Investigation of the pharmacological profiles of dinuclear metal complexes as novel, potent and selective cytotoxic agents against ras-transformed cells.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 897–906

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Investigation of the pharmacological profiles of dinuclear metal complexes as novel, potent and selective cytotoxic agents against ras-transformed cells ˘ a,∗ , A. Tansu Koparal a , Kadriye Benkli b R. Beklem Bostancıoglu a b

Anadolu University, Faculty of Sciences, Department of Biology, 26470 Eskis¸ehir, Turkey Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskisehir, Turkey

a r t i c l e

i n f o

a b s t r a c t

Article history:

Around the world scientists try to design successful cures against still incurable dis-

Received 10 December 2013

eases, especially cancers. New targets for prevention and new agents for therapy

Received in revised form

need to be identified. We synthesized novel metal complexes [Au(L1)(L2)Pt]Cl2 and

3 March 2014

[Ru(L1)2(L2)Pt]Cl2 for determining their cytotoxic and apoptotic effects. The complexes are

Accepted 5 March 2014

synthesized by using 1,8-diaminonaphthalene (L1), and bis-1,4-di[([1,10]phenanthroline-5-

Available online 15 March 2014

il)aminomethyl]cyclohexane (L2) as ligands. This is the first study to examine these metals and these molecules in cancer treatment. We elucidated the effects of test compounds

Keywords:

with embryonic rat fibroblast-like cells (F2408) and H-ras oncogene activated embryonic

Au(III)

rat fibroblast-like cancer cells (5RP7). Results showed that our complexes are more effec-

Ru(II)

tive than cisplatin to kill ras-transformed cells. Although the [Au(L1)(L2)Pt]Cl2 compound

Pt(II)

showed a cytotoxic potency higher than [Ru(L1)2(L2)Pt]Cl2 against cancer cells, it proved

Antitumoral activity

to be almost five times less effective in decreasing cell viability over healthy cells. Au(III)

Apoptosis

compound selectively targets the cancer cells but not the healthy cells.

Selective toxicity

1.

Introduction

It has been known for many years that metal ions such as gold(III), ruthenium(III) or platinum(II) exert a wide range of biological activities including the suppression of cell proliferation, anti-tumorigenic activity, prevention against HIV or malaria, and various apoptotic effects. Today, platinum-based chemotherapy is routinely employed in the clinic as cancer chemotherapy (Carcelli et al., 2013; Grazul and Budzisz, 2009).

∗

© 2014 Elsevier B.V. All rights reserved.

The similarity between platinum(II) and isoelectronic gold(III) suggests that certain complexes of the latter metal centre may also have promising cytotoxicity in cancer cell (Martínez et al., 2010). Human tumours harbour multiple genetic alterations in genes controlling cell growth, differentiation and survival. These genetic changes comprise activation of oncogenes and inactivation of tumour suppressor genes. One of the most frequently activated oncogenes in human cancer is the Ras gene family. So, in humans, about 30% of the tumours carry

Corresponding author. Tel.: +90 222 3350580/4839; fax: +90 222 3204910. ˘ E-mail addresses: [email protected] (R.B. Bostancıoglu), [email protected] (A.T. Koparal), [email protected] (K. Benkli).

http://dx.doi.org/10.1016/j.etap.2014.03.003 1382-6689/© 2014 Elsevier B.V. All rights reserved.

