Journal of Inorganic Biochemistry 142 (2015) 118–125

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio

A monofunctional platinum(II)-based anticancer agent from a salicylanilide derivative: Synthesis, antiproliferative activity, and transcription inhibition Beilei Wang a,b, Zhigang Wang a,b, Fujin Ai a,b, Wai Kin Tang a, Guangyu Zhu a,b,⁎ a b

Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong Special Administrative Region Shenzhen Research Institute of City University of Hong Kong, Shenzhen, China

a r t i c l e

i n f o

Article history: Received 19 August 2014 Received in revised form 8 October 2014 Accepted 8 October 2014 Available online 18 October 2014 Keywords: Cisplatin Platinum-based anticancer agents Transcription inhibition DNA binding

a b s t r a c t Cationic monofunctional platinum(II)-based anticancer agents with a general formula of cis-[Pt(NH3)2(N-donor) Cl]+ have recently drawn significant attention due to their unique mode of action, distinctive anticancer spectrum, and promising antitumor activity both in vitro and in vivo. Understanding the mechanism of action of novel monofunctional platinum compounds through rational drug design will aid in the further development of active agents. In this study, we synthesized and evaluated a monofunctional platinum-based anticancer agent SA–Pt containing a bulky salicylanilide moiety. The antiproliferative activity of SA–Pt was close to that of cisplatin. Mechanism studies revealed that SA–Pt entered HeLa cells more efficiently than cisplatin, blocked the cell cycle at the S-phase, and induced apoptosis. The compound bound to DNA as effectively as cisplatin, but did not block RNA polymerase II-mediated transcription as strongly as cisplatin, indicating that once the compound formed Pt-DNA lesions, the salicylanilide group was more easily recognized and removed. This study not only enriches the family of monofunctional platinum-based anticancer agents but also guides the design of more potent monofunctional platinum complexes. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The serendipitous discovery of the antitumor activity of cisplatin 45 years ago began a new era for metal-based anticancer drug development [1]. Thousands of platinum compounds were subsequently synthesized and tested for anticancer activity [2]. Two additional platinum compounds, carboplatin and oxaliplatin, were approved by the US FDA, and a couple of more were approved in China and Japan [3]. Extensive studies have been conducted to reveal the mechanism of action of platinum drugs, with the aim of gaining the information needed to design the next generation of platinum-based chemotherapeutics [4,5]. It is now generally agreed that the major cytotoxicity of platinum drugs stems from their binding to DNA to form Pt-DNA cross-links that inhibit transcription and trigger apoptosis [6]. In the search for novel platinum compounds with improved antitumor profiles, enhanced accumulation, and reduced resistance compared to cisplatin, “non-traditional” platinum compounds that do not obey the original structure–activity relationships have recently attracted attention [7]. Among them, monofunctional platinum-based anticancer agents with ⁎ Corresponding author at: Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong Special Administrative Region. Tel.: + 852 34426857; fax: +852 3442 0522. E-mail address: [email protected] (G. Zhu).

http://dx.doi.org/10.1016/j.jinorgbio.2014.10.003 0162-0134/© 2014 Elsevier Inc. All rights reserved.

the general formula of cis-[Pt(NH3)2(L)Cl]+, where L is an N-heterocycle, have displayed promising antitumor activity. Mechanism studies have revealed that monofunctional platinum compounds bind well to DNA and effectively inhibit transcription both in vitro and in live mammalian cells [8,9]. In these cases, the choice for L is usually small N-heterocyclic amine ligands with a molecular weight of less than 200, such as pyridine (for pyriplatin) or phenanthridine (for phenanthriplatin, Fig. 1). The properties of N-heterocyclic ligands have impact on the biological activity of monofunctional platinum compounds. For example, in pyriplatin, pyridine faces towards the 5′-end of the platinated strand and acts as a steric block to inhibit RNA polymerase II (Pol II)-mediated transcription [10]. In phenanthriplatin, the plane of phenanthridine is approximately perpendicular to that of platinum [9]. Compared with bifunctional cisplatin, monofunctional compounds display a distinct mechanism of action and a different antitumor profile [9,11,12]. Questions remain regarding the choice of the N-heterocycle ligand to tune anticancer activity and influence the mechanism of monofunctional platinum complexes. For instance, if the ligand is a more bulky Nheterocycle with a molecular weight greater than 400, will the corresponding monofunctional platinum compound's antitumor activity be identical to that of cisplatin? Will the compound efficiently enter cells and trigger apoptosis? What differences, if any, are there in the DNA binding ability of the compound versus those of cisplatin? To what extent does such a compound inhibit transcription in live mammalian cells? Will the

B. Wang et al. / Journal of Inorganic Biochemistry 142 (2015) 118–125

119

Fig. 1. Chemical structures of monofunctional platinum(II) anticancer agents, pyriplatin, phenanthriplatin, and SA–Pt.

