Synthesis, structural characterization and cell death-inducing effect of novel palladium(II) and platinum(II) saccharinate complexes with 2-(hydroxymethyl)pyridine and 2-(2-hydroxyethyl)pyridine on cancer cells in vitro Ferda Ari a , Nazlihan Aztopal a , Ceyda Icsel b , Veysel T. Yilmaz b , Emel Guney b , Orhan Buyukgungor c , Engin Ulukaya d,⇑ a Department of Biology, Faculty of Arts and Sciences, Uludag University, 16059 Bursa, Turkey b Department of Chemistry, Faculty of Arts and Sciences, Uludag University, 16059 Bursa, Turkey c Department of Physics, Faculty of Arts and Sciences, Ondokuz Mayis University, 55159 Samsun, Turkey d Department of Medical Biochemistry, Medical School, Uludag University, 16059 Bursa, Turkey article info Article history: Received 18 June 2013 Revised 16 August 2013 Accepted 23 August 2013 Available online xxxx Keywords: Palladium(II) and platinum(II) complexes Cancer Cytotoxicity Apoptosis abstract Four palladium(II) and platinum(II) saccharinate (sac) complexes with 2-(hydroxymethyl)pyridine (2-hmpy) and 2-(2-hydroxyethyl)pyridine (2-hepy), namely trans-[Pd(2-hmpy) 2 (sac) 2 ]H 2 O(1), trans- [Pt(2-hmpy) 2 (sac) 2 ]3H 2 O(2), trans-[Pd(2-hepy) 2 (sac) 2 ](3) and trans-[Pt(2-hepy) 2 (sac) 2 ](4), have been synthesized and characterized by elemental analysis, UV–vis, IR and NMR. Single crystal X-ray analysis reveals that the metal(II) ions in each complex are coordinated by two sac and two 2-hmpy or 2-hepy ligands with a trans arrangement. Anticancer effects of 1–4 were tested against four different cancer cell lines (A549 and PC3 for lung cancer, C6 for glioblastoma, and Hep3B for liver cancer). Cytotoxicity was first screened by the MTT assay and the results were further confirmed by the ATP assay. The mode of cell death was determined by both histological and biochemical methods. Among the metal complexes, complex 2 resulted in relatively stronger anti-growth effect in a dose-dependent manner (3.13–200 lM), compared to the others, by inducing apoptosis. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Saccharin (1,1-dioxo-1,2-benzothiazol-3-one or o-benzosulfi- mide) is most widely used synthetic sweetener. Saccharin is read- ily deprotonated forming the anionic form, saccharinate (sac), which has several potential donor atoms such as an imino nitro- gen, a carbonyl and two sulfonyl oxygen atoms. The coordination chemistry of saccharinate is interesting, owing to its coordination ability, forming complexes from mononuclear species to coordina- tion polymers. 1 Although a large number of metal complexes of sac were reported, their biological evaluations received less attention. Only a few metal–sac complexes showing promising cytotoxic activity appeared in this field. For instance, a platinum(II) complex, K[Pt(sac) 3 (H 2 O)], has testes against HeLa cells and the IC 50 value for the complex was 6.8 lM, close to that obtained for cisplatin. 2 The cytotoxic activity of a series of new gold(I) and gold(III) complexes of sac was studied using A2780 cells and the gold(I) complexes were found more cytotoxic. 3 As a result of our ongoing research, we recently reported several palladium(II) and plati- num(II) sac complexes with pyridine-based ligands. Anticancer activities of some of these complexes were also investigated. For example, the cytotoxic activity of [Pd(bpma)Cl](sac)2H 2 O (bpma = bis(2-pyridylmethyl)amine) on A549 and C6 cells is com- parable to cisplatin, while the platinum analogue of this complex has no cytotoxicity. 4 On the other hand, the palladium(II) and plat- inum(II) sac complexes of 2,2 0 :6 0 ,2 00 -terpyridine (terpy) were found to be highly cytotoxic on a number of cancer cells such as MCF-7, MDA-MB-231, A549, H1299, PC-3 and 5RP7. 5–7 The interaction of the palladium(II) sac complexes of terpy complexes with fish sperm (FS) DNA clearly indicated that these complexes act as dual-function metallointercalators and bind to FS-DNA strongly by both intercalation and coordination. 8 Taking into account the promising activity of palladium(II) and platinum(II) sac complexes against cancer, we have recently synthesized new palladium(II) and platinum(II) sac with 2-(hydroxy- methyl)pyridine (2-hmpy) and 2-(2-hydroxyethyl)pyridine (2-hepy), namely trans-[Pd(2-hmpy) 2 (sac) 2 ]H 2 O (1), trans-[Pt(2-hmpy) 2 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.08.050 ⇑ Corresponding author. Address: Medical School of Uludag University, Depart- ment of Clinical Biochemistry, 16059 Bursa, Turkey. Tel.: +90 224 295 3913; fax: +90 224 442 8245. E-mail address: [email protected](E. Ulukaya). Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc Please cite this article in press as: Ari, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.08.050
Synthesis, structural characterization and cell death-inducingeffect of novel palladium(II) and platinum(II) saccharinatecomplexes with 2-(hydroxymethyl)pyridine and2-(2-hydroxyethyl)pyridine on cancer cells in vitro
0968-0896/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmc.2013.08.050
⇑ Corresponding author. Address: Medical School of Uludag University, Depart-ment of Clinical Biochemistry, 16059 Bursa, Turkey. Tel.: +90 224 295 3913; fax:+90 224 442 8245.
