Toxicology in Vitro 25 (2011) 652–656

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Toxicology in Vitro

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Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatinderivatives against human cancer cell lines and primary human hepatocytes

Zdenek Dvorák a,⇑, Pavel Štarha b, Zdenek Trávnícek b

a Regional Centre of Advanced Technologies and Materials, Department of Cell Biology and Genetics, Faculty of Science, Palacky University, 17. listopadu 12, 771 46Olomouc, Czech Republicb Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacky University, 17. listopadu 12, 771 46 Olomouc, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 October 2010Accepted 3 January 2011Available online 11 January 2011

Keywords:Human hepatocytesCytotoxicity6-Benzylaminopurine derivativeCarboplatin derivativeCDK inhibitor

0887-2333/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.tiv.2011.01.002

Abbreviations: A2780, human ovarian carcinomaovarian carcinoma cisplatin-resistant cell line; A5carcinoma; A9opy, E-2-[1-(9-anthryl)-3-oxo-3-prop-2methyl; cbdc, dianion of cyclobutane-1,1-dicarboxylickinase; dach, trans-1,2-diaminocyclohexane; DMF, N,Ncoordinated dimethyl sulfoxide molecule; G361, hmelanoma; HeLa, human negroid cervix epithelioCaucasian osteosarcoma; ipram, isopropylamine; Kleukaemia; Ln, variously substituted 6-benzylaminchloro-6-(3-methoxybenzyl)amino-9-isopropylpurineethylamino)-6-(4-methoxybenzyl)amino-9-isopropylpdimethoxybenzyl)amino-9-isopropylpurine; L11,zyl)amino-9-isopropylpurine; MCF7, human Caucasmeim, 1-methylimidazole; mepz, 1-methylpyrazole;2-yl)-2,5-diphenyltetrazolium bromide; ros, 2-(1-et(benzyl)amino-9-isopropylpurine.⇑ Corresponding author. Tel.: +420 58 5634903; fax

E-mail address: moulin@email.cz (Z. Dvorák).

A series of seven platinum(II) cyclobutane-1,1-dicarboxylato (cbdc) complexes {[Pt(cbdc)(Ln)2], 1–7},derived from carboplatin by a substitution of two NH3 molecules for two 2,6,9-trisubstituted 6-benzyl-aminopurine-based N-donor ligands (Ln), was studied by the MTT assay for their in vitro cytotoxic activityagainst seven human cancer cell lines, i.e. lung carcinoma (A549), cervix epithelioid carcinoma (HeLa),osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma(A2780) and its cisplatin-resistant analogue (A2780cis), and against two primary cultures of human hepa-tocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in somecases even more than cisplatin. The best results were achieved for complexes 1 (IC50 = 17.4 ± 2.0 lM)and 2 (IC50 = 14.8 ± 2.1 lV) against HOS cells, 1 (IC50 = 15.1 ± 6.8 lM), 2 (IC50 = 13.6 ± 5.2 lM) and 6(IC50 = 19.0 ± 6.6 lM) against MCF7, 6 (IC50 = 6.4 ± 0.1 lM) against A2780, and 1–6 (IC50 = 15.6 ± 4.0,12.9 ± 3.7, 15.8 ± 3.8, 16.6 ± 5.5, 22.1 ± 2.5, and 5.6 ± 1.7 lM, respectively) against A2780cis. Viability ofhuman hepatocytes was not declined by the tested complexes up to the concentration of 50 lM (for 1,3–7) and 20 lM (for 2; caused by lower solubility of this complex).

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1. Introduction words, mitigation of negative side-effects compensates for lower

Carboplatin, diamminecyclobutane-1,1-dicarboxylatoplatinum(II)complex, [Pt(cbdc)(NH3)2], followed cisplatin, cis-[PtCl2 (NH3)2], asa representative of the second generation of platinum-based anti-cancer drugs (Kelland and Farrell, 2000). Although the carrier ligand,i.e. NH3, is identical in the case of both metallotherapeutic drugs, car-boplatin is known to be less toxic as compared with cisplatin. In other

ll rights reserved.

cell line; A2780cis, human49, human Caucasian lung-enylpyridine; AM, acetoxy-acid; CDK, cyclin-dependent0-dimethylformamide; dmso,uman Caucasian malignant

id carcinoma; HOS, human-562, chronic myelogenousopurine derivatives; L8, 2-; L9, 2-(1-ethyl-2-hydroxy-urine; L10, 2-chloro-6-(2,4-2-chloro-6-(2-methoxyben-

ian breast adenocarcinoma;MTT, 3-(4,5-dimethylthiazol-hyl-2-hydroxyethylamino)-6-

: +420 58 5634905.

activity of carboplatin, which is very important for the applicationof this substance in cancer therapy.

