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Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes
Toxicology in Vitro 25 (2011) 652–656
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Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes Zdeneˇk Dvorˇák a,⇑, Pavel Štarha b, Zdeneˇk Trávnícˇek b a ´ University, 17. listopadu 12, 771 46 Regional Centre of Advanced Technologies and Materials, Department of Cell Biology and Genetics, Faculty of Science, Palacky Olomouc, Czech Republic b ´ University, 17. listopadu 12, 771 46 Olomouc, Czech Republic Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacky
a r t i c l e
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Article history: Received 4 October 2010 Accepted 3 January 2011 Available online 11 January 2011 Keywords: Human hepatocytes Cytotoxicity 6-Benzylaminopurine derivative Carboplatin derivative CDK inhibitor
a b s t r a c t 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-benzylaminopurine-based N-donor ligands (Ln), was studied by the MTT assay for their in vitro cytotoxic activity against 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 hepatocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in some cases 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 of human 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). Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Carboplatin, diamminecyclobutane-1,1-dicarboxylatoplatinum(II) complex, [Pt(cbdc)(NH3)2], followed cisplatin, cis-[PtCl2 (NH3)2], as a representative of the second generation of platinum-based anticancer drugs (Kelland and Farrell, 2000). Although the carrier ligand, i.e. NH3, is identical in the case of both metallotherapeutic drugs, carboplatin is known to be less toxic as compared with cisplatin. In other
Abbreviations: A2780, human ovarian carcinoma cell line; A2780cis, human ovarian carcinoma cisplatin-resistant cell line; A549, human Caucasian lung carcinoma; A9opy, E-2-[1-(9-anthryl)-3-oxo-3-prop-2-enylpyridine; AM, acetoxymethyl; cbdc, dianion of cyclobutane-1,1-dicarboxylic acid; CDK, cyclin-dependent kinase; dach, trans-1,2-diaminocyclohexane; DMF, N,N0 -dimethylformamide; dmso, coordinated dimethyl sulfoxide molecule; G361, human Caucasian malignant melanoma; HeLa, human negroid cervix epithelioid carcinoma; HOS, human Caucasian osteosarcoma; ipram, isopropylamine; K-562, chronic myelogenous leukaemia; Ln, variously substituted 6-benzylaminopurine derivatives; L8, 2chloro-6-(3-methoxybenzyl)amino-9-isopropylpurine; L9, 2-(1-ethyl-2-hydroxyethylamino)-6-(4-methoxybenzyl)amino-9-isopropylpurine; L10, 2-chloro-6-(2,4dimethoxybenzyl)amino-9-isopropylpurine; L11, 2-chloro-6-(2-methoxybenzyl)amino-9-isopropylpurine; MCF7, human Caucasian breast adenocarcinoma; meim, 1-methylimidazole; mepz, 1-methylpyrazole; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; ros, 2-(1-ethyl-2-hydroxyethylamino)-6(benzyl)amino-9-isopropylpurine. ⇑ Corresponding author. Tel.: +420 58 5634903; fax: +420 58 5634905. E-mail address: [email protected] (Z. Dvorˇák). 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.01.002
words, mitigation of negative side-effects compensates for lower activity of carboplatin, which is very important for the application of 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-fluoro5-bromobenzyl)amino-9-isopropylpurine (coordinated in the complex 1), L2 = 2-chloro-6-(3,4-dichlorobenzyl)amino-9-isopropylpurine (2), L3 = 2-chloro-6-(3-bromobenzyl)amino-9-isopropylpurine (3), L4 = 2-chloro-6-(2-trifluoromethylbenzyl)amino-9isopropylpurine (4), L5 = 2-chloro-6-(3-trifluoromethylbenzyl) amino-9-isopropylpurine (5) and L6 = 2-chloro-6-(4-trifluoromethylbenzyl) amino-9-isopropylpurine (6) (Dvorˇák et al., 2010). These complexes were tested by the AM assay for their in vitro cytotoxicity against 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 several times more cytotoxic as compared with carboplatin (Dvorˇák et al., 2010). The above-mentioned statements encouraged us to prepare one more carboplatin derivative with 6-benzylaminopurine-based Ndonor ligand, [Pt(cbdc)(L7)2] (7). L7 symbolizes highly effective
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and selective CDK inhibitor 2-(3-hydroxypropylamino)-6-(benzyl)amino-9-isopropylpurine (bohemine), which was formerly, together with similar 6-benzylaminopurine-based CDK inhibitor 2-(1-ethyl-2-hydroxyethylamino)-6-(benzyl)amino-9-isopropylpurine (roscovitine), reported as a suitable N-donor ligand of the cytotoxic active platinum(II)-dichlorido and oxalato complexes ˇ et al., 2005 and Trávnícˇek et al., 2010). Moreover, we also (Malon decided to study deeply the biological activity of all the complexes 1–7 and to broaden the number of human cancer cell lines to assess in vitro cytotoxicity of the complexes. As it is discussed below, the tested platinum(II) complexes were in several cases more in vitro cytotoxic than cisplatin. This positive finding led us to investigate how the prepared substances 1–7 affect healthy noncancer cells in the hepatotoxicity test against primary cultures of human hepatocytes.
2. Materials and methods 2.1. Materials Collagen-coated culture dishes were purchased from BD Biosciences (Le Pont de Claix, France). All the chemicals and solvents were purchased from commercial sources, namely Sigma–Aldrich Co., Acros Organics Co., Lachema Co. and Fluka Co., and they were used as received. [Pt(cbdc)(dmso)2] and 2-(hydroxypropylamino)6-(benzyl)amino-9-isopropylpurine (bohemine, L7) were prepared by slightly modified synthetic procedures described by Bitha et al. (1990), and Oh et al. (1999), respectively.
2.1.1. Platinum(II) complexes 1–7 The synthesis and characterization of the complexes [Pt(cbdc)(Ln)2] (n = 1–6 for the complexes 1–6, see also Fig. 1) were reported by Dvorˇák et al. (2010). The complex [Pt(cbdc)(L7)2] (7; Fig. 1) was prepared according to the procedure described in the same literature source. Briefly, [Pt(cbdc)(dmso)2] reacted in the distilled water/isopropyl alcohol mixture (1:1, v/v) with two molar equivalents of bohemine (L7). The light grey product, which formed in two days of stirring at 90 °C, was filtered off and washed by distilled water and isopropyl alcohol.