Please cite this article in press as: Ari, F.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.08.050
Ferda Ari a, Nazlihan Aztopal a, Ceyda Icsel b, Veysel T. Yilmaz b, Emel Guney b, Orhan Buyukgungor c,Engin Ulukaya d,⇑a Department of Biology, Faculty of Arts and Sciences, Uludag University, 16059 Bursa, Turkeyb Department of Chemistry, Faculty of Arts and Sciences, Uludag University, 16059 Bursa, Turkeyc Department of Physics, Faculty of Arts and Sciences, Ondokuz Mayis University, 55159 Samsun, Turkeyd Department of Medical Biochemistry, Medical School, Uludag University, 16059 Bursa, Turkey
a r t i c l e i n f o
Article history:Received 18 June 2013Revised 16 August 2013Accepted 23 August 2013Available online xxxx
Keywords:Palladium(II) and platinum(II) complexesCancerCytotoxicityApoptosis
a b s t r a c t
Four palladium(II) and platinum(II) saccharinate (sac) complexes with 2-(hydroxymethyl)pyridine(2-hmpy) and 2-(2-hydroxyethyl)pyridine (2-hepy), namely trans-[Pd(2-hmpy)2(sac)2]�H2O (1), trans-[Pt(2-hmpy)2(sac)2]�3H2O (2), trans-[Pd(2-hepy)2(sac)2] (3) and trans-[Pt(2-hepy)2(sac)2] (4), have beensynthesized and characterized by elemental analysis, UV–vis, IR and NMR. Single crystal X-ray analysisreveals that the metal(II) ions in each complex are coordinated by two sac and two 2-hmpy or 2-hepyligands with a trans arrangement. Anticancer effects of 1–4 were tested against four different cancer celllines (A549 and PC3 for lung cancer, C6 for glioblastoma, and Hep3B for liver cancer). Cytotoxicity wasfirst screened by the MTT assay and the results were further confirmed by the ATP assay. The mode ofcell death was determined by both histological and biochemical methods. Among the metal complexes,complex 2 resulted in relatively stronger anti-growth effect in a dose-dependent manner (3.13–200 lM),compared to the others, by inducing apoptosis.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Saccharin (1,1-dioxo-1,2-benzothiazol-3-one or o-benzosulfi-mide) is most widely used synthetic sweetener. Saccharin is read-ily deprotonated forming the anionic form, saccharinate (sac),which has several potential donor atoms such as an imino nitro-gen, a carbonyl and two sulfonyl oxygen atoms. The coordinationchemistry of saccharinate is interesting, owing to its coordinationability, forming complexes from mononuclear species to coordina-tion polymers.1
Although a large number of metal complexes of sac werereported, their biological evaluations received less attention. Onlya few metal–sac complexes showing promising cytotoxic activityappeared in this field. For instance, a platinum(II) complex,K[Pt(sac)3(H2O)], has testes against HeLa cells and the IC50 valuefor the complex was 6.8 lM, close to that obtained for cisplatin.2
The cytotoxic activity of a series of new gold(I) and gold(III)
complexes of sac was studied using A2780 cells and the gold(I)complexes were found more cytotoxic.3 As a result of our ongoingresearch, we recently reported several palladium(II) and plati-num(II) sac complexes with pyridine-based ligands. Anticanceractivities of some of these complexes were also investigated. Forexample, the cytotoxic activity of [Pd(bpma)Cl](sac)�2H2O(bpma = bis(2-pyridylmethyl)amine) on A549 and C6 cells is com-parable to cisplatin, while the platinum analogue of this complexhas no cytotoxicity.4 On the other hand, the palladium(II) and plat-inum(II) sac complexes of 2,20:60,200-terpyridine (terpy) were foundto be highly cytotoxic on a number of cancer cells such as MCF-7,MDA-MB-231, A549, H1299, PC-3 and 5RP7.5–7 The interaction ofthe palladium(II) sac complexes of terpy complexes with fishsperm (FS) DNA clearly indicated that these complexes act asdual-function metallointercalators and bind to FS-DNA stronglyby both intercalation and coordination.8
Taking into account the promising activity of palladium(II) andplatinum(II) sac complexes against cancer, we have recentlysynthesized new palladium(II) and platinum(II) sac with 2-(hydroxy-methyl)pyridine (2-hmpy) and 2-(2-hydroxyethyl)pyridine (2-hepy),namely trans-[Pd(2-hmpy)2(sac)2]�H2O (1), trans-[Pt(2-hmpy)2
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(sac)2]�3H2O (2), trans-[Pd(2-hepy)2(sac)2] (3) and trans-[Pt(2-hepy)2(sac)2] (4) (Fig. 1). The complexes were fully characterized byelemental analysis, UV–vis, IR and NMR spectroscopic techniques.The crystal and molecular structures of 1 and 2 were determined byX-ray diffraction. In addition, we have investigated the growth inhib-iting effects and cell death mode of these complexes against four dif-ferent cell lines (A549, PC3, C6 and Hep3B). The results of the presentstudy have showed that complex 2 demonstrated significant growth-inhibiting effects against the all cell lines. Apoptosis-like cell deathmode was found to be responsible for the anti-growth effect of com-plex 2 on these tumor types.