compound be recognized more effectively by cellular repair machineries? To address these questions, we designed, synthesized, and characterized a monofunctional platinum-based anticancer agent from a bulky N-donor ligand. Profound biological studies were conducted to reveal the detailed mode of action for this compound. Such information enriches our understanding of monofunctional platinum-based anticancer agents and guides the design of the next generation of monofunctional platinum complexes with improved anticancer activity. 2. Materials and methods 2.1. Materials and measurements All chemicals and solvents were purchased from commercial suppliers and applied directly in the experiments without further purification. (2,2′:6′,2″-Terpyridine)platinum(II) {[((terpy)PtCl)Cl]} was synthesized according to a previously published paper and characterized by 1H NMR and MS (mass spectrometry) (data not shown) [13]. 1H and 13C NMR spectra were recorded on a Bruker DPX-400 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm) relative to internal standard residual solvent peaks. 1H NMR coupling constants (J) are reported in Hertz (Hz), and multiplicity is indicated as follows: s (singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of doublet). Mass spectra were obtained on a PC Sciex API 150 EX ESI-MS (electrospray ionization mass spectrometry) system. Platinum contents were analyzed by a PerkinElmer PE Optima 2100 DV ICP-OES (inductively coupled plasmaoptical emission spectrometry). Concentrations of plasmid DNA were ascertained by a NanoDrop UV–vis (UV–visible) spectrophotometer at 260 nm. Other UV–vis spectra were measured on a Shimadzu 1700 spectrometer. Emission spectra were obtained using a Horiba FluoroMax-4 spectrometer. 2.2. General synthesis procedure 2.2.1. Synthesis of 6-(2-chloro-4-nitrophenoxy)quinolone (2) To 1-bromo-2-chloro-4-nitrobenzene (1, 2.90 g, 12.26 mmol) in DMF (dimethylformamide), 6-hydroxyquinoline (1.78 g, 12.26 mmol, 1 equiv.) and K2CO3 (5.01 g, 36.78 mmol, 3 equiv.) were added, and the mixture was left to stir under nitrogen atmosphere. The reaction was kept at 100 °C until completion (12–24 h). The reaction progress was monitored by TLC (ethyl acetate/petroleum ether = 1/5). Once the reaction was complete, the solvent was removed using rotary vacuum evaporation, and 30 mL H2O was added. The aqueous phase was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to get pure yellow solid 2 without further purification (3.3 g, yield: 90%). 1H NMR (400 MHz, DMSO-d6) δ 8.90 (dd, J = 4.2, 1.7 Hz, 1H), 8.51 (d, J = 2.7 Hz, 1H), 8.37–8.30 (m, 1H), 8.18 (dd, J =9.1, 2.8 Hz, 1H), 8.13 (d, J = 9.1 Hz, 1H), 7.71 (d, J = 2.7 Hz, 1H), 7.65 (dd, J = 9.1, 2.8 Hz, 1H), 7.56 (dd, J = 8.3, 4.2 Hz, 1H), 7.22 (d, J =9.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 158.1, 152.7, 150.8, 145.8, 143.6, 136.1, 132.3,

129.2, 126.7, 125.1, 124.6, 123.6, 122.7, 119.5, 116.1. MS (ESI+) m/z: [M + H]+ calculated for C15H9ClN2O3: 301.04, found: 301.2.

2.2.2. Synthesis of 3-chloro-4-(quinolin-6-yloxy)aniline (3) To a solution of 6-(2-chloro-4-nitrophenoxy)quinoline (3.0 g, 10 mmol) dissolved in 35 mL methanol, NH4Cl solution (2.7 g, 50 mmol in 25 mL H2O) was added. Then zinc power (1.95 g, 30 mmol) was added to the mixture, and the reaction was refluxed for about 6 h. The reaction progress was monitored by TLC (ethyl acetate/petroleum ether = 1/2), and once the reaction was complete, the mixture was filtered. To the filtrate was added 15 mL H2O. The aqueous phase was extracted with ethyl acetate (3 × 30 mL), and the combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue obtained was purified by silica gel column chromatography using petroleum ether/ethyl acetate (1/2) as eluent to afford target compound 3 as yellow solid (1.65 g, yield: 65%). 1H NMR (400 MHz, DMSO-d6) δ 8.75 (dd, J = 4.2, 1.6 Hz, 1H), 8.22 (d, J =7.6 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.48 (dd, J = 9.2, 2.8 Hz, 1H), 7.44 (dd, J = 8.3, 4.2 Hz, 1H), 7.06–7.00 (m, 2H), 6.76 (d, J = 2.6 Hz, 1H), 6.60 (dd, J = 8.7, 2.6 Hz, 1H), 5.41 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.8, 149.1, 148.1, 144.6, 139.9, 135.5, 131.4, 129.2, 126.4, 124.4, 122.3, 121.8, 114.9, 114.3, 109.4. MS (ESI+) m/z: [M + H]+ calculated for C15H11ClN2O: 271.07, found: 271.2.

2.2.3. Synthesis of 3,5-dichloro-N-(3-chloro-4-(quinolin-6-yloxy)phenyl)2-hydroxybenzamide (4) To a suspension of 3,5-dichlorosalicylic acid (0.92 g, 4.44 mmol) in 30 mL dry dichloromethane, O-(7-azabenzotriazol-1-yl)-N,N,N′, N′-tetramethyluronium hexafluorophosphate (HATU, 5.9 g, 3.5 equiv.) and N,N-diisopropylethylamine (2.0 g, 3.5 equiv.) were added under N2, and the mixture was stirred at room temperature for 3 h. Then 3-chloro-4-(quinolin-6-yloxy)aniline (1.0 g, 3.7 mmol, 1.1 equiv.) was added to the mixture and stirred at room temperature for 12–24 h. The reaction was diluted with dichloromethane (30 mL), and the mixture was washed with water (20 mL) and brine (3 × 20 mL). The organic layer was dried with Na2SO4, filtered, and concentrated in vacuo. The crude residue obtained was purified by silica gel column chromatography using dichloromethane/methanol (15/1) as eluent to afford target compound 4 as yellow solid (1.39 g, yield: 82%). 1H NMR (400 MHz, DMSO-d6) δ 12.44 (br, 1H), 10.84 (s, 1H), 8.86–8.76 (m, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.13–8.00 (m, 3H), 7.80 (d, J = 2.3 Hz, 1H), 7.71 (dd, J = 8.9, 2.4 Hz, 1H), 7.57 (dd, J = 9.2, 2.7 Hz, 1H), 7.50 (dd, J =8.3, 4.2 Hz, 1H), 7.34 (d, J = 8.9 Hz, 1H), 7.27 (d, J = 2.7 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6 ) δ 166.8, 155.4, 155.0, 149.6, 147.8, 144.7, 136.0, 135.8, 133.5, 131.6, 129.2, 127.0, 125.5, 123.6, 123.2, 123.0, 122.8, 122.4, 122.2, 119.5, 111.5. MS (ESI+ ) m/z: [M + H] + calculated for C 22H13 Cl3N 2 O3 : 459.01, found: 459.2.