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[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; cm1): 526m (PtN), and 563vs (PtO). IR (KBr; cm1): 3122m, 3068m (CHar), 2970m, 2932m, 2875m (CHal), 1661s (COox), 1612vs (CN), and 1551s (CC). Raman (cm1): 3057s (CHar), 2978s, 2938vs, 2914vs (CHal), 1661w (COox), and 1611vs (CN). 1H NMR (400 MHz, DMFd7, 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 CHNSO Elemental Analyzer (Thermo Scientific). IR spectra were recorded on a Nexus 670 FT-IR spectrometer (Thermo Nicolet) at 400– 4000 cm1 (KBr pellets) and 150–600 cm1 (Nujol technique). Raman spectra were recorded using an NXR FT-Raman Module (Thermo Nicolet) between 150 and 3750 cm1. 1H, 13C and 195Pt NMR spectra and 1H–1H gs-COSY, 1H–13C gs-HMQC, 1H–13C gs-HMBC two dimensional correlation experiments of the DMF-d7 solutions were measured at 300 K on a Bruker 300 device. 1H spectra were also, together with 1H–15N gs-HMBC experiments, recorded at 340 K. 1H and 13C spectra were adjusted against the signals of tetramethylsilane (Me4Si). 195Pt spectra were calibrated against K2PtCl6 in D2O found at 0 ppm. 1H–15N gs-HMBC experiments were obtained at natural abundance and calibrated against the residual signals of DMF adjusted to 8.03 ppm (1H) and 104.7 ppm (15N). The splitting of proton resonances in the reported 1H spectra is defined as 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 Collection of Cell Cultures (ECACC). The following cell lines were employed in the current study: human ovarian carcinoma cells (A2780; ECACC No. 93112519), human ovarian carcinoma cisplatin-resistant cells (A2780cis; ECACC No. 93112517), human Caucasian malignant melanoma (G361; ECACC No. 88030401), human 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 the ECACC instructions. Briefly, culture medium was DMEM (for cell lines HOS, MCF7, HeLa, A549), RPMI1640 (for cell lines A2780 and A2780cis) and McCoy´s (for cell line G361). The medium was supplemented with penicillin, streptomycin and 10% of foetal bovine serum. The cells were maintained at 37 °C and 5% CO2 in a humidified incubator. 2.4. Primary cultures of human hepatocytes
Fig. 1. The proposed structure of the complexes [Pt(cbdc)(Ln)2] (1–7), given with specification of the R1 and R2 substituents of the 6-benzylamino-9-isopropylpurine moiety.
Human hepatocytes were isolated from liver tissue, resected from multiorgan donors. A tissue acquisition protocol was in
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accordance with the requirements issued by a local ethical commission in the Czech Republic. Human liver tissues used in this study were obtained from two donors: LH31 (male, 28 years) and LH32 (male, 70 years). Hepatocytes were isolated by two-step collagenase perfusion and the cells were plated on collagen-coated culture dishes using cell density of 14 104 cells/cm2 (Pichard´s Garcia et al., 2002). The culture medium was Williams and HAM F-12 (1:1) supplemented with penicillin, streptomycin, ascorbic acid, linoleic acid, holo-transferin, ethanolamine, glucagon, insulin, dexamethasone, pyruvate, glucose, glutamine, amphotericin. The medium was enriched for plating with 2% foetal calf serum (v/v). The medium was exchanged for a serum-free medium the day after and the culture was stabilized for additional 24 h. Thereafter, the cells were ready for treatments. The cultures were maintained at 37 °C and 5% CO2 in a humidified incubator. 2.5. Cytotoxicity assays Human cancer cell lines and primary cultures of human hepatocytes were treated with the tested compounds for 24 h, using multi-well culture plates of 96 wells (Vrzal et al., 2010). The following compounds – cisplatin, oxaplatin, carboplatin and 1–7 were applied to the cells up to the concentration of 50 lM. In parallel, the cells were 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 incubated with MTT for 3–4 h, and after removal of the medium and washing the cells with PBS, formazan dye was dissolved in DMSO containing 1% of ammonia. Absorbance was measured spectrophotometrically at 540 nm (TECAN, Schoeller Instruments LLC). The data were expressed as the percentage of viability, when 100% and 0% represent the treatments with DMF and Triton X-100, respectively. The data on human hepatocytes were obtained from two independent cultures (obtained from two different donors). The data from cancer cell lines were acquired from three independent cell passages. 3. Results 3.1. Synthesis and characterization of the complex [Pt(cbdc)(L7)2] (7) The complex 7 was prepared according to the formerly described synthetic procedure employed for the synthesis of the complexes 1–6 (Dvorˇák et al., 2010), as described in Section 2.1.1 (Fig. 1). The characterization of the compound, by the methods summarized in Section 2.2., proved a composition corresponding to the [Pt(cbdc)(L7)2] formula. The results of the detailed NMR spectroscopic study indicated that the cbdc anion is bidentatecoordinated to the metal centre through two oxygen atoms and both L7 molecules are bound to the Pt(II) atom through their N7 atoms. 3.2. Cytotoxicity in human cancer cell lines We examined cytotoxicity of the prepared platinum(II) complexes and three standard platinum-derived cytotoxic compounds (i.e. cisplatin, oxaliplatin, carboplatin) in seven different commercial human cancer cell lines, including A2780, A2780cis, G361, MCF7, A549, HOS and HeLa. The obtained results (IC50 ± SD values given in lM) are summarized in Figs. 2–4, as well as in Table S1 of Supplementary material. No cytotoxic effects of the tested compounds were found in human A549 cells. Due to the limited solubility of the compounds, the estimate IC50 values were >50 lM for the complexes 1, 3–7, and cisplatin. The IC50 value for complex 2 was >20 lM (Fig. 2A). The cytotoxicity of the tested compounds was weak in HeLa cancer cells. With the exception of cisplatin
Fig. 2. Cytotoxicity of the tested compounds against the A549, HeLa and HOS cell lines. The cells were plated at 96-well dishes and cultured according to the manufacturer instructions. The tested compounds were applied to the cells for 24 h in 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. The cytotoxicity 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) HeLa cells and (Panel C) HOS cells. The data are expressed as a mean ± SD from three independent experiments.