2. Materials and methods
2.1. Materials and measurements
All chemicals used in the experiments were purchased com-mercially and used without further purification. Elemental analy-ses for C, H, and N were performed using a Costech elementalanalyser. UV–vis spectra were measured on a Perkin Elmer Lambda35 spectrophotometer. IR spectra were recorded on a Thermo Nico-let 6700 FT-IR spectrophotometer as KBr pellets in the frequencyrange 4000–400 cm�1. 1H NMR and 13C NMR spectra were re-corded on a Varian Mercuryplus spectrometer at 400 MHz inDMSO-d6 using TMS as internal reference.
2.2. Synthesis of the palladium(II) and platinum(II) complexes
Complexes 1–4 were synthesized by the following method.trans-[M(L)2Cl2] complexes (L = 2-hmpy or 2-hepy and M = PdII orPtII) were prepared as reported previously by our research group.9
Then, Na(sac) (1 mmol, 0,24 g) dissolved in 5 mL water was addedto the solutions of trans-[M(L)2Cl2] in water–EtOH at 60 �C. The yel-low polycrystalline products were filtered off and dried at roomtemperature (rt) X-ray quality yellow-orange crystals of complexes1 and 2 were obtained by slow evaporation of the solutions ofthese complexes in DMSO at rt.
The intensity data for the complexes 1 and 2 were collectedusing a STOE IPDS 2 diffractometer, with graphite-monochromatedMo Ka radiation (k = 0.71073 Å). The structures were solved by di-rect methods and refined on F2 with the SHELX-97 program.10 Allnon-hydrogen atoms were found from the difference Fourier mapand refined anisotropically. The C-bound H atoms were placedgeometrically and O-bound H atoms were located in difference
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maps. The details of data collection, refinement and crystallo-graphic data are summarized in Table 1.
2.4. Cell culture and chemicals
Complexes 1–4 were dissolved in DMSO as a stock solution.Further dilutions were made in culture medium and used at differ-ent concentrations ranging from 3.13 to 200 lM. Non-small celllung cancer cell lines A549 and PC3, human hepatoma Hep3Band rat C6 glioma cell lines were cultured in RPMI 1640 mediumsupplemented with penicillin G (100 U/ml), streptomycin(100 lg/ml), L-glutamine, and 10% fetal calf serum at 37 �C in ahumidified atmosphere containing 5% CO2.
2.5. Determination of cytotoxic activity
2.5.1. The MTT viability assayA549, PC3, Hep3B and C6 cells were seeded in 200 ll culture
medium in triplicates at a density of 1 � 104 cells per well of a96-well plate. The untreated cells received only the medium with-out using any drugs for control (full viability). All of the cells weretreated with different concentration of the complexes for 48 h.Each experiment was carried out twice in triplicates.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide (MTT) viability assay was performed as previouslydescribed.11 MTT was first prepared as a stock solution of 5 mg/ml in phosphate buffer (PBS, pH 7.2) and was filtered. At theend of the treatment period (48 h), 20 ll of MTT solution(5 mg/ml PBS, pH 7.2) was added to each well. After incubationfor 4 h at 37 �C, 100 ll of solubilizing buffer (10% sodium dodecylsulfate dissolved in 0.01 N HCl) was added to each well. Afterovernight incubation, the absorbance (Abs) was read by an ELISAplate reader at 570 nm to determine the cell viability. % Viabilityof treated cells was calculated by dividing the absorbance oftreated cells with the absorbance of control cells and then bymultiplying with 100.
Table 1Crystallographic data and structure refinement for trans-[Pd(hmpy)2(sac)2] (1) andtrans-[Pt(hmpy)2(sac)2]�2(DMSO) (2)
Complex 1 2
Formula C26H22N4O8PdS2 C30H34N4O10PtS4
M 689.00 933.94T (K) 298(2) 298(2)k (Å) 0.71073 0.71073Crystal system Triclinic TriclinicSpace group P�1 P�1a (Å) 11.1475(3) 8.1404(5)b (Å) 16.4808(4) 10.0798(6)c (Å) 17.6719(4) 11.7077(6)a (�) 62.765(2) 71.084(4)b (�) 76.366(3) 80.703(5)c (�) 82.186(2) 75.754(5)V (Å3) 2803.91(12) 877.32(9)Z 4 1Dcalcd (g cm�3) 1.632 1.768l (mm�1) 0.867 4.297F(000) 1392 464h Range (�) 1.88–26.50 1.85–26.50Index range �13 6 h 6 13,
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2.5.2. The ATP viability assayThe seeding of cells and treatment conditions as well as the
calculation of viability was performed similar to that of the MTTassay (see above). The ATP assay was based on the highly sensitive‘firefly’ reaction to determine the level of cellular ATP as a surro-gate marker for the number of alive cells.12 At the end of the treat-ment period (48 h), the ATP assay was used for luminometricmeasurement of cell growth (viability) according to the standardprotocol with a little modification of the manufacturer (ATP Biolu-minescent Somatic Cell Assay Kit, Sigma, St. Louis, MO, USA). TheATP was extracted from the cells by the addition of 50 ll of tumorcell extraction reagent to each well. After mixing thoroughly, themicroplates were allowed to stand on the bench for 20 min at roomtemperature before 50 ll medium from each well was transferredto a white plate. Next, the 50 ll luciferin–luciferase countingreagent was added. The microplates were measured using a countintegration time of 1 s at luminometer (Bio-Tek, Vermont, USA).
