and

propidium iodide (PI) were purchased from Sigma (St. Louis, MO). Annexin V-FITC (AnnV)

was obtained from Santa Cruz Biotechnology (Dallas, TX). Acridine orange (AO) was from

Labo-Moderna (Paris, France). 518A2, SW480, 8505C, A253 and MCF-7 cells were regularly

cultivated in HEPES-buffered RPMI-1640 medium with 10% FCS, 2 mM L-glutamine,

0.01% sodium pyruvate and antibiotics (culture medium) at 37 °C in a humidified atmosphere

with 5% CO2. For viability determination cells were seeded at 1–2 × 103 / well in 96-well

plates and 1.5 × 105 / well in 6-well plates for flow cytometric analysis.

2.5. Viability evaluation by SRB, CV and MTT tests

Viability of adherent cells was estimated by SRB, CV and MTT tests, while for nonadherent

cells only MTT was used. Cells were treated with different concentrations of experimental

drugs for an indicated time, and the determination of viability was done exactly as described

previously [38–40]. The absorbance was measured in an automated microplate reader (LKB

5060-006, LKB, Vienna, Austria) at 540 nm, and background at 670 nm was subtracted. Cell

viability was expressed as a percentage of the control value (untreated cells), which was

arbitrarily set to 100% and presented as mean ± SD.

2.6. AnnexinV-FITC/PI, apostat and acridin orange staining

MCF-7 cells were treated with an IC50 dose of ruthenium(II) complex 1 for 72 h and then

staining with AnnV-FITC/PI or apostat (R&D Systems, Minneapolis, MN, USA) was done

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according to the instructions of the manufacturer. For detection of autophagy, cells were

stained with AO (1 μg/mL) for 15 min at 37 °C, subsequently washed and resuspended in

PBS. Analysis was done with a CyFlow® Space Partec using PartecFloMax® software

(Partec GmbH, Münster, Germany).

2.7. CFSE staining

MCF-7 cells were stained for 10 min with 1 μM of CFSE at 37 °C, and then cultivated in the

presence of an IC50 dose of complex 1 for 72 h. Then the cells were washed, detached,

resuspended in PBS and analyzed with a CyFlow® Space Partec using PartecFloMax®

software (Partec GmbH, Münster, Germany).

2.8. Immune cell isolation and in vitro treatments

Mixed populations of immune spleen cells (SC) were obtained from age- and sex-matched

healthy C57BL/6 mice. Directive 2010/63/EU on the protection of animals was used for

experimental and other scientific purposes, which were approved by the Ethical Committee

for the Use of Laboratory Animals of the Institute for Biological Research "Siniša Stanković"

(application No. 03-01/14). To obtain single cell suspensions, spleens were mechanically

disrupted by gentle teasing through a 40 μm nylon mesh filter (BD Bioscience, Bedford, MA,

USA) and the suspension of SC was collected by centrifugation. Erythrocytes were lyzed

using lysis buffer (eBioscience, San Diego, CA, USA). Samples of conditioned medium used

for cytokine detection were obtained by seeding SC (5 × 106 per well) in 24-well culture

plates (Sarstedt, Numbrecht, Germany) at 37 °C in a 5% CO2 incubator. Cells were cultured

for 48 h in RPMI-1640 medium (25 mM HEPES, 2 mM L-glutamine) supplemented with 5%

fetal calf serum (FCS, PAA Chemicals, Pasching, Austria), 5 μM/mL of β-mercaptoethanol,

100 U/mL penicillin and 100 mg/mL streptomycin (complete medium) in the presence or

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absence of graded concentrations of 1 or 2 and were stimulated with 1 μg/mL concanavalin A

(ConA).

2.9. Determination of cytokine secretion

Cytokine concentrations in the cell free culture supernatants were determined by sandwich

ELISA using MaxiSorp plates (Nunc, Rochild, Denmark) and anti-cytokine paired antibodies

according to the manufacturer’s instructions. Samples were analyzed in triplicate for murine

IL-17 (BD Pharmingen, San Diego, CA,USA), IFN-γ and IL-10 (eBioscience). The

absorbance at 450 nm was determined with a microplate reader. The results were calculated

using standard curves made on the basis of known concentrations of the relevant recombinant

cytokines.