(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
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Fig. 4. Cytotoxicity of the tested compounds against the A2780 and A2780cis cell lines. The cells were plated at 96-well dishes and cultured according to the manufacturer instructions. The tested compounds were applied to the cells for 24 h in 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. The cytotoxicity 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 are expressed as a mean ± SD from three independent experiments.
3.3. Cytotoxicity in primary cultures of human hepatocytes In none of the primary human hepatocyte cultures, the IC50 values were reached by the tested compounds. This was due to the limited solubility of these compounds. Therefore, the estimated IC50 values for all the tested complexes were IC50 > 50 lM, with the exception of carboplatin (IC50 > 1 lM) and 2 (IC50 > 20 lM). 4. Discussion
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 manufacturer instructions. The tested compounds were applied to the cells for 24 h in concentrations ranging from 0.01 to 50 lM. As the positive and negative control, TritonX100 (1% v/v) and vehicle (DMF; 0.1% v/v) were used, respectively. The cytotoxicity was assessed by the MTT test, and the values of IC50 were calculated. The bar graphs show the IC50 values against (Panel A) G361 cells and (Panel B) MCF7 cells. The data are expressed as a mean ± SD from three independent experiments.
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-cytotoxic within the tested concentration range (Fig. 2C). The best cytotoxic potency of the tested compounds was attained in the case of G361 (Fig. 3A) and MCF7 (Fig. 3B) cells. The exception was the complex 7, which was not cytotoxic in either cell line in the concentration up to 50 lM. We also examined comparative effects of the tested compounds in cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) human ovarian carcinoma cell line. Cisplatin exerted significantly higher cytotoxicity in A2780 cells (IC50 = 11.5 ± 1.6 lM) as compared to A2780cis cells (IC50 = 30.3 ± 6.1 lM), as expected. Beside the complex 7, which was not cytotoxic, all the complexes displayed significant cytotoxicity in both A2780 and A2780cis cells (Fig. 4). We have also found that none of the employed cancer cell lines were sensitive to oxaliplatin (IC50 > 50 lM) and carboplatin (IC50 > 1 lM) up to the concentration given by the compound solubility.
In the present work, we tested in vitro cytotoxicity of the [Pt(cbdc)(Ln)2] complexes (1–7) and compared the obtained results with those of cisplatin, oxaliplatin and carboplatin. The cytotoxicity was evaluated in seven commercial human cancer cell lines (A2780, A2780cis, G361, MCF7, A549, HOS and HeLa) derived from various types of cancer (vide supra) and in two primary cultures of human hepatocytes. The cells were challenged for 24 h with the tested compounds and the MTT test was used to assess the resulting cytotoxicity. We have found out that oxaliplatin (IC50 > 50 lM) and carboplatin (IC50 > 1 lM) did not cause significant cell damage in any cell line used, because the cytotoxic potential of oxaliplatin and carboplatin was restricted by their limited solubility. Therefore, it makes sense to test platinum-based derivatives with increased solubility and clinically reasonable cytotoxicity in vitro. Indeed, in the current paper we describe different in vitro cytotoxicity of the tested platinum-derivatives. The tested complexes were not cytotoxic against human Caucasian lung carcinoma cells A549, and very weak cytotoxicity was observed against human negroid cervix epithelioid carcinoma cells (HeLa) and human Caucasian osteosarcoma cells (HOS). In contrast, significant dose-dependent cytotoxicity of the tested compounds was observed against human Caucasian malignant melanoma cells (G361), human Caucasian breast adenocarcinoma cells (MCF7) and human ovarian carcinoma cell line (A2780) and its cisplatin-resistant variant (A2780cis). The cytotoxic effect of the studied compounds 1–7 in primary cultures of human hepatocytes, an in vitro model considered as the most suitable for studies of xenobiotic cytotoxicity and metabolism, was studied as well. We used primary human hepatocytes obtained from two human liver donors. The obtained results of in vitro hepatotoxicity revealed that the studied platinum(II)
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complexes 1–7 did not affect healthy human hepatocytes up to the concentration of 50 lM. The presented platinum(II) cyclobutane-1,1-dicarboxylato complexes follow the recently reported highly in vitro cytotoxic ˇ et al., 2005 and oxalato dichlorido {cis-[PtCl2(Ln)2]; Malon {[Pt(ox)(Ln)2]; Štarha et al., 2010; Trávnícˇek et al., 2010; Vrzal et al., 2010} complexes prepared in our laboratory. The cis[PtCl2(Ln)2] complexes were tested against the G361, HOS, MCF7 and K-562 (not used in this work) cancer cells. We performed a comparison of the calculated IC50(cisplatin)/IC50(complex) ratio of the formerly tested dichlorido and oxalato complexes with the cyclobutan-1,1-dicarboxylato ones reported in this work to better describe and compare in vitro cytotoxicity of these types of complexes. The ratio of the most active cis-[PtCl2(ros)2] complex with the coordinated CDK inhibitor roscovitine equals 3.0 (it means that the tested complex is three times more active than cisplatin) ˇ et al., 2005). against G361 and HOS and 5.0 against MCF7 (Malon The oxalato complexes were tested against the same cancer cell lines as herein described complexes 1–7 (Trávnícˇek et al., 2010; Vrzal et al., 2010). Again we can calculate the ratio of the in vitro cytotoxicity against respective cancer cell line of cisplatin versus the appropriate oxalato complex, which equals 1.7 against G361 for [Pt(ox)(L8)2], 1.7 against HeLa for [Pt(ox)(L9)2], 5.4 against MCF7 for [Pt(ox)(L10)2], 9.5 against HOS for [Pt(ox)(L11)2], 3.6 against 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 are higher (it means more in vitro cytotoxic active) as compared with 1–7, in particular with 0.6 against G361 for 6, 1.0 against HeLa for 4, 1.4 against MCF7 for 2, 2.3 against HOS for 2, 1.8 against A2780 for 6 and 5.4 against A2780cis for 6. Another important finding is that the studied platinum(II) complexes 1–7 overcome cisplatin resistance. It was demonstrated by the IC50(A2780cis)/IC50(A2780) ratio, which is equal to 2.6 for cisplatin and 0.9 (1), 1.1 (2), 1.4 (3), 1.2 (4), 1.2 (5) and 0.9 (6). We can compare these results with those reported in the literature sources for different in vitro cytotoxic mononuclear platinum(II) complexes. The above-mentioned [Pt(ox)(L8)2] complex has the IC50(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 pyrophosphato (pyro) complex [Pt(pyro)(dach)] was determined to be 2.4 (Bose et al., 2008). As for six platinum(II) dichlorido complexes with 2-aminomethylpyrrolidine-based ligands, their IC50(A2780cis)/IC50(A2780) ratios equal 1.1–4.9 (Diakos et al., 2009). The last example, which could be mentioned concerning in vitro cytotoxicity against A2780 and A2780cis cells, is cis-[PtCl2(A9opy)] with the IC50(A2780cis)/IC50(A2780) ratio of 1.4 (Marqués-Gallego et al., 2009). On the other hand, the effectiveness of tested compounds against cisplatin-resistant cells is not, in principle, general, since cisplatin resistance comprises multiple mechanisms, including decreased drug uptake, increased efflux, increased inactivation by glutathione, increased excision of DNA adducts etc. In conclusion, we demonstrated that the tested carboplatin derivatives 1–7 may be classified as tentatively promising anticancer drugs, because they were found to be toxic against human cancer cells but not against healthy human hepatic cells. Further, the platinum(II) complexes of the general formula [Pt(cbdc)(Ln)2] significantly more effectively inhibit growth of both A2780 and A2780cis cancer cells as compared with several recently reported mononuclear platinum(II) complexes. Conflict of interest We declare no conflict of interest.
