European Journal of Medicinal Chemistry 86 (2014) 1e11

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Singly protonated dehydronorcantharidin silver coordination polymer induces apoptosis of lung cancer cells via reactive oxygen speciesmediated mitochondrial pathway Senpeng Li a, 1, Shuo Zhang b, 1, Xing Jin a, Xuejie Tan c, Jianfang Lou c, Xiumei Zhang a, Yunxue Zhao a, * a b c

Department of Pharmacology, School of Medicine, Shandong University, Jinan 250012, PR China Department of Geriatrics, Shandong University Qilu Hospital, Jinan, Shandong 250012, PR China School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan 250353, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 September 2013 Received in revised form 11 July 2014 Accepted 14 August 2014 Available online 15 August 2014

Silver complexes have been shown to possess antimicrobial and anticancer properties. Ag-SP-DNC, a novel silver and singly protonated dehydronorcantharidin complex, was synthesized in our previous study. In this study, we offer evidence that Ag-SP-DNC elicits a reactive oxygen species (ROS)-mediated mitochondrial apoptosis in lung cancer cells. Ag-SP-DNC inhibited the growth of A549 cells by inducing G2/M phase cell cycle arrest and apoptosis. Ag-SP-DNC induced apoptosis was associated with the levels of intracellular ROS. The further study revealed that Ag-SP-DNC disrupted the mitochondrial membrane potential, induced the caspase-3 activation and led to the translocation of apoptosis inducing factor and endonucleaseG to the nucleus. These findings have important implications for the development of silver complexes for anticancer applications. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Ag-SP-DNC Lung cancer Apoptosis Reactive oxygen species Mitochondria

1. Introduction Metal-based drugs have many activities and are widely used in the treatment of a variety of diseases [1,2]. Cisplatin and its close analogues carboplatin and oxaliplatin, as the leading metal-based drugs, are used to treat about half of all patients receiving chemotherapy for cancer [3,4]. Broader application of platinumbased anticancer drugs, however, is limited by intrinsic or acquired drug resistance and high incidences of serious side effects [5]. Extensive efforts have been made to synthesize and test new metal-based anticancer agents, with the promise that compounds with improved anticancer activity and fewer toxic side effects will be discovered. Lung cancer, predominantly non-small cell lung cancer (NSCLC), is the most common cancer and the most common cause of cancer death in the world [6]. Current standard therapies

Abbreviations: Ag-SP-DNC, poly[[[m3-(5,6-h):kO2:kO2-(±)-(1S,2S,3R,4R)-3carboxy-7-oxabicyclo[2.2.1]hept-5-ene-2-carboxylato]silver(I)] monohydrate]; ROS, reactive oxygen species. * Corresponding author. E-mail address: [email protected] (Y. Zhao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2014.08.052 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

include surgical resection, chemotherapy, and radiation therapy. Unfortunately, these therapies rarely cure the disease, and the overall 5-year survival rate is still only 15% [7,8]. Chemotherapy is the standard frontline treatment option for the majority of patients with locally advanced or metastatic lung cancer [9,10]. Platinumbased compounds are the most commonly used chemotherapeutic agents in the treatment of advanced stage lung cancer patients [11,12]. Platinum resistance is a major limitation in the treatment of advanced lung cancer [13]. Silver complexes exhibit distinct biological activities that could be exploited to develop effective therapeutic agents including antimicrobial and anticancer drugs [14,15]. The remarkable example of a silver-based drug is silver sulphadiazine that has been used topically as an antibacterial agent in the treatment of burns and wounds [16]. N-Heterocyclic carbenes (NHCs) are stable singlet carbenes that can act as excellent two electron donor ligands towards almost any element in the periodic table, and NHCs can bind to transition metals, and in practice, the complexes of silver and NHCs have been synthesized as novel potential anticancer agents [1,17,18]. Eloy and co-workers reported that silver-NHCs induce cancer cell death independent of the caspase cascade via the mitochondrial AIF pathway [19]. The connection between

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inflammation and cancer has been extensively proven [20]. The complexes of silver and anti-inflammatory agents were synthesized, and these compounds were found to induce apoptosis of cancer cells by Banti et al. [21]. Coumarins were shown to be potent cytotoxic agents, capable of killing cancer cells, and Thati et al. investigated the in vitro anticancer properties of a silver complex of 4-hydroxy-3-nitro-coumarin-bis(phenanthroline) in human malignant cancer cells. Results showed that this complex was more active than cisplatin [22]. Many other silver complexes have been reported to be active against cancer cells [23e26]. However, the precise mechanism of the anticancer action of silver complexes is not well characterized. Cantharidin and norcantharidin display anticancer activity against a broad range of tumor cells lines [27]. Meanwhile, their carbonyl oxygen and bridge oxygen atoms are apt to coordinate with silver ions. Therefore, the molecular design and application in anticancer research of silver-cantharidin and norcantharidin complexes are the interesting aspects of bioinorganic chemistry and metal-based drugs research. In our previous report, a novel class of silver and singly protonated dehydronorcantharidin complex poly[[[m3-(5,6h):kO2:kO2-(±)-(1S,2S,3R,4R)-3-carboxy-7-oxabicyclo[2.2.1]hept5-ene-2-carboxylato]silver(I)] monohydrate] was synthesized in high yield [28]. The complex was characterized using elemental analysis and single-crystal X-ray diffraction. The crystal structure indicates a two-dimensional coordination polymer with a repeating unit consisting of an Ag-SP-DNC cluster (Fig. 1). In this study, we investigate the anticancer effects of Ag-SP-DNC on human lung cancer cells A549, in an attempt to find a potential agent for the treatment of lung cancer from silver complexes and explore the possible underlying mechanisms.