898


Ras mutations (Duursma and Agami, 2003). The oncogenic forms of Hras, Kras and Nras are detected in specific tumour types. For example, Hras mutations are high in bladder carcinomas, gliomas, adenocarcinomas of the lung, oral cancers, squamous cell carcinoma, cervical cancer, breast and colon cancer (Karnoub and Weinberg, 2008; Murugan et al., 2012; Vageli et al., 1996). The oncogene H-ras plays an important role in tumour growth, maintenance and metastasis (Perkins et al., 2003). Thus, efforts for finding new compounds that are capable of killing cancer cells carrying Ras mutations are becoming increasingly important. Induction of apoptosis in cancer cells is a key targeting approach for most anti-tumour therapies including chemotherapy, g-irradiation, ´ immunotherapy, or cytokines (Czene et al., 2002; Srdic-Raji c´ et al., 2011), which all are dependent on agents that trigger the apoptotic pathway, thus inhibiting the development of cancer. Cisplatin is one of the most successful drug for the treatment of testicular and ovarium tumours, whereas carboplatin and oxaplatin being the second generation platinum drugs, several drugs based on metallic compounds such as gallium, germanium, early-transition metal complexes such as titanium, vanadium, molybdenum, and also late-transition metal complexes especially ruthenium, platinum and gold have all shown considerable potential for anticancer activity via apoptosis (De Vizcaya-Ruiz et al., 2000; Kim et al., 2006). In recent years, new metal complexes have been identified as very promising class of antitumor active compounds (Buschini et al., 2009; Carcelli et al., 2013; Palanichamy et al., 2012). DNA is an important potential biological target for many metalbased anticancer agents. These complexes readily interact with DNA and they can form stable mono-functional adducts with DNA. These adducts affect the DNA confirmation and are recognized by downstream cellular mechanisms (Nováková et al., 2009). Exposure to cytotoxic compounds like cisplatin leads to increase in P53 protein levels. P53 may play a dual role after exposure to cytotoxic treatment and then it might also cause cisplatin resistance (Brabec and Kasparkova, 2005; Wu et al., 2013). Additionally, several studies showed that Au(III) effects the permeability of the cells and the function of ion channels, receptors and enzymes (Suwalsky et al., 2010). Furthermore, instead of hampering the DNA replication, Au(III) drugs have been found to induce tumour cell death by inhibiting the function of intracellular proteins and/or altering the ˘ et al., 2012; Wein normal mitochondrial function (Bostancıoglu et al., 2011). The toxicity of the anti-cancer drugs is an important parameter that correlates with the life quality of patients due to the severe side effects following the administration. The studies to modulate the toxicity levels of anticancer drugs have led to the development of transition metals and phenanthroline-based compounds. 1,10-Phenanthroline (phen) is known to be the parent of an important class of chelating agents and has structural features such as rigid planar chemistry, hydrophobicity, and electron-poor heteroaromatic coordinate system, the nitrogen atoms of which are placed to act cooperatively in cation binding. Some metal complexes containing 1,10-phenanthroline are also known to intercalate into the DNA. Additionally, several metal complexes with 1,10-phenanthroline and natural products incorporating this heterocyclic nucleus

possess interesting anticancer properties (Bencini and Lippolis, 2010; Dotsenko et al., 2011; Liu and Sun, 2012; Palanichamy et al., 2012; Sun et al., 2012; Zhao et al., 1998). In the present work, we investigated the anticancer activity of dinuclear [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 complexes including 1,8-diaminonaphthalene (L1), and bis1,4-di[([1,10]phenanthroline-5-il)aminomethyl]cyclohexane (L2) as ligands. Our work is the first study to examine the dinuclear complexes with these ligands and these molecules in cancer treatment.

2.

Materials and methods

2.1.

Chemical synthesis

RuCl3 ·(H2 O)n, LiCl, NaAuCl4 ·H2 O and ligands were purchased from Aldrich organics. All other reagents used were purchased from commercial suppliers without further purification. The compounds were checked for purity by TLC on silica gel 60 F254 (Merck). Melting points were determined by using a Gallenkamp MPD350.BM2.5 digital melting point apparatus. Spectroscopic data were recorded on the following instruments: elemental analyses were performed on a CHNS-O Carlo Erba EA 1108 elemental analyzer; IR with a Shimadzu 470 IR spectrophotometer; 1 H NMR spectra (ı(ppm) Hz) were run on a Varian (300 MHz) spectrometer in CDCl3 or d6-DMSO; Fast atom bombardment (FAB) was recorded with a Finnigan Mat 95 mass spectrometer with meta-nitrobenzylalcohol as matrix. The compounds were checked for purity by TLC on silica gel 60 F254 (Merck). In this research, [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 complexes were synthesized using 1,8-diaminonaphthalene (L1), and bis-1,4-di[([1,10]phenanthroline-5-il)aminomethyl] cyclohexane (L2) as ligands. The characterization of the intermediate and final compounds arising from this work was carried out with a variety of spectroscopic methods (1 H NMR, IR, MS, and elemental analysis). A 2:1 molar ratio L(2 mmol)/RuCl3 ·nH2 O was dissolved in DMF (8 ml) and the mixture refluxed under argon for 8 h. The reaction mixture was cooled down, then acetone was added and the mixture kept 24 h at 0 ◦ C. The precipitate was filtered off, washed with water and diethyl ether, and dried in vacuum desiccator. Purification of the compounds was monitored by thin layer chromatography and 1 H NMR. [Ru(L1)]Cl2 complex was synthesized according to classical literature methods (Pyle et al., 1989). To prepare [Ru(L1)(L2)](PF6 )2 ; [Ru(L1)]Cl2 (1 mmol) and bis-1,4-di[([1,10] phenanthroline-5-il)aminomethyl]cyclohexane (L2) (1 mmol) in MeOH was heated to reflux under argon for 6 h. After cooling to room temperature, MeOH was evaporated. The complex was precipitated as a dark colour solid by the addition of saturated aqueous NH4 PF6 . The product was filtered and washed with water and diethyl ether. The complex was dried in vacuum for 24 h and stored in a desiccator. Recrystallization from appropriate acetonitrile/diethyl ether or acetone/toluene was used for purification. The compound was characterized by IR, 1 H NMR and mass spectroscopy. The mixture of [Ru(L1)(L2)](PF6 )2 solution in EtOH and K2 PtCl4 in H2 O was stirred at room temperature for 12 h. The