2.6. Apoptosis assays
2.6.1. Detection of caspase-cleaved cytokeratin 18Apoptosis was assayed by measuring the level of caspase-
cleaved keratin 18 (ccK18, M30) by a commercially availableimmunoassay kit (M30-Apoptosense ELISA kit, Peviva AB, Sweden)according to the manufacturer’s instructions. This kit measures thelevels of the CK18-Asp396 neo-epitope (M30), which is a well-known marker of apoptosis. 1 � 104 cells were seeded per well ofa 96-well plate in 200 ll culture medium in triplicates. Cells weretreated for 48 h with complex 2 (200 lM). Cisplatin (12.6 lM,clinically-relevant dose) was used as a comparing agent for theeffect of complex 2. Each experiment was carried out twice in trip-licates. At the end of the treatment period, the cells were lysedwith 10% NP-40 for 10 min on a shaker. The content of identicalwells were pooled and centrifuged at 2000 rpm for 10 s to removethe debris. All samples were placed into wells coated with a mousemonoclonal antibody as a catcher. After washing, a horseradishperoxidase conjugated antibody (M30) was used for detection.The absorbance was determined with an ELISA reader at 450 nm(FLASH Scan S12, Eisfeld, Germany).
2.6.2. Measurement of active caspase-3 and cleaved PARP levelsA549, PC3 and Hep3B cells (1 � 106) were seeded in 25 cm2
flasks and treated with complex 2 for 48 h in order to detect activecaspase-3 and cleaved PARP (poly(ADP-ribose) polymerase) levels,which are the markers for apoptosis. After treatment, cells werewashed in ice-cold PBS, and lysed in lysis buffer (Cell Signaling,MA, USA), containing protease inhibitors (Sigma, St. Louis, MO,USA) and 1 mM of PMSF (phenylmethylsulfonyl fluoride). Cellswere extracted at 4 �C for 5 min, and centrifuged at 4 �C for10 min at 14,000g. The level of cleaved PARP was estimated usingthe PARP Cleavage [214/215] Human ELISA Kit (Invitrogen Corpo-ration, Camarillo, CA, USA) and Caspase-3 (Active) Human ELISAKit (Invitrogen Corporation, Camarillo, CA, USA) according to theprotocols described in manufacturer’s instructions. For determina-tion of active caspase-3, 100 ll of cell lysates were incubated in themicroplate wells provided in the kit at room temperature for 2 h.The samples were aspirated and washed 4 times with washingbuffer and incubated with 100 ll of detection antibody (Anti-ac-tive caspase-3) for 1 h at room temperature. After removal of theantibody solution, the wells were washed again and incubatedwith 100 ll of HRP anti-rabbit antibody for 30 min at room tem-perature. After the aspiration of the anti-rabbit antibody, blue colorwas developed by adding 100 ll of stabilized chromogen solutionfor 20 min at room temperature. The reaction was stopped afterthe addition of 100 ll of stopping solution. For determination ofcleaved PARP levels, 50 ll from each lysate was incubated with
4 F. Ari et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Anti-cleaved PARP (detection antibody) for 3 h. After washing, eachsample in a well was incubated with a secondary antibody, IgG-HRP solution for 30 min. Stabilized chromogen was added to eachwell for another 30 min followed by addition of stop solution. Theabsorbance of each well was read at 450 nm using a microplatereader. The experiment is representative of two independentexperiments.
translocate the outside of the cell membrane, which is an earlyevent in apoptotic cells. Annexin-V-FITC is able to bind to PS,allowing the apoptotic cells visible. A549, PC3, Hep3B and C6 cellswere seeded in a 96-well plate at the density of 1 � 104 cells perwell, and then the cells were treated with complex 2 (200 lM)for 12 h. Also paclitaxel (3.13 lM) was used as a positive controlfor apoptosis as this agent is considered as an appropriate apopto-sis-inducer.13 Cisplatin (12.6 lM) was used for comparison withcomplex 2. After the treatment, cells were stained with Annexin-V-FITC and propidium iodide using the Annexin-V-Fluos kit (Roche,Germany). Annexin-V-FITC and propidium iodide was diluted 1:50from stock with incubation buffer to yield a working solution. AlsoHoechst dye 43332 (20 lg/ml, 1:10) was added in this solution toobserve all the cells (alive and dead). Then the cells were incubatedafter added 50 ll working solution to each well for 30 min at roomtemperature. Apoptotic cells were then visualized under fluores-cence microscope.
2.7. Statistical analyses
All statistical analyses were performed using the SPSS 20.0 sta-tistical software for Windows. The significance was calculatedusing one-way analysis of variance (ANOVA). A value of p <0.05was considered statistically significant. Results were expressed asmean ± SD (standard deviation).
Figure 2. Molecular structure of trans-[Pd(hmpy)2(sac)2] (1). C–H hydrogen atomswere omitted for clarity.