Because cell morphology is an important indicator of cell behavior, we also took photos for the cells after treating for 48 h. It was found that cells became rounded and retracted following treatment of Ag-SP-DNC (Fig. 3). Actin is one of the most abundant and common cytoskeletal proteins for cell growth, motility and maintenance of cell shape. It has been recognized that control of the actin cytoskeleton must be coordinated with control of cell cycle events. Alterations in actin organization and dynamics also play a role in apoptosis [29e31]. Therefore, the formation of actin cytoskeleton in A549 cells was assessed by staining with TRITCphalloidin, which specifically binds F-actin. As shown in Fig. 3B, F-actin assembly in A549 cells was disrupted after Ag-SP-DNC (10 mM) treatment for 24 h.

2. Results and discussion

2.4. Ag-SP-DNC induces apoptosis in lung cancer cells

2.1. Ag-SP-DNC inhibits the proliferation of human lung cancer cells

We further tested whether apoptosis induction could contribute to growth inhibitory function of Ag-SP-DNC in A549 cells. The ability of Ag-SP-DNC to induce apoptosis in A549 cells was thus evaluated by treatment of cells with Ag-SP-DNC for 48 h and subsequently subjecting the cells to staining with annexin V and propidium iodide (PI) and flow cytometry analysis. As shown in Fig. 4A, B, Ag-SP-DNC (10 mM) treatment for 48 h caused 35.3% annexin Vpositive cells as compared to controls which showed only 1.0% annexin V-positive cells. The results indicate that Ag-SP-DNC can induce apoptosis in lung cancer cells. As positive control, cisplatin treatment for 48 h caused 41.6% annexin V-positive cells (Fig. 4C).

Initially, we evaluated the growth inhibitory effect of Ag-SP-DNC in lung cancer cells A549 using Sulforhodamine B (SRB) assay. Table 1 shows the 72-h IC50 of Ag-SP-DNC, dehydronorcantharidin acid (DNCA) and dehydronorcantharidin (DNC). Ag-SP-DNC is able to inhibit proliferation of A549 cells with IC50 of 7.41 ± 0.78 mM. By contrast, DNCA and DNC are inactive with no significant inhibition of proliferation being observed at concentration up to 100 mM. Furthermore, the IC50 values obtained for cisplatin were shown to be similar to Ag-SP-DNC in our experiments. 2.2. Ag-SP-DNC induces G2/M phase arrest We further examined how Ag-SP-DNC suppressed the growth of lung cancer cells. The effect of Ag-SP-DNC on cell cycle distribution was analyzed and the results of a typical experiment are shown in Fig. 2. As determined by flow cytometry, the exposure of A549 cells to Ag-SP-DNC (10 mM) after 24 h, resulted in a clear increase of the percentage of cells in G2/M phase when compared with the control. Ag-SP-DNC treatment caused 25.8% cells in G2/M phase as

Fig. 1. Chemical structures of Ag-SP-DNC, DNCA, and DNC.

Table 1 The effect of Ag-SP-DNC on the growth of human lung cancer cells. Compound

A549 cells growth inhibition, IC50 (mM, Mean ± SD. n ¼ 3)

Ag-SP-DNC DNCA DNC Cisplatin

7.41 ± 0.78 >100 >100 6.59 ± 0.63

compared to control showing 14.2%. Conversely, G1 phase cell population was decreased to 50.2% as compared to control having 66.2%. These results suggest the role of cell cycle arrest in Ag-SPDNC-induced growth inhibition of human lung cancer cells. 2.3. Ag-SP-DNC induces morphological change and actin depolymerization in lung cancer cells

2.5. Protective effect of glutathione on Ag-SP-DNC-induced apoptosis in A549 cells Glutathione (g-glutamyl-L-cysteinylglycine, GSH) is the most abundant non-protein thiol compound widely distributed in cells. The GSH detoxification system follows a critical homeostatic mechanism in mammalian cells [32,33]. Platinum compounds (cisplatin, carboplatin, and oxaliplatin) are the most commonly used chemotherapeutic agents in the treatment of advanced stage lung cancer patients, and the GSH metabolic pathway is directly involved in the detoxification or inactivation of platinum drugs [12,34]. In our experiments, cisplatin decreased the intracellular GSH levels (Fig. 5A); treatment with outside source GSH (2 mM) significantly suppressed the appearance of Annexin V-positive cells in cisplatin treated cells (Fig 4C). We suspected that Ag-SP-DNC may deplete intracellular GSH in lung cancer cells and enhance its cytotoxicity. Therefore, the GSH levels of A549 cells were determined. Fig. 5A shows that, when A549 cells were treated with Ag-SP-DNC (10 mM) for 24 h, there was about 70% loss of intracellular GSH compared to the control. Because depleting the GSH level

