A novel pyrazolone-based derivative induces apoptosis in human esophageal cells via reactive oxygen species (ROS) generation and caspase-dependent mitochondria-mediated pathway.

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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 6 7 3 4 5 8 9 10 11 12 13 14 1 9 6 2 17 18 19 20 21 22 23 24 25 26 27 28

A novel pyrazolone-based derivative induces apoptosis in human esophageal cells via reactive oxygen species (ROS) generation and caspase-dependent mitochondria-mediated pathway Jing Zhao a, Li Zhang b, Jinyao Li a, Ting Wu a, Meifang Wang a, Guancheng Xu b, Fuchun Zhang a, Lang Liu b, Jianhua Yang a,c,1, Surong Sun a,⇑,1 a

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, PR China Institute of Applied Chemistry, Xinjiang University, Urumqi 830046, PR China c Texas Children’s Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, TX 77030, USA b

a r t i c l e

i n f o

Article history: Received 18 April 2014 Received in revised form 3 February 2015 Accepted 6 February 2015 Available online xxxx Keywords: Pyrazolone-based derivative Cadmium Eca-109 cells Apoptosis Mitochondrial pathway

a b s t r a c t Pyrazolone complexes have strong bio-activity but the anti-tumor mechanism of pyrazolone-based metal complexes is not fully understood. In this study, the inhibitory effect and possible mechanism of a novel pyrazolone-based derivative compound (Cd–PMPP-SAL) on human esophageal cancer cells were investigated. We found that Cd–PMPP-SAL inhibited the proliferation of Eca-109 cells in a dose-dependent manner and induced the apoptosis in the cells. Interestingly, Cd–PMPP-SAL promoted the production of ROS, loss of mitochondrial membrane potential, PARP cleavage and activation of caspase-3/9. These results suggest Cd–PMPP-SAL-induced apoptosis might be mediated by the increased production of ROS and caspase-dependent mitochondria-mediated pathway. These results suggest that Cd–PMPP-SAL is a potential candidate for the treatment of esophageal cancer. Ó 2015 Published by Elsevier Ireland Ltd.

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1. Introduction

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Metal complexes have been considered as potential sources of anticancer drugs for years. Some platinum-based anticancer drugs such as cisplatin, carboplatin, and oxaliplatin have been widely used. However, these drugs always accompany with serious side effects and drug resistance. Therefore, it is urgent to develop anticancer drugs with high efficacy and low toxicity [1]. It has been reported that anticancer drugs generally show their antitumor activities through inducing apoptosis of cancer cells, such as nonplatinum complexes [2,3]. Up to now, many transition-metal elements such as Zn, Cu and Co have been investigated in the development of non-platinum-based drugs. The 4-acyl pyrazolone derivatives, especially the Schiff based derivatives, are useful organic ligands for construction of novel complexes with diverse structures due to the multiple active coordination sites and the tautomeric effect of enol form and keto form [4–9]. Attention has been paid to pyrazolone-based metal complexes due to the advantages including stability, easy synthesis, easy alteration, and abundant bio-activity [10]. These compounds

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⇑ Corresponding author. Tel.: +86 09918582077; fax: +86 09918583517. 1

have antibacterial [11,12], antiviral [13] and anticancer activity [4,14,15]. Acylpyrazolone-Cu(II) had been shown to be able to induce the apoptosis in KB cells and KBv200 cells via reactive oxygen species (ROS)-independent mitochondrial pathway [16]. Cadmium (Cd) is a kind of environmental pollutant which can lead to lung cancer through ingestion or inhalation of Cd containing substances [17]. However, it has been reported that DSF-Cd selectively inhibit proteasome activity in tumor cells, and induce the apoptosis in the cancer cells [18]. Cd complexes, combined with indole propionic acid and 3,5-di-amino benzoic acid o-vanillin Schiff Base, had been shown to be able to induce the apoptosis in MCF-7 and MDA-MB-231 cells, whereas it had a weak inhibitory effect on the growth of non-cancer MCF-10A cells [19]. Due to Cd and Cu are closely situated and have similar physicochemical properties, we hypothesized that Cd could form complexes with pyrazolone in a manner similar to that of Cu and this complex could induce apoptosis in cancer cells. In this study, we synthesized a novel pyrazolone derivatives Cd–PMPP-SAL through the interaction between PMPP-SAL and Cd(II). The inhibitory effect and mechanism of Cd–PMPP-SAL on Eca-109 cells was studied in vitro. Our results suggest that Cd–PMPP-SAL inhibits the proliferation of Eca-109 cells through inducing the apoptosis.