precipitate was filtered and washed several times with water and diethyl ether, and dried in vacuum desiccator. All steps were used to synthesize [Au(L1)(L2)Pt]Cl2 are shown in Scheme 1. The elucidation of the structures of the synthesized compounds was performed by IR, 1 H NMR and mass spectroscopy and elemental analyses. [Ru(L1)2 (L2)Pt]Cl2 : Anal. Calcd. For C52 H50 Cl2 N10 PtRu: C, 52.84%; H, 4.26%; N, 11.85%. Found: C, 52.81%; H, 4.35%; N, 11.28%. Selected IR (KBr)max (cm−1 ): 3445, 3148, 3100, 3087 (Ar N H, C H), 1640, 1632, 1456, 1392 (C N, C C), 327 (Pt Cl), 287 (Pt N). 1 H NMR(300 MHz) (DMSO-d6 ) d(ppm): 3.18–3.26 (4H, m), 3.97–4.14 (2H, m), 4.20–4.48 (8H, m), 7.55–8.13 (13H, m), 8.50–8.70 (7H, m), 8.80–8.92 (8H, m), 9.10–9.21 (2H, m). MS (ES): m/z: 1212 [M+1]. [Au(L1)(L2)Pt]Cl2 : Anal. Calcd. For C42 H42 Cl2 N8 PtAu: C, 44.97%; H, 3.77%; N, 9.99%. Found: C, 44.91%; H, 3.85%; N, 9.88%. IR (KBr)max (cm−1 ): 3425, 3147, 3076 (Ar N H, C H), 1625, 1619, 1442 (C N, C C), 324 (Pt Cl), 288 (Pt N). 1 H NMR (300 MHz) (DMSO-d6 ) d(ppm): 3.33–3.38 (4H, m), 4.11–4.16 (2H, m), 4.21–4.45 (8H, m), 7.63–8.12 (13H, m), 8.54–8.70 (7H, m), 8.89–8.93 (8H, m), 9.15–9.27 (2H, m). MS (ES): m/z: 1308 [M+1].

for 12 h. The assay was performed as previously described ˘ et al., 2012). (Bostancıoglu

2.2.3.

Determination of cytotoxicity by MTT assay

3-(4,5-Dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was used to evaluate viability of cells (Mosmann, 1983). Compounds were dissolved freshly in dimethyl sulfoxide (DMSO) and screened at a range of concentrations in fresh liquid medium using cancerous and normal cells. Both cell lines were cultured in DMEM with 10% FCS in the presence of 0.5–160 ␮M of [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 . After 48 h incubation, MTT assay was performed as previously ˘ et al. (2012). Cisplatin is used as described by Bostancıoglu positive control, due to its previous use for the treatment or palliation of many types of cancers including non-small cell lung, ovarian and testicular tumours (Alderden et al., 2006).

2.2.2.

Statistical data analysis

Cells and cell culture assays

Rat embryonic fibroblast-like cells (F2408) and H-ras oncogeneactivated rat embryonic fibroblast-like cancer cell line (5RP7) were obtained from JCRB (Japan Cenn Bank). Cells were maintained as a monolayer in Dulbecco’s Modified Eagle’s Media containing 10% (v/v) Foetal Bovine Serum (Sigma), PenicilinStreptomycin (Sigma) and sodium hydrogen carbonate. Cells were incubated at 37 ◦ C in a humidified atmosphere of 5% (v/v) CO2 in air.