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Fig. 2. Ag-SP-DNC induces cell cycle arrest at G2/M stage in A549 cells. A549 cells were treated with Ag-SP-DNC (10 mM) for 24 h. Cells were then stained with Propidium iodide (PI), and nuclei were analyzed for DNA content by flow cytometry. A Total of 10,000 cells were analyzed from each sample, and the percentage of cells within G1, S, and G2/M were determined. (A) A fluorescence pattern of PI stained cells with or without Ag-SP-DNC treatment. (B)The statistical data of cell cycle distribution. Each bar represents the mean (±SD n ¼ 3) **p < 0.01 compared with control.

Fig. 3. Effects of Ag-SP-DNC on cell morphology and F-actin in A549 cells. (A) The A549 cells were treated with Ag-SP-DNC (10 mM) for 48 h. The representative fields were photographed at 100 magnification. (B) The F-actin was detected by TRITC-phalloidin. The A549 cells were treated with Ag-SP-DNC (10 mM) for 24 h. Each group was tested in triplicate and the representative data are shown.

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Fig. 4. Flow cytometric analysis of cells stained with Annexin V-FITC and PI. The A549 Cells were treated with Ag-SP-DNC (5, 10 mM), Ag-SP-DNC (10 mM) þ GSH (2 mM), Ag-SP-DNC (10 mM) þ zVAD-fmk (20 mM), cisplatin (5, 10 mM) and cisplatin (10 mM) þ GSH (2 mM) for 48 h and then harvested and processed by annexin V-FITC and PI staining followed by flow cytometry analysis. (A) The fluorescence pattern of annexin V-FITC and PI-stained A549 cells after 48 h treatment. (B) and (C) Percentages of annexin V positive cells for different treatments. Each bar represents the mean (±SD n ¼ 3). *p < 0.05, **p < 0.01, comparing with control. ##p < 0.01, comparing with Ag-SP-DNC (10 mM). #p < 0.01, comparing with cisplatin (10 mM).

in cells is associated with the apoptosis of cells after treated A549 cells with Ag-SP-DNC, we hypothesized that treatment with outside source GSH would protect cells from Ag-SP-DNC induced apoptosis by raising intracellular GSH levels. As shown in Fig. 4, when GSH (2 mM) and Ag-SP-DNC (10 mM) were added to the cells simultaneously, the annexin V positive cells decreased from 35.3% to 2.6% (with GSH). The results indicate that the intracellular GSH plays a crucial role for A549 cells to detoxify the Ag-SP-DNC.

2.6. Ag-SP-DNC promotes the generation of reactive oxygen species We then focused on understanding the cause of depleting GSH by Ag-SP-DNC. GSH is synthesized sequentially by glutamate cysteine ligase (GCL) and GSH synthase. GCL is composed of a light modifier submit (GCLM) as well as a heavy catalytic submit (GCLC), and GCLC is the key subunit of the rate-limiting enzyme in GSH synthesis [35,36]. Based on the results of GSH depleting by Ag-SPDNC, we examined the expression of GCLC by western blot in A549

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Fig. 4. (continued).

Fig. 5. Effects of Ag-SP-DNC on the expression of GSH and GCLC. (A) GSH levels in A549 cells were detected by Glutathione Quantification Kit. The mean concentration of control was taken as 100%, and other values were calculated relative to this. Each bar represents the mean (±SD n ¼ 3) of triplicate determinations. **p < 0.01 versus control. (B) Western blot analyses for levels of GCLC in A549 cells. The A549 cells were treated with Ag-SP-DNC for 24 h. Cell lysates were prepared from the treated cells, and western blot analyses were performed. b-actin was used as control. Each group was tested in triplicate and the representative data are shown.

cells. As shown in Fig 5B, the expression of GCLC was not reduced in the presence of Ag-SP-DNC. The results suggest that Ag-SP-DNC do not affect the GSH synthesis. GSH provides important antioxidant defense, and GSH depletion is commonly observed when cells are oxidatively stressed. Reactive oxygen species (ROS) are a byproduct of normal metabolism, including free radicals such as the superoxide anion, hydroxyl and lipid radicals, as well as oxidizing nonradical species such as hydrogen peroxide, peroxynitrite, and singlet oxygen. They often cause cellular damage and lead to cell death and tissue injury, especially at high concentrations. The antioxidant defense mechanism to minimize cellular damage relies on the interactions between cellular constituents and ROS [37,38]. Cisplatin was observed to generate more intracellular ROS in lung cancer cells A549 (Fig. 6). We were particularly interested in whether GSH is responsible for controlling the intracellular ROS

levels and whether ROS are responsible for apoptosis and depleting GSH in Ag-SP-DNC treated cells. Therefore, we determined the ROS levels by means of fluorescence of DCFH-DA. As indicated in Fig. 6, Ag-SP-DNC (10 mM) treatment resulted in a 2.5-fold increase of intracellular ROS levels as compared with control; the ROS were eliminated by adding outside source GSH.