Acknowledgements This work was financially supported by the Grant Agency of the Czech Republic (Grant Nos.: GACR503/10/0579 and GACR304/10/ 0149), the Ministry of Education, Youth and Sports of the Czech Republic (a Grant No.: MSM6198959218), Faculty of Science of Palacky University in Olomouc (a Grant No.: PrF_2010_018), and by the Operational Program Research and Development for Innovations – European Social Fund (a Grant No.: CZ.1.05/2.1.00/ 03.0058). We thank Mr. Lukáš Dvorˇák for his help with the synthesis of the complexes, Ms. Radka Novotná for IR and Raman spectra measurements and Dr. Igor Popa for NMR spectra measurements and interpretation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tiv.2011.01.002. References Bitha, P., Morton, G.O., Dunne, T.S., Delos Santos, E.F., Lin, Y., Boone, S.R., Haltiwanger, R.C., Pierpoint, C.G., 1990. (Malonato)bis[sulfinylbis[methane]S]platinum(II) compounds: versatile synthons for a new general synthesis of antitumor symmetrical and dissymmetrical (Malonato)platinum(II) complexes. Inorganic Chemistry 29, 645–652. Bose, R.N., Maurmann, L., Mishur, R.J., Yasui, L., Gupta, S., Grayburn, W.S., Hofstetter, H., Salley, T., 2008. Non-DNA-binding platinum anticancer agents: cytotoxic activities of platinum-phosphato complexes towards human ovarian cancer cells. PNAS 105, 18314–18319. Diakos, C.I., Zhang, M., Beale, P.J., Fenton, R.R., Hambley, T.W., 2009. Synthesis, characterisation and in vitro cytotoxicity studies of a series of chiral platinum(II) complexes based on the 2-aminomethylpyrrolidine ligand: X-ray crystal structure of [PtCl2(R-dimepyrr)] (R-dimepyrr = N-dimethyl-2(R)aminomethylpyrrolidine). European Journal of Medicinal Chemistry 44, 2807– 2814. Dvorˇák, L., Popa, I., Štarha, P., Trávnícˇek, Z., 2010. In vitro cytotoxic active platinum(ii) complexes derived from carboplatin and involving purine derivatives. European Journal of Inorganic Chemistry 3441, 3448. Kelland, L.-R., Farrell, N.-P., 2000. Platinum-based Drugs in Cancer Therapy. Humana Press, Totowa, New Jersey. ˇ , M., Trávnícˇek, Z., Marek, R., Strnad, M., 2005. Synthesis, spectral study and Malon cytotoxicity of platinum(II) complexes with 2, 9-disubstituted-6benzylaminopurines. Journal of Inorganic Biochemistry 99, 2127–2138. Marqués-Gallego, P., Kalayda, G.V., Jaehde, U., den Dulk, H., Brouwer, J., Reedijk, J., 2009. Cellular accumulation and DNA platination of two new platinum(II) anticancer compounds based on anthracene derivatives as carrier ligands. Journal of Inorganic Chemistry 103, 791–796. Oh, C.H., Lee, S.C., Lee, K.S., Woo, E.R., Hong, C.Y., Yang, B.S., Baek, D.J., Cho, J.H., 1999. Synthesis and biological activities of C-2, N-9 substituted 6-benzylaminopurine derivatives as cyclin-dependent kinase inhibitor. Archiv der Pharmazie 332, 187–190. Pantoja, E., Gallipoli, A., van Zutphen, S., Tooke, D.M., Spek, A.L., Navarro-Ranninger, C., Reedijk, J., 2006. In vitro antitumor activity and interaction with DNA model bases of cis-[PtCl2(iPram)(azole)] complexes and comparison with their trans analogues. Inorganica Chimica Acta 359, 4335–4342. Pichard-Garcia, L., Gerbal-Chaloin, S., Ferrini, J.B., Fabre, J.M., Maurel, P., 2002. Use of long-term cultures of human hepatocytes to study cytochrome P450 gene expression. Methods in Enzymology 357, 311–321. Štarha, P., Trávnícˇek, Z., Popa, I., 2010. Platinum(II) oxalato complexes with adeninebased carrier ligands showing significant in vitro antitumor activity. Journal of Inorganic Biochemistry 104, 639–647. Trávnícˇek, Z., Štarha, P., Popa, I., Vrzal, R., Dvorˇák, Z., 2010. Roscovitine-based CDK inhibitors acting as N-donor ligands in the platinum(II) oxalato complexes: preparation, characterization and in vitro cytotoxicity. European Journal of Medicinal Chemistry 45, 4609–4614. Vrzal, R., Štarha, P., Dvorˇák, Z., Trávnícˇek, 2010. Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(ii) and palladium(ii) oxalato complexes with adenine derivatives as carrier ligands. Journal of Inorganic Biochemistry 104, 1130–1132.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes Zdeneˇk Dvorˇák a,⇑, Pavel Štarha b, Zdeneˇk Trávnícˇek b a ´ University, 17. listopadu 12, 771 46 Regional Centre of Advanced Technologies and Materials, Department of Cell Biology and Genetics, Faculty of Science, Palacky Olomouc, Czech Republic b ´ University, 17. listopadu 12, 771 46 Olomouc, Czech Republic Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacky
a r t i c l e
i n f o
Article history: Received 4 October 2010 Accepted 3 January 2011 Available online 11 January 2011 Keywords: Human hepatocytes Cytotoxicity 6-Benzylaminopurine derivative Carboplatin derivative CDK inhibitor