One of our recent papers describes a series of the platinum(II)cyclobutane-1,1-dicarboxylato complexes of the [Pt(cbdc)(Ln)2]general composition, where n = 1–6 and L1 = 2-chloro-6-(2-fluoro-5-bromobenzyl)amino-9-isopropylpurine (coordinated in thecomplex 1), L2 = 2-chloro-6-(3,4-dichlorobenzyl)amino-9-isopro-pylpurine (2), L3 = 2-chloro-6-(3-bromobenzyl)amino-9-isopropyl-purine (3), L4 = 2-chloro-6-(2-trifluoromethylbenzyl)amino-9-isopropylpurine (4), L5 = 2-chloro-6-(3-trifluoromethylbenzyl)amino-9-isopropylpurine (5) and L6 = 2-chloro-6-(4-trifluorometh-ylbenzyl) amino-9-isopropylpurine (6) (Dvorák et al., 2010). Thesecomplexes were tested by the AM assay for their in vitro cytotoxicityagainst the K-562 and MCF7 human cancer cells. The complex 3(IC50 = 4.5 ± 1.0 lM) was evaluated as more active than cisplatin(IC50 = 4.7 lM) against the K-562 cells, while the complexes 1–4(IC50 = 9.0 ± 2.3, 4.3 ± 0.2, 5.0 ± 0.3, and 7.9 ± 2.3 lM, respectively)exceeded the in vitro cytotoxic activity of cisplatin (IC50 = 10.9 lM)against the MCF7 cell line. Moreover, all the compounds were severaltimes more cytotoxic as compared with carboplatin (Dvorák et al.,2010).

The above-mentioned statements encouraged us to prepare onemore carboplatin derivative with 6-benzylaminopurine-based N-donor ligand, [Pt(cbdc)(L7)2] (7). L7 symbolizes highly effective

Z. Dvorák et al. / Toxicology in Vitro 25 (2011) 652–656 653

and selective CDK inhibitor 2-(3-hydroxypropylamino)-6-(benzy-l)amino-9-isopropylpurine (bohemine), which was formerly, to-gether with similar 6-benzylaminopurine-based CDK inhibitor2-(1-ethyl-2-hydroxyethylamino)-6-(benzyl)amino-9-isopropyl-purine (roscovitine), reported as a suitable N-donor ligand of thecytotoxic active platinum(II)-dichlorido and oxalato complexes(Malon et al., 2005 and Trávnícek et al., 2010). Moreover, we alsodecided to study deeply the biological activity of all the complexes1–7 and to broaden the number of human cancer cell lines to as-sess in vitro cytotoxicity of the complexes. As it is discussed below,the tested platinum(II) complexes were in several cases morein vitro cytotoxic than cisplatin. This positive finding led us toinvestigate how the prepared substances 1–7 affect healthy non-cancer cells in the hepatotoxicity test against primary cultures ofhuman hepatocytes.

2. Materials and methods

2.1. Materials

Collagen-coated culture dishes were purchased from BD Biosci-ences (Le Pont de Claix, France). All the chemicals and solventswere purchased from commercial sources, namely Sigma–AldrichCo., Acros Organics Co., Lachema Co. and Fluka Co., and they wereused as received. [Pt(cbdc)(dmso)2] and 2-(hydroxypropylamino)-6-(benzyl)amino-9-isopropylpurine (bohemine, L7) were preparedby slightly modified synthetic procedures described by Bithaet al. (1990), and Oh et al. (1999), respectively.