2.7. Ag-SP-DNC disrupts mitochondrial membrane potential in lung cancer cells Mitochondria play a central role in the life and death of cells, and they are known to be a major source and target of oxidative stress. Mitochondria maintain homeostasis in the cell by interacting with ROS and responding adequately to different stimuli. An abnormal cellular ROS balance can be activated by the structural injury of

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Fig. 6. Effect of Ag-SP-DNC on intracellular ROS in A549 cells and the protective effect of GSH. The A549 cells were treated with Ag-SP-DNC (5, 10 mM), Ag-SP-DNC (10 mM) þ GSH (2 mM) or cisplatin (10 mM) for 24 h. The mean fluorescence intensity of control was taken as 100%, and other values were calculated relative to this. Each bar represents the mean (±SD n ¼ 3) of triplicate determinations. **p < 0.01 versus control. ##p < 0.01, comparing with Ag-SP-DNC (10 mM).

mitochondria. Furthermore, excess ROS production can induce mitochondrial damage [39e41]. The damage of mitochondrial integrity and the consequent loss of mitochondrial membrane potential (DJm) are the early events in the initiation and activation of apoptotic cascades [42]. To determine whether Ag-SP-DNC induce mitochondrial disruption in A549 cells, we examined the depolarization of mitochondrial membrane by measuring the fluorescence remission shift (red to green) of the DJm sensitive cationic JC-1 dye in A549 cells. Cells were treated with Ag-SP-DNC (5, 10 mM) for 24 h and subsequently processed and stained with JC-1 dye and analyzed by flow cytometry. Ag-SP-DNC (10 mM) treated cells showed an increase in green/red fluorescence intensity indicating increased mitochondrial membrane depolarization (Fig. 7). When GSH (2 mM) and Ag-SP-DNC (10 mM) were added to the cells simultaneously, the DJm was similar to the normal control cells. The results indicate that the induction of apoptosis by Ag-SP-DNC in lung cancer cells is closely associated with mitochondrial function disruption. As positive control, cisplatin also disrupted mitochondrial membrane potential in lung cancer cells (Fig. 7).

2.8. Ag-SP-DNC induces caspase-dependent apoptosis in lung cancer cells A critical process in apoptosis is the activation of a family of cysteine proteases, termed caspases. Two overlapping caspasedependent apoptotic pathways have been identified: the death receptor pathway (extrinsic pathway) and the mitochondrial pathway (intrinsic pathway). Both of these pathways converge on caspase-3 activation, and therefore detection of active caspase-3 in cells and tissues is an important method for caspase-dependent apoptosis. The disruption of mitochondrial membrane potential results in the release of cytochrome c from the mitochondrial intermembrane space into the cytoplasm, which initiates apoptosome-formation with initiator caspase-9 and subsequent activation of caspase-3 [43e45]. Cisplatin caused activation of caspase-3 in lung cancer cells A549 (Fig. 8). To determine whether apoptosis induced by AgSP-DNC is a mitochondria-dependent caspase pathway, we examined the effect of the activation of caspase-3 by flow cytometry using specific antibodies that recognize the particularly cleaved and activated form after Ag-SP-DNC treatment. As shown in Fig. 8, increase in the cleaved and activated form of caspase-3 was observed in the treated cells as compared to the control cells. N-benzyloxycarbonylVal-Ala-Asp-fluoromethylketone (zVAD-fmk) is a cell-permeant pan caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases and can inhibit caspase-dependent apoptosis [46,47]. To explore whether Ag-SP-DNC-induced cell death depends

on caspase activity, we employed the broad-spectrum caspase inhibitor zVAD-fmk. Cells were cultured with Ag-SP-DNC (with or without zVAD-fmk) for 48 h. Then the cells were collected and stained with Annexin V and PI. Fig 4 shows that zVAD-fmk (20 mM) treatment decreased cell apoptosis in Ag-SP-DNC-treated cells. The annexin V positive cells decreased from 35.3% to 10.1% (with zVADfmk). The results indicate that caspase activation is involved in AgSP-DNC induced cell apoptosis.

2.9. Ag-SP-DNC causes intracellular Ca2þ elevation in lung cancer cells Calcium is a ubiquitous second messenger involved in many cellular processes, including regulation of transcription, metabolism, proliferation, and cell death. Due to many effects of calcium, the intracellular calcium concentration is tightly regulated, and calcium overload can induce cell apoptosis. Mitochondria can be considered as a firewall that controls the Ca2þ concentration in the cell and in cytoplasmic microdomains by tuning the frequency of oscillatory Ca2þ signals and by blunting the spread of cytosolic Ca2þ waves [48,49]. Our results have shown that Ag-SP-DNC disrupt mitochondrial function and induce apoptosis. To determine the role of calcium signaling in Ag-SP-DNC-induced apoptosis, A549 cells were treated with Ag-SP-DNC for 24 h. Subsequently, Ca2þ was measured with a calcium indicator dye, Fluo-3/AM. We found that treatment with Ag-SP-DNC or cisplatin resulted in an elevation of Ca2þ in the cells (Fig. 9). The results suggest that Ag-SP-DNC-induced apoptosis might be associated with its induction of Ca2þ elevation.