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3. Results and discussion
3.1. Synthesis and characterization
The starting complexes of trans-[M(L)2Cl2] (L = 2-hmpy or 2-hepy and M = PdII or PtII) were prepared by the reaction of 2-hmpyor 2-hepy with Na2[PdCl4] or K2[PtCl4].9 Then, complexes 1–4 weresynthesized by the sac ligand substitution of chlorides in the start-ing complexes. Complexes 1 and 2 were obtained as single crystals,but attempts for the crystallization of 3 and 4 failed. The C, H and Ncontents of the complexes agree well with calculated values. Allcomplexes are air-stable and obtained in high yields (over 55%).All complexes are highly soluble in DMSO and DMF, but not solublein the other common solvents.
Table 3Selected bond lengths [Å] and angles [�] for trans-[Pt(hmpy)2(sac)2]�2(DMSO) (2)
Figure 4. The anti-growth effect after the treatment with varying doses of complexes for 48 h against different cancer cell lines by the MTT assay as described in the materialsand methods. ⁄ Denotes statically significant differences in comparison with control (p <0.05).
Figure 5. The anti-growth effect after the treatment with varying doses of thecomplex 2 for 48 h against different cancer cell lines by the ATP assay as describedin the materials and methods. ⁄ Denotes statically significant differences incomparison with control (p <0.05).
Table 4The IC50 and IC90 values of the complex 2 and carboplatin
Cell lines IC50a (lM) IC90 (lM)
A549 Complex 2 117.5 192.4Carboplatin 70.5 >85.1
PC3 Complex 2 72.2 95.6Carboplatin 85.1 >85.1
Hep3B Complex 2 35.0 82.1Carboplatin 6.8 28.7
C6 Complex 2 38.9 90.9Carboplatin 7.6 42.7
a IC50 is defined as the concentration inhibiting 50% of cell growth (viability) afterthe treatment with complex 2 and carboplatin for 48 h on the basis of the ATPviability assay.
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The FTIR spectra of complexes 1–4 display the main absorptionbands of the 2-hmpy, 2-hepy and sac ligands. The broad and in-tense bands centered over 3400 cm�1 are attributed to the m(OH)vibrations of the hydroxyl group of water molecules or 2-hmpyand 2-hepy ligands, while the aromatic and aliphatic CH stretchesappear in the frequency range 2850–3115 cm�1 as weak bands.The sharp bands around 1670 cm�1 are due to the absorption ofthe carbonyl group of sac. The asymmetric (mas) and symmetric(ms) stretchings of the SO2 group appear as two strong bands atca. 1270 and 1172–1155 cm�1, respectively. All complexes givewell-resolved 1H NMR spectra. The atom numbering is given in
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Figure 1. In the spectra of 1–4, the pyridine protons of the 2-hmpyand 2-hepy ligands appear as four signals in the range d = 9.20–7.30 ppm, while the chemical shifts for the phenyl protons of sacare observed in the range of 7.795–7.57 ppm as multiplets. Thehydroxyl group protons are observed as a triplet at d = 6.07–5.92 ppm for 1 and 2, and 4.95–4.70 ppm for 3 and 4, indicatingthe strong shielding compared to trans-[M(L)2Cl2] complexes(L = 2-hmpy or 2-hepy and M = PdII or PtII).9 On the other hand,the resonances of the methylene protons of complexes 1 and 2appear at d = ca. 5.83–5.63 ppm, while the ethylene protons of 3and 4 are observed at 4.47–4.12 and 3.88–3.79 ppm. In the 13CNMR spectra of the complexes, the carbonyl group of the sac ligandoccurs at ca. d 165 ppm, while the chemical shifts for the ph and pycarbons are observed in the range of 164.5–120.0 ppm. Themethylene carbons in 1 and 2 occur at around 64 ppm, while theethylene carbons of 3 and 4 resonance at ca. 59 and 43 ppm.
Figure 6. M30 levels (U/L) 48 h after the treatment with complex 2 (200 lM), and cisplatin (12.6 lM). M30 detection was performed in the cell culture medium by ELISA asexplained in the materials and methods. ⁄ Denotes statically significant differences in comparison with control (p <0.05).
Figure 7. Effect of complex 2 on caspase-3 activity (A) and PARP cleavage (B). The cells were treated with 200 lM complex 2 for 48 h.
6 F. Ari et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3.2. Description of the crystal structures of 1 and 2
The molecular structures of complexes 1 and 2 are shown inFigures 2 and 3, respectively, while selected bond distances andangles are listed in Tables 2 and 3. Complex 1 crystallizes in tri-clinic ðP�1Þ crystal system and its unit cell contains two indepen-dent molecules of the complex. The two [Pd(2-hmpy)2(sac)2]molecules are almost identical. The palladium(II) ion in complex1 shows the usual square-planar coordination with two trans 2-hmpy ligands and two trans sac anions. The phenyl rings of thetwo sac ligands are coplanar with a dihedral angle of 2.81�, whilethe py rings of two 2-hmpy ligands make a dihedral angle of6,92 and 5,68�. In order to reduce the steric hindrance, the phenyland py rings tend to be oriented nearly perpendicularly to eachother and the dihedral angle between the py and sac rings is ca.86�. The coordination geometry shows a slight distortion and theN–Pd–N angles are almost close to those of the ideal square-plane(Table 2). The Pd–N(sac) bond distances range from 2.020(8) to2.054(8) Å, which are in the range of the palladium(II) complexesof sac.4,14–17 The Pd–N(2-hmpy) bond distances are in the rangeof 2.007(10)–2.051(8) Å, being similar to those observed intrans-[Pd(2-hmpy)2Cl2].9 The molecules of 1 is linked into one-dimensional chains by strong O–H� � �O hydrogen bonds involvingthe hydroxyl hydrogens of 2-hmpy and sulfonyl oxygen atoms ofsac. These chains are further connected by O–H� � �O hydrogenbonds between the hydroxyl hydrogens and sulfonyl and carbonyloxygen atoms, forming a two-dimensional supramolecular network.
