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Cytotoxicity and Antiviral Activity of Palladium(II) and Platinum(II) Complexes with 2-(Diphenylphosphino)Benzaldehyde 1-Adamantoylhydrazone

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Cytotoxicity and Antiviral Activity of Palladium(II) and Platinum(II) Complexes with 2-(Diphenylphosphino)Benzaldehyde 1-Adamantoylhydrazone Turkish Journal of Biology Turk J Biol (2016) 40: 661-669 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1503-23 Cytotoxicity and antiviral activity of palladium(II) and platinum(II) complexes with 2-(diphenylphosphino)benzaldehyde 1-adamantoylhydrazone 1, 2 3 3 2 Vera SIMIĆ *, Stoimir KOLAREVIĆ , Ilija BRČESKI , Dejan JEREMIĆ , Branka VUKOVIĆ-GAČIĆ 1 National Control Laboratory, Medicines and Medical Devices Agency of Serbia, Belgrade, Serbia 2 Chair of Microbiology, Center for Genotoxicology and Ecogenotoxicology, Faculty of Biology, University of Belgrade, Belgrade, Serbia 3 Faculty of Chemistry, University of Belgrade, Belgrade, Serbia Received: 09.03.2015 Accepted/Published Online: 26.08.2015 Final Version: 18.05.2016 Abstract: Metal coordination compounds have an important role in the development of novel drugs. Using the resazurin microtitration assay we assessed the cytotoxicity and antiviral activity of the ligand 2-(diphenylphosphino)benzaldehyde 1-adamantoylhydrazone and its Pd(II) and Pt(II) complexes. Cytotoxicity was tested in A549 human lung adenocarcinoma epithelial cells. We observed that the ligand displayed a more pronounced cytotoxic activity than the platinum-based drug, carboplatin. Morphological evaluation of A549 cells treated with the ligand by acridine orange and ethidium bromide double staining revealed the presence of signs of apoptosis. Antiviral activity against poliovirus type 1 was assessed by examination of the cytopathic effect (CPE) in Hep-2 cells. Cells that were exposed to the 19 µM ligand before infection displayed a maximal significant reduction (by 24.42 ± 1.49%) of the CPE. This was likely due to the inhibition of virus receptors and prevention of viral adsorption. Treatment with 17 µM Pt(II) complex after viral infection caused a maximal significant reduction (by 30.52 ± 3.12%) of the CPE, presumably through an effect on viral replication. The results indicate that the ligand should be viewed as a potential anticancer agent. The ligand and the Pt(II) complex show promising results for further investigation of antiviral activity. Key words: Cytotoxicity, antiviral activity, 2-(diphenylphosphino)benzaldehyde hydrazone ligands, cancer cell lines, apoptosis 1. Introduction benzaldehyde 1-adamantoylhydrazone and its Pd(II) and The clinical use of metal coordination compounds has Pt(II) complexes (Figure 1) in human larynx carcinoma improved considerably in recent years in clinical therapy. Hep-2 cells and healthy human lung MRC-5 cells (Đorđević Platinum-based drugs (cisplatin, carboplatin, and et al., 2014). The antiviral activities of Pt(II) and Pd(II) oxaliplatin) have a wide use in the treatment of human complexes with 2-(diphenylphosphino)benzaldehyde malignancies, such as ovarian, testicular, head and neck, 1-adamantoylhydrazone ligand have not been studied. and lung cancers. Considerable effort has been devoted Several studies have shown that palladium complexes to the development of new anticancer metal coordination have cytotoxic activities similar to standard platinum- compounds that impede the development of resistance and based drugs (Gao et al., 2010). Some palladium complexes produce fewer side effects during chemotherapy (Frezza et have better growth-inhibition effects on human nonsmall- al., 2010). cell lung cancer in vitro than cisplatin (Ulukaya et al., Uncovering the anticancer properties of phosphines 2011). has led to the development and biological evaluation of Ligand 2-(diphenylphosphino)benzaldehyde 1-adam- numerous metal complexes with phosphine ligands (Starosta antoylhydrazone and its Pd(II) and Pt(II) complexes et al., 2011; Galassi et al., 2012; Milenković et al., 2013a, contain an adamantyl group, which changes the therapeutic 2013b). Metal complexes with 2-(diphenylphosphino) profile of the pharmacologically active molecules (Liu benzaldehyde (dpba) hydrazone ligands also have et al., 2011). Compounds with the adamantyl group are improved cytotoxic activity (Malešević et al, 2006). In widely used for prophylaxis and treatment of influenza our previous work we investigated the biological activity infections (Monto, 2003) and also possess antiviral of the novel synthesized ligand 2-(diphenylphosphino) potency against viruses of the family Picornaviridae (De * Correspondence: [email protected] 661 SIMIĆ et al. / Turk J Biol a b Figure 1. Chemical structures of a) ligand and b) palladium(II) and platinum(II) complexes (M = Pd, Pt). Palma et al., 2008). Poliovirus, a member of the genus 2.2. Test compounds Enterovirus (family Picornaviridae), is a causative agent 