Novel Selenosemicarbazone Metal Complexes Exert Anti-Tumor Effect Via Alternative, Caspase-Independent Necroptotic Cell Death

Send Orders for Reprints to [email protected] Medicinal Chemistry, 2014, 10, 000-000

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Novel Selenosemicarbazone Metal Complexes Exert Anti-tumor Effect via Alternative, Caspase-independent Necroptotic Cell Death Manja Zeca, Tatjana Srdi-Rajib, Ana Krivokuab, Radmila Jankovib, Tamara Todorovic, Katarina Anelkovic and Sinia Radulovib* a

Institute for Medical Research Belgrade, University of Belgrade, Tadeua Koukog 1, Belgrade, Serbia; bInstitute for Oncology and Radiology of Serbia, Department of Experimental Oncology, Pasterova 14, Belgrade, Serbia; cFaculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia Abstract: The synthesis and chemical characterization of the novel 2,6-diacetylpyridine-bis(selenosemicarbazone) metal complexes of Zn(II), Cd(II) and Ni(II) were published previously. Here we report first evidence on anti-proliferative activity of the complexes and molecular patterns that underlie it. The complexes and the corresponding ligand are shown to be cytotoxic on the panel of nine, malignant and non-malignant cell lines, with the exception of Ni(II) complex that did not achieve IC50 value on any of the cell lines tested. Further experiments on the selected cell lines including A 549, MRC-5, EA.hy 926 and HeLa, have shown that the complexes posses unambiguous property of inducing necrosis in the cells treated for 6 hours, with the ligand and Zn(II) complex being the most active on all cell lines. On the contrary, only small portion of early apoptotic events was detected, under the same experimental condition. This was in complete concordance with the results obtained from Western blot analysis of the treated cells that showed no or slight increase of the protein amounts of two crucial apoptotic mediators: Cytochrome C and Caspase III. We propose the model, under which tested complexes induce necroptosis in treated cells, a recently described type of cell death with necrotic morphological features and acting via caspase independent pathway, and without elevated amounts of intracellular ROS. Endothelial EA.hy 926 cells have proven to be extremely sensitive on the necrosis-inducing effect of the complexes, which could indicate potential anti-angiogenic effect of the novel complexes that is to be investigated.

Keywords: A 549, EA.hy 926, Metal complexes, Necroptosis, ROS, Selenosemicarbazone, Zinc (II) complex. INTRODUCTION Metal compounds have been intriguing field for research in the field of cancer therapeutics since cisplatin [cisdiamminedichloroplatinum(II)] and carboplatin [cis-diammine(1,1-cyclobutanedicarboxylato) platinum(II)] were discovered [1, 2]. Although metal compounds have a priori been considered as toxic, the use of cisplatin remains the first line for the treatment of solid tumors. However, there is an increasing incidence of resistance to cisplatin [3, 4], in addition to side effects caused by cisplatin therapy [5, 6], which can be life-threatening, such as kidney toxicity [7]. Thus, there is a strong need for further development of novel compounds with high specificity for tumor in contrast with normal tissue cells, and with promising anti-proliferative as well as anti-metastatic potential in the treatment of solid tumors. Heterocyclic compounds are of interest for medicinal chemists due to their versatile biological activity. Semicarbazones are a class of organic heterocyclic compounds derived from condensation reaction between aldehyde/ketone and semicarbazides, which are urea derivatives. Semicarabazones have been widely investigated for their anti-microbial, *Address correspondence to this author at the National Cancer Research Center, Department of Experimental Pharmacology, Pasterova 14, 11 000 Belgrade, Serbia; Tel: + 381 11 2067434; Fax: + 381 11 2067292; E-mail: [email protected] 1573-4064/14 $58.00+.00

anti-viral, anti-bacterial, anti-malarial activity [8, 9] and recently for anti-tumor activity as well [10, 11]. The well-known thiosemicarabazone compound Triapine (3-aminopyridine-2carboxaldehyde thiosemicarbazone) investigated for its ribonucleotide reductase inhibitory activity, has reached Phase 2 clinical trial [12]. In comparison with its chalcogen analogues, selenosemicarbazones have been shown as the most potent anti-tumor compounds [13], and became an intriguing field for further development and studies. The rationale for investigating the selenium compounds furthermore lies in its demonstrated chemopreventive action [14, 15] through the cell cycle perturbation, induction of apoptosis and inhibition of migration and invasiveness in vitro [16]. It is suggested that chemopreventive role of selenium compounds is derived from its ability to induce formation of reactive oxygen species (ROS), which in turn induce apoptosis and cell cycle perturbation [17]. In general, induction of apoptosis represents one of the major directions in the design of novel therapeutic strategies for the cancer treatment. Although the apoptosis was considered as the unique organized cell death, programmed mechanisms underlying the other types of cell death such as necrosis, were discovered recently and studied extensively [18]. Furthermore, it was shown that selenium compounds effectively cause cell death through the mechanisms other than apoptosis, such as necrosis or even autophagy [19]. Thus the necrosis and un© 2014 Bentham Science Publishers