2.1.1. Platinum(II) complexes 1–7The synthesis and characterization of the complexes

[Pt(cbdc)(Ln)2] (n = 1–6 for the complexes 1–6, see also Fig. 1) werereported by Dvorák et al. (2010). The complex [Pt(cbdc)(L7)2] (7;Fig. 1) was prepared according to the procedure described in thesame literature source. Briefly, [Pt(cbdc)(dmso)2] reacted in thedistilled water/isopropyl alcohol mixture (1:1, v/v) with two molarequivalents of bohemine (L7). The light grey product, which formedin two days of stirring at 90 �C, was filtered off and washed by dis-tilled water and isopropyl alcohol.

Fig. 1. The proposed structure of the complexes [Pt(cbdc)(Ln)2] (1–7), given withspecification of the R1 and R2 substituents of the 6-benzylamino-9-isopropylpurinemoiety.

[Pt(cbdc)(L7)2] (7): Anal. Calc. for C42H54N12O6Pt: C, 49.6; H, 5.4;N, 16.5. Found: C, 49.9; H, 5.3; N, 16.1%. IR (Nujol; cm�1): 526m(PtN), and 563vs (PtO). IR (KBr; cm�1): 3122m, 3068m (CHar),2970m, 2932m, 2875m (CHal), 1661s (COox), 1612vs (CN), and1551s (CC). Raman (cm�1): 3057s (CHar), 2978s, 2938vs, 2914vs(CHal), 1661w (COox), and 1611vs (CN). 1H NMR (400 MHz, DMF-d7, ppm): d 8.74 (s, 1H, C8H), 8.66 (br, 1H, N6H), 7.51 (dd, 2H,J = 7.3 Hz, 1.5 Hz, C11H, C15H), 7.27 (tt, 2H, J = 7.3 Hz, 1.5 Hz, C12H,C14H), 7.20 (tt, 1H, J = 7.3 Hz, 1.5 Hz, C13H), 6.72 (t, 1H, J = 6.2 Hz,N2H), 4.74 (d, 2H, J = 6.0 Hz, C9H), 4.72 (sp, 1H, J = 6.8 Hz, C16H),4.49 (br, 1H, C21H), 3.59 (t, 2H, J = 6.2 Hz, C21H), 3.42 (q, 2H,J = 6.2 Hz, C19H), 2.89 (t, 4H, J = 8.2 Hz, C25H, C27H), 1.77 (t, 2H,J = 8.2 Hz, C26H), 1.75 (t, 2H, C20H), 1.54 (d, 6H, J = 6.8 Hz, C17H,C18H). 13C NMR (400 MHz, DMF-d7, ppm): d 177.70 (C22, C23),160.58 (C2), 153.53 (C6), 151.01 (C4), 140.62 (C10), 139.17 (C8),128.82 (C12, C14), 128.30 (C11, C15), 127.21 (C13), 111.45 (C5),60.71 (C21), 56.73 (C24), 48.30 (C16), 44.62 (C9), 39.45 (C19), 33.45(C20), 31.38 (C25, C27), 22.06 (C17, C18), 15.85 (C26). 15N NMR(400 MHz, DMF-d7, ppm): d 179.9 (N9), 127.1 (N7), 89.8 (N6), 89.6(N2). 195Pt NMR (400 MHz, DMF-d7, ppm): d-1611.

2.2. Characterization of [Pt(cbdc)(L7)2] (7)

Elemental analyses were performed on a Fisons EA-1108 CHNS-O Elemental Analyzer (Thermo Scientific). IR spectra were recordedon a Nexus 670 FT-IR spectrometer (Thermo Nicolet) at 400–4000 cm�1 (KBr pellets) and 150–600 cm�1 (Nujol technique). Ra-man spectra were recorded using an NXR FT-Raman Module (Ther-mo Nicolet) between 150 and 3750 cm�1. 1H, 13C and 195Pt NMRspectra and 1H–1H gs-COSY, 1H–13C gs-HMQC, 1H–13C gs-HMBCtwo dimensional correlation experiments of the DMF-d7 solutionswere measured at 300 K on a Bruker 300 device. 1H spectra werealso, together with 1H–15N gs-HMBC experiments, recorded at340 K. 1H and 13C spectra were adjusted against the signals of tet-ramethylsilane (Me4Si). 195Pt spectra were calibrated againstK2PtCl6 in D2O found at 0 ppm. 1H–15N gs-HMBC experiments wereobtained at natural abundance and calibrated against the residualsignals of DMF adjusted to 8.03 ppm (1H) and 104.7 ppm (15N). Thesplitting of proton resonances in the reported 1H spectra is definedas s = singlet, d = doublet, t = triplet, q = quadruplet, sp = septuplet,br = broad band, dd = doublet of doublets, tt = triplet of triplets.