2.10. Ag-SP-DNC induced lung cancer cells apoptosis involves AIF and EndoG proapoptotic factors working independently of caspases Apoptosis might proceed through the activation of both caspase-dependent and -independent pathways. The proapoptotic mitochondrial proteins apoptosis inducing factor (AIF) and endonucleaseG (EndoG) are well-described death effectors working independently of caspases during cell death. On apoptotic stimuli, AIF and EndoG translocate from mitochondria to the nucleus, inducing chromatin condensation and DNA fragmentation [50,51]. So the translocation of AIF and EndoG to the nucleus was analyzed by western blot. As shown in Fig. 10, the translocation of AIF and EndoG to the nucleus was detected after treatment of A549 cells with Ag-SP-DNC. These findings indicate that mitochondriareleased proapoptotic proteins, AIF and EndoG, are important factors in the Ag-SP-DNC-induced lung cancer cell apoptosis.

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Fig. 7. Effect of Ag-SP-DNC on mitochondrial membrane potential in A549 cells. Cells were treated with Ag-SP-DNC or cisplatin for 24 h, and then harvested and processed by JC-1 staining followed by flow cytometry analysis. Each bar represents the mean (±SD n ¼ 3). *p < 0.05, **p < 0.01, comparing with control. ##p < 0.01, comparing with Ag-SP-DNC (10 mM).

Fig. 8. Effect of Ag-SP-DNC on caspase-3 activation in A549 cells. The A549 cells were treated with Ag-SP-DNC or cisplatin for 24 h. Cells were then harvested and stained with cleaved caspase-3 (Asp175) antibody (Alexa fluor 488 conjugate). The fluorescence intensity was determined with flow cytometry. Each bar represents the mean (±SD n ¼ 3) of triplicate determinations. **p < 0.01 versus control.

Fig. 9. Effect of Ag-SP-DNC on intracellular free Ca2þ ([Ca2þ]i) in A549 cells. The A549 cells were treated with Ag-SP-DNC or cisplatin. After 24 h, cells were then harvested and stained with Fluo-3/AM. The fluorescence intensity was determined with flow cytometry. Each bar represents the mean (±SD n ¼ 3) of triplicate determinations. **p < 0.01 versus control.

2.11. Effects of Ag-SP-DNC on the expression of Bcl-2 and p53 in lung cancer cells To elucidate the molecular mechanisms involved in the observed apoptosis alterations, we investigated the effects of AgSP-DNC on the expression of proteins important for mitochondria mediated apoptosis. Cancer cells deploy diverse strategies to limit or evade apoptosis that frequently involve perturbation of the Bcl-2

intrinsic apoptotic pathway. Bcl-2 is a membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 can prevent cells from apoptosis, while loss of Bcl-2 may lead to mitochondrial dysfunction and apoptosis [52,53]. p53, a crucial tumor suppressor, plays an important role in the prevention of cancer development by mediating cell cycle arrest and apoptosis through its downstream targets. Upon cellular stress, p53 can mediates cell apoptosis by induction of pro-apoptotic proteins

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Fig. 10. Western blot analysis of nuclear fractions for AIF and EndoG. Histone H3 was used as loading control. The A549 cells were treated with Ag-SP-DNC for 24 h. Cells were then harvested and isolated the nuclei with nuclear extract kit. Western blot analyses were subsequently performed. Each group was tested in triplicate and the representative data are shown.

belonging to the Bcl-2 family, such as Bax and PUMA. p53 has also been shown to promote the release of AIF and EndoG from mitochondria [54e56]. We found that Ag-SP-DNC was able to decrease the levels of Bcl-2 in A549 cells. In contrast, p53 expression was increased after treated the cells with Ag-SP-DNC (Fig. 11). The upregulation of p53 expression and down-regulation of Bcl-2 expression, are correlated with mitochondrial membrane depolarization, caspase-3 activation and translocation of AIF and EndoG to the nucleus.

Fig. 11. Western blot analyses for levels of Bcl-2 and p53 in A549 cells. The A549 cells were treated with Ag-SP-DNC for 24 h. Cell lysates were prepared from the treated cells, and western blot analyses were performed. b-actin was used as control. Each group was tested in triplicate and the representative data are shown.

dimethyl sulfoxide (DMSO) to give a stock solution with a concentration of 20 mM. In experiments involving the addition of compounds to cells, the stock solutions were diluted in sterile culture medium. An equivalent amount of DMSO diluted in culture medium was added to control cultures. Electrospray ionization quadrupole time-of-flight mass spectrometry, (ESI-QTOF-MS) showed that the coordination polymer Ag-SP-DNC is dissociated into its monomer in solution. So the concentration is determined on the basis of the molar mass of its monomer (C8H7O5Ag, MW ¼ 291.01).