2.10. Statistical analysis

Results are presented as mean ± standard deviation (SD) obtained in independent

experiments. Each experiment was repeated at least three times. The significance of the

changes was evaluated by Two-tailed Student’s t-test. Statistical evaluation of the results was

made with Statistica version 6.0 (StatSoft, Tulsa, OK, USA) and conducted at the 0.05

significance level.

3. Results and discussion

3.1. Synthesis and characterization

[Ru(η6-p-cym)Cl(dpa)][PF6] (dpa = 2,2’-dipyridylamine; 1) was obtained by an adapted

literature procedure (Scheme 1) [35]. Dichlorido(p-cymene)ruthenium(II) dimer was reacted

with dpa(CH2)4COOEt in the presence of [PF6]– forming the cationic complex [Ru(η

6-p-

cym)Cl{dpa(CH2)4COOEt}][PF6] (2) in a good yield. The ruthenium(II) complexes 2 was

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characterized by elemental analysis, IR and multinuclear (1H,

13C,

31P) NMR spectroscopy as

well as single-crystal X-ray structure analysis.

Scheme 1. Synthetic route of 1 and 2 [35].

ESI-MS of 2, recorded in methanol as solvent, gave evidence for the molecular composition.

Namely, the [M–PF6]+ ion with the characteristic expected isotope pattern was detected. In the

IR spectrum of 2 the band at 297 cm–1

is assigned to the Ru–Cl vibration [32,41]. The

vibrations at 1244 cm–1

and 1181 cm–1

are typical bands for carbon–oxygen single bonds,

while a strong absorption at 1730 cm–1

is assigned to the ester function [42]. Vibrations of the

alkyl groups are found near 2966 cm–1

. Less intense bands near 3015 cm–1

can be assigned to

heteroaromatic and aromatic vibrations of C–H bonds of the pyridine ring and the p-cymene

ligand.

The N-coordination of the dpa-derived ligand generates a strong downfield shift of all

proton resonances of the pyridine ring which were found in the 1H NMR spectrum

from 7.2 to 8.7 ppm (free ligand: 6.8 to 8.3 ppm). The hydrogen atoms of the p-cym

ligand gave chemical shifts similar to literature values [43]. The same trend is observed

in the 13

C NMR spectrum in which the chemical shifts of the carbon atoms bound to

the coordinating nitrogen atom are found at 154 and 158 ppm (free ligand: 148 and 156

ppm).

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Crystals of [RuCl(η6-p-cym){dpa(CH2)4COOEt}][PF6] (2) suitable for X-ray

diffraction analysis were obtained from the reaction solution at room temperature. The

compound crystallized as discrete cations and anions with relatively short C–H∙∙∙F

interatomic distances (2.39–2.73 Å). The molecular structure of 2 is shown in Figure 1

along with selected structural parameters, given in the figure caption. Complex 2

exhibits a half sandwich, “piano stool” structure. Thus, the coordination sphere of

ruthenium(II) is built up by an η6-p-cym, a chlorido as well as an N

N-κ

2N,N’ ligand.

The structure can be considered as a slightly distorted octahedron, because the angles

at the ruthenium(II) atoms are close to 90° (81.09(6)–86.81(4)°). The Ru–Cl

(2.3805(4) Å) and Ru–N bond lengths (2.101(1) and 2.107(1) Å) are in the expected

range (median Ru–Cl: 2.414 Å, lower/higher quartile: 2.389/2.442 Å, n = 5542; n –

number of observations) [35,44].

Figure 1. Molecular structure of the cation in [RuCl(η6-p-cym){dpa(CH2)4COOEt}][PF6].

Hydrogen atoms are omitted for clarity. The ellipsoids are shown with a probability of 50%.