120

B. Wang et al. / Journal of Inorganic Biochemistry 142 (2015) 118–125

2.2.4. Synthesis of cis-{Pt(NH3)2[3,5-dichloro-N-(3-chloro-4-(quinolin-6yloxy)phenyl)-2-hydroxybenzamide]Cl}NO3 (SA–Pt) Nitrate salt of complex SA–Pt was prepared through a modification of a reported method [14], and the details are given as follows. AgNO3 (56.6 mg, 0.33 mmol, 1.0 equiv.) was added to a solution of cisplatin (0.1 g, 0.33 mmol) in 30 mL DMF, and the reaction was stirred at 55 °C under protection from light. After 16 h, the mixture was centrifuged at 4500 rpm, 4 °C for 20 min to remove AgCl precipitate. To the supernatant, 3,5-dichloro-N-(3-chloro-4-(quinolin-6-yloxy)phenyl)-2hydroxybenzamide (4, 137.9 mg, 0.297 mmol, 0.9 equiv. of cisplatin) was added at one port, and the reaction was carried out at 55 °C with stirring for 16 h. The reaction mixture was evaporated under reduced pressure and the residue was dissolved in 35 mL of MeOH. Unreacted cisplatin was removed in a form of yellow powder by centrifugation at 4500 rpm, 25 °C for 20 min. The supernatant was stirred vigorously, and 100 mL diethyl ether was subsequently added to precipitate the desired compound as solid. The compound was washed twice with 30 mL of diethyl ether, and purified by recrystallization in methanol and diethyl ether. The final product was isolated by centrifugation and dried under reduced pressure (105 mg, yield: 45%). 1H NMR (400 MHz, DMSO-d6 ) δ 12.45 (s, 1H), 11.18 (s, 1H), 9.58 (d, J = 9.5 Hz, 1H), 9.19–9.09 (m, 1H), 8.57 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 2.4 Hz, 1H), 8.02 (d, J = 2.5 Hz, 1H), 7.94 (s, 1H), 7.91 (dd, J = 9.5, 2.8 Hz, 1H), 7.80 (d, J = 2.4 Hz, 1H), 7.75 (dd, J = 8.9, 2.4 Hz, 1H), 7.63 (dd, J = 8.4, 5.3 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 2.8 Hz, 1H), 4.54 (s, 3H), 4.40 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 168.4, 158.3, 156.9, 155.2, 148.6, 145.3, 139.8, 137.4, 134.7, 132.3, 131.7, 127.9, 127.4, 125.0, 124.8, 124.5, 124.4, 124.2, 124.0, 123.0, 119.3, 111.7. MS (ESI+) m/z: [M]+ calculated for [C22H19Cl4N4O3Pt]+: 723.98, found: 724.0. Anal. calcd. for C22H19Cl4N5O6Pt: C 33.60, H 2.44, N 8.91. Found: C 33.13, H 2.94, N 8.96. 2.3. PARP [poly(ADP-ribose) polymerase] and PARG [poly(ADP-ribose) glycohydrolase] assays PARP assay was carried out according to a published protocol [15]. PARG activity was ascertained using a HT Universal Colorimetric PARG Assay Kit (Trevigen, MD, USA). A negative control without PARG enzyme provided the 100% reference absorbance. ADP-ribosylation reaction was occurred by adding PARP-1 and activated DNA to histones that coated on the wells and incubating at room temperature for 30 min. After the formation of poly(ADP-ribose) (PAR), compounds 4 and SA–Pt were added to each well together with PARG and incubated for 2 h at room temperature for hydrolysis of PAR. The final concentration of DMF was 1% in this assay and in the following biological assays. The reaction was stopped by removing the solution and washing strip wells twice with PBS (phosphate buffered saline) containing 0.1% Triton X-100 and twice with PBS, then 50 μL Strep-HRP was added to each well. After incubation at room temperature for 30 min, the wells were washed again. The final product was detected by incubation with TACS-Sapphire for 15 min at room temperature in the dark. An equal volume of freshly prepared 5% phosphoric acid was added to stop the reaction, which generated a yellow color stable for up to 60 min at ambient temperature. The absorbance was read at 450 nm on a Biotek Microplate Reader. 2.4. Cell-based assays 2.4.1. Cell lines and cell culture Human cancer cells HeLa (cervical), A549, A549cDDP (non-smallcell lung), MDA-MB-231, MDA-MB-436, MCF-7 (breast), and A2780 (ovarian), as well as human normal lung fibroblasts MRC-5 were used. All the cells were grown in a humidified incubator at 37 °C under 5% CO2. A549, A549cDDP, HeLa, MCF-7, and MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin,