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Medicinal Chemistry, 2014, Vol. 10, No. ??

derlying signaling pathways have become another interesting targets for the development of novel therapeutics. Aside targeting apoptosis, there are several more cancer hallmarks that should be taken into account when designing novel anti-cancer strategies [20]. The fact that cancer cells exert elevated levels of ROS in comparison with normal cell population is widely known. Reactive oxygen species may contribute to the carcinogenesis in the dual manner: acting as pro-angiogenic factors via VEGF signaling or inducing cell death through DNA damage and disturbed DNA repair [21, 22]. Moreover, there is a common over-expression of some redox-state modulating proteins such as thioreduxin reductase (TR1) in cancer cells like lung cancer cells A 549 [23]. Thus, modulation of ROS amounts selectively in the population of cancer and endothelial cells may represent an interesting approach in designing novel chemopreventive therapeutics, especially if considering inherent susceptibility of cancer cells to oxidative signals. Our group has previously reported anti-tumor properties of zinc (II), cadmium (II) and nickel (II) complexes with 2formylpyridine selenosemicarbazone (Hfpsesc). Hfpsesc complexes’ demonstrated the possibility of exerting cytotoxic effects through induction of inner mitochondrial pathway in tumor HeLa and MDA-MB-361 cell lines [10], as well as strong anti-angiogenic activity on human and mouse endothelial EA.hy 926 and MS1 cells [11]. Novel metal complexes of the ligand 2,6-diacetylpyridine-bis(selenosemicarbazone) (H2dapsesc) were synthesized and chemically characterized [24]. These complexes are structural analogous to the Hfpsesc metal complexes. In this work we perform extensive biological screening of the zinc (II), nickel (II) and cadmium (II) complexes of H2dapsesc. We have examined their cytotoxic properties towards a panel of tumor as well as normal cell lines. In the continuation of our work, we have analyzed anti-proliferative characteristics of the complexes through their ability to induce cell cycle perturbation and apoptosis, with the emphasis on the activity of the complexes on lung tissue cells, malignant and nonmalignant A 549 and MRC-5 cell line, respectively. Molecular events underlying observed anti-tumor activity of the complexes were further examined through their impact on some of the well known cancer hallmarks, such as p21 and MMP-9 in NSCLC (non-small cell lung cancer) A 549 and highly invasive MCF-7 cells, respectively. METHODS Materials Mouse monoclonal antibody against cytochrome C was purchased from BD Pharmingen San Diego, CA, USA; mouse monoclonal antibody against caspase III was purchased from Calbiochem, Merck Biosciences, Germany; Mouse monoclonal antibody against -actin was purchased from Sigma Chemicals Co, USA. All other reagents were purchased from Sigma Chemicals Co, USA. Metal Complexes of Selenosemicarbazones The synthesis and characterization of the novel ligand 2,6-diacetylpyridine-bis(selenosemicarbazone) (H2dapsesc) compound L, and its complexes with Zn(II): [Zn(dapsesc)] -

Zec et al.