a b s t r a c t 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-benzylaminopurine-based N-donor ligands (Ln), was studied by the MTT assay for their in vitro cytotoxic activity against 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 hepatocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in some cases 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 of human 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). Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Carboplatin, diamminecyclobutane-1,1-dicarboxylatoplatinum(II) complex, [Pt(cbdc)(NH3)2], followed cisplatin, cis-[PtCl2 (NH3)2], as a representative of the second generation of platinum-based anticancer drugs (Kelland and Farrell, 2000). Although the carrier ligand, i.e. NH3, is identical in the case of both metallotherapeutic drugs, carboplatin is known to be less toxic as compared with cisplatin. In other
Abbreviations: A2780, human ovarian carcinoma cell line; A2780cis, human ovarian carcinoma cisplatin-resistant cell line; A549, human Caucasian lung carcinoma; A9opy, E-2-[1-(9-anthryl)-3-oxo-3-prop-2-enylpyridine; AM, acetoxymethyl; cbdc, dianion of cyclobutane-1,1-dicarboxylic acid; CDK, cyclin-dependent kinase; dach, trans-1,2-diaminocyclohexane; DMF, N,N0 -dimethylformamide; dmso, coordinated dimethyl sulfoxide molecule; G361, human Caucasian malignant melanoma; HeLa, human negroid cervix epithelioid carcinoma; HOS, human Caucasian osteosarcoma; ipram, isopropylamine; K-562, chronic myelogenous leukaemia; Ln, variously substituted 6-benzylaminopurine derivatives; L8, 2chloro-6-(3-methoxybenzyl)amino-9-isopropylpurine; L9, 2-(1-ethyl-2-hydroxyethylamino)-6-(4-methoxybenzyl)amino-9-isopropylpurine; L10, 2-chloro-6-(2,4dimethoxybenzyl)amino-9-isopropylpurine; L11, 2-chloro-6-(2-methoxybenzyl)amino-9-isopropylpurine; MCF7, human Caucasian breast adenocarcinoma; meim, 1-methylimidazole; mepz, 1-methylpyrazole; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; ros, 2-(1-ethyl-2-hydroxyethylamino)-6(benzyl)amino-9-isopropylpurine. ⇑ Corresponding author. Tel.: +420 58 5634903; fax: +420 58 5634905. E-mail address: [email protected] (Z. Dvorˇák). 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.01.002
words, mitigation of negative side-effects compensates for lower activity of carboplatin, which is very important for the application of 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-fluoro5-bromobenzyl)amino-9-isopropylpurine (coordinated in the complex 1), L2 = 2-chloro-6-(3,4-dichlorobenzyl)amino-9-isopropylpurine (2), L3 = 2-chloro-6-(3-bromobenzyl)amino-9-isopropylpurine (3), L4 = 2-chloro-6-(2-trifluoromethylbenzyl)amino-9isopropylpurine (4), L5 = 2-chloro-6-(3-trifluoromethylbenzyl) amino-9-isopropylpurine (5) and L6 = 2-chloro-6-(4-trifluoromethylbenzyl) amino-9-isopropylpurine (6) (Dvorˇák et al., 2010). These complexes were tested by the AM assay for their in vitro cytotoxicity against 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 several times more cytotoxic as compared with carboplatin (Dvorˇák et al., 2010). The above-mentioned statements encouraged us to prepare one more carboplatin derivative with 6-benzylaminopurine-based Ndonor ligand, [Pt(cbdc)(L7)2] (7). L7 symbolizes highly effective
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and selective CDK inhibitor 2-(3-hydroxypropylamino)-6-(benzyl)amino-9-isopropylpurine (bohemine), which was formerly, together with similar 6-benzylaminopurine-based CDK inhibitor 2-(1-ethyl-2-hydroxyethylamino)-6-(benzyl)amino-9-isopropylpurine (roscovitine), reported as a suitable N-donor ligand of the cytotoxic active platinum(II)-dichlorido and oxalato complexes ˇ et al., 2005 and Trávnícˇek et al., 2010). Moreover, we also (Malon decided to study deeply the biological activity of all the complexes 1–7 and to broaden the number of human cancer cell lines to assess in vitro cytotoxicity of the complexes. As it is discussed below, the tested platinum(II) complexes were in several cases more in vitro cytotoxic than cisplatin. This positive finding led us to investigate how the prepared substances 1–7 affect healthy noncancer cells in the hepatotoxicity test against primary cultures of human hepatocytes.
2. Materials and methods 2.1. Materials Collagen-coated culture dishes were purchased from BD Biosciences (Le Pont de Claix, France). All the chemicals and solvents were purchased from commercial sources, namely Sigma–Aldrich Co., Acros Organics Co., Lachema Co. and Fluka Co., and they were used as received. [Pt(cbdc)(dmso)2] and 2-(hydroxypropylamino)6-(benzyl)amino-9-isopropylpurine (bohemine, L7) were prepared by slightly modified synthetic procedures described by Bitha et al. (1990), and Oh et al. (1999), respectively.
2.1.1. Platinum(II) complexes 1–7 The synthesis and characterization of the complexes [Pt(cbdc)(Ln)2] (n = 1–6 for the complexes 1–6, see also Fig. 1) were reported by Dvorˇák et al. (2010). The complex [Pt(cbdc)(L7)2] (7; Fig. 1) was prepared according to the procedure described in the same literature source. Briefly, [Pt(cbdc)(dmso)2] reacted in the distilled water/isopropyl alcohol mixture (1:1, v/v) with two molar equivalents of bohemine (L7). The light grey product, which formed in two days of stirring at 90 °C, was filtered off and washed by distilled water and isopropyl alcohol.