3. Conclusion In conclusion, our study revealed that Ag-SP-DNC induces ROSmediated mitochondrial dependent apoptosis in lung cancer cells. Ag-SP-DNC treatment induced ROS generation, decreased the expression of Bcl-2 and increased the expression of p53 in A549 cells. The ROS elevation and the disequilibrium expression of antiapoptotic and pro-apoptosis proteins led to mitochondrial membrane potential disruption and calcium overload. Then the caspase3 was activated, and the AIF and EndoG translocated from mitochondria to the nucleus. Both caspase-dependent and caspaseindependent apoptosis were induced by Ag-SP-DNC in lung cancer cells. The properties of Ag-SP-DNC in activating multiple apoptotic pathways may be exploited in anticancer applications. 4. Materials and methods 4.1. Materials TRITC-phalloidin, Propidium iodide (PI), and reduced glutathione (GSH), were purchased from SigmaeAldrich. Annexin-V FITC apoptosis kit was purchased from Invitrogen. Nuclear Extract Kit, Glutathione Quantification Kit, 2,7-dichlorodihydro fluorescent diacetate (DCFH-DA), JC-1 staining kit and Fluo-3/AM were purchased from Beyotime Institute Biotechnology. The caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk) was purchased from Promega. Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate), Bcl-2 rabbit mAb, Histone H3 (3H1) Rabbit mAb (HRP Conjugate), AIF (D39D2) XP® Rabbit mAb and EndonucleaseG Antibody were purchased from Cell Signaling Technology. p53 rabbit pAb was purchased from Bioworld Technology. b-actin rabbit pAb was purchased from Beijing Biosynthesis Biotechnology Co. Ltd. 4.2. Preparation of solutions of compounds The Ag complex was obtained from the reaction of DNC and AgNO3 (1:1 M ratio) in water at room temperature and characterized using elemental analysis and single-crystal X-ray diffraction, as described previously [28]. The compounds were dissolved in

4.3. Cell culture Lung cancer cells (A549 cells) were purchased from Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences. The cells were cultured in F12K medium supplemented with 10% heat inactivated FBS, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin and maintained at 37  C in a humidified atmosphere of 5% CO2. 4.4. SRB assay A549 cells (3000 cells) were seeded on 96-well microtitre plates in F12K medium with 10% FBS and incubated overnight. The cell culture medium was replaced by the different dose of compounds solution, and then the cells were cultured for another 72 h. The supernatant medium from the 96 well plates was thrown away, and the cells were fixed with 10% TCA for at least 1 h at 4  C. The cells were then washed five times with the distilled water and next dried by the air. The cells were then dyed with 100 ml of 0.4% SRB, for about 30 min. After dying, the plates were again washed to remove the dye with 1% acetic acid, and allowed to air dry overnight. Tris base solution (150 ml, 10 mM) was added to each well and the absorbance was measured at 560 nm using a plate reader.

4.5. Cell cycle analysis Cell cycle analysis was conducted as described previously [57]. Cells were seeded on 6 well plates in F12K medium with 10% FBS overnight. Then Ag-SP-DNC was added to the cells, which were then cultured for another 24 h. Cells were collected with trypsinEDTA and washed three times with PBS. The cells were resuspended and fixed for at least 2 h at 20  C with 70% ethanol. After washing twice with PBS, cells were incubated at 25  C with 200 mg/ml RNase A for 30 min. The resulting cells were incubated with 50 mg/ml PI for 30 min at 4  C. The treated cells were subjected to flow cytometry and the percentage of cells at each phase of the cell cycle was analyzed.

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4.6. Immunofluorescence and confocal microscopic analysis for actin filaments F-actin of A549 cells was detected using fluorescent phalloidin and analyzed by confocal microscopy, as described previously [57]. Cells were seeded to the glass coverslips in cell culture dishes (3.5 cm) overnight. Ag-SP-DNC was then added and cells were cultured for 24 h. After fixing with 4% paraformaldehyde, cells were treated with 0.1% Triton X-100 and blocked with 1% BSA. Cells were incubated with TRITC-conjugated phalloidin for 60 min and examined under Confocal Laser Scanning Microscope.

4.7. Analysis of cell apoptosis The ability of Ag-SP-DNC to induce cell apoptosis of A549 cells was quantified by annexin V and PI staining and flow cytometry, as described previously [57]. Briefly, after treatment with Ag-SP-DNC or cisplatin for 48 h, cells were collected and washed with PBS twice, and subjected to annexin V and propidium iodide staining using annexin-V FITC apoptosis kit following the step-by-step protocol provided by the manufacturer. After staining, flow cytometry was performed for the quantification of apoptotic cells.

4.8. GSH measurement A549 cells were seeded into the cell culture dish plate (6 cm) for overnight and then incubated with Ag-SP-DNC or cisplatin. After 24 h, the cells were trypsinized and the total number of cells was counted. GSH levels were determined using Glutathione Quantification Kit. 5,50-dithiobis (2-nitrobenzoic acid) (DTNB) and GSH react to generate 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG) [58]. Because 2-nitro-5-thiobenzoic acid is a yellow colored product, GSH concentration in a sample solution can be determined by 412 nm absorbance with a multiwell plate reader.