compound 1, Cd(II): [Cd(dapsesc)] - compound 2, and the crystal structure of Ni(II) complex [Ni(hcn)] - compound 3 with a modified H2dapsesc ligand, were reported earlier [24]. The complexes were obtained after the complexation of the ligand with the corresponding salts: Zn(CH3COO)2· 2H2O compound 4, Cd(CH3 COO)2· 2H2O - compound 5, and Ni(CH3 COO)2· 4H2O - compound 6, respectively. The complexation of the H2dapsesc ligand with the Ni(II) corresponding salt, resulted in the modification of the symmetrical ligand side chains and elimination of one-side hydrogen selenide, which resulted in formation of the Ni(II) complex with 2-{1-[6-(1-selenosemicarbazonoethyl)-2-pyridyl]ethylidene} hydrazine carbonitrile (H2hcn) [24]. The chemical strucutures of the compounds are presented in the (Fig. 1). MTT - Cytotoxicity Assay Cytotoxicity of the investigated compounds was determined by using MTT assay (Sigma) [25]. Cells were seeded in 96-well cell culture plates (NUNC) in culture medium. Due to the different morpho-physiological features of the different cell lines, cells were seeded in the concentration of 4000 c/w for MDA-MB-361, MDA-453 cells, 5000 c/w for A 549, MRC-5, FemX, HeLa and MCF-7 cells, 6000 c/w for EA.hy 926 cell line and 7000 c/w for LS-174 cell line. Stock solutions of investigated compounds were made in DMSO at concentrations of 10 mM (final concentration of DMSO per well did not exceed 1%) and afterwards diluted with nutrient medium to desired final concentrations (in range of 1-100
M). All samples were done in pentaplicate. After incubation period of 24 h at 37°C, in 5% CO2 in humidified atmosphere, 20 mL of MTT solution, 5 mg/mL in phosphate buffer solution (PBS) pH 7.2 were added to each well. Samples were incubated for 4 h at 37°C, with 5% CO2 in humidified atmosphere. Formazan crystals were dissolved in 100 mL 10% sodium dodecyl sulphate (SDS) in 0.01 M HCl, and absorbance was recorded on an enzyme-linked immunosorbent assay (ELISA) reader after 24 h at a wavelength of 570 nm. Results are finally expressed as IC50 values (concentration of investigated agent that decreases the number of viable cells by 50% in treated cell population compared to nontreated control) determined using sigmoidal dose-response (variable slope) curve fitting (four-parameter logistic equation) on GraphPad Prism 5.01 software. Cell Cycle Analysis DNA content in fixed A 549, MDA-453 and MRC-5 cells was analyzed by quantitative analysis of cell cycle phase distribution performed by flow-cytometric analysis, after staining of the cells with propidium iodide (PI) (SigmaAldrich, US) [26]. All tested cell lines were seeded in sixwell plates, and were continually exposed for 48 h to the compounds. Afterwards, cells were trypsinized, collected, washed twice with ice-cold PBS, and fixed for 30 min in 70% EtOH. Fixed cells were washed again with PBS, and following the last spinning, PBS was discarded and cells were incubated with 0.1 mL of RNase A (1 mg/mL) for 30 min at 37°C. The addition of 400 mg/mL of PI per specimen has preceded flow-cytometric analysis of the cells. Cell cycle phase distribution were analyzed using a fluorescence activated sorting cells (FACS) Calibur Becton Dickinson flow cytometer and Cell Quest computer software.

Induction of Necroptosis by New Metal Complexes

Medicinal Chemistry, 2014, Vol. 10, No. ??

N

N

N HN H2N

N

NH Se

N

N

N

Se

NH2

H2dapsesc

H2N

N

N N

M Se

3

Se

N NH2

H2N

N Ni

N

Se N

M = Zn; [Zn(dapsesc)] M = Cd; [Cd(dapsesc)]

[Ni(hcn)]

Fig. (1). Structure of the ligand H2dapsesc (L), Zn (II) (1), Cd (II) (2), and Ni (II) (3) complexes used in this work. The complexes were synthesized previously and biological activity was determined throughout this report.

Cell Death Analysis Induction of different cell death types in A 549, MRC-5, HeLa and EA.hy 926 cells treated with investigated compounds, was evaluated by Annexin V-FITC apoptosis detection kit (BD Biosciences, Pharmingen San Diego, CA, USA, Cat No 556570). Briefly, 1106 cells/mL treated with 50 μM of investigated compounds for 6 h, were washed twice with cold PBS and then resuspended in 200 mL binding buffer (containing 10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Following the transfer of 100 mL of the solution (containing 1105 cells) to a 5 mL culture tube, 5
L of both Annexin V-FITC and propidium-iodide (PI) was added. Cells were gently vortexed and incubated for 15 min at 25°C in the dark. After that, 400 mL of binding buffer was added to each sample. Cells were analyzed using a FACS Calibur Becton Dickinson flow cytometer and Cell Quest computer software. Western blot Analysis Preparation of Cell Lysates and Immunoblot After the continual period of treatment of 6 h, protein samples derived from treated A 549 and MRC-5 cells were extracted for immunoblot assay. The cells were washed twice with ice-cold PBS, and lysed in Cell Lysis Buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerolphosphate, 1 mM Na3PO4, 1 mg/mL Leupeptin, 1 mM PMSF). Cell lysates were centrifuged then normalized by the Lowry assay; afterwards equal amounts of protein were separated by sodium dodecyl sulfate (SDS)polyacrylamaide gel electrophoresis (PAGE). The proteins were transferred to nitrocellulose membranes and the membranes were blocked with TBST containing 5% non-fat milk. Following the overnight incubation of the membranes with primary antibodies overnight at 4°C, the membranes were washed with TBST containing 0.1% Tween 20, and incubated with determined dilutions of goat antimouse peroxidase-conjugated secondary antibodies for 60 min at room temperature. The blots were probed with an enhanced chemiluminescence (ECL) substrate (Pierce, Thermo scientific) and exposed to Hyperfilm ECL to visualize the immunoreactive bands. Preparation of the Cytosol S-100 Fraction from Cancer Cells Cells were treated and harvested for the preparation of S100 fraction [27]. Briefly, cells were harvested by centrifugation and the cell pellet was resuspended in 5 vol of ice-