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[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; cm1): 526m (PtN), and 563vs (PtO). IR (KBr; cm1): 3122m, 3068m (CHar), 2970m, 2932m, 2875m (CHal), 1661s (COox), 1612vs (CN), and 1551s (CC). Raman (cm1): 3057s (CHar), 2978s, 2938vs, 2914vs (CHal), 1661w (COox), and 1611vs (CN). 1H NMR (400 MHz, DMFd7, 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 CHNSO Elemental Analyzer (Thermo Scientific). IR spectra were recorded on a Nexus 670 FT-IR spectrometer (Thermo Nicolet) at 400– 4000 cm1 (KBr pellets) and 150–600 cm1 (Nujol technique). Raman spectra were recorded using an NXR FT-Raman Module (Thermo Nicolet) between 150 and 3750 cm1. 1H, 13C and 195Pt NMR spectra and 1H–1H gs-COSY, 1H–13C gs-HMQC, 1H–13C gs-HMBC two dimensional correlation experiments of the DMF-d7 solutions were measured at 300 K on a Bruker 300 device. 1H spectra were also, together with 1H–15N gs-HMBC experiments, recorded at 340 K. 1H and 13C spectra were adjusted against the signals of tetramethylsilane (Me4Si). 195Pt spectra were calibrated against K2PtCl6 in D2O found at 0 ppm. 1H–15N gs-HMBC experiments were obtained at natural abundance and calibrated against the residual signals of DMF adjusted to 8.03 ppm (1H) and 104.7 ppm (15N). The splitting of proton resonances in the reported 1H spectra is defined as 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 Collection of Cell Cultures (ECACC). The following cell lines were employed in the current study: human ovarian carcinoma cells (A2780; ECACC No. 93112519), human ovarian carcinoma cisplatin-resistant cells (A2780cis; ECACC No. 93112517), human Caucasian malignant melanoma (G361; ECACC No. 88030401), human 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 the ECACC instructions. Briefly, culture medium was DMEM (for cell lines HOS, MCF7, HeLa, A549), RPMI1640 (for cell lines A2780 and A2780cis) and McCoy´s (for cell line G361). The medium was supplemented with penicillin, streptomycin and 10% of foetal bovine serum. The cells were maintained at 37 °C and 5% CO2 in a humidified incubator. 2.4. Primary cultures of human hepatocytes
Fig. 1. The proposed structure of the complexes [Pt(cbdc)(Ln)2] (1–7), given with specification of the R1 and R2 substituents of the 6-benzylamino-9-isopropylpurine moiety.
Human hepatocytes were isolated from liver tissue, resected from multiorgan donors. A tissue acquisition protocol was in
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accordance with the requirements issued by a local ethical commission in the Czech Republic. Human liver tissues used in this study were obtained from two donors: LH31 (male, 28 years) and LH32 (male, 70 years). Hepatocytes were isolated by two-step collagenase perfusion and the cells were plated on collagen-coated culture dishes using cell density of 14 104 cells/cm2 (Pichard´s Garcia et al., 2002). The culture medium was Williams and HAM F-12 (1:1) supplemented with penicillin, streptomycin, ascorbic acid, linoleic acid, holo-transferin, ethanolamine, glucagon, insulin, dexamethasone, pyruvate, glucose, glutamine, amphotericin. The medium was enriched for plating with 2% foetal calf serum (v/v). The medium was exchanged for a serum-free medium the day after and the culture was stabilized for additional 24 h. Thereafter, the cells were ready for treatments. The cultures were maintained at 37 °C and 5% CO2 in a humidified incubator. 2.5. Cytotoxicity assays Human cancer cell lines and primary cultures of human hepatocytes were treated with the tested compounds for 24 h, using multi-well culture plates of 96 wells (Vrzal et al., 2010). The following compounds – cisplatin, oxaplatin, carboplatin and 1–7 were applied to the cells up to the concentration of 50 lM. In parallel, the cells were 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 incubated with MTT for 3–4 h, and after removal of the medium and washing the cells with PBS, formazan dye was dissolved in DMSO containing 1% of ammonia. Absorbance was measured spectrophotometrically at 540 nm (TECAN, Schoeller Instruments LLC). The data were expressed as the percentage of viability, when 100% and 0% represent the treatments with DMF and Triton X-100, respectively. The data on human hepatocytes were obtained from two independent cultures (obtained from two different donors). The data from cancer cell lines were acquired from three independent cell passages. 3. Results 3.1. Synthesis and characterization of the complex [Pt(cbdc)(L7)2] (7) The complex 7 was prepared according to the formerly described synthetic procedure employed for the synthesis of the complexes 1–6 (Dvorˇák et al., 2010), as described in Section 2.1.1 (Fig. 1). The characterization of the compound, by the methods summarized in Section 2.2., proved a composition corresponding to the [Pt(cbdc)(L7)2] formula. The results of the detailed NMR spectroscopic study indicated that the cbdc anion is bidentatecoordinated to the metal centre through two oxygen atoms and both L7 molecules are bound to the Pt(II) atom through their N7 atoms. 3.2. Cytotoxicity in human cancer cell lines We examined cytotoxicity of the prepared platinum(II) complexes and three standard platinum-derived cytotoxic compounds (i.e. cisplatin, oxaliplatin, carboplatin) in seven different commercial human cancer cell lines, including A2780, A2780cis, G361, MCF7, A549, HOS and HeLa. The obtained results (IC50 ± SD values given in lM) are summarized in Figs. 2–4, as well as in Table S1 of Supplementary material. No cytotoxic effects of the tested compounds were found in human A549 cells. Due to the limited solubility of the compounds, the estimate IC50 values were >50 lM for the complexes 1, 3–7, and cisplatin. The IC50 value for complex 2 was >20 lM (Fig. 2A). The cytotoxicity of the tested compounds was weak in HeLa cancer cells. With the exception of cisplatin
Fig. 2. Cytotoxicity of the tested compounds against the A549, HeLa and HOS cell lines. The cells were plated at 96-well dishes and cultured according to the manufacturer instructions. The tested compounds were applied to the cells for 24 h in 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. The cytotoxicity 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) HeLa cells and (Panel C) HOS cells. The data are expressed as a mean ± SD from three independent experiments.