E-mail address: [email protected] (S. Sun). Jianhua Yang and Surong Sun are the co-corresponding authors.

http://dx.doi.org/10.1016/j.cbi.2015.02.004 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: J. Zhao et al., A novel pyrazolone-based derivative induces apoptosis in human esophageal cells via reactive oxygen species (ROS) generation and caspase-dependent mitochondria-mediated pathway, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/ j.cbi.2015.02.004

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2. Materials and methods

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2.1. Reagents and cell culture

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1-Phenyl-3-methyl-5-pyrazolone (PMP), salicylic hydrazide (SAL), Ca(OH)2, propionyl chloride, Cd(CH3COO)22H2O, hydrochloric, glacial acetic acid and ethanol are all analytical grade and used without further purification, dioxane is used after purification. 1Phenyl-3-methyl-4-propionyl-5-pyrazolone (PMPP) and the ligand N-(1-phenyl-3-methyl-4-propylene-5-pyrazolone)-salicylidene hydrazide (PMPP-SAL) were prepared and purified according to the methods described in the literature [20,21]. Scheme 1 represents the ligand used in this work. 3-(4,5-Dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 20 ,70 -dichlorofluorescin diacetate (DCFH-DA), 3,30 -dihexyloxacarbocyanine iodide (DiOC6(3)), N-Acetyl Cysteine (NAC) and Hoechst 33258 were purchased from Sigma Chemical Co. Annexin V-FITC Apoptosis Detection Kit was obtained from Calbiochem Co. Antibodies against caspase-3, -7, -9, PARP and b-actin were obtained from Cell Signaling Technology Co. Anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology Co. The Caspase Activity Assay Kit, caspase-3 inhibitor Z-DEVD-FMK, caspase-9 inhibitor Z-LEHD-FMK and pancaspase inhibitor Z-VAD-FMK were purchased from Beyotime Co., Ltd. (Shanghai, China). Other routine laboratory reagents were obtained from commercial sources of analytical or HPLC grade. The human esophageal cancer Eca-109 cells and the normal human gastric epithelial GES cells, obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering in Xinjiang University, were cultured in RPMI-1640 medium (HyClone, Thermo) containing 10% FBS (Gibco), 1% penicillin– streptomycin (100 U/mL penicillin and 100 lg/mL streptomycin), and 1% glutamine in 25 cm2 tissue culture flasks under humidified 5% CO2 atmosphere at 37 °C.

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2.2. Synthesis of the complex Cd–PMPP-SAL

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The ligand PMPP-SAL (1.5 mmol 0.547 g) was dissolved in 25 mL of hot anhydrous ethanol followed by a drop-wise addition of an ethanolic solution of Cd(CH3COO)22H2O (1.5 mmol 0.3998 g)

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with constant stirring. The mixture was further refluxed for 3 h, a white precipitate was separated by filtration, washed with ethanol and dried naturally. Anal. calcd. for CdC20H18N4O3 (F.w.: 474.78): C, 50.55; H, 3.82; N, 11.80. Found: C, 50.44; H, 3.91; N, 12.18%.

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2.3. Cell viability assay

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The Eca-109 and GES cells were harvested during logarithmic growth phase and seeded in 96-well plates at a density of 3 104 cells/mL in a final volume of 100 lL. Twenty-four hours after incubation, cells were treated with various concentrations of Cd–PMPPSAL (0, 10, 30, 60, 120 lM) and control (DMSO, 0.1% in culture media) for 72 h., Then 20 lL of MTT (stock solution 5 mg/mL of saline) was added to each well for 4 h followed by removal of the supernatant, and solubilization of MTT crystals with 100 lL of anhydrous DMSO in each well. The percentage of cell growth inhibition was calculated using the following formula: Cell growth inhibition (%) = (1 mean experimental absorbance/mean control absorbance) 100%. Experiments were performed in triplicate.