cold buffer A (20 mM Hepes-KOH [pH 7.5], 10 mM KCI, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol [DTT], and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]), supplemented with protease inhibitors (5 mg/mL pepstatin A, 10 mg/mL leupeptin, 2 mg/mL aprotinin). The nuclei were centrifuged at 1000g for 10 min at 4°C. The supernatant was further centrifuged at 105g for 1 h, and the resulting supernatant (S-100 fraction) was used for Cytochrome C analysis. Quantification of Reactive Oxygen Species Accumulation The production of reactive oxygen intermediates (ROI) was measured by Flow Cytometry, by using mono-color dihydrorhodamine (dRh) 123 staining in A 549, MRC-5 and EA.hy 926 cells treated for 3 h. The fluorescent marker dRh 123, prepared as a 5 mM stock solution in dimethyl sulfoxide, was purchased from Molecular Probes. The dihydrorhodamine 123 was applied at a concentration of 1 μM. Following the treatment, the cells were stained with dihydrorhodamine 123 for 20 min [28]. Subsequently, cells were washed twice in PBS and green fluorescence was analyzed with FACS Calibur using Cell Quest Pro Software. Gene Expression Analyses RNA Extraction For this experiment A 549 and MCF-7 cells were seeded in the six-well plates, in the duplicates of 2106 c/w. Cells were treated, harvested and collected. Total RNA was extracted from cell cultures using TRI Reagent® BD kit (Sigma) according to the manufacturer`s recommendations. After homogenization and phase separation by chlorophorm and centrifugation, RNA remains in aqueous phase from which is precipitated by mixing with isopropyl alchocol and dissolved in RNase-free water. The RNA extracts were separated by electrophoresis (Pharmacia Biotech) on 2% agarose gel stained with ethidium bromide to determine the size and integrity of the nucleic acids. RNA bands were visualized on UV transilluminator (Hoefer). RNA concentration was determined by using spectrophotometry (Eppendorf BioPhotometer), on the basis of absorbance values at a wavelength of 260 nm. A260/A280 ratio was used to determine purity of isolated RNA. RNA samples were stored in RNase-free water at - 80°C until analyzed. cDNA Synthesis To prepare primary complementary DNA (cDNA) with random primers by RT-PCR, 2
g total RNA was used as

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Zec et al.

Dunn’s Multiple Comparison Test was performed in order to analyse each two samples independently. For p
0,05 the results were considered significant.

template for MultiScribeTM Reverse Transcriptase (50 UL1) in a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). The reaction was conducted according to manufacturer`s recommendations at a final volume of 20 L. It proceeded at 25°C for 10 min, 37°C for 120 min, and by inactivation at 85°C for 5 min. The cDNA was stored at 20°C until the samples were processed.

RESULTS Cytotoxicity Assay The first screening of the newly synthesized compounds was performed on the panel of nine cell lines. Cytotoxicity assay was performed on the following human cancer cell lines: lung cancer cells A 549 - adenocarcinoma epithelial cells, epithelial breast cancer cell lines MDA-MB-361 and MDA-453, highly metastatic breast cancer MCF-7 cell line, cervical cancer HeLa cells, melanoma cells FemX, and colorectal cancer cells LS-174; and on the human non-malignant lung fibroblast MRC-5 and endothelial EA.hy 926 cell lines, as well. Cells were treated with the ligand, compounds 1-3 and corresponding salts for 24 h.