MDA-MB-436 cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids, and 2 mM L-glutamine. A2780 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium supported with 10% FBS, 2 mM Lglutamine, and 1% penicillin/streptomycin. MRC-5 cells were grown in minimum essential medium (MEM) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids, and 2 mM Lglutamine. Cells were grown in tissue culture flasks until they were 85–95% confluent prior to trypsinization and splitting. 2.4.2. Compound treatment and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2-H-tetrazolium bromide (MTT) assay Cells were plated into 96-well microtiter plates at 1500 to 3000 cells per well, depending on the cell type, in the above-mentioned corresponding culture medium. Cells were allowed to attach and grow for 48 h before treated with various concentrations of test agents in culture medium and exposed for additional 72 h. After this period of time, the original medium was removed, a volume of 160 μL fresh medium without FBS together with 40 μL MTT stock solution (5 mg/mL in PBS) was added to each well and cells were incubated at 37 °C for 2–4 h. The MTT-containing medium was then removed completely, followed by the addition of 200 μL DMSO in each well. Cell viabilities were determined by reading the absorbance at 550 nm and 730 nm using a BioTek PowerWave XS microplate reader, and 50% growth inhibitory concentrations (IC50) were calculated using the results from at least two independent experiments. 2.4.3. Intracellular uptake of platinum HeLa cells were seeded in 10 cm culture dishes in DMEM and incubated until the confluency reached about 90%. Then the medium was changed to fresh culture medium containing 10 μM cisplatin or SA–Pt and cells were allowed to grow for 6 h. The medium was subsequently removed and cells were washed with 3 mL ice-cold PBS for three times to remove excess amount of test compounds. The cells were harvested by scraping, washed with 3 mL ice-cold PBS for three times, and spun down at 1500 rpm, 4 °C for 5 min. Cell number was confirmed by counting. Then the cell suspension in PBS was centrifuged, and the supernatant was discarded. To the cell pellet were added 300 μL HNO3 and 200 μL H2O2 to digest the cells at 37 °C for 24 h. Platinum content was measured by ICP-OES. 2.4.4. Cell cycle distribution and apoptosis induction HeLa cells were seeded in 60 mm dishes in DMEM (10% FBS, 1% penicillin/streptomycin) and incubated for 24 h until the confluency reached about 80%. Then the medium was changed to fresh culture medium containing 0, 0.47, 0.93, 1.86, or 3.73 μM cisplatin, SA–Pt, or ligand 4, and cells were incubated for another 24 h. The medium was subsequently removed and cells were washed with 1 mL ice-cold PBS to remove excess amount of agents. The cells were harvested by trypsinization, resuspended in 5 mL ice-cold PBS, counted, and spun down at 1500 rpm, 4 °C for 5 min, and then resuspended to a concentration of 1 × 106 cells/mL in PBS. A volume of 0.5 mL cell suspension was taken out and 4.5 mL cold 70% EtOH was added to fix cells at 4 °C overnight. The cells were subsequently centrifuged, washed with 5 mL cold PBS for three times, and resuspended in 1 mL propidium iodide (PI) staining solution (0.1% Triton-X 100, 200 μg/mL RNase A, and 20 μg/mL PI in PBS). The cells were then incubated under protection from light at 37 °C for 15 min, and cell cycle distribution was analyzed immediately using a flow cytometer (BD FACS Calibur). The data were acquired and analyzed by ModFit 1.2 software. For apoptosis analysis, HeLa cells were treated with test agents, harvested, washed in PBS, and counted. Cells were then resuspended in 1× annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) at a density of 1 × 106 cells/mL. A volume of 5 μL FITCannexin V conjugate and 1 μL 100 μg/mL PI solution were added to each 100 μL of cell suspension. The cells were incubated at room temperature for 15 min, and then 400 μL 1 × annexin-binding buffer was

B. Wang et al. / Journal of Inorganic Biochemistry 142 (2015) 118–125

added. The cells were mixed gently and kept on ice. Cell apoptosis was analyzed immediately by flow cytometry, which measures the fluorescence emission at 530 nm and 575 nm using 488 nm excitation.

2.5. DNA binding assay 2.5.1. Plasmid DNA binding assay Plasmid DNA binding assay was carried out following a previously reported protocol [8]. pGLuc plasmid (80 μg) was treated with different concentrations of SA–Pt or cisplatin in a buffer (24 mM NaHepes pH 7.4) for 16 h at 37 °C. A control plasmid was treated in parallel without platinum compounds. Upon completion, the plasmids in the reaction mixture were precipitated by ethanol and washed twice with 70% ice-cold ethanol. The plasmid pellets were dissolved in a TE buffer (10 mM Tris–HCl, 2 mM EDTA, pH 7.4). Platinum contents were analyzed by ICP-OES. The DNA concentrations were determined by a NanoDrop UV–vis spectrophotometer.

2.5.2. Calf thymus (CT)-DNA binding studies The concentration of CT-DNA in base pairs was determined by measuring the absorbance at 260 nm (ε = 6600 M− 1 cm− 1). Dyebound CT-DNA solution was prepared by mixing certain concentrations of dye with CT-DNA in a binding buffer (50 mM Tris, 200 mM NaCl, 10% DMF, pH = 7.5) for 12 h at 4 °C. The dye-bound DNA solution was stored at 4 °C and used within a week. Fluorescent competitive binding assays were conducted by adding increasing concentrations of Pt complexes to dye-DNA complex. In the case of EB (ethidium bromide), the fluorescent spectra of EB-DNA complex ([EB] = 3 μM, [CT-DNA] = 12 μM) were measured at 530–720 nm using an excitation wavelength of 510 nm. In other experiments, Hoechst 33342-DNA complex ([Hoechst 33342] = 3 μM, [CTDNA] = 12 μM) was excited at 330 nm while 4′,6-diamidino-2phenylindole (DAPI)-DNA complex ([DAPI] = 2 μM, [CT-DNA] = 20 μM) was excited at 370 nm, and the emission at 400–600 nm was monitored. Due to the intrinsic fluorescence of SA–Pt under the excitation of 330 nm and 370 nm, the fluorescence of Hoechst 33342 (3 μM) and DAPI (2 μM) in the presence of SA–Pt (15 μM) was measured to realize the interference of SA–Pt with the dyes. The results suggested that the influence of SA–Pt on the fluorescent properties of Hoechst and DAPI was not significant (data not shown).