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2.4. Assessment of apoptosis (Hoechst 33258 staining)

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After treatment with or without 25 lM of Cd–PMPP-SAL, Eca-109 cells were washed twice with ice-cold PBS. Then the cells were incubated in 1 mL of Hoechst 33258 (Sigma, USA) at 37 °C for 5 min (final concentration, 0.5 lg/mL), washed again, and observed under fluorescence microscope (Leica Dmirb, Germany) in random microscopic fields at 400 magnification.

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2.5. Transmission electron microscopy

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The Eca-109 cells were treated with Cd–PMPP-SAL for the indicated concentration. The collected cells were fixed with PBS containing 2.5% glutaraldehyde followed by treatment with PBS containing 1% OSO4. The samples were dehydrated in graded alcohol, embedded, and sectioned. Ultrathin sections were stained with uranyl acetate and lead citrate, followed by examination by a JEM1200 transmission electron microscope (JEOL, Japan).

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Scheme 1.


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Fig. 1. The TG–DSC curves of Cd–PMPP-SAL. Scanning rate 10 °C/min. 153

2.6. DNA fragmentation assay

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After exposure to different concentrations of Cd–PMPP-SAL and DMSO solvent for 24 h, 5 106 cells were harvested, washed twice with ice cold PBS, and centrifuged at 1500 rpm for 3 min. The cell pellets were resuspended in 0.5 mL of DNAzol reagent (Invitrogen) and incubated at room temperature for 2 h. After centrifugation at 12,000 rpm for 15 min, the supernatant was transferred to a 1.5 mL microcentrifuge tube. 0.5 mL of ice-cold dehydrated alcohol was added to precipitate the DNA. The DNA was separated in 2% agarose gel at 60 V for 2 h, which was then visualized via UV transilluminator.

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2.7. Annexin V-FITC/PI assay

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Eca-109 cells at an initial density of 2 105 cells/mL were incubated with different concentrations of Cd–PMPP-SAL and an equal amount of DMSO for control cells for 24 h at 37 °C. At the end of the

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incubation, cells were harvested for apoptosis analysis using Annexin V-FITC Apoptosis Detection Kit (Calbiochem Co.) according to the manufacturer’s protocols. Cell analysis and data acquisition were performed using a FACSCalibur flow cytometer.

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2.8. Caspase activity assays

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Eca-109 cells were treated with different concentrations of Cd–PMPP-SAL and an equal amount of DMSO for control cells for 24 h. Caspase-3 and caspase-9 activities were assessed according to the manufacturer’s instructions (Beyotime, Shanghai, China). Cell lysate (100 lg total protein) was added to a reaction mixture, which contained colorimetric substrate peptides specific to caspase-3 or -9. The reaction was incubated at 37 °C for 2 h or overnight. Caspase activity was determined by measuring OD 405 and following formula: Caspase activity = (OD inducer/OD control). To further confirm whether Cd–PMPP-SAL-induced apoptosis was affected by caspase-3 and -9, the Eca-109 cells were harvested during logarithmic growth phase and seeded in 96-well plates at a density of 1 105 cells/mL in a final volume of 100 lL. Eca-109 cells were preincubated with the pancaspase inhibitor Z-DEVDFMK, Z-LEHD-FMK for 1 h before treatment with 25 lM Cd–PMPP-SAL, respectively. After 24 h of treatment, 20 lL of MTT was added to each well for 4 h and the percentage of cell growth inhibition was calculated.

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2.9. Determination of mitochondrial transmembrane potential (DWm)

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Eca-109 cells were initially seeded at 1 106 in 25 cm2 tissue culture flasks and then treated with various concentrations of Cd–PMPP-SAL or DMSO solvent for 24 h. After drug treatment, cells were loaded with the probe DiOC6 (20 nM) for 30 min at 37 °C before cytometric analysis. The cells were harvested and resuspended in PBS. Measurement of the retained DiOC6 in 1 104 cells of each sample was performed in a FACSCalibur flow cytometer.