2.3. Human cancer cell lines

Human cancer cell lines were purchased from European Collec-tion of Cell Cultures (ECACC). The following cell lines were em-ployed in the current study: human ovarian carcinoma cells(A2780; ECACC No. 93112519), human ovarian carcinoma cis-platin-resistant cells (A2780cis; ECACC No. 93112517), humanCaucasian malignant melanoma (G361; ECACC No. 88030401), hu-man Caucasian breast adenocarcinoma (MCF7; ECACC No.86012803), human Caucasian lung carcinoma (A549; ECACC No.86012804), human Caucasian osteosarcoma (HOS; ECACC No.87070202) and human negroid cervix epithelioid carcinoma (HeLa;ECACC No. 93021013). The cells were cultured according to theECACC instructions. Briefly, culture medium was DMEM (for celllines HOS, MCF7, HeLa, A549), RPMI1640 (for cell lines A2780and A2780cis) and McCoys (for cell line G361). The medium wassupplemented with penicillin, streptomycin and 10% of foetal bo-vine serum. The cells were maintained at 37 �C and 5% CO2 in ahumidified incubator.

2.4. Primary cultures of human hepatocytes

Human hepatocytes were isolated from liver tissue, resectedfrom multiorgan donors. A tissue acquisition protocol was in

654 Z. Dvorák et al. / Toxicology in Vitro 25 (2011) 652–656

accordance with the requirements issued by a local ethical com-mission in the Czech Republic. Human liver tissues used in thisstudy were obtained from two donors: LH31 (male, 28 years) andLH32 (male, 70 years). Hepatocytes were isolated by two-step col-lagenase perfusion and the cells were plated on collagen-coatedculture dishes using cell density of 14 � 104 cells/cm2 (Pichard-Garcia et al., 2002). The culture medium was Williams and HAMsF-12 (1:1) supplemented with penicillin, streptomycin, ascorbicacid, linoleic acid, holo-transferin, ethanolamine, glucagon, insulin,dexamethasone, pyruvate, glucose, glutamine, amphotericin. Themedium was enriched for plating with 2% foetal calf serum (v/v).The medium was exchanged for a serum-free medium the day afterand the culture was stabilized for additional 24 h. Thereafter, thecells were ready for treatments. The cultures were maintained at37 �C and 5% CO2 in a humidified incubator.

2.5. Cytotoxicity assays

Human cancer cell lines and primary cultures of human hepato-cytes were treated with the tested compounds for 24 h, using mul-ti-well culture plates of 96 wells (Vrzal et al., 2010). The followingcompounds – cisplatin, oxaplatin, carboplatin and 1–7 were appliedto the cells up to the concentration of 50 lM. In parallel, the cellswere treated with vehicle (DMF; 0.1%, v/v) and Triton X-100 (1%,v/v) to assess the minimal (i.e. positive control) and maximal (i.e.negative control) cell damage, respectively. Cells were incubatedwith MTT for 3–4 h, and after removal of the medium and washingthe cells with PBS, formazan dye was dissolved in DMSO contain-ing 1% of ammonia. Absorbance was measured spectrophotometri-cally at 540 nm (TECAN, Schoeller Instruments LLC). The data wereexpressed as the percentage of viability, when 100% and 0% repre-sent the treatments with DMF and Triton X-100, respectively. Thedata on human hepatocytes were obtained from two independentcultures (obtained from two different donors). The data from can-cer cell lines were acquired from three independent cell passages.