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Complex 2 was crystallized as a DMSO solvate (Fig. 3). In 2, theplatinum(II) ion lies on a crystallographic inversion center andcoordinated by two 2-hmpy and two sac ligands in a trans fashion,forming a distorted square-planar geometry. As observed in 1, bothsac and py rings are perpendicular to the coordination plane in or-der to reduce steric hindrance. The Pt–N(2-hmpy) bond distancesare in agreement with those found in trans-[Pt(2-hmpy)2Cl2],9
while the Pt–N(sac) bond distances are typical of those previouslyreported platinum(II) complexes containing the sac ligand.4,9,14–17
The sulfonyl group of the DMSO molecules interacts with thehydroxyl hydrogen of the 2-hmpy lignads. The molecules of 2 areconnected by p(sac)� � �p(sac) stacking interactions of 3.631(4) Åand packing of the molecules are further reinforced by the rela-tively weak weak C–H� � �O hydrogen bonds (C� � �O = 3.23–3.42 Å).
3.3. Cytotoxic activities of the complexes by the MTT and ATPviability assays
Cytotoxic effect of the complexes of 1–4 on lung cancer (A549and PC3), human hepatoma Hep3B and rat C6 glioma cell lineswere first evaluated by the MTT viability assay after treating cellswith the complexes at different concentrations (3.13–200 lM) for48 h. As shown in Figure 4, among complexes, only the complex2 significantly inhibited the growth of the cells in a dose-depen-dent manner (p <0.05). Because the complex 2 was found to havethe most promising cytotoxic activity, only this complex was stud-ied further for the elucidation of cell death mode.
Figure 8. Fluorescence Imaging for Apoptosis. Cells were treated with complex 2 (200 lM), and the clinically-relevant doses of cisplatin (12.6 lM) and paclitaxel (3.13 lM) for12 h. (A) A549 cell line. (B) PC3 cell line. (C) Hep3B cell line. (D) C6 cell line. Yellow arrows show the early apoptotic cells while white arrows show the late apoptotic cells.
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The cytotoxic effect of the complex 2 was also assessed by theATP viability assay to confirm the results of the MTT assay. Asshown in Figure 5, the complex 2 significantly reduced the viabilitylevels, especially at relatively higher doses (p <0.05). IC50 and IC90
values were calculated on the basis of the results of the ATP assayand also compared with carboplatin as a platin-based anticanceragent shown in Table 4.
Pd and Pt complexes are considered to be of high significance aspotent chemotherapeutic agents,18–20 and there is a significantsimilarity between the coordination chemistry of these com-plexes.21 Therefore, comparisons of the cytotoxic activities forthese two metal complexes are often made. For example, in astudy, the anti-cancer activities of Pd and Pt complexes have beencompared against different cancer cell lines and it was concludedthat the palladium complexes are less cytostatic than their plati-num analogous.22 The results from the studies presented hereinshowed that, complex 2 demonstrated better growth-inhibiting ef-fects than Pd (II) complexes (complex 1 and 3) and the other Pt(II)complex (complex 4), against A549, PC3, Hep3B and C6 cells. Mar-ques et al. showed in their study that distinct Pt complexes maydisplay different cytotoxic and anti-proliferative effect due to thestructural features of compounds.23 In addition, the results of thepresent work also confirm that the platinum(II) complex contain-ing two planar heterocyclic 2-hmpy ligands results in activationof the trans geometry and greatly enhanced cytotoxic potency asobserved earlier for [Pt(2-hepy)2Cl2].9 In the last two decade, anumber of trans-configured platinum(II) complexes have shownsignificant anticancer activity.24–30
Please cite this article in press as: Ari, F.; et al. Bioorg. Med. Chem. (201
3.4. Detection of caspase-cleaved cytokeratin 18
The apoptosis inducing effect of the complex 2 was investigatedby the M30 assay. Cisplatin (12.6 lM) was used as a positive con-trol for comparison with complex 2. C6 cells do not have any cyto-keratin 18. Therefore, this cell line was not used for the detection ofM30 assay. Figure 6 shows that M30 levels (a marker of apoptosis)were not changed in PC3 and Hep3B after 48 h treatment withcomplex 2 (200 lM). In addition, cisplatin did not exhibit anysignificant increase in these cell lines. However, it is noticeable thatM30 levels were increased in A549 cells after the treatment withcomplex 2 and Cisplatin. This shows that complex 2 induces apop-totic cell death in A549 cells. However, PC3 and Hep3B cells did notexhibit any increase in M30 (caspase-cleaved cytokeratin 18)levels, implying that the mode of cell death may either be differentfrom apoptosis or anti-growth effect was resulted from cytostaticeffect, not from cytotoxic effect, or other reasons regarding thegenomic status of the cells might play a role. Regarding the geno-mic status, it was reported that in the breast cancer cell linesexpressing low amount of vimentin, the cytokeratin levels mayalso be low.31 Also in another study, it was shown that Hep3B cellline expresses vimentin at high level.32 Hence, the M30 assay maynot be an ideal test to detect apoptosis in such cell lines.