2.2.1.

Detection of apoptosis by DNA laddering

Chromosomal DNA from drug-treated cells was extracted using DNA ladder kit (Roche) in accordance with the manufacturer’s protocols. Briefly, the cells were cultured in 25 cm2 flasks, total volume 5 ml of medium per flask, in the absence or presence of IC50 value of [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 compounds for 12 h at 37 ◦ C. After incubation, the cell layer was rinsed twice with 5 ml cold PBS: to extract DNA, samples were incubated in binding/lysis buffer for 10 min at RT. The lysate was mixed with isopropanol and pipetted into filter tube. The samples were centrifuged at 8000 rpm and the flow-through was discarded. Followed by adding wash buffer to the filter tube, the tubes were centrifuged as described before. The DNA was recovered by centrifugation. Gel electrophoresis was carried out in 1% agarose at 90 V for 50 min. DNA fragments were visualized under UV on the gel stained with ethidium bromide.

2.3. 2.2.

899

Detection of apoptosis by DAPI staining

Degradation of DNA into specific fragmentation pattern (consist of apoptotic bodies) is characteristic feature of apoptosis (Daniel and DeCoster, 2004). To explore whether the compounds induced apoptosis, we determined the apoptotic nuclei and condensed chromatin by morphological DAPI staining. DAPI is a DNA-staining agent and it binds to grooves on the surface of the DNA helix. Apoptotic nuclei can be identified by the reduced nuclear size, condensed chromatin gathering at the periphery of the nuclear membrane, or a total fragmented morphology of nuclear bodies (Daniel and DeCoster, 2004). After 24 h of seeding, cells were treated with various doses of [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 and cisplatin

The SPSS software has been used for the statistical analyses of assessment of the MTT assays. Data were evaluated using one-way ANOVA followed by Tukey test. A value of p < 0.05 was considered significant.

3.

Results

3.1.

Chemistry

In this research, [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 complexes have been synthesized by using 1,8-diaminonaphthalene (L1), and bis-1,4-di[([1,10]phenanthroline-5-il)aminomethyl]cyclohexane (L2) as ligands. The characterization of the intermediate and final compounds arising from this work was carried out by a variety of spectroscopic methods including 1 H NMR, IR, mass, and elemental analysis. IR spectrums of the ligands showed two bands between 3127 and 2786 cm−1 due to n(N H), 1654–1635 cm−1 due to n(C N) which are different from the spectrum of [Ru(L1)2 (L2)Pt]Cl2 . In general, the carbonyl stretches are relatively insensitive to changes in the metal centre, and its coordination environment on the carbonyl stretch is a secondary effect. In the 1 H NMR spectra, the peaks of aromatic protons for [Ru(L1)2 (L2)Pt]Cl2 were observed about 6.54–10.12 ppm as multiplets. Due to greater aromatic planar and stronger deshielding effect, the phenanthroline protons show large downfield shifts. Elemental analyses results and especially the M+1 values in ES/MS spectra are as expected.

3.2. Inhibition of proliferation by new metal complexes on cells Cell viability was assessed by using the MTT reduction assay that measures mitochondrial dehydrogenase activity. The assay detects cleavage of 3-(4,5-dimethyl-2thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by active

900


+ L

N

NaAuCl4.2H2O

Au N

H2O, 30oC

N

N'

N'

N'

N

N

LiCl, DMF

Ru N

Cl N

N'

N'

N'

N'

N

B

=

N'

N

L

N'

N

N'

N

3+

Pt Cl

N'

N'

N'

N'

Cl Pt Cl

N

N

N'

N'

N

Ru N

N'

Cl

N

N

N'

N

N

Cl

Ru

RuCl3.nH2O

Au

N'

Au

N

H2O, 30oC

Cl

N

H2O, 30oC

K2PtCl2.2H2O

N

N

K2PtCl2.2H2O

L

Cl

N H

N H

N

N'

H2N

=H N 2

Scheme 1 – Synthesis of the compounds.