(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
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Fig. 4. Cytotoxicity of the tested compounds against the A2780 and A2780cis cell lines. The cells were plated at 96-well dishes and cultured according to the manufacturer instructions. The tested compounds were applied to the cells for 24 h in 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. The cytotoxicity 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 are expressed as a mean ± SD from three independent experiments.
3.3. Cytotoxicity in primary cultures of human hepatocytes In none of the primary human hepatocyte cultures, the IC50 values were reached by the tested compounds. This was due to the limited solubility of these compounds. Therefore, the estimated IC50 values for all the tested complexes were IC50 > 50 lM, with the exception of carboplatin (IC50 > 1 lM) and 2 (IC50 > 20 lM). 4. Discussion
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 manufacturer instructions. The tested compounds were applied to the cells for 24 h in concentrations ranging from 0.01 to 50 lM. As the positive and negative control, TritonX100 (1% v/v) and vehicle (DMF; 0.1% v/v) were used, respectively. The cytotoxicity was assessed by the MTT test, and the values of IC50 were calculated. The bar graphs show the IC50 values against (Panel A) G361 cells and (Panel B) MCF7 cells. The data are expressed as a mean ± SD from three independent experiments.
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-cytotoxic within the tested concentration range (Fig. 2C). The best cytotoxic potency of the tested compounds was attained in the case of G361 (Fig. 3A) and MCF7 (Fig. 3B) cells. The exception was the complex 7, which was not cytotoxic in either cell line in the concentration up to 50 lM. We also examined comparative effects of the tested compounds in cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) human ovarian carcinoma cell line. Cisplatin exerted significantly higher cytotoxicity in A2780 cells (IC50 = 11.5 ± 1.6 lM) as compared to A2780cis cells (IC50 = 30.3 ± 6.1 lM), as expected. Beside the complex 7, which was not cytotoxic, all the complexes displayed significant cytotoxicity in both A2780 and A2780cis cells (Fig. 4). We have also found that none of the employed cancer cell lines were sensitive to oxaliplatin (IC50 > 50 lM) and carboplatin (IC50 > 1 lM) up to the concentration given by the compound solubility.
In the present work, we tested in vitro cytotoxicity of the [Pt(cbdc)(Ln)2] complexes (1–7) and compared the obtained results with those of cisplatin, oxaliplatin and carboplatin. The cytotoxicity was evaluated in seven commercial human cancer cell lines (A2780, A2780cis, G361, MCF7, A549, HOS and HeLa) derived from various types of cancer (vide supra) and in two primary cultures of human hepatocytes. The cells were challenged for 24 h with the tested compounds and the MTT test was used to assess the resulting cytotoxicity. We have found out that oxaliplatin (IC50 > 50 lM) and carboplatin (IC50 > 1 lM) did not cause significant cell damage in any cell line used, because the cytotoxic potential of oxaliplatin and carboplatin was restricted by their limited solubility. Therefore, it makes sense to test platinum-based derivatives with increased solubility and clinically reasonable cytotoxicity in vitro. Indeed, in the current paper we describe different in vitro cytotoxicity of the tested platinum-derivatives. The tested complexes were not cytotoxic against human Caucasian lung carcinoma cells A549, and very weak cytotoxicity was observed against human negroid cervix epithelioid carcinoma cells (HeLa) and human Caucasian osteosarcoma cells (HOS). In contrast, significant dose-dependent cytotoxicity of the tested compounds was observed against human Caucasian malignant melanoma cells (G361), human Caucasian breast adenocarcinoma cells (MCF7) and human ovarian carcinoma cell line (A2780) and its cisplatin-resistant variant (A2780cis). The cytotoxic effect of the studied compounds 1–7 in primary cultures of human hepatocytes, an in vitro model considered as the most suitable for studies of xenobiotic cytotoxicity and metabolism, was studied as well. We used primary human hepatocytes obtained from two human liver donors. The obtained results of in vitro hepatotoxicity revealed that the studied platinum(II)
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complexes 1–7 did not affect healthy human hepatocytes up to the concentration of 50 lM. The presented platinum(II) cyclobutane-1,1-dicarboxylato complexes follow the recently reported highly in vitro cytotoxic ˇ et al., 2005 and oxalato dichlorido {cis-[PtCl2(Ln)2]; Malon {[Pt(ox)(Ln)2]; Štarha et al., 2010; Trávnícˇek et al., 2010; Vrzal et al., 2010} complexes prepared in our laboratory. The cis[PtCl2(Ln)2] complexes were tested against the G361, HOS, MCF7 and K-562 (not used in this work) cancer cells. We performed a comparison of the calculated IC50(cisplatin)/IC50(complex) ratio of the formerly tested dichlorido and oxalato complexes with the cyclobutan-1,1-dicarboxylato ones reported in this work to better describe and compare in vitro cytotoxicity of these types of complexes. The ratio of the most active cis-[PtCl2(ros)2] complex with the coordinated CDK inhibitor roscovitine equals 3.0 (it means that the tested complex is three times more active than cisplatin) ˇ et al., 2005). against G361 and HOS and 5.0 against MCF7 (Malon The oxalato complexes were tested against the same cancer cell lines as herein described complexes 1–7 (Trávnícˇek et al., 2010; Vrzal et al., 2010). Again we can calculate the ratio of the in vitro cytotoxicity against respective cancer cell line of cisplatin versus the appropriate oxalato complex, which equals 1.7 against G361 for [Pt(ox)(L8)2], 1.7 against HeLa for [Pt(ox)(L9)2], 5.4 against MCF7 for [Pt(ox)(L10)2], 9.5 against HOS for [Pt(ox)(L11)2], 3.6 against 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 are higher (it means more in vitro cytotoxic active) as compared with 1–7, in particular with 0.6 against G361 for 6, 1.0 against HeLa for 4, 1.4 against MCF7 for 2, 2.3 against HOS for 2, 1.8 against A2780 for 6 and 5.4 against A2780cis for 6. Another important finding is that the studied platinum(II) complexes 1–7 overcome cisplatin resistance. It was demonstrated by the IC50(A2780cis)/IC50(A2780) ratio, which is equal to 2.6 for cisplatin and 0.9 (1), 1.1 (2), 1.4 (3), 1.2 (4), 1.2 (5) and 0.9 (6). We can compare these results with those reported in the literature sources for different in vitro cytotoxic mononuclear platinum(II) complexes. The above-mentioned [Pt(ox)(L8)2] complex has the IC50(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 pyrophosphato (pyro) complex [Pt(pyro)(dach)] was determined to be 2.4 (Bose et al., 2008). As for six platinum(II) dichlorido complexes with 2-aminomethylpyrrolidine-based ligands, their IC50(A2780cis)/IC50(A2780) ratios equal 1.1–4.9 (Diakos et al., 2009). The last example, which could be mentioned concerning in vitro cytotoxicity against A2780 and A2780cis cells, is cis-[PtCl2(A9opy)] with the IC50(A2780cis)/IC50(A2780) ratio of 1.4 (Marqués-Gallego et al., 2009). On the other hand, the effectiveness of tested compounds against cisplatin-resistant cells is not, in principle, general, since cisplatin resistance comprises multiple mechanisms, including decreased drug uptake, increased efflux, increased inactivation by glutathione, increased excision of DNA adducts etc. In conclusion, we demonstrated that the tested carboplatin derivatives 1–7 may be classified as tentatively promising anticancer drugs, because they were found to be toxic against human cancer cells but not against healthy human hepatic cells. Further, the platinum(II) complexes of the general formula [Pt(cbdc)(Ln)2] significantly more effectively inhibit growth of both A2780 and A2780cis cancer cells as compared with several recently reported mononuclear platinum(II) complexes. Conflict of interest We declare no conflict of interest.