Selected structural parameters (bond lengths in Å, angles in °): Ru1–Cl1 2.3805(4), Ru1–N2

2.107(1), Ru1–N3 2.101(1), Cl1–Ru1–N1 86.81(4), Cl1–Ru1–N3 85.34(4), N1–Ru1–N3

81.09(6).

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3.2. The effect of complexes 1 and 2 on tumor cell viability

In the present study, investigations of the anticancer properties of the

two ruthenium(II) complexes 1 and 2 were performed. To test the effectiveness of the

ruthenium(II) complexes, 518A2, SW480, 8505C, A253 and MCF-7 cells were cultivated

with a wide range of doses for 96 h and cell viability was estimated by SRB assay. As

presented in Table 2 and Figure S1.A, complex 1 displayed a higher potential to down-

regulate tumor cell growth than complex 2 affecting all investigated tumor cell lines while the

activity of 2 is restricted only to caspase 3 deficient breast cancer cell line MCF-7 [45,46].

However, both complexes are less active than cisplatin and some recently described

ruthenium(II) complexes in a more appropriate ligand environment against this specific cell

line [47]. Analogously to cisplatin and [RuCl(η6-p-cym)(en)]

+, as described by Sedler et al.

[48,49], also for 1 could be expected that hydrolysis of the Ru–Cl bond will occur in the cell

(due to lower concentration of Cl– ions in comparison to extracellular matrix) yielding active

aqua species. To confirm the results obtained by SRB, additional viability assessment by CV

and MTT tests was done and same experimental setting was applied varying time of drug

exposure (Table 2, Figure S1.B). CV dye stains DNA and RNA, while MTT is an enzyme-

based method for determination of mitochondrial dehydrogenase activities, both in the living

cells. Achieved results showed discrepancy between these two assays. IC50 values obtained of

these cell lines varied from 22 to 40 M depending on the applied test confirming high

efficacy of complex 1. According to National Cancer Institute guidelines the compound with

an IC50 value < 30 μg/mL is considered active [50]. The effective concentration of

ruthenium(II) complex 1 is in that range (IC50 = 40 µM corresponds to 28.6 µg/mL). The

lowest IC50 value was observed by MTT test indicating that 1 targeted mitochondrial

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respiration. Due to the higher activity of compound 1 this agent was selected for further

analysis of the mechanism of drug action on MCF-7 as the most sensitive cell line.

Table 2. IC50 values (µM)a of the ruthenium(II) complexes 1, 2 and cisplatin.

Compound SRB (96 h, treatment)

518A2 SW480 8505C A253 MCF-7

1 68.9 ± 2.4 63.1 ± 1.8 85.6 ± 3.5 181.1 ± 6.3 35.2 ± 1.1

2 > 200 > 200 > 200 > 200 176.2 ± 4.5

cisplatin 1.5 ± 0.2 3.2 ± 0.2 5.0 ± 0.2 0.8 ± 0.1 2.0 ± 0.1

MCF-7

CV (h, treatment) MTT (h, treatment)

48 72 96 48 72 96

1 65.9 ± 3.1 36.6 ± 1.7 22.7 ± 1.3 >100 61.5 ± 2.0 40.8 ± 1.5

amean values ± SD (standard deviation) from three experiments.

3.3. The effect of complex 1 on cellular proliferation and cell death

To further explore the cause of decreased viability cells were treated with an IC50 dose of 1

for 72 h and cellular proliferation was determined by flow cytometric analysis (Figure 2.A).

Contrarily to control were entire cell population was divided, accumulation of nondivided

cells exposed to 1 was observed. Inhibition of proliferation was synchronized with the

appearance of both early (Ann+PI

-) and late apoptotic cells (Ann

+PI

+) indicating that

ruthenium(II) complex 1 acts through inhibition of cell proliferation and subsequent apoptotic

cell death (Figure 2.B). Detected apoptosis was not caused by caspase activation (Figure 2.C),

and only slight potentiation of autophagic process was observed (Figure 2.D) indicating their

irrelevance for the antitumor activity of ruthenium(II) complex 1. This is expected because, as

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mentioned above, the tested cell line MCF-7 is caspase 3 deficient [45,46]. Although this

caspase plays a critical role in realization of apoptosis and is commonly activated by

numerous death signals, its deficiency did not disturb the completion of dying process

triggered by complex 1. In addition, special sensitivity of these cells to applied drug indicate

possibility that defect in caspase cascade is connected with some intracellular specificity

making them a good target for this ruthenium(II) potential experimental therapeutic.