3.5. Detection of active caspase-3 and cleaved PARP
Caspase-3 also known as CPP32 (32 kDa cysteine protease) orapopain is a cysteine protease with aspartate specificity and a
8 F. Ari et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
well-characterized effector of apoptosis. Caspase-3 was synthe-sized as an inactive proenzyme, where upon cleavage at Asp175/Ser176, is converted to the active enzyme. As caspase-3 activationfollowed by PARP cleavage is a key event in the process of apopto-sis, and that these two events are used as markers for apoptosisinduction we sought to determine the role of complex 2 on cas-pase-3 activation and PARP cleavage. The effect of complex 2 oncaspase-3 activation and PARP cleavage was shown in Figure 7.
As can be seen in Figure 7A, caspase-3 activity was not en-hanced in PC3 and Hep3B cells after 48 h with complex 2(200 lM). In contrast, we found that complex 2 induced caspase-3 activity (�twofold) in A549 cells. This apoptosis-inducing effectalso confirmed the caspase-cleaved cytokeratin 18 increases in thiscell line demonstrated above for the complex 2. Taking into ac-count the results of caspase-3 activity, increased cleaved-PARP lev-els (twofold) were observed only in A549 cell line. Confirming theapoptosis once again in A549 cells (Fig. 7B).
Previously reported that several different Pt(II) and Pt(IV) com-plexes were induced caspase-dependent apoptosis in different can-cer cell lines.33 Kim et al.34 reported that Hep3B cells may undergoapoptosis through caspase activation-independent apoptotic path-way when apoptosis is induced by cisplatin. We suggest that com-plex 2 as a Pt-based agent may induce apoptotic cell death throughthe caspase-independent pathway in Hep3B and PC3 cell lines.
3.6. Detection of phosphatidylserine (PS) translocation
The results of the M30 assay above showed that the mode of thecell death (cytotoxicity) induced by the complex 2 might not havebeen due to the induction of apoptosis in PC3, Hep3B and C6 cellsbecause of the lack of any increase in M30 levels. Therefore, weexamined the mode of cell death histologically by fluorescenceimaging on the basis of phosphatidylserine (PS) translocation. Cellswere treated with complex 2, Cisplatin and paclitaxel for 12 h. Weobserved pyknotic nucleus (a marker of apoptosis) in all cell linesby staining with Hoechst dye 43332. Early apoptotic cells exposingphosphatidylserine on the cell membrane surface are observedwith green staining and late apoptotic cells were stained in bothgreen and red.
In A549 cell line, apoptotic cell death was evident from pyknoticnuclei, as well as from the presence of Annexin-V-FITC stainingpositivity and PI staining negativity. In the C6 cell line, all the trea-ted cells were positive for both staining (Annexin-V-FITC and PI)(Fig. 8A and D), implying either apoptosis-like cell death (due topyknotic nucleus at the same time) or other kind of cell death(e.g., aponecrosis). In PC3 and Hep3B cell lines, treated cells wereobserved to be at both early stage of apoptosis (positivity forAnnexin-V-FITC staining and negativity for PI staining) and at latestage of apoptosis (positivity for Annexin-V-FITC and positivity forPI staining) at the same time point (Fig. 8B, C). The presence of bothearly and late stage apoptotic cells together implies that apoptosisoccurs in these cell lines relatively at a sooner time point.
Cisplatin did not show apoptosis inducing effect when com-pared with untreated control in A549 cells although an early stageof apoptosis were observed in PC3 cells (Fig. 8A and B). This impliesthat the cytotoxic effect of the compounds also depends on the celltype. In addition, Hep3B and C6 cells exhibited both of them (earlyand late stage of apoptosis) at the same time point (Fig. 8C and D).However, paclitaxel induced pyknotic changes in nucleus, suggest-ing apoptosis, in all cell lines. We confirmed both early apoptotic(positivity for Annexin-V-FITC staining and negativity for PI stain-ing) and late apoptotic (positivity for Annexin-V-FITC staining andpositivity for PI staining) cells (Fig. 8A–D).
In conclusion, the Pt complex (complex 2), but not the corre-sponding Pd complex, had a strong anti-cancer activity againstcancer cells (A549, PC3, Hep3B and C6) in a dose-dependent
Please cite this article in press as: Ari, F.; et al. Bioorg. Med. Chem. (201
manner. The anti-growth effect was found to be resulted fromapoptosis, depending on the cell type used.
Supplemenatry data
CCDC 942988 and 942989 contain the supplementary crystallo-graphic data for complexes 1 and 2, respectively. These data can beobtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic DataCentre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected].
Acknowledgement
We appreciate the Research Fund of University of Uludag forproviding us with the kits/chemicals.