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Scheme 2. The predicted structure of complex Cd–PMPP-SAL.


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Fig. 2. The effect of Cd–PMPP-SAL on the proliferation of Eca-109 and GES cells. Eca-109 and GES cells were treated with various concentrations of Cd–PMPP-SAL for 72 h, and then cell viability was measured by MTT assay. All data were expressed as mean ± SD of three experiments and each experiment included triplicate repeats. ⁄P < 0.05, ⁄⁄⁄ P < 0.001 vs. control (DMSO, 0.1% in culture media).

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DiOC6 was excited at 488 nm, and the fluorescence was analyzed at 525 nm (FL-1) after logarithmic amplification.

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2.10. Quantitative analysis of intracellular ROS

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Eca-109 cells were initially seeded at 1 106 in 25 cm2 tissue culture flasks and then treated with various concentrations of Cd–PMPP-SAL or DMSO solvent for 24 h, in the presence or absence of NAC. After drug treatment, cells were trypsinized, collected by centrifugation, washed once with PBS, and resuspended in PBS supplemented with 20 lM 20 ,70 -dichlorofluorescin-diacetate (DCFH-DA; Molecular Probes). After incubation for 15 min at room temperature, the fluorescence was measured by flow cytometry on a FACSCalibur flow cytometer. DCFH-DA was excited at 488 nm, and fluorescence was analyzed at 525 nm. Average ROS production (relative to the level of vehicle-treated controls) was calculated from three individual wells in at least three independent plates.

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2.11. Western blot analysis

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After exposure to different concentrations of Cd–PMPP-SAL and DMSO solvent for 24 h, both adherent and floating cells were collected and lysed in ice-cold RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM NaF, 1% Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM Na3VO4, 1% Triton X-100, 10 mg/mL Aprotinin and 10 mg/mL Leupeptin) for 30 min. The lysates were centrifuged at 12,000 rpm for 10 min and the supernatant was collected. Equal amount of protein lysates were electrophoresed on a 12% SDS polyacrylamide gel and transferred onto nitrocellulose filter (NC) membrane. After blocking with 5% nonfat dry milk suspended in TBST (Tris-Buffered Saline and Tween-20) for 2 h, membranes were individually incubated with the appropriate primary antibodies overnight followed by secondary antibodies for 2 h at room temperature. Protein bands were detected using ECL assay kit (Beyotime, Jiangsu, China) and exposed using a Kodak medical X-ray processor (Kodak, USA).

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2.12. Statistical analysis

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Student t tests were used to determine the significant differences between the treatment and the control groups, and P < 0.05 was considered to be biologically and statistically significant. All of the experiments were conducted in three independent replications.

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Fig. 3. Morphological changes of the apoptotic Eca-109 cells after Cd–PMPP-SAL treatment. (A) The Eca-109 cells were treated with 25 lM Cd–PMPP-SAL for 24 h, the morphologic changes were observed under a fluorescent microscope after Hoechst 33258 staining. (B) The cellular ultrastructure was examined by transmission electron microscope.


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Fig. 4. Cd–PMPP-SAL induced apoptosis in Eca-109 Cells. (A) Cd–PMPP-SAL induced DNA ladder formation in Eca-109 cells. After exposure to Cd–PMPP-SAL (0–50 lM) for 24 h, cells were harvested and DNA was extracted. Apoptosis-characteristic ladder-like patterns of DNA fragments consisting of approximately 180–200 bp were observed in Eca-109 cells, with a concentration-dependent progressive accumulation. M, molecular marker. (B) Detection of apoptosis using Annexin V-FITC/PI staining. After treatment with increasing concentrations of Cd–PMPP-SAL for 24 h, Eca-109 cells were analyzed by FACSCalibur flow cytometer. Horizontal axis represents Annexin V-FITC intensity and vertical axis shows PI staining. The lines divide each plot into four quadrants: lower left quadrant (Annexin VPI), living cells; lower right quadrant (Annexin V+PI), early apoptotic cells; upper left quadrant (Annexin VPI+), necrotic cells; upper right quadrant (Annexin V+PI+), late apoptotic cells. (C) Summary data (mean ± SD) of % early apoptotic cells from three independent experiments are shown. ⁄P < 0.05, ⁄⁄P < 0.01 vs. control (0.1% DMSO in culture media). (D) Cd–PMPP-SAL induced the cleavage of PARP in Eca-109 cells. Cells were exposed to the indicated concentration of Cd–PMPP-SAL for 24 h. Total protein was extracted from treated and untreated cells and the cleavage of PARP was detected by Western blot.