2.6. Transcription assay Transfection of the plasmids into HeLa cells was carried out as described previously [8]. In short, HeLa cells were seeded in 96-well plates at a density of 4000 cells per well. The cells were allowed to grow to reach 50% confluence, and were subsequently washed with 100 μL antibiotics-free media. Transient transfection of the cells was carried out in quadruplicate using Lipofectamine 2000. Platinated plasmids (250 ng) and Lipofectamine 2000 (0.625 μL) were diluted in Opti-MEM (62.5 μL) separately. After 5 min of incubation, the two solutions were mixed together and incubated for another 20 min. Then, 250 μL antibiotic-free media were added to the mixture to yield master transfection solutions. The solutions (75 μL) were delivered into each well and the cells were incubated for 2 h. Then the trasfection solutions were removed and the cells were washed twice with antibiotics-free media (100 μL). This time point was defined as 0 h. The culture medium was collected at 8, 16, 24, 34, and 44 h and replaced with fresh medium at each time point. The collected media were kept at 4 °C. GLuc expression levels were quantitated by measuring the luminescence of the expressed GLuc luciferase in the collected media as previously reported [15].

121

3. Results and discussion 3.1. Compound design To address the aforementioned questions, a monofunctional platinum(II) compound containing a bulky N-donor group was required. The design of the ligand was based on the following premises: (1) the ligand should have proper hydrophobicity to facilitate the cellular uptake of the platinum complex; (2) the ligand should have a relatively high molecular weight for revealing the biological difference; (3) the ligand should contain a single N-donor group to facilitate the synthesis and purification of the complex; and (4) the ligand may be biologically active to promote the activity of the platinum complex. To meet our demands, we chose one of the salicylanilide (SA) derivatives, 3,5-dichloro-N-(3-chloro-4-(quinolin-6-yloxy)phenyl)-2-hydroxybenzamide (4), as the ligand and the corresponding monofunctional platinum(II) compound was designated as SA–Pt (Fig. 1). Ligand 4 contained a quinoline moiety for the convenient complexation of the platinum moiety. Derivatives of salicylanilide have shown a variety of pharmacological uses, including antibacterial, antifungal, and enzyme inhibitory activities [16,17]. 3.2. Synthesis and characterization of SA–Pt The ligand was synthesized according to the following procedure (Scheme 1): 6-(2-chloro-4-nitrophenoxy)quinoline (2) was obtained by reacting 1-bromo-2-chloro-4-nitrobenzene (1) with 6-hydroxyquinoline, and 2 was reduced to 3-chloro-4-(quinolin-6yloxy)aniline (3). Ligand 4 was subsequently synthesized by conjugating 3 with 3,5-dichlorosalicylic acid using a coupling agent. SA–Pt was then prepared in a single step, by reacting cis-[Pt(NH3)2Cl(H2O)]+ with 4. SA–Pt showed poor solubility in aqueous solution, but dissolved well in methanol, ethanol, DMSO, and DMF. The final product was characterized by 1H, 13C NMR spectroscopy, ESI-MS, and elemental analysis (see Experimental; Figs. S7 and S8). 3.3. Antiproliferative activity of the compound The antiproliferative activity of cisplatin, 4, and SA–Pt was assessed against a panel of human cancer cells, including A2780 (ovarian), HeLa (cervical), MCF-7 (breast), MDA-MB-231 (breast), MDA-MB-436 (breast), and A549 (lung), along with human normal cell MRC-5 (lung fibroblast). MTT assay was used to determine cell viability after 72 h treatment with the test agents, and the results are summarized in Table 1. Cisplatin displayed antiproliferative activity against both cancer and normal cells, as usual, and the IC50 values were in the range of 1.2– 15.1 μM. Ligand 4 showed moderate antiproliferative activity against cancer cells, although the IC50 values were 1.8–22.6 times higher than those of cisplatin. Ligand 4 is therefore biologically active. The antiproliferative activity of 4 was much lower than that of cisplatin in MRC-5 normal cells. Encouragingly, SA–Pt exhibited antiproliferative activity close to the level of cisplatin against human cancer cells, and the antiproliferative activity was much higher than that of ligand 4. Thus, the conjugation of 4 with the platinum moiety resulted in significantly increased antiproliferative activity. Additionally, the antiproliferative activity of SA–Pt was 1.8 times lower than that of cisplatin in normal cells. These results indicated that although a bulky N-donor group 4 was present in the monofunctional platinum(II) compound with a formula of cis-[Pt(NH3)2(N-donor)Cl]+, its antiproliferative activity was identical to that of cisplatin. SA–Pt was also active against A549cDDP, the cisplatin-resistant lung cancer cells (Table 1). 3.4. Cellular uptake To reveal the efficiency of SA–Pt in entering cells, a cellular uptake study was conducted. HeLa cells were treated with 10 μM cisplatin or

122

B. Wang et al. / Journal of Inorganic Biochemistry 142 (2015) 118–125

Scheme 1. Synthetic route for SA–Pt. Reagents and conditions: (i) 6-hydroxyquinoline, DMF, K2CO3, 100 °C; (ii) Zn/NH4Cl; (iii) 3,5-dichlorosalicylic acid, HATU; (iv) cis-[Pt(NH3)2 Cl(H2O)]+, 55 °C. The details for the synthesis are given in 2.2 General Synthesis Procedure.

SA–Pt for 6 h before the platinum content of the whole cell was determined by ICP-OES. Cisplatin displayed a level of 74 ± 19 pmol Pt/106 cells. The cellular uptake of SA–Pt was 2.4-times higher than that of cisplatin with a level of 177 ± 53 pmol Pt/106 cells (Fig. 2). The presence of hydrophobic ligand 4 in the monofunctional platinum compound facilitated its efficient entrance into cells.

depicted in Fig. 4. In the control group that was not treated with any test agent, 4.1% of the cells were in early apoptosis, and the number increased to 26.7%, 15.6%, and 9.5% for cisplatin, SA–Pt, and 4, respectively. The fractions of necrotic cells upon treatment with cisplatin and SA–Pt were 2.3% and 2.6%, respectively. Thus, SA–Pt was able to effectively induce apoptosis but not necrosis in HeLa cells.