Acknowledgements This work was financially supported by the Grant Agency of the Czech Republic (Grant Nos.: GACR503/10/0579 and GACR304/10/ 0149), the Ministry of Education, Youth and Sports of the Czech Republic (a Grant No.: MSM6198959218), Faculty of Science of Palacky University in Olomouc (a Grant No.: PrF_2010_018), and by the Operational Program Research and Development for Innovations – European Social Fund (a Grant No.: CZ.1.05/2.1.00/ 03.0058). We thank Mr. Lukáš Dvorˇák for his help with the synthesis of the complexes, Ms. Radka Novotná for IR and Raman spectra measurements and Dr. Igor Popa for NMR spectra measurements and interpretation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tiv.2011.01.002. References Bitha, P., Morton, G.O., Dunne, T.S., Delos Santos, E.F., Lin, Y., Boone, S.R., Haltiwanger, R.C., Pierpoint, C.G., 1990. (Malonato)bis[sulfinylbis[methane]S]platinum(II) compounds: versatile synthons for a new general synthesis of antitumor symmetrical and dissymmetrical (Malonato)platinum(II) complexes. Inorganic Chemistry 29, 645–652. Bose, R.N., Maurmann, L., Mishur, R.J., Yasui, L., Gupta, S., Grayburn, W.S., Hofstetter, H., Salley, T., 2008. Non-DNA-binding platinum anticancer agents: cytotoxic activities of platinum-phosphato complexes towards human ovarian cancer cells. PNAS 105, 18314–18319. Diakos, C.I., Zhang, M., Beale, P.J., Fenton, R.R., Hambley, T.W., 2009. Synthesis, characterisation and in vitro cytotoxicity studies of a series of chiral platinum(II) complexes based on the 2-aminomethylpyrrolidine ligand: X-ray crystal structure of [PtCl2(R-dimepyrr)] (R-dimepyrr = N-dimethyl-2(R)aminomethylpyrrolidine). European Journal of Medicinal Chemistry 44, 2807– 2814. Dvorˇák, L., Popa, I., Štarha, P., Trávnícˇek, Z., 2010. In vitro cytotoxic active platinum(ii) complexes derived from carboplatin and involving purine derivatives. European Journal of Inorganic Chemistry 3441, 3448. Kelland, L.-R., Farrell, N.-P., 2000. Platinum-based Drugs in Cancer Therapy. Humana Press, Totowa, New Jersey. ˇ , M., Trávnícˇek, Z., Marek, R., Strnad, M., 2005. Synthesis, spectral study and Malon cytotoxicity of platinum(II) complexes with 2, 9-disubstituted-6benzylaminopurines. Journal of Inorganic Biochemistry 99, 2127–2138. Marqués-Gallego, P., Kalayda, G.V., Jaehde, U., den Dulk, H., Brouwer, J., Reedijk, J., 2009. Cellular accumulation and DNA platination of two new platinum(II) anticancer compounds based on anthracene derivatives as carrier ligands. Journal of Inorganic Chemistry 103, 791–796. Oh, C.H., Lee, S.C., Lee, K.S., Woo, E.R., Hong, C.Y., Yang, B.S., Baek, D.J., Cho, J.H., 1999. Synthesis and biological activities of C-2, N-9 substituted 6-benzylaminopurine derivatives as cyclin-dependent kinase inhibitor. Archiv der Pharmazie 332, 187–190. Pantoja, E., Gallipoli, A., van Zutphen, S., Tooke, D.M., Spek, A.L., Navarro-Ranninger, C., Reedijk, J., 2006. In vitro antitumor activity and interaction with DNA model bases of cis-[PtCl2(iPram)(azole)] complexes and comparison with their trans analogues. Inorganica Chimica Acta 359, 4335–4342. Pichard-Garcia, L., Gerbal-Chaloin, S., Ferrini, J.B., Fabre, J.M., Maurel, P., 2002. Use of long-term cultures of human hepatocytes to study cytochrome P450 gene expression. Methods in Enzymology 357, 311–321. Štarha, P., Trávnícˇek, Z., Popa, I., 2010. Platinum(II) oxalato complexes with adeninebased carrier ligands showing significant in vitro antitumor activity. Journal of Inorganic Biochemistry 104, 639–647. Trávnícˇek, Z., Štarha, P., Popa, I., Vrzal, R., Dvorˇák, Z., 2010. Roscovitine-based CDK inhibitors acting as N-donor ligands in the platinum(II) oxalato complexes: preparation, characterization and in vitro cytotoxicity. European Journal of Medicinal Chemistry 45, 4609–4614. Vrzal, R., Štarha, P., Dvorˇák, Z., Trávnícˇek, 2010. Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(ii) and palladium(ii) oxalato complexes with adenine derivatives as carrier ligands. Journal of Inorganic Biochemistry 104, 1130–1132.