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Fig. 5. Cd–PMPP-SAL induced the activation of caspase. (A) Eca-109 cells were treated with different concentrations of Cd–PMPP-SAL (0–50 lM) for 24 h. Spectrophotometry was used to determine the activities of caspase-3 and -9. (B) Cd–PMPP-SAL induced the activation of caspase-3, -7, -9 in Eca-109 cells. Cells were exposed to the indicated concentration of Cd–PMPP-SAL for 24 h. Immunobloting of caspase-3, -7, and -9, while b-actin was probed as the protein loading reference. (C) Eca-109 cells were preincubated with the pancaspase inhibitor Z-DEVD-FMK, Z-LEHD-FMK for 1 h before treatment with 25 lM Cd–PMPP-SAL for 24 h, followed by the MTT assay. (D) Eca-109 cells were preincubated with the pancaspase inhibitor Z-VAD-FMK (20 lM) for 1 h before treatment with Cd–PMPP-SAL for 24 h, followed by the annexin V–PI assay. ⁄ P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 vs. control.

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3. Results

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3.1. Elemental analysis of the complex

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We analyzed the component of the complex Cd–PMPP-SAL after reaction of PMPP-SAL and Cd(CH3COO)22H2O. Consistent with the theoretical prediction, the elemental analysis results show that the complex is composed of metal and ligand stoichiometry at 1:1 ratio.

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3.2. Thermal analysis of the complex

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The typical TG-DSC curves of complex Cd–PMPP-SAL are shown in Fig. 1. The TG curve indicates that the complex is thermally stable up to 340 °C, implying that the complex does not contain solvent molecule. The thermal decomposition takes place in one stage in the temperature range of 340–540 °C. The observed mass loss (72.46%) is attributed to the decomposition of one ligand molecule, which is consistent with the theoretical value (72.95%). Weight constancy is attained at around 540 °C. The end product has the observed mass of 27.54%, which is comparable to CdO with the calculated value of 27.05%. The result further demonstrates that the complex is composed of Cd(II) ion and PMPP-SAL at ratio 1:1.

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3.3. IR spectra

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The IR spectrum of the ligand shows a medium intensity band at around 3249/cm, which is assigned to the t(OAH) and t(NAH) stretching vibrations [22]. The strong bands at 1663 and 1614/cm correspond to the t(C@O) of the hydrazide and t(C@O)

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of the pyrazolone ring vibration, respectively [23]. These results suggest that the ligand PMPP-SAL exists in the keto form (see Scheme 1) in the solid state, which is consistent with the X-ray crystal structure analysis [15]. Nevertheless the t(C@O) and t(NAH) bands in the complex are both absent. Meanwhile, two new bands are observed at around 1440/cm due to t(CAO) and 510/cm due to t(MAO). There are two new bands at 1578 and 1607/cm due to t(C@NAN@C) of hydrazone. In addition, a weak new band at 470/cm for the complex is assigned to t(MAN). All of these demonstrate that the ligand undergoes isomerization from keto form to the enol form during the coordination, and then lose two protons to coordination with Cd(II) atom as double negative charged units. The t(CAOH) stretching vibration at 1308/cm indicates that the phenolic oxygen does not coordinate with the central metal.