3.5. Cell cycle arrest ability

3.7. Inhibitory effect against PARP-1 and PARG

Given that SA–Pt kills cancer cells as effectively as cisplatin and enters cells more efficiently than cisplatin, it is tempting to speculate that the compound may arrest the cell cycle and greatly induce apoptosis. We studied the cell cycle arrest ability of SA–Pt (Fig. 3). HeLa cells were treated with cisplatin or SA–Pt for 24 h before flow-cytometric measurements. Cisplatin displayed a typical concentration-dependent cell cycle arrest at S-phase [18]. Cells treated with various concentrations of SA–Pt also exhibited large S-phase populations after 24 h, and the effect was also concentration-dependent. For example, upon treatment with 3.7 μM SA–Pt, 85% of the cells stopped at the S-phase. We measured the ability of 4 to arrest the cell cycle. The compound did not significantly disturb cell cycle distribution, possibly due to the low concentrations used, considering its low antiproliferative activity. This result confirmed that both cisplatin and SA–Pt arrested the cell cycle at the S phase.

A group of SA analogs had inhibitory effects against poly(ADPribose) glycohydrolase (PARG) and poly(ADP-ribose) polymerase-1 (PARP-1) [20], and metal-based anticancer agents also inhibited PARP1 [21–23]. For instance, cisplatin inhibited the catalytic activity of PARP-1 with an IC50 of 12.3 μM [21]. We tested the inhibitory effects of SA–Pt and 4 against PARP-1 and PARG using Trevigen PARP and PARG assay kits [24], respectively. The concentration-dependent inhibitory effects of the compounds against PARP-1 are plotted in Fig. 5. Ligand 4 had a weak inhibitory effect against recombinant PARP-1. Notably, SA–Pt displayed strong inhibition on the catalytic ability of PARP-1 with an IC50 value of 8.2 μM. For instance, in the presence of 10 μM SA–Pt, the activity of the PARP-1 enzyme decreased to a level of 36.6%. Conversely, both SA–Pt and 4 only presented weak inhibitory effects against recombinant PARG. The IC50 values of SA–Pt and 4 against PARG were 41.5 μM and N62.5 μM, respectively (Fig. S9).

3.6. Apoptosis induction

3.8. Plasmid DNA binding ability

We examined the ability of SA–Pt to induce apoptosis in HeLa cells. The cells were treated with 50 μM cisplatin, SA–Pt, or 4 for 24 h and a double-staining fluorescent assay using propidium iodide (PI) and a conjugate of Annexin V-FITC (fluorescein isothiocyanate) was performed to quantitate early apoptosis, dead, early necrosis, and viable cells [19]. The fraction of each status for treated and untreated cells is

To examine the DNA binding ability of SA–Pt, a luciferase-encoding plasmid DNA (pGLuc) was incubated with various concentrations of cisplatin or SA–Pt. The number of platinum per plasmid in the reaction 250 200

Table 1 Antiproliferative activities of SA–Pt in comparison to those of cisplatin and 4 in human cancer and normal cell lines by IC50 (μM).a Cell name

Type

Cisplatin

4

SA–Pt

A2780 HeLa MCF-7 MDA-MB-231 MDA-MB-436 A549 A549cDDP MRC-5

Ovarian cancer Cervical cancer Breast cancer Breast cancer Breast cancer Lung cancer Lung cancer Lung fibroblast

1.2 4.6 11.6 15.1 3.6 4.5 9.6 4.2

27.2 ± 1.1 27.8 ± 1.8 42.9 ± 6.2 27.6 ± 13.6 46.8 ± 3.2 31.7 ± 2.0 23 ± 1.9 N50

0.9 4.7 20.5 15.4 3.8 8.0 17.9 7.4

a

± ± ± ± ± ± ± ±

0.3 0.6 4.2 1.9 0.2 2.1 0.9 1.2

± ± ± ± ± ± ± ±

150 100

0.1 3.8 3.2 5.9 2.1 0.7 6.9 0.7

MTT assay was used to measure cell viability after 72 h treatment with test agents.

50 0

Fig. 2. Cellular uptake of cisplatin and SA–Pt in HeLa cells. Data were obtained from the average of four independent experiments.

B. Wang et al. / Journal of Inorganic Biochemistry 142 (2015) 118–125

123

Fig. 5. Concentration-dependent inhibitory effect against recombinant PARP-1 by SA–Pt and 4.

versus the bound platinum per plasmid is plotted in Fig. 6. Our result showed that SA–Pt was able to bind to plasmid DNA as efficiently as cisplatin in a concentration-dependent fashion. Although SA–Pt contains a salicylanilide ligand that may have a steric effect capable of influencing the DNA binding efficiency of the compound, SA–Pt still bound to DNA as well as cisplatin.