Real Time PCR Amplification All target transcripts were detected using quantitative Real time PCR (qRT-PCR) and Taqman assays which include TaqMan® Gene Expression Assays and TaqMan® Gene Expression Master Mix. TaqMan® Gene Expression Assays (Inventoried: part number 4331182, Non-inventoried: part number 4351372) consist of a 20 mix of unlabeled PCR primers and TaqMan® MGB probes (FAM™ dyelabeled). To exclude variations arising from different inputs of total mRNA to the reaction, data on MMP-9 and p21 were normalized to an internal housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for which data was obtained using TaqMan GAPDH control reagents (Applied Biosystems). GAPDH served as internal control for A 549 and MCF-7 cell lines. PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems). All primers and probes for GAPDH, p21 and MMP-9 were from Applied Biosystems. The thermal cycling conditions include an initial denaturation step at 95°C for 10 min followed by 40 cycles of denaturation (15 sec at 95°C) and annealing/extension (1 min at 60°C) in a final volume of 20 l. All the reactions were performed in triplicate and the data were averaged from the values obtained in each reaction. Non-template controls were also included in all amplifications. The fluorescence of the double stranded products was monitored in real time. Statistical Analyses

Compound 3 i.e. nickel complex, and the corresponding salt, have shown to be extremely non-efficient, since these did not induce death of 50% of treated cells in proposed time, in any of the investigated cell lines (Table 1). On the other hand, compound 2 i.e. cadmium complex, has shown greater cytotoxic activity with IC50 values gaining 2-30 μM, with the exception of HeLa cells where compound 2 has reached IC50 value of 90 μM approximately (Table 1). The corresponding cadmium salt has shown cytotoxic activity with IC50 values similar to the one obtained for the cadmium complex on the particular cell lines. In addition, ligand has shown anti-tumor activity with IC50 values around 30 μM for all tested cell lines, except for the non-malignant EA.hy 926 and MRC-5 cells, where obtained IC50 values were 4 and 14 μM, respectively (Table 1). If taking into consideration proven cytotoxic activity of the ligand, one could conclude that the demonstrated efficiency of the compound 2 in killing treated cells may be attributed to the ligand itself.

The data are represented as mean values±SD (standard deviation). The statistical analyses were performed by using GraphPad Prism Software 5.01. The statistical significance was calculated by non-parametric two-tailed Mann-Whitney U test and Kruskal-Wallis analysis of variance. For KruskalWallis analyses of the multiple sample differences, post-hoc

In comparison with other compounds, compound 1 i.e. zinc complex has shown moderate cytotoxic activity on all tested cell lines, with IC50 values ranging from 20-60 μM. HeLa cells have proven to be insensitive again, and compound 1 did not reach IC50 value in the proposed ranged of concentrations. In contrast with cadmium salt, zinc salt has

Table 1. IC50[
M] values obtained after the 24 h of continual treatment with the compounds. A 549

MRC-5

MDA-361

MDA-453

FemX

LS-174

EA.hy 926

HeLa

MCF-7

Ligand

32,15

14,16

42,63

71,33

>100

31,6

3,9

>100

43,68

Zn (II) complex

30,32

17,88

34,65

32,97

12,93

17,71

11,21

>100

62,15

Cd (II) complex

22

18,44

11,83

25,03

1,58

15,22

7,93

89,81

30,99

Ni (II) complex

>100

>100

>100

>100

>100

>100

>100

>100

>100

Zn(CH3COO) 2·2H2O

>100

>100

>100

22,04

>100

>100

>100

>100

>100

Cd(CH3COO) 2·2H2O

26,50

12,7

42,08

>100

8,17

28,3

14,57

>100

98,99

Ni(CH3COO)2 ·4H2O

>100

>100

>100

>100

>100

>100

>100

>100

>100

Cisplatin*

n.d.

n.d.

14,7

7,6

2,1

11,7

31,4

6,9

3,4

*IC50 values for cisplatin determined after 48 hours of incubation; n.d.=not determined

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Fig. (2). Effect of 2,6-diacetylpyridine-bis(selenosemicarbazone) metal complexes on cell cycle progression of tumor cells and fibroblasts. Cell cycle arrest and subsequent apoptosis induced by ligand, compounds 1-3 and cisplatin in A 549, MRC-5 and MDA-453 cells are presented in Figure 2. Percent of cells corresponding to the subG1 (a), G0/G1 (b), S (c) and G2/M (d) phases of cell cycle progression is presented in the Figure 2. The cultures were treated for 48 h at the concentrations of investigated compounds corresponding to the 0.5 IC50 value, stained with PI and analyzed for apoptotic cell populations and for alternations in cell cycle phase distribution, by flow cytometry. Apoptotic cells are characterized by DNA fragmentation, which is represented by a "subG1" peak on the histogram of cell cycle, and varies significantly between different cell lines treated. The experiments were performed in triplicates, and representative results are shown. * p