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3.4. The predicted structure of complex

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According to the results obtained by the elemental analysis, IR analysis and thermal analysis, we speculate that Cd(II) ion in Cd–PMPP-SAL is coordinated with a double negatively charged ligand with ONO donor set and the N atom from the adjacent pyrazolone ring of another ligand, forming a one-dimensional helical chain structure. The predicted structures are illustrated in Scheme 2.

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3.5. Effect of Cd–PMPP-SAL on the proliferation of cancer cells

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We investigated the effect of Cd–PMPP-SAL on the proliferation of cancer cells by MTT assay. We observed that Cd–PMPP-SAL inhibited cell proliferation of Eca-109 cells in a dose-dependent

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manner (Fig. 2). We found that the half-maximal inhibitory concentration (IC50) of Cd–PMPP-SAL was 29.86 lM. We also determined the effect of Cd–PMPP-SAL on the growth of the normal human gastric epithelial GES cells. The results showed that Cd– PMPP-SAL inhibited the growth of GES cells in a dose-dependent manner. However, the inhibitory effect of Cd–PMPP-SAL on GES cells is much lower compared to that of Eca-109 cells. The IC50 value to GES cells was 142.6 lM, which is about five times higher than the IC50 value of Eca-109 cells. We further compared the antiproliferative activity of Cd–PMPP-SAL to cisplatin (DDP). Interestingly, the activity of DDP to Eca-109 cells was lower than that of Cd–PMPP-SAL. The IC50 value of DDP was 45.34 lM, which is about two folds higher than the IC50 value of Cd–PMPP-SAL. DDP (IC50: 177 lM) and Cd–PMPP-SAL had similar cytotoxicity to GES cells. These data suggest that Cd–PMPP-SAL has higher cytotoxicity to cancer cells than to normal cells.

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3.6. Cd–PMPP-SAL induces apoptosis in Eca-109 cells

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3.6.1. Morphological changes of Eca-109 cells after Cd–PMPP-SAL treatment To analyze the morphological changes, Eca-109 cells were stained with Hoechst 33258 in the absence and presence of 25 lM of Cd–PMPP-SAL for 24 h, and observed under the fluorescence microscope. Control cells without Cd–PMPP-SAL treatment showed even distribution of the staining and round homogeneous nuclei feature. However, Cd–PMPP-SAL treated cells showed bright staining and condensed or fragmented nuclei which are stereotypical feature of apoptotic cells. Moreover, some nuclei were turgescent and enlarged (Fig. 3A). The ultrastructural alterations of apoptotic cells were observed under electron microscope. As shown in Fig. 3B, the control cells display normal cell morphology. In contrast, Cd–PMPP-SAL-treated Eca-109 cells showed typical apoptotic features including chromatin condensation and margination at the nuclear periphery. These results suggest that Cd– PMPP-SAL induces apoptosis in Eca-109 cells.

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3.6.2. Induction of apoptosis in Eca-109 Cells by Cd–PMPP-SAL treatment To evaluate the effect of Cd–PMPP-SAL on the induction of apoptosis, Eca-109 cells were treated with different concentrations of Cd–PMPP-SAL for 24 h. DNA was extracted from treated and untreated cells and DNA fragmentation was detected by gel electrophoresis. The results showed that Cd–PMPP-SAL-treatment induced a typical ladder pattern of DNA fragmentation in a dosedependent manner (Fig. 4A). We further investigated the apoptosis using Annexin V/PI staining by flow cytometry. After Cd–PMPP-SAL treatment for 24 h, the percent of early apoptotic Eca-109 cells was dose-dependently increased compared with the control group (from 2.31 ± 0.21% to 17.97 ± 0.91%) (Fig. 4B and C). Importantly, dose-dependent PARP cleavage was observed in the Cd–PMPPSAL treated Eca-109 cells by Western blotting assay (Fig. 4D). Taken together, these results indicate that Cd–PMPP-SAL induces apoptosis in Eca-109 cells.