mitochondria in viable cells to insoluble formazan product. Both of the new compounds caused a dose- and time-dependent decrease in cell survival following the 24 and 48 h recovery period, resulting in 50% cell death of 5RP7 (rat transformed fibroblast cancer cell line) at 1 ␮M [Au(L1)(L2)Pt]Cl2 and 10 ␮M [Ru(L1)2 (L2)Pt]Cl2 after 24 h. Treatment of the F2408 cells with 5 ␮M and 20 ␮M [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 respectively, for 24 h decreased the MTT-reducing activity to 50%. Although the [Au(L1)(L2)Pt]Cl2 compound showed a cytotoxic potency higher than [Ru(L1)2 (L2)Pt]Cl2 against 5RP7, it proved to be almost five times less effective in decreasing cell viability over F2408 (healthy cells). On the other hand, under the same experimental conditions, cisplatin – the most widely used anticancer metallodrug – was also evaluated. IC50 average values of our new compounds were found to be about 15–20 fold lower than cisplatin on H-ras oncogene activated cells (Fig. 1A–F).

3.3.

Detection of apoptosis DAPI staining

To determine whether apoptosis is involved in [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 induced toxicity, DNA fragmentation and condensation, one hallmark of apoptosis was checked (Danial and Korsmeyer, 2004). Cisplatin was known to induce apoptotic cell death in cancer cells (Kim et al., 2006). Both of the cell lines were exposed to compounds and cisplatin for 12 h and then cells were stained with DAPI. As shown Figs. 2 and 3, DAPI staining showed that apoptotic morphological features such as cell shrinkage, condensation and apoptotic bodies were present in cisplatin, [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 treated cells, while no significant changes in control and in solvent group were observed. Furthermore, [Au(L1)(L2)Pt]Cl2 triggered the apoptosis to a greater extent than the [Ru(L1)2 (L2)Pt]Cl2 did in both of the cell lines, as indicated by the apoptotic bodies.


901

Fig. 1 – (A and B). Comparison between cytotoxic activity of Cisplatin, [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 on 5RP7 (A-C-E) and F2408 (B-D-F) cell lines as determined in the MTT assay. Dose-response curves of the anti-proliferative effect of compounds for MTT assays performed after 24 and 48 h exposures. The results are expressed as the mean SD. * Indicates significant difference from the control group by the Tukey test (p < 0.05). Cisplatin are used as a positive control.

3.4.

Detection of apoptosis By DNA laddering

Chromosomal DNA fragmentation at internucleosomal sites is the earliest nuclear hallmark and the most extensive way of studying the biochemical event of apoptosis. Internucleosomal DNA fragmentation is frequently used to show existence

of apoptosis in biomedical and pharmacological research (Zhu and Wang, 1997). After we analyzed the DNA fragmentation in the form of a laddering pattern, we further confirmed the apoptosis inducer role of [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 on both healthy and transformed cancer cells. Fig. 4 demonstrates that the classical 180-base pair integers of the DNA

902


Fig. 2 – The effect of [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 on apoptosis detection in 5RP7 were assessed by fluorescence staining with DAPI, a substance which specifically binds DNA. (A) Control cells and (B) DMSO solvent control cells. (C1) 5RP7 cells were treated with Cisplatin 10 ␮М, (C2) Cisplatin 20 ␮М, (C3) Cisplatin 40 ␮М, (C4) Cisplatin 80 ␮М, (D1) [Au(L1)(L2)Pt]Cl2 5 ␮М, (D2) [Au(L1)(L2)Pt]Cl2 10 ␮М, (D3) [Au(L1)(L2)Pt]Cl2 20 ␮М, (E1) [Ru(L1)2 (L2)Pt]Cl2 5 ␮М, (E2) [Ru(L1)2 (L2)Pt]Cl2 10 ␮М, (E2) [Ru(L1)2 (L2)Pt]Cl2 20 ␮М. Images shown are representative of independent triplicate assays. Scale bar, 20 ␮m.

fragmentation are typical of apoptosis. It is evident that [Au(L1)(L2)Pt]Cl2 could induce nucleosomal ladder intensities of the ladders were higher than in the [Ru(L1)2 (L2)Pt]Cl2 .

4.