4.9. Measurement of intracellular reactive oxygen species (ROS) The levels of intracellular reactive oxygen species (ROS) were determined using 2,7-dichlorodihydro fluorescent diacetate (DCFH-DA), as described previously [59]. A549 cells were plated into a 6 well plate for 24 h prior to the experiment. On the following day, cells were treated with Ag-SP-DNC or cisplatin. After 24 h incubation, cells were washed with PBS for three times and subsequently treated with 10 mM DCFH-DA. After incubating for 30 min, the cells were washed by PBS, trypsinized, and collected by centrifugation. Cells (30,000) in 200 mL PBS solution from each well were transferred to a 96 well black microplate. The fluorescence intensity of cells was measured with a fluorescence spectrophotometer, with excitation and emission wavelengths of 488 and 525 nm, respectively.

4.10. Mitochondrial membrane potential assay JC-1(5,50,6,60-tetrachloro-1,10,3,30tetraethylbenzamidazolocarbocyanin iodide) is most widely applied for detecting mitochondrial depolarization occurring in the early stages of apoptosis. Mitochondrial membrane potential (DJm) analysis was conducted and modified as described previously [57]. Briefly, A549 cells were treated with Ag-SP-DNC or cisplatin for 24 h, at the end of treatment, cells were harvested. Cells were incubated with JC-1 at 37  C for 20 min. Stained cells were washed with PBS twice and analyzed by flow cytometry.

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4.11. Measurement of intracellular cleaved caspase-3 Intracellular cleaved caspase-3 was determined by flow cytometry with cleaved Caspase-3 (Asp175) Antibody. After treatment with Ag-SP-DNC or cisplatin for 24 h, the cells were trypsinized, collected by centrifugation, washed twice with PBS and fixed with 4% paraformaldehyde. After treated with 0.1% Triton X-100 and blocked with 1% BSA, cells were incubated with cleaved caspase-3 (Asp175) antibody (Alexa fluor 488 conjugate) for 30 min. Stained cells were washed with PBS twice and analyzed by flow cytometry. 4.12. Measurement of intracellular free Ca2þ Intracellular free Ca2þ levels in A549 cells were measured using Ca specific fluorescent probe Fluo-3/AM, as described recently with minor modifications [60]. A549 cells were treated with Ag-SPDNC or cisplatin for 24 h, at the end of treatment, cells were harvested. Cells were loaded with 5 mM Fluo-3/AM for 60 min at room temperature. After incubation, cells were harvested and washed twice with PBS and analyzed by flow cytometry. 2þ

4.13. Western blot analysis Cells were washed twice with ice-cold PBS and lysed with RIPA buffer containing protease inhibitors. After centrifugation at 13,000 g for 15 min, protein concentrations of the lysates were determined by the micro-BCA protein assay kit. The total cellular protein extracts were boiled with 5 Laemmli sample buffer and separated by SDS-PAGE and transferred to PVDF membrane. Membranes were blocked with 5% non-fat dry milk in TBS containing 0.1% Tween 20 for 1 h at room temperature and incubated with appropriate antibodies overnight at 4  C. Blots were washed three times in TBS-T buffer, followed by incubation with the appropriate HRP-linked secondary antibodies for 1 h at room temperature. The specific proteins in the blots were visualized using the enhanced chemiluminescence reagent. 4.14. Nuclei isolation Isolation of nuclei was carried out using the nuclear extract kit according to the manufacturer's protocol. Translocation of AIF and EndoG to the nucleus was analyzed by western blot. Briefly, after treatment with Ag-SP-DNC for 24 h, cells were trypsinized, collected by centrifugation and washed with PBS. Cell pellets were resuspended in hypotonic buffer and incubated for 15 min at 4  C. Cells were permeabilized with detergent and centrifuged (5 min, 13,000 g, 4  C) and the supernatants were removed (nonnucleic fraction). Pellets were resuspended in lysis buffer, incubated for 30 min at 4  C, and centrifuged (10 min, 13,000 g, 4  C). The supernatants contain the nuclear proteins. 4.15. Statistical analysis The statistical analysis was done using Student's t test with the SPSS 16.0 statistical program. p < 0.05 was accepted as significant, and p < 0.01 was regarded as highly significant. Conflict of interest The authors declare that there is no conflict of interest. Authors' contributions Conception and design: Y. Zhao, X. Tan.

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S. Li et al. / European Journal of Medicinal Chemistry 86 (2014) 1e11