Figure 2. Influence of 1 on MCF-7 cells: A inhibition of cell proliferation, B apoptosis

induction, C caspase activation and D the presence of autophagic vesicles.

3.4. The effects of 1 and 2 on cell viability of normal mouse splenocytes

Apart from the observed capability to affect caspase deficient tumor cells MCF-7, a

particularly important finding of this study is that 1 does not disturb the function of immune

cells. Cells of the adaptive immune system may also be implicated in the induction of tumor

cell death. In view of the results obtained on tumor cells with the two ruthenium(II)

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complexes 1 and 2, an evaluation of the selectivity of these compounds was carried out by

studying their action against nonmalignant immune cells. As stimulation of lymphocytes with

T specific mitogen ConA mimics the T cell receptor engagement by its cognate antigens

leading to T cell activation and cytokine production, unfractionated primary mouse SC

stimulated with ConA were used as a model of in vitro T lymphocyte responses. In order to

evaluate the effect of 1 and 2 on the viability of activated cells, SC were stimulated in vitro

with ConA for 48 h in the presence of these compounds in concentrations ranging from 6.25

to 50 µM. Importantly, as revealed by MTT assay, both compounds were completely nontoxic

for nonmalignant ConA-activated immune cells in all concentrations tested (Figure 3.A)

indicating a potential selectivity of 1 toward MCF-7 cancer cell lines.

Figure 3. The effect of 1 and 2 on the viability and cytokine production in mouse spleen cells

stimulated with ConA. Eritrolyzed splenocytes were obtained from healthy mice, activated

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with ConA (2.5g/mL) and grown in cell culture in the presence or absence of 1 and 2 (6.25 -

50M) for 48 h. MTT test was performed in order to determine the viability of the cells (A).

Cell-free supernatants were collected, and ELISA was performed for determining the levels of

secreted cytokines, IFN-, IL-17 (C) and IL-10 (D).

3.5. The effects of 1 and 2 on cytokine secretion by mouse splenocytes

A further goal was to evaluate potential immunomodulatory effects of both ruthenium(II)

complexes. For that purpose, the secretion of Th1 signature cytokine IFN-, Th17 signature

cytokine IL-17 and Th2 related IL-10 into culture supernatants of ConA-stimulated SC in the

presence of various single concentrations of the complexes was determined. The results

showed that neither 1 nor 2 influenced the secretion of IFN-(Figure 3.B), IL-17 (Figure 3.C)

or IL-10 (Figure 3.D), since all of the cytokines tested remained unchanged upon the

treatment.

The immune system plays an integral role in almost every aspect of tumorigenesis, including

tumor initiation, prevention and progression as well as the response to therapeutics [51]. In

recent years, it has become apparent that the beneficial effects of various conventional

therapeutic modalities, including chemotherapy, are associated with the rescue of an immune

response against the tumor. Therefore, in order to find most effective approaches, which at the

same time have fewer side effects, it is of primary interest to investigate in parallel direct

effects of the compound on tumor cells and immune cells. Although immune surveillance and

cytotoxic actions of NK cells and CD8+ T cells are considered essential for prevention of

tumor development and restraint of its progression [52], Th cells are assumed to play a major

role in anti-tumor immunity through inducing and directing the effector cellular immune

responses as well as inflammatory responses [53,54]. Thus, in several types of solid tumors

Th1 cells and their cytokines, especially IFN-γ, are associated with more potent anti-tumor

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immune responses [55]. Moreover, IFN-γ was identified as the major factor responsible for

Th1-induced dendritic cell tumoricidal activity [56]. Th17 cells and their major cytokine, IL-

17, also play dynamic roles in inflammation and tumor immunity. Tumor-infiltrating Th17

cells can have either pro- or antitumor activities depending on the type of neoplasm [57].