Fig. 2. Cytotoxicity of the tested compounds against the A549, HeLa and HOS celllines. The cells were plated at 96-well dishes and cultured according to themanufacturer instructions. The tested compounds were applied to the cells for 24 hin concentrations ranging from 0.01 to 50 lM. As the positive and negative control,Triton-X100 (1% v/v) and vehicle (DMF; 0.1% v/v) were used, respectively. Thecytotoxicity was assessed by the MTT test, and the values of IC50 were calculated.The bar graphs show the IC50 values against (Panel A) A549 cells, (Panel B) HeLacells and (Panel C) HOS cells. The data are expressed as a mean ± SD from threeindependent experiments.

3. Results

3.1. Synthesis and characterization of the complex [Pt(cbdc)(L7)2] (7)

The complex 7 was prepared according to the formerly de-scribed synthetic procedure employed for the synthesis of thecomplexes 1–6 (Dvorák et al., 2010), as described in Section 2.1.1(Fig. 1). The characterization of the compound, by the methodssummarized in Section 2.2., proved a composition correspondingto the [Pt(cbdc)(L7)2] formula. The results of the detailed NMRspectroscopic study indicated that the cbdc anion is bidentate-coordinated to the metal centre through two oxygen atoms andboth L7 molecules are bound to the Pt(II) atom through their N7atoms.

3.2. Cytotoxicity in human cancer cell lines

We examined cytotoxicity of the prepared platinum(II) com-plexes and three standard platinum-derived cytotoxic compounds(i.e. cisplatin, oxaliplatin, carboplatin) in seven different commercialhuman cancer cell lines, including A2780, A2780cis, G361, MCF7,A549, HOS and HeLa. The obtained results (IC50 ± SD values givenin lM) are summarized in Figs. 2–4, as well as in Table S1 of Sup-plementary material. No cytotoxic effects of the tested compoundswere found in human A549 cells. Due to the limited solubility ofthe compounds, the estimate IC50 values were >50 lM for the com-plexes 1, 3–7, and cisplatin. The IC50 value for complex 2 was>20 lM (Fig. 2A). The cytotoxicity of the tested compounds wasweak in HeLa cancer cells. With the exception of cisplatin

(IC50 = 39.9 ± 4.6 lM), 1 (IC50 = 47.0 ± 1.5 lM) and 4 (IC50 = 40.2 ±4.2 lM), the estimated IC50 values for the tested compounds

Fig. 3. Cytotoxicity of the tested compounds against the G361 and MCF7 cell lines.The cells were plated at 96-well dishes and cultured according to the manufacturerinstructions. The tested compounds were applied to the cells for 24 h in concen-trations ranging from 0.01 to 50 lM. As the positive and negative control, Triton-X100 (1% v/v) and vehicle (DMF; 0.1% v/v) were used, respectively. The cytotoxicitywas assessed by the MTT test, and the values of IC50 were calculated. The bar graphsshow the IC50 values against (Panel A) G361 cells and (Panel B) MCF7 cells. Thedata are expressed as a mean ± SD from three independent experiments.

Fig. 4. Cytotoxicity of the tested compounds against the A2780 and A2780cis celllines. The cells were plated at 96-well dishes and cultured according to themanufacturer instructions. The tested compounds were applied to the cells for 24 hin concentrations ranging from 0.01 to 50 lM. As the positive and negative control,Triton-X100 (1% v/v), and vehicle (DMF; 0.1% v/v) were used, respectively. Thecytotoxicity was assessed by the MTT test, and the values of IC50 were calculated.The bar graphs show the IC50 values in A2780 and A2780cis cells. The data areexpressed as a mean ± SD from three independent experiments.

Z. Dvorák et al. / Toxicology in Vitro 25 (2011) 652–656 655

exceeded their solubility (Fig. 2B). Similarly, in osteosarcoma cells,the significant cytotoxic effects were detected only for cisplatin(IC50 = 34.2 ± 6.4 lM), 1 (IC50 = 17.4 ± 2.0 lM) and 2 (IC50 = 14.8 ±2.1 lM), whereas the other compounds were found to be non-cyto-toxic within the tested concentration range (Fig. 2C). The best cyto-toxic potency of the tested compounds was attained in the case ofG361 (Fig. 3A) and MCF7 (Fig. 3B) cells. The exception was thecomplex 7, which was not cytotoxic in either cell line in the con-centration up to 50 lM.