Development of methodology: S. Li, S Zhang, Xing Jin. Analysis and interpretation of data: S. Li, S Zhang, Y. Zhao. Writing, review, and/or revision of the manuscript: J. Lou, Xing Jin, Y. Zhao. Supervision of study: X. Zhang. Acknowledgments This work was supported by the National Natural Science Foundation of China (81102468 to Y. Zhao) and Science & Technology Development Projects of Shandong Province in China (No. 2011GGX10706 to X. Tan). References [1] C.H. Wang, W.C. Shih, H.C. Chang, Y.Y. Kuo, W.C. Hung, T.G. Ong, W.S. Li, Preparation and characterization of amino-linked heterocyclic carbene palladium, gold, and silver complexes and their use as anticancer agents that act by triggering apoptotic cell death, J. Med. Chem. 54 (2011) 5245e5249. [2] C.X. Zhang, S.J. Lippard, New metal complexes as potential therapeutics, Curr. Opin. Chem. Biol. 7 (2003) 481e489. [3] B. Biersack, A. Ahmad, F.H. Sarkar, R. Schobert, Coinage metal complexes against breast cancer, Curr. Med. Chem. 19 (2012) 3949e3956. [4] G. Zhu, M. Myint, W.H. Ang, L. Song, S.J. Lippard, Monofunctional platinumDNA adducts are strong inhibitors of transcription and substrates for nucleotide excision repair in live mammalian cells, Cancer Res. 72 (2012) 790e800. [5] M.A. Fath, I.M. Ahmad, C.J. Smith, J. Spence, D.R. Spitz, Enhancement of carboplatin-mediated lung cancer cell killing by simultaneous disruption of glutathione and thioredoxin metabolism, Clin. Cancer Res. 17 (2011) 6206e6217. [6] D. Samanta, J. Kaufman, D.P. Carbone, P.K. Datta, Long-term smoking mediated down-regulation of Smad3 induces resistance to carboplatin in non-small cell lung cancer, Neoplasia 14 (2012) 644e655. [7] L. Hu, C. Wu, X. Zhao, R. Heist, L. Su, Y. Zhao, B. Han, S. Cao, M. Chu, J. Dai, J. Dong, Y. Shu, L. Xu, Y. Chen, Y. Wang, F. Lu, Y. Jiang, D. Yu, H. Chen, W. Tan, H. Ma, J. Chen, G. Jin, T. Wu, D. Lu, D.C. Christiani, D. Lin, Z. Hu, H. Shen, Genome-wide association study of prognosis in advanced non-small cell lung cancer patients receiving platinum-based chemotherapy, Clin. Cancer Res. 18 (2012) 5507e5514. [8] R. Ramlau, V. Gorbunova, T.E. Ciuleanu, S. Novello, M. Ozguroglu, T. Goksel, C. Baldotto, J. Bennouna, F.A. Shepherd, S. Le-Guennec, A. Rey, V. Miller, N. Thatcher, G. Scagliotti, Aflibercept and docetaxel versus docetaxel alone after platinum failure in patients with advanced or metastatic non-small-cell lung cancer: a randomized, controlled phase III trial, J. Clin. Oncol. 30 (2012) 3640e3647. [9] H. West, D. Harpole, W. Travis, Histologic considerations for individualized systemic therapy approaches for the management of non-small cell lung cancer, Chest 136 (2009) 1112e1118. [10] X. Wu, Y. Ye, R. Rosell, C.I. Amos, D.J. Stewart, M.A. Hildebrandt, J.A. Roth, J.D. Minna, J. Gu, J. Lin, S.C. Buch, T. Nukui, J.L. Ramirez Serrano, M. Taron, A. Cassidy, C. Lu, J.Y. Chang, S.M. Lippman, W.K. Hong, M.R. Spitz, M. Romkes, P. Yang, Genome-wide association study of survival in non-small cell lung cancer patients receiving platinum-based chemotherapy, J. Natl. Cancer Inst. 103 (2011) 817e825. [11] R. Pirker, J.R. Pereira, A. Szczesna, J. von Pawel, M. Krzakowski, R. Ramlau, I. Vynnychenko, K. Park, C.T. Yu, V. Ganul, J.K. Roh, E. Bajetta, K. O'Byrne, F. de Marinis, W. Eberhardt, T. Goddemeier, M. Emig, U. Gatzemeier, Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX): an open-label randomised phase III trial, Lancet 373 (2009) 1525e1531. [12] P. Yang, J.O. Ebbert, Z. Sun, R.M. Weinshilboum, Role of the glutathione metabolic pathway in lung cancer treatment and prognosis: a review, J. Clin. Oncol. 24 (2006) 1761e1769. [13] E.S. Kim, J.J. Lee, G. He, C.W. Chow, J. Fujimoto, N. Kalhor, S.G. Swisher, I.I. Wistuba, D.J. Stewart, Z.H. Siddik, Tissue platinum concentration and tumor response in non-small-cell lung cancer, J. Clin. Oncol. 30 (2012) 3345e3352. [14] S. Ray, R. Mohan, J.K. Singh, M.K. Samantaray, M.M. Shaikh, D. Panda, P. Ghosh, Anticancer and antimicrobial metallopharmaceutical agents based on palladium, gold, and silver N-heterocyclic carbene complexes, J. Am. Chem. Soc. 129 (2007) 15042e15053. [15] C.N. Banti, S.K. Hadjikakou, Anti-proliferative and anti-tumor activity of silver(I) compounds, Metallomics 5 (2013) 569e596. [16] S.F. Sucena, R.E. Paiva, C. Abbehausen, I.B. Mattos, M. Lancellotti, A.L. Formiga, P.P. Corbi, Chemical, spectroscopic characterization, DFT studies and antibacterial activities in vitro of a new gold(I) complex with rimantadine, Spectrochim. Acta. A Mol. Biomol. Spectrosc. 89 (2012) 114e118. [17] S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke, Novel benzyl-substituted N-heterocyclic carbene-silver acetate complexes: synthesis, cytotoxicity and antibacterial studies, Metallomics 3 (2011) 74e88.

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