Although the exact role of these cells in tumor growth and progression is still controversial

[58], several studies have suggested that the increase in the number of Th17 cells is associated

with lower stages of the malignant diseases and less metastasis [59,60]. Moreover,

inflammation in the presence of Th17 cells appears to initiate, maintain, and enhance

protective anti-tumor immunity in some cases. Th17 cells may contribute to protective tumor

immunity through recruitment and induction of Th1-type effector cells to the tumor [61].

Many anticancer therapies cause immunosuppression and lympho-depletion that may

destabilize immune cell defense against cancer. There are numerous conflicting data about

cisplatin-induced immunosuppression on one side and on the other, preclinical and clinical

evidence about its immunomodulatory potential through improved recruitment and

proliferation of immune T cell effectors in the tumor microenvironment [62-64]. In the light

of these data, our finding of specific-targeting of tumor cells by ruthenium(II) complex 1

without disturbance of immune cell viability and function represents the important advantage

of this kind of therapeutic.

4. Conclusions

Herein, the synthesis of [Ru(6-p-cym)Cl{dpa(CH2)4COOEt}][PF6] (2) is described. 2 was

characterized by spectroscopic methods and X-ray structural analysis. Structural analog

[Ru(6-p-cym)Cl(dpa)][PF6] (1) and 2 were tested for their in vitro antitumoral potential. The

most efficient complex 1 showed higher activity against MCF-7 cells (the caspase 3 deficient

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cell line, more resistant to chemotherapy). Decreased viability was due to blockade of cell

division and subsequent apoptotic cell death without activation of caspases. Outstandingly, in

the present study it was demonstrated that cells of the adaptive immune system were

completely resistant to antiproliferative/cytotoxic effects of 1 suggesting a possibility for a

tumor-specific targeting. Therefore, present data are of importance for the potential

application of the ruthenium(II) complex 1 as antitumor agent as it might directly affect

tumor, specially the caspase 3 deficient MCF-7 cell line, but without modulation of the

immune response thus favoring antitumor milieu.

Abbreviations

8505C anaplastic thyroid tumor cell line

518A2 melanoma cell line

A253 head and neck tumor cell line

AnnV annexin V-FITC

AO acridine orange

CFSE carboxyfluorescein diacetate succinimidyl ester

ConA concanavalin A

CV crystal violet

dpa 2,2’-dipyridylamine

DMSO dimethyl sulfoxide

DMF dimethylformamide

FCS fetal calf serum

HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

IFN- interferon

IL-10 interleukin 10

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IL-17 interleukin 17

MCF-7 breast tumor cell line

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS phosphate-buffered saline

p-cym p-cymene

PI propidium iodide

py pyridine

SC spleen cells

SRB sulforhodamine B

SW480 colon cancer cell line

Acknowledgements

The authors would like to acknowledge financial support from the European Union and the

Free State of Saxony (project No. 100099597) and the Ministry of Education, Science and

Technological Development of the Republic of Serbia (project No. 173013). We would like to

thank Dr. Peter Lönnecke (Leipzig University) for the X-ray structural measurement of

complex 2.

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Figure Captions

1. Figure 1. Molecular structure of the cation in [RuCl(η6-p-cym){dpa(CH2)4COOEt}][PF6].

Hydrogen atoms are omitted for clarity. The ellipsoids are shown with a probability of

50%. Selected structural parameters (bond lengths in Å, angles in °): Ru1–Cl1 2.3805(4),

Ru1–N2 2.107(1), Ru1–N3 2.101(1), Cl1–Ru1–N1 86.81(4), Cl1–Ru1–N3 85.34(4), N1–

Ru1–N3 81.09(6).

2. Figure 2. Influence of 1 on MCF-7 cells: A inhibition of cell proliferation, B apoptosis

induction, C caspase activation and D the presence of autophagic vesicles.