We also examined comparative effects of the tested compoundsin cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) hu-man ovarian carcinoma cell line. Cisplatin exerted significantlyhigher cytotoxicity in A2780 cells (IC50 = 11.5 ± 1.6 lM) as com-pared to A2780cis cells (IC50 = 30.3 ± 6.1 lM), as expected. Besidethe complex 7, which was not cytotoxic, all the complexes dis-played significant cytotoxicity in both A2780 and A2780cis cells(Fig. 4).

We have also found that none of the employed cancer cell lineswere sensitive to oxaliplatin (IC50 > 50 lM) and carboplatin(IC50 > 1 lM) up to the concentration given by the compoundsolubility.

3.3. Cytotoxicity in primary cultures of human hepatocytes

In none of the primary human hepatocyte cultures, the IC50 val-ues were reached by the tested compounds. This was due to thelimited solubility of these compounds. Therefore, the estimatedIC50 values for all the tested complexes were IC50 > 50 lM, withthe exception of carboplatin (IC50 > 1 lM) and 2 (IC50 > 20 lM).

4. Discussion

In the present work, we tested in vitro cytotoxicity of the[Pt(cbdc)(Ln)2] complexes (1–7) and compared the obtained resultswith those of cisplatin, oxaliplatin and carboplatin. The cytotoxicitywas evaluated in seven commercial human cancer cell lines(A2780, A2780cis, G361, MCF7, A549, HOS and HeLa) derived fromvarious types of cancer (vide supra) and in two primary cultures ofhuman hepatocytes. The cells were challenged for 24 h with thetested compounds and the MTT test was used to assess the result-ing cytotoxicity.

We have found out that oxaliplatin (IC50 > 50 lM) and carbo-platin (IC50 > 1 lM) did not cause significant cell damage in any cellline used, because the cytotoxic potential of oxaliplatin and carbo-platin was restricted by their limited solubility. Therefore, it makessense to test platinum-based derivatives with increased solubilityand clinically reasonable cytotoxicity in vitro. Indeed, in the currentpaper we describe different in vitro cytotoxicity of the tested plat-inum-derivatives.

The tested complexes were not cytotoxic against human Cauca-sian lung carcinoma cells A549, and very weak cytotoxicity was ob-served against human negroid cervix epithelioid carcinoma cells(HeLa) and human Caucasian osteosarcoma cells (HOS). In contrast,significant dose-dependent cytotoxicity of the tested compoundswas observed against human Caucasian malignant melanoma cells(G361), human Caucasian breast adenocarcinoma cells (MCF7) andhuman ovarian carcinoma cell line (A2780) and its cisplatin-resis-tant variant (A2780cis).

The cytotoxic effect of the studied compounds 1–7 in primarycultures of human hepatocytes, an in vitro model considered asthe most suitable for studies of xenobiotic cytotoxicity and metab-olism, was studied as well. We used primary human hepatocytesobtained from two human liver donors. The obtained results ofin vitro hepatotoxicity revealed that the studied platinum(II)

656 Z. Dvorák et al. / Toxicology in Vitro 25 (2011) 652–656

complexes 1–7 did not affect healthy human hepatocytes up to theconcentration of 50 lM.