2-(Diphenylphosphino)benzaldehyde 1-adamantoylhydr- of paralytic poliomyelitis. The Hep-2 cell line is a suitable azone (HL) (43.1 mM, referred to as compound ‘1’); host cell model with an elevated sensitivity to poliovirus Pd(II) complex with 2-(diphenylphosphino)benzaldehyde infection. Hep-2 cells provide a high yield of poliovirus 1-adamantoylhydrazone, [Pd(L)Cl], (34.3 mM, compound and exhibit a visible and reliable level of cytopathic effect ‘2’); and Pt(II) complex with 2-(diphenylphosphino) (CPE) (Johnston and Siegel, 1990; WHO, 1997). benzaldehyde 1-adamantoylhyd-razone, [Pt(L)Cl], (19.8 mM, compound ‘3’) were used at the indicated In this study we examined the cytotoxicity of concentrations. ligand 2-(diphenylphosphino)benzaldehyde 1-adam- antoylhydrazone and its Pt(II) and Pd(II) complexes 2.3. Virus As a starting virus suspension, we used the poliovirus in A549 human lung adenocarcinoma cells, and their reference standard, which contains a mixture of poliovirus antiviral activity against poliovirus type 1 in Hep-2 cells types 1, 2, and 3. Antipoliovirus sera types 2 and 3 were using the resazurin microtitration assay (UptiBlue assay). added to the poliovirus reference standard. Poliovirus types 2 and 3 were neutralized at 37 °C for 3 h. After 2. Materials and methods neutralization of poliovirus types 2 and 3, the content of 2.1. Reagents and chemicals poliovirus type 1 was determined by the microtitration Minimal essential medium (MEM, GIBCO), penicillin- assay in Hep-2 cell culture, using the Kärber formula. The streptomycin (Pen Strep, 10,000 U/mL penicillin, 10,000 results were expressed as a tissue culture infection dose µg/mL streptomycin, GIBCO), nonessential amino acids per milliliter (TCID50/mL) (WHO, 1997). The stock virus (MEM NEAA, GIBCO), L-glutamine (2 mM, GIBCO), suspension was aliquoted and stored at –20 °C until use. fetal bovine serum (FBS, GIBCO), nutrient medium Dilution of the virus suspension was prepared in nutrient (MEM, 1% Pen Strep, 1% MEM NEAA, 1% L-glutamine, medium supplemented with 2% FBS. supplemented with FBS), trypsin-EDTA (0.05%, GIBCO), 2.4. Cell cultures UptiBlue (Uptima, Interchim), acridine orange (10 mg/mL, A549 human lung adenocarcinoma epithelial cells and Sigma), ethidium bromide (10 mg/mL, Sigma), Dulbecco’s Hep-2 human larynx carcinoma cells were cultivated in a nutrient medium supplemented with 5%–10% FBS at 37 phosphate-buffered saline (DPBS, GIBCO), dimethyl °C and 5% CO . The confluent monolayer was trypsinized sulfoxide (DMSO, Sigma-Aldrich), cisplatin (0.5 mg/mL, 2 with 0.05% trypsin-EDTA. The cells were resuspended Cisplatin, Medac), carboplatin (10 mg/mL, Carboplatin- in a nutrient medium supplemented with 10% FBS and Teva, Teva), oxaliplatin (5 mg/mL, Eloxatin, Sanofi- placed into cell culture 96-well microtiter plates (Nunc), at 6 Aventis), interferon α-2a (3 × 10 IU/0.5 mL, Roferon-A, a concentration of 1 × 105 cells/mL. The microtiter plates Roche), poliovirus reference standard (2nd International were incubated for 24 h at 37 °C and 5% CO2 humidified Reference Reagent 2004 for Live Attenuated Poliovirus air. The quality of the cell monolayer was confirmed with (Sabin), National Institute for Biological Standards and an inverted microscope (Freshney, 2005). Control, code: 02/306), and antipoliovirus sera (3rd 2.5. Cytotoxicity assay in A549 cells International Standard Anti-Poliovirus Serum Types 1, The cytotoxic potential of test compounds 1, 2, and 3 was 2, and 3, National Institute for Biological Standards and assayed in the A549 lung adenocarcinoma epithelial cell Control, code: 82/585) were used. line, using UptiBlue (Vega-Avila and Pugsley, 2011). 662 SIMIĆ et al. / Turk J Biol The A549 cell line is chosen for the cytotoxicity and emits red fluorescence by intercalation into DNA. examination due to a high potential for resistance The A549 cells were treated with the IC50 concentration development to cisplatin (Cetintas et al., 2012). The of ligand for 48 h. After the treatment, A549 cells were cytotoxic drugs cisplatin, carboplatin, and oxaliplatin washed with DPBS and trypsinized. The cells were stained served as positive controls. Two-fold serial dilutions with 2 µL of AO/EB solution (100 µg/mL AO and 100 µg/ were prepared in nutrient medium supplemented with mL EB) and observed under a fluorescence microscope 10% FBS, starting from 336.5 µM, 268 µM, and 155.2 µM (Leica) at 400× magnification within 30 min before for compounds 1, 2, and 3, respectively, and from 416 the fluorescence color started to fade. According to the µM, 672 µM, and 628 µM for cisplatin, carboplatin, and fluorescence emission and the morphological aspect of oxaliplatin, respectively. Diluted compounds were added chromatin condensation of stained nuclei, the cells were to microtiter plates with an A549 cell monolayer. The distinguished as described by Baskić et al. (2006). cells were incubated for 48 h at 37 °C and
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