Discussion

Creating metal complexes as anticancer drugs is a promising approach for hampering tumour progression, yet not easy

because the accumulation of metal ions in the body can cause undesirable effects. As the main aim of chemotherapy is destruction of tumour cells without any undue influence on healthy cells, it should not be forgotten that although metals cause desirable effects like alleviating cell division, they are also potentially carcinogenic (Grazul and Budzisz, 2009). Our results show that [Au(L1)(L2)Pt]Cl2 selectively targets the transformed cancer cells but not the healthy ones

903


Fig. 3 – The effect of [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 on apoptosis detection in F2408 were assessed by fluorescence staining with DAPI, a substance which specifically binds DNA. (A) Control cells and (B) DMSO solvent control cells. (C1) F2408 cells were treated with Cisplatin 10 ␮М, (C2) Cisplatin 20 ␮М, (C3) Cisplatin 40 ␮М, (C4) Cisplatin 80 ␮М, (D1) [Au(L1)(L2)Pt]Cl2 5 ␮М, (D2) [Au(L1)(L2)Pt]Cl2 10 ␮М, (D3) [Au(L1)(L2)Pt]Cl2 20 ␮М, (E1) [Ru(L1)2 (L2)Pt]Cl2 5 ␮М, (E2) [Ru(L1)2 (L2)Pt]Cl2 10 ␮М, (E2) [Ru(L1)2 (L2)Pt]Cl2 20 ␮М. Images shown are representative of independent triplicate assays. Scale bar, 20 ␮m.

Table 1 – IC50 Values of [Au(L1)(L2)Pt]Cl2 , [Ru(L1)2 (L2)Pt]Cl2 and Cisplatin on 5RP7 and F2408. 24 h

[Au(L1)(L2)Pt]Cl2 [Ru(L1)2 (L2)Pt]Cl2 Cisplatin

48 h

5RP7

F2408

5RP7

1 ␮M 10 ␮M 20–40 ␮M

5 ␮M 20 ␮M 80 ␮M

0.5–1 ␮M 5–10 ␮M 2.5–5 ␮M

F2408 0.5–1 ␮M 2.5 ␮M 2.5 ␮M

904


Fig. 4 – Measurement of apoptosis on F2408 and 5RP7 cells after the IC50 values of compounds [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 for 12 h, by visualization of internucleosomal DNA ladders. Line M represents results obtained with DNA size markers, lines c and c (control) with untreated cells, lanes p and p (pozitive control) with cells treated with etaposite 25 ␮M, lines 1 and 1 for cells treated with [Au(L1)(L2)Pt]Cl2 (5 ␮M for F2408-1 ␮M for 5RP7), lines 2 and 2 for cells treated with [Ru(L1)2 (L2)Pt]Cl2 (20 ␮M for F2408-10 ␮M for 5RP7).

(Fig. 1). Additionally, significant increase in the levels of apoptosis was observed, directly proportional to the exposure of the cells to these compounds (Fig. 2). Furthermore, at low concentrations, [Au(L1)(L2)Pt]Cl2 proved to be more toxic and apoptotic than [Ru(L1)2 (L2)Pt]Cl2 and reference cisplatin (Figs. 1–3). In the presented study, embryonic rat fibroblast-like cells (F2408) and H-ras oncogene activated embryonic rat fibroblastlike cancer cell line (5RP7) were chosen. Pozzatti et al. (1986) demonstrated that second-passage rat embryo cells transformed by the ras oncogene alone are both tumorigenic and highly metastatic when injected into nude mice. Storer et al. (1988) studies also suggest that the phenotypic conversion of normal cells to tumorigenic cells with experimental metastatic potential by ras and myc oncogenes can be completed within 3–4 cell divisions after transfection. Activated ras oncogene transfection into suitable recipient cells has been shown to induce both a tumorigenic and a metastatic phenotype (Boylan et al., 1990; Thorgeirsson et al., 1985). We previously published the synthesis, characterization, cytotoxicity, anti-tumour properties and the mitochondrialdependent apoptotic effects of the Au(III) and Pt(II) complexes of 1,10-phenanthroline. The results shown in this report indicate that metal complexes with phenanthroline, especially with Au(III), are able to induce apoptosis in lung cancer cells (Bostancıoglu et al., 2012). Additionally, our group studied Au(III) and Pt(II) metal complexes of 1,10-phenanthroline5,6-dione on rat glioma C6 cells. Especially, an inhibition in the cancer cell proliferation was observed as the Au(III) metal complex being more potent than Pt(II) metal complex (Dikmen et al., 2011). Here, we further investigate the dinuclear complexes of [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 by using 1,8-diaminonaphthalene (L1), and bis-1,4di[([1,10]phenanthroline-5-il)aminomethyl]cyclohexane (L2) as ligands. We observed that these complexes have cytotoxic and apoptotic activity especially in transformed cancer cells. Our results showed that dinuclear metal