The presented platinum(II) cyclobutane-1,1-dicarboxylato com-plexes follow the recently reported highly in vitro cytotoxicdichlorido {cis-[PtCl2(Ln)2]; Malon et al., 2005 and oxalato{[Pt(ox)(Ln)2]; Štarha et al., 2010; Trávnícek et al., 2010; Vrzalet al., 2010} complexes prepared in our laboratory. The cis-[PtCl2(Ln)2] complexes were tested against the G361, HOS, MCF7and K-562 (not used in this work) cancer cells. We performed acomparison of the calculated IC50(cisplatin)/IC50(complex) ratio ofthe formerly tested dichlorido and oxalato complexes with thecyclobutan-1,1-dicarboxylato ones reported in this work to betterdescribe and compare in vitro cytotoxicity of these types of com-plexes. The ratio of the most active cis-[PtCl2(ros)2] complex withthe coordinated CDK inhibitor roscovitine equals 3.0 (it means thatthe tested complex is three times more active than cisplatin)against G361 and HOS and 5.0 against MCF7 (Malon et al., 2005).The oxalato complexes were tested against the same cancer celllines as herein described complexes 1–7 (Trávnícek et al., 2010;Vrzal et al., 2010). Again we can calculate the ratio of the in vitrocytotoxicity against respective cancer cell line of cisplatin versusthe appropriate oxalato complex, which equals 1.7 against G361for [Pt(ox)(L8)2], 1.7 against HeLa for [Pt(ox)(L9)2], 5.4 againstMCF7 for [Pt(ox)(L10)2], 9.5 against HOS for [Pt(ox)(L11)2], 3.6against A2780 for [Pt(ox)(L8)2] and 9.5 against A2780cis for[Pt(ox)(L8)2]. It has to be mentioned that all of these ratios arehigher (it means more in vitro cytotoxic active) as compared with1–7, in particular with 0.6 against G361 for 6, 1.0 against HeLafor 4, 1.4 against MCF7 for 2, 2.3 against HOS for 2, 1.8 againstA2780 for 6 and 5.4 against A2780cis for 6.

Another important finding is that the studied platinum(II) com-plexes 1–7 overcome cisplatin resistance. It was demonstrated bythe IC50(A2780cis)/IC50(A2780) ratio, which is equal to 2.6 for cis-platin and 0.9 (1), 1.1 (2), 1.4 (3), 1.2 (4), 1.2 (5) and 0.9 (6). Wecan compare these results with those reported in the literaturesources for different in vitro cytotoxic mononuclear platinum(II)complexes. The above-mentioned [Pt(ox)(L8)2] complex has theIC50(A2780cis)/IC50(A2780) ratio of 1.0, those of cis-[PtCl2(ipram)(-meim)] and cis-[PtCl2(ipram)(mepz)] complexes are equal to 1.9,and 2.3, respectively (Pantoja et al., 2006), while that of the pyro-phosphato (pyro) complex [Pt(pyro)(dach)] was determined to be2.4 (Bose et al., 2008). As for six platinum(II) dichlorido complexeswith 2-aminomethylpyrrolidine-based ligands, their IC50(A2780-cis)/IC50(A2780) ratios equal 1.1–4.9 (Diakos et al., 2009). The lastexample, which could be mentioned concerning in vitro cytotoxic-ity against A2780 and A2780cis cells, is cis-[PtCl2(A9opy)] with theIC50(A2780cis)/IC50(A2780) ratio of 1.4 (Marqués-Gallego et al.,2009). On the other hand, the effectiveness of tested compoundsagainst cisplatin-resistant cells is not, in principle, general, sincecisplatin resistance comprises multiple mechanisms, including de-creased drug uptake, increased efflux, increased inactivation byglutathione, increased excision of DNA adducts etc.

In conclusion, we demonstrated that the tested carboplatinderivatives 1–7 may be classified as tentatively promising antican-cer drugs, because they were found to be toxic against human can-cer cells but not against healthy human hepatic cells. Further, theplatinum(II) complexes of the general formula [Pt(cbdc)(Ln)2] sig-nificantly more effectively inhibit growth of both A2780 andA2780cis cancer cells as compared with several recently reportedmononuclear platinum(II) complexes.

Conflict of interest

We declare no conflict of interest.

Acknowledgements

This work was financially supported by the Grant Agency of theCzech Republic (Grant Nos.: GACR503/10/0579 and GACR304/10/0149), the Ministry of Education, Youth and Sports of the CzechRepublic (a Grant No.: MSM6198959218), Faculty of Science of Pa-lacky University in Olomouc (a Grant No.: PrF_2010_018), and bythe Operational Program Research and Development for Innova-tions – European Social Fund (a Grant No.: CZ.1.05/2.1.00/03.0058). We thank Mr. Lukáš Dvorák for his help with the synthe-sis of the complexes, Ms. Radka Novotná for IR and Raman spectrameasurements and Dr. Igor Popa for NMR spectra measurementsand interpretation.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.tiv.2011.01.002.

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