3.9. Interaction with calf thymus DNA (CT-DNA) To test if SA–Pt was able to non-covalently interact with CT-DNA, fluorescent competitive binding assays were carried out. Firstly, SA–Pt was added to ethidium bromide (EB)-bound CT-DNA and the fluorescence of EB-DNA was monitored. EB is a classic DNA intercalator and any complex that competitively replaces EB from DNA resulted in a fluorescent decrease [25]. The addition of SA–Pt to EB-DNA complex did not lead to significant change on the fluorescent intensity, indicating that SA–Pt did not intercalate into DNA base pairs (Fig. S10A). [(terpy)PtCl] Cl (terpy = 2,2′:6′,2″-terpyridine), a known DNA intercalator, was used as a positive control in this assay and a significant decrease of fluorescent intensity was observed (Fig. S10B) [26], showing its ability of intercalation. Secondly, we studied if SA–Pt could act as a DNA groove binder using Hoechst 33342 and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) [25,27–29]. Upon continuous addition of SA– Pt into Hoechst-bound DNA, the fluorescent intensity of Hoechst decreased significantly, indicating the displacement of Hoechst 33342 from the minor grove of DNA double helix by SA–Pt (Fig. S11A). The same effect was observed when DAPI was utilized, confirming that SA–Pt was able to bind into DNA minor grooves (Fig. S11B).

Fig. 3. Concentration-dependent effects of (A) cisplatin, (B) SA–Pt, and (C) 4 on cell cycle distribution of HeLa cells after treatment for 24 h.

Fig. 4. Flow-cytometric analysis of PI/annexin V staining of HeLa cells treated with 50 μM cisplatin, SA–Pt, and 4 for 24 h.

Fig. 6. DNA binding of cisplatin and SA–Pt to plasmid DNA.

124

B. Wang et al. / Journal of Inorganic Biochemistry 142 (2015) 118–125

as cisplatin, and the transcription level recovered significantly after 44 h, indicating the removal of SA–Pt-DNA lesions by cellular repair machineries (Fig. 7B). When the data were re-plotted to compare the transcription levels at the same time interval, cisplatin did show stronger transcription inhibition after 8 h than SA–Pt, and the transcription inhibition by SA–Pt was also weaker than cisplatin after 44 h. To further quantitate the transcription inhibition profiles of cisplatin and SA–Pt in HeLa cells, we calculated the D50 value, defined as the number of platinum per plasmid required to reduce the transcription level to 50% of the control (Table 2). A higher D50 value indicated weaker transcription inhibition ability, which was likely due to the repair of platinum-DNA damage. For cisplatin, the D50 value increased from 6.6 at 8 h to 9.5 at 44 h, and for SA–Pt, the value increased from 13.0 at 8 h to 23.3 at 44 h. These results further confirmed that SA–Pt did not inhibit transcription as strongly as cisplatin, and that the DNA lesions from SA–Pt were more easily removed than those from cisplatin. Therefore, although SA–Pt entered cells more efficiently than cisplatin, blocked the cell cycle, and induced apoptosis, the compound was not able to strongly inhibit transcription due to the repair of SA–Pt-DNA lesions, and this effect lowered its antiproliferative activity. 4. Conclusions

Fig. 7. Transcription inhibition profiles of cisplatin and SA–Pt in HeLa cells. Data are expressed as transcription inhibition effect of (A) cisplatin and (B) SA–Pt after 8, 16, 24, 34, and 44 h.

3.10. Transcription inhibition Next, we studied the transcription inhibition profile of SA–Pt in live mammalian cells. pGLuc plasmids encoding a secreted version of Gaussia luciferase were utilized. The plasmids containing different levels of platinum-DNA lesions were transfected into cells, and the levels of secreted luciferase at different time intervals after transfection were monitored by a luciferase assay. The expression level was normalized to a control plasmid without platinum-DNA lesions. The presence of platinum-DNA lesions would therefore inhibit the expression level of mRNA and the corresponding amount of protein. We have utilized this system to study the transcription inhibition as well as transcriptioncoupled repair of different types of metal-DNA cross-links [8,30]. Timeand concentration-dependent transcription inhibition profiles of SA–Pt and cisplatin in HeLa cells are shown in Fig. 7. Cisplatin displayed strong transcription inhibition after 8 h, and the transcription level recovered after 44 h, indicating the repair of cisplatin-DNA cross-links (Fig. 7A). SA–Pt showed a transcription inhibition effect after 8 h, not as strongly

Table 2 D50 values of platinated pGLuc plasmids assayed at different time intervals after transfection in HeLa cells.a Time after transfection

Cisplatin

SA–Pt

8h 16 h 24 h 34 h 44 h

6.6 6.8 7.6 9.0 9.5

13.0 13.9 18.3 21.9 23.3

a

D50 value is defined as the number of Pt lesions per plasmid required to reduce transcription level to 50% of the control.

In summary, we first designed, synthesized, and characterized SA– Pt, a monofunctional platinum(II) anticancer agent bearing an SA derivative as the bulky N-donor ligand. Detailed studies on the mechanism of action of SA–Pt were subsequently conducted. SA–Pt was active against the proliferation of a panel of human cancer cells. In HeLa cells, SA–Pt entered cells more effectively than cisplatin, and arrested the cell cycle at the S-phase. The compound induced apoptosis and inhibited the catalytic activity of PARP-1. Further studies showed that SA–Pt bound to DNA as efficiently as cisplatin. A transcription assay, however, showed that SA–Pt did not inhibit transcription as strongly as cisplatin at different time intervals in the cancer cells tested, indicating that SA–Pt-DNA lesions were more easily removed by DNA damage repair machineries. If SA–Pt exhibited antiproliferative activity in a similar way compared to cisplatin, including the steps 1) uptake; 2) activation; 3) DNA binding; and 4) cellular processing, SA–Pt exceeded or equalized the ability of cisplatin in steps 1 and 3, respectively, but was removed more efficiently than cisplatin once bound to DNA, at least in the cell lines that we used. If the activation step of SA–Pt, which was not revealed in this study, was more or less similar to that of cisplatin, the cellular processing of SA–Pt-DNA lesions, especially regarding the cells' ability to remove the lesions more effectively, may account for its identical in vitro antiproliferative activity compared to that of cisplatin, although the cells were able to take more SA–Pt. This information sheds additional light on the design of more active monofunctional platinum(II) anticancer agents. For example, choosing a suitable ligand that can strongly inhibit transcription but is hardly removed once the compound binds to DNA and that can ensure the effective cellular entrance is pivotal to conferring the compound's higher antiproliferative activity. Further study of the activation/deactivation of SA–Pt and its repair mechanism should offer more information on how SA–Pt is activated and SA–Pt-DNA lesions are recognized and removed. Abbreviations DAPI DMEM EB FBS HATU MEM PAR PARG