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3.7. Mechanism of the apoptosis in Eca-109 induced by Cd–PMPP-SAL

caspase-3, -7 and -9 was significantly increased in Eca-109 cells after treatment with 50 lM Cd–PMPP-SAL for 24 h (Fig. 5B). Consistently, the addition of the caspase-3 and -9 inhibitors (Z-DEVD-FMK and Z-LEHD-FMK) partially decreased the inhibitory effect of Cd–PMPP-SAL on the growth of Eca-109 cells (Fig. 5C). The pan-caspase inhibitor Z-VAD-FMK (20 lM) was also used to determine whether Cd–PMPP-SAL-induced apoptosis was caspasedependent. We found that Z-VAD-FMK significantly decreased Cd–PMPP-SAL-induced apoptosis (Fig. 5D). These results suggest that Cd–PMPP-SAL-induced apoptosis is partially caspasedependent.

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3.7.1. Cd–PMPP-SAL induces apoptosis in Eca-109 cells via the activation of caspase To investigate whether Cd–PMPP-SAL-induced apoptosis in Eca109 cells was dependent on the activation of caspase-3 and -9, we measured the activity of caspase-3 and -9 using a caspase activity assay kit and the expression of activated caspase-3, -7 and -9 by Western blotting analysis. In this assay, we found that the activity of both caspase-3 and -9 was upregulated by the Cd–PMPP-SAL treatment (Fig. 5A). Furthermore, the expression of activated

3.7.2. Cd–PMPP-SAL-induced DWm loss in Eca-109 cells Mitochondria plays a key role in the regulation of apoptosis, and changes in DWm reflect mitochondrial dysfunction [16]. In this study, DWm changes induced with Cd–PMPP-SAL was detected by DiOC6 (20 nM) staining followed by flow cytometry. The results showed that Cd–PMPP-SAL induced the decrease of DWm in a dose-dependent manner. The percentage of cells with DWm loss in Eca-109 cells increased from 3.43% to 32.6 9% after Cd–PMPPSAL treatment (Fig. 6).

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Fig. 6. Cd–PMPP-SAL induced changes in the mitochondrial membrane potential. (A) After treatment with 15, 25, and 50 lM Cd–PMPP-SAL for 24 h, Eca-109 cells were stained with 20 nM DiOC6, and a decrease in the mitochondrial membrane potential was determined by monitoring the uptake of DiOC6 using flow cytometry. The X and Y axes represent the DiOC6 fluorescence and number of cells, respectively. Cells with low DiOC6 fluorescence were expressed as the percentage of total cell counts. (B) Quantitative data of (A). ⁄⁄⁄P < 0.001 vs. control.


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Fig. 7. Cd–PMPP-SAL induced ROS generation in the Eca-109 cells. (A) Effect of Cd–PMPP-SAL on ROS generation of Eca-109 cells. Eca-109 cells were treated with different concentrations of Cd–PMPP-SAL for 24 h with or without pretreatment of ROS scavenger NAC (1 mM) for 1 h. ROS generation was determined by measuring DCF fluorescence intensity using flow cytometry. (B) Effect of NAC on early apoptosis of Eca-109 cells upon Cd–PMPP-SAL treatment. Eca-109 cells were treated with different concentrations of Cd–PMPP-SAL for 24 h with or without pretreatment of ROS scavenger NAC (1 mM) for 1 h. The treated and untreated cells were stained with Annexin V/PI and analyzed by flow cytometry. The cells in lower right quadrant (Annexin V+PI) were considered as ‘‘Early apoptotic cells’’. ⁄P < 0.05, ⁄⁄P < 0.01 vs. the control.

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3.7.3. Cd–PMPP-SAL-induced intracellular ROS generation ROS is usually related to mitochondrion-dependent cell injury [24]. The ROS generation in Eca-109 cells treated by different concentration of Cd–PMPP-SAL were determined using the fluorescent probe DCFH-DA followed by flow cytometry. Fig. 7A showed that treatment with 25 lM Cd–PMPP-SAL significantly increased the level of ROS at 24 h compared with the control group (from 0.17 ± 0.12 to 32.5 ± 1.15). Consistently, the addition of the ROS scavenger NAC (1 mM) completely decreased ROS regardless of the concentration of Cd–PMPP-SAL. We further found that the pretreatment with NAC for 1 h significantly decreased 25 lM Cd–PMPP-SAL-induced early apoptosis of Eca-109 cells (Fig. 7B). Taken together, these results indicate that Cd–PMPP-SAL-induced apoptosis is partially dependent on ROS generation.