3. Figure 3. The effect of 1 and 2 on the viability and cytokine production in mouse spleen

cells stimulated with ConA. Eritrolyzed splenocytes were obtained from healthy mice,

activated with ConA (2.5g/mL) and grown in cell culture in the presence or absence of 1

and 2 (6.25 - 50M) for 48 h. MTT test was performed in order to determine the viability

of the cells (A). Cell-free supernatants were collected, and ELISA was performed for

determining the levels of secreted cytokines, IFN-, IL-17 (C) and IL-10 (D).

Scheme Caption

1. Scheme 1. Synthetic route of 1 and 2 [35].

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Table Captions

1. Table 1. Crystal data and structure refinement for 2

2. Table 2. IC50 values (µM)a of the ruthenium(II) complexes 1, 2 and cisplatin.

amean values ± SD (standard deviation) from three experiments.

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Graphical abstract

Synthesis and characterization of [Ru(6-p-cym)Cl{dpa(CH2)4COOEt}][PF6] (cym = cymene;

dpa = 2,2’-dipyridylamine; 2) as well as its antitumor activity and that of [Ru(6-p-

cym)Cl(dpa)][PF6] (1) is described. The most prominent activity was observed for 1 against

caspase deficient MCF-7 breast cancer cells without affect to the immune cells and cytokine

production.

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Table 1. Crystal data and structure refinement for 2

Compound 2

Chemical formula C27H35N3O2F6PClRu

Formula weight 715.07

Crystal system Monoclinic

Space group P21/c

a (Å) 10.3288(1)

b (Å) 18.8479(2)

c (Å) 15.8486(2)

α (°) 90

β (°) 108.705(1)

γ (°) 90

V (Å3) 2922.38(6)

Z 4

Dcalc (g cm-3

) 3.200

μ (mm-1

) 0.725

F(000) 752.0

Crystal size (mm3) 0.13 × 0.11 × 0.11

Data collection

Monochromator graphite

Radiation, Mo Kα (Å) 0.71073

Temperature (K) 200

θ Range (°) 2.9–27.5

Index range –13 ≤ h ≤ 13

–24 ≤ k ≤ 24

–20 ≤ l ≤ 20

Tmin/ Tmax 0.907/ 0.921

Number of measured reflections 48139

Number of independent reflections 6708

Refinement

Refinement on F2

Data/restraints/parameters 6708 / 0 / 370

R[F2 > 4σ(F

2)] 0.025

wR(F2)

a 0.054

Goodness-of-fit on F2 0.995

Δρmin/Δρmax (e Å−3

) −0.48/ 0.53 a w = 1/[σ

2(Fo

2) + (0.0178P)

2 + 3.5701P] where P = (Fo

2 + 2Fc

2)/3

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Table 2. IC50 values (µM)a of the ruthenium(II) complexes 1, 2 and cisplatin.

Compound SRB (96 h, treatment)

518A2 SW480 8505C A253 MCF-7

1 68.9 ± 2.4 63.1 ± 1.8 85.6 ± 3.5 181.1 ± 6.3 35.2 ± 1.1

2 > 200 > 200 > 200 > 200 176.2 ± 4.5

cisplatin 1.5 ± 0.2 3.2 ± 0.2 5.0 ± 0.2 0.8 ± 0.1 2.0 ± 0.1

MCF-7

CV (h, treatment) MTT (h, treatment)

48 72 96 48 72 96

1 65.9 ± 3.1 36.6 ± 1.7 22.7 ± 1.3 >100 61.5 ± 2.0 40.8 ± 1.5

amean values ± SD (standard deviation) from three experiments.

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Highlights

[Ru(6-p-cym)Cl(dpaR)][PF6], dpa = 2,2’-dipyridylamine; R = H (1), (CH2)4COOEt (2).

Ru(II) complex 1 is more active against tumor cells than 2.

1 is the most efficient against caspase 3 deficient MCF-7 breast cancer cells.

1 is not active against cells of the adaptive immune system.

1 does not exert a general immunosuppressive effect on cytokine production.