4′,6-diamidino-2-phenylindole, dihydrochloride Dulbecco's modified Eagle's medium ethidium bromide fetal bovine serum O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate minimum essential medium poly(ADP-ribose) poly(ADP-ribose) glycohydrolase

B. Wang et al. / Journal of Inorganic Biochemistry 142 (2015) 118–125

PARP Pol II RPMI

poly(ADP-ribose) polymerase RNA polymerase II Roswell Park Memorial Institute

Acknowledgments This research was supported by the National Natural Science Foundation of China (Grant No. 21371145) and City University of Hong Kong (Project 9680087). Appendix A. Supplementary data Figs. S1–S11. This material is available free of charge via the Internet at http://dx.doi.org/10.1016/j.jinorgbio.2014.10.003. References [1] [2] [3] [4] [5] [6] [7] [8] [9]

B. Rosenberg, L. Van Camp, T. Krigas, Nature 205 (1965) 698–699. R.A. Alderden, M.D. Hall, T.W. Hambley, J. Chem. Educ. 83 (2006) 728–734. X. Wang, Z. Guo, Chem. Soc. Rev. 42 (2013) 202–224. M.A. Fuertes, C. Alonso, J.M. Pérez, Chem. Rev. 103 (2003) 645–662. D. Gibson, Dalton Trans. (2009) 10681–10689. L. Kelland, Nat. Rev. Cancer 7 (2007) 573–584. K.S. Lovejoy, S.J. Lippard, Dalton Trans. (2009) 10651–10659. G. Zhu, M. Myint, W.H. Ang, L. Song, S.J. Lippard, Cancer Res. 72 (2012) 790–800. G.Y. Park, J.J. Wilson, Y. Song, S.J. Lippard, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 11987–11992. [10] D. Wang, G. Zhu, X. Huang, S.J. Lippard, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 9584–9589.

125

[11] W.J. Guo, Y.M. Zhang, L. Zhang, B. Huang, F.F. Tao, W. Chen, Z.J. Guo, Q. Xu, Y. Sun, Autophagy 9 (2013) 996–1008. [12] G. Ma, Y. Min, F. Huang, T. Jiang, Y. Liu, Chem. Commun. 46 (2010) 6938–6940. [13] Y.C. Lo, T.P. Ko, W.C. Su, T.L. Su, A.H.J. Wang, J. Inorg. Biochem. 103 (2009) 1082–1092. [14] L.S. Hollis, S.J. Lippard, Inorg. Chem. 22 (1983) 2708–2713. [15] Z. Wang, H. Qian, S.M. Yiu, J. Sun, G. Zhu, J. Inorg. Biochem. 131 (2014) 47–55. [16] C. Liechti, U. Séquin, G. Bold, P. Furet, T. Meyer, P. Traxler, Eur. J. Med. Chem. 39 (2004) 11–26. [17] M. Kratky, J. Vinsova, Curr. Pharm. Des. 17 (2011) 3494–3505. [18] B. Wang, H. Qian, S.M. Yiu, J. Sun, G. Zhu, Eur. J. Med. Chem. 71 (2014) 366–373. [19] G. Cafeo, G. Carbotti, A. Cuzzola, M. Fabbi, S. Ferrini, F.H. Kohnke, G. Papanikolaou, M.R. Plutino, C. Rosano, A.J.P. White, J. Am. Chem. Soc. 135 (2013) 2544–2551. [20] J.D. Steffen, D.L. Coyle, K. Damodaran, P. Beroza, M.K. Jacobson, J. Med. Chem. 54 (2011) 5403–5413. [21] F. Mendes, M. Groessl, A.A. Nazarov, Y.O. Tsybin, G. Sava, I. Santos, P.J. Dyson, A. Casini, J. Med. Chem. 54 (2011) 2196–2206. [22] N. Lease, V. Vasilevski, M. Carreira, A. de Almeida, M. Sanaú, P. Hirva, A. Casini, M. Contel, J. Med. Chem. 56 (2013) 5806–5818. [23] M. Serratrice, F. Edafe, F. Mendes, R. Scopelliti, S.M. Zakeeruddin, M. Grätzel, I. Santos, M.A. Cinellu, A. Casini, Dalton Trans. 41 (2012) 3287–3293. [24] H.K. Rhee, S.Y. Lim, M.J. Jung, Y. Kwon, M.H. Kim, H.Y.P. Choo, Bioorg. Med. Chem. 17 (2009) 7537–7541. [25] S.U. Rehman, Z. Yaseen, M.A. Husain, T. Sarwar, H.M. Ishqi, M. Tabish, PLoS One 9 (2014) e93913. [26] K.W. Jennette, S.J. Lippard, G.A. Vassiliades, W.R. Bauer, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 3839–3843. [27] K. Suntharalingam, O. Mendoza, A.A. Duarte, D.J. Mann, R. Vilar, Metallomics 5 (2013) 514–523. [28] A.Y. Chen, C. Yu, B. Gatto, L.F. Liu, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 8131–8135. [29] A.K. Williams, S.C. Dasilva, A. Bhatta, B. Rawal, M. Liu, E.A. Korobkova, Anal. Biochem. 422 (2012) 66–73. [30] G. Zhu, L. Song, S.J. Lippard, Cancer Res. 73 (2013) 4451–4460.