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4. Discussion

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Pyrazolone can form various complexes with different metals which generally have strong bio-activity [3]. New transition-metal complexes are increasingly being considered as potential anticancer drugs [25,26]. It has been demonstrated that pyrazolone-based metal complexes can induce apoptosis in KB cells and multidrug resistant KBv200 cells [16]. In this study, we found that Cd– PMPP-SAL inhibited the proliferation of Eca-109 cells and the IC50 value was 29.86 lM. The inhibitory effect of Cd–PMPP-SAL on Eca-109 cells is higher than that of DDP. Although Cd–PMPPSAL showed inhibitory effect on the proliferation of normal GES cells, the IC50 value is much higher than that of cancer cells, suggesting that Cd–PMPP-SAL is the potential candidate for the treatment of cancer. We further found that the inhibition of cancer cell growth is due to the increased apoptosis induced by Cd–PMPPSAL treatment. Apoptosis can be induced by catalyzing ROS to execute oxidative modification of cellular components, interfering with intracellular redox balance, and/or modulating redox related signal pathways [27]. Apoptosis is also associated with other intracellular cascade reactions, such as degrading mitochondrial transmembrane potential and up-regulating Caspase-3 [28]. It has been shown that a Cu(II) Schiff based complex could induce apoptosis of HeLa cells through activation of caspase-3 and caspase-9 [29]. Mitochondria plays an important role in the induction of apoptosis, including the loss of mitochondrial membrane potential (DWm) and the change of intracellular redox state [30–34]. We found that the mitochondrial membrane potential was decreased after Cd–PMPP-SAL treatment. The activity of caspase-3 and -9 and the levels of activated caspase-3, -7 and -9 in Eca-109 cells were

significantly increased after Cd–PMPP-SAL treatment. Moreover, the addition of caspase-3 and -9 inhibitors reduced the inhibitory effect of Cd–PMPP-SAL on the growth of cancer cells and the apoptosis induced by Cd–PMPP-SAL treatment. Taken together, the activation of caspase-9 upon Cd–PMPP-SAL treatment clearly showed the involvement of mitochondrial pathway in Cd–PMPPSAL-induced apoptosis. Previous studies demonstrate that increased ROS level could lead to the loss of mitochondrial transmembrane potential [24,35]. We found that the level of ROS was increased after Cd– PMPP-SAL treatment. Furthermore, the pretreatment with the ROS scavenger NAC decreased the ROS generation and apoptosis induced by Cd–PMPP-SAL. These results suggest that the ROS generation contributes to the mitochondria-mediated caspasedependent apoptosis induced by Cd–PMPP-SAL. In summary, our data suggest that Cd–PMPP-SAL inhibits the growth of cancer cells through inducing apoptosis, which is partially dependent on mitochondrial-mediated caspase-dependent pathway. Cd–PMPP-SAL is a potential candidate for cancer treatment.

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Acknowledgments

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This work were supported by the grant from the Science Foundation of Xinjiang (No. 2013211A018), and the Graduate Research and Innovation Project of Xinjiang (No. XJGRI2013043).

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References

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SAPK pathway.

Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells.

mitochondria pathway in p53-independent manner.

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Bax signaling pathway.

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Mitochondrial Dysfunction and the Caspases Dependent Pathway.

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Substituent effects on reactive oxygen species (ROS) generation by hydroquinones.

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Ouabain elicits human glioblastoma cells apoptosis by generating reactive oxygen species in ERK-p66SHC-dependent pathway.

Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release.

Generation of Reactive Oxygen Species (ROS) and Pro-Inflammatory Signaling in Human Brain Cells in Primary Culture.

GGsTOP increases migration of human periodontal ligament cells in vitro via reactive oxygen species pathway.