References and notes
1. Baran, E. J.; Yilmaz, V. T. Coord. Chem. Rev. 2006, 250, 1980.2. Cavicchioli, M.; Massabni, A. C.; Castellano, E. E.; Sabeh, L. P. B.; Costa-Neto, C.
Yilmaz, V. T. J. Cancer Res. Clin. Oncol. 2011, 137, 1425.6. Ulukaya, E.; Ari, F.; Dimas, K.; Ikitimur, E. I.; Guney, E.; Yilmaz, V. T. Eur. J. Med.
Chem. 2011, 46, 4957.7. Coskun, M. D.; Ari, F.; Yilmaztepe Oral, A.; Sarimahmut, M.; Kutlu, H. M.;
Yilmaz, V. T.; Ulukaya, E. Bioorg. Med. Chem. 2013, 21, 4698.8. Icsel, C.; Yilmaz, V. T. DNA Cell Biol. 2013, 32, 165.9. Icsel, C.; Yilmaz, V. T.; Ari, F.; Ulukaya, E.; Harrison, W. T. A. Eur. J. Med. Chem.
2013, 60, 386.10. Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112.11. Ulukaya, E.; Ozdikicioglu, F.; Oral, A. Y.; Demirci, M. Toxicol. In Vitro 2008, 22,
232.12. Andreotti, P. E.; Cree, I. A.; Kurbacher, C. M.; Hartmann, D. M.; Linder, D.; Harel,
G. Cancer Res. 1995, 55, 5276.13. Park, S. J.; Wu, C. H.; Gordon, J. D.; Zhong, X.; Emami, A.; Safa, A. R. J. Biol. Chem.
2004, 279, 51057.14. Guney, E.; Yilmaz, V. T.; Kazak, C. Polyhedron 2010, 29, 1285.15. Guney, E.; Yilmaz, V. T.; Sengul, A.; Buyukgungor, O. Inorg. Chim. Acta 2010, 363,
438.16. Guney, E.; Yilmaz, V. T.; Buyukgungor, O. Inorg. Chim. Acta 2010, 363, 2416.17. Guney, E.; Yilmaz, V. T.; Buyukgungor, O. Polyhedron 2011, 30, 1968.18. Garoufis, A.; Hadjikakou, S. K.; Hadjiliadis, N. Coord. Chem. Rev. 2009, 253, 1384.19. Komeda, S.; Takayama, H.; Suzuki, T.; Odani, A.; Yamori, T.; Chikuma, M.
Metallomics 2013, 5, 461.20. Ari, F.; Ulukaya, E.; Sarimahmut, M.; Yilmaz, V. T. Bioorg. Med. Chem. 2013, 21,
3016.21. Rau, T.; van Eldik, R. Metal Ions in Biological Systems. In Platinum and Other
Metal Coordination Compounds in Cancer Chemotherapy; Sigel, H., Sigel, A., Eds.;Marcel Dekker: New York, 1996; pp 339–378.
22. Karaküçük-_Iyidogan, A.; Tas�demir, D.; Oruç-Emre, E. E.; Balzarini, J. Eur. J. Med.Chem. 2011, 46, 5616.
23. Marques, M. P. M.; Girao, T.; Pedroso De Lima, M. C.; Gameiro, A.; Pereira, E.;Garcia, P. Biochim. Biophys. Acta 2002, 1589, 63.
24. Natile, G.; Coluccia, M. Coord. Chem. Rev. 2001, 216–217, 383.25. Coluccia, M.; Natile, G. Anti-Cancer Agents Med. Chem. 2007, 7, 111.26. Novakova, O.; Kasparkova, J.; Malina, J.; Natile, G.; Brabec, V. Nucleic Acids Res.
2003, 31, 6450.27. Farrell, N.; Ha, T. T. B.; Souchard, J. P.; Wimmer, F. L.; Cros, S.; Johnson, N. P. J.
Med. Chem. 1989, 32, 2240.28. Aris, S. M.; Farrell, N. P. Eur. J. Inorg. Chem. 2009, 10, 1293.29. Kasparkova, J.; Novakova, O.; Farrell, N.; Brabec, V. Biochemistry 2003, 42, 792.30. Zakovska, A.; Novakova, O.; Balcarova, Z.; Bierbach, U.; Farrell, N.; Brabec, V.
Eur. J. Biochem. 1998, 254, 547.31. Sommers, C. L.; Walker-Jones, D.; Heckford, S. E.; Worland, P.; Valverius, E.;
Clark, R.; McCormick, F.; Stampfer, M.; Abularach, S.; Gelmann, E. P. Cancer Res.1989, 49, 4258.
33. Kaluderovic, G. N.; Mijatovic, S. A.; Zmejkovski, B. B.; Bulatovic, M. Z.; Gomez-Ruiz, S.; Mojic, M. K.; Steinborn, D.; Miljkovic, D. M.; Schmidt, H.; Stosic-Grujicic, S. D.; Saboe, T. J.; Maksimovic-Ivanic, D. D. Metallomics 2012, 4, 979.
34. Kim, J. S.; Lee, J. M.; Chwae, Y. J.; Kim, Y. H.; Lee, J. H.; Kim, K.; Lee, T. H.; Kim, S.J.; Park, J. H. Biochem. Pharmacol. 2004, 67, 1459.