complexes – both Au(III) and Pt(II) – are more effective than mononuclear complexes of Au(III) and Pt(II). Furthermore, Au(III) complex has selective toxicity against transformed cancer cells. This observation is of great importance, especially because the chemotherapeutics, which are being used for the treatment of several cancer types nowadays, have many side effects that cause decline in patient’s life quality. Ideally, chemotherapeutic drugs should specifically target only cancer cells and should provide a basis for selective ablation of cancer cells by inducing cytotoxicity at the target location, with minimum collateral damage to normal/healthy cells (Kosmider et al., 2004; Nell et al., 2009). Our compounds’ IC50 values can be seen in Table 1. There have been a number of reports highlighting the use of transition metal complexes as anticancer agents (Bencini and Lippolis, 2010). The clinical success of cisplatin is limited due to its significant side effects. These limitations have stimulated a search for other transition metal complexes which are more effective, but bear fewer side effects. There are many examples, such as Pérez et al. (1999) reported that trans-PtCl2 (isopropylamine)(dimethylamine) and trans-PtCl2 (isopropylamine)(butylamine) complexes induce apoptosis in cis-DDP resistant murine keratinocytes transformed by the H-ras oncogene. Ruthenium compounds are the most widely developed alternatives, and some of ruthenium complexes have entered clinical trials. These compounds exhibit only low side effects (Kandioller et al., 2009). Nell et al. (2009) exhibited modified complex of [Au(dppe)]Cl2 is capable of selectively killing cancer cells. A series of new gold(I) and gold(III) complexes were synthesized and characterized; in addition, anti-proliferative effect was investigated on A2780S and cisplatin resistant cells by Maiore et al. (2011). Gold(III) compounds, [Au(Phen)Cl2 ]PF6 , [Au(DPQ)Cl2 ]PF6 , and [Au(DPQC)Cl2 ]PF6 , (Phen = 1,10[Au(DPPZ)Cl2 ]PF6 , phenanthroline,DPQ = dipyrido[3,2d:2 ,3 -f]quinoxaline, DPPZ = dipyrido[3,2-a:2 ,3 -c] phenazine, DPQC = dipyrido[3,2d:2 ,3 -f] cyclohexyl quinoxaline) that exhibited anticancer activity in both cisplatin sensitive and cisplatin resistant ovarian cancer cells (Palanichamy et al., 2012). There is a scarcity of data concerning the anticancer activity of 1,10-phenanthroline (phen) and its derivatives (Bencini and Lippolis, 2010; Cusumano et al., 2006; Deegan et al., 2007; Wesselinova et al., 2009). Messori et al. (2007) reported that gold(III) phen complex is quite cytotoxic. Wein et al. (2011) synthesized a gold(III) complex possessing 5,6-dimethyl1,10-phenanthroline and evaluated for its potential use an anticancer therapeutic. The stability of gold(III) anticancer agents is therefore an important determinant of clinical efficacy. Our complexes are very stable and they can easily be dissolved in DMSO at small volumes.

5.

Conclusion

Our results indicate that [Au(L1)(L2)Pt]Cl2 and [Ru(L1)2 (L2)Pt]Cl2 complexes might be promising candidates for potential anticancer therapeutics due to their specific selective toxicity on transformed cancer cells. Especially the Au(III)/Pt(II) dinuclear complexes should be researched


thoroughly. Further research is currently under progress in our laboratory to elucidate the mechanism of inhibition of cell proliferation in different cancer cells, and to improve the potency of these chemicals through modifications with different organometallics.

Conflict of interest The authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgements This work was financed by a grant from the Scientific and Technological Research Council of Turkey (TUBITAK-SBAG-108S206) and NOVARTISNovartis (2007 Novartis Pharmaceutical and Medicinal Chemistry Drug Design and Development Research Support). The authors are grateful to Anadolu University, Commission of Scientific Research Projects for financial support to Project 070304. Authors also thank deeply to Assoc. Prof. Dr. Caghan Kizil (Helmholtz Association, German Center for Neurodegenerative Diseases (DZNE) Dresden, Germany) for proof-reading of the manuscript.

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