Article Type : Original Article
DIPHENYL DITELLURIDE-INDUCED CELL CYCLE ARREST AND APOPTOSIS: A RELATION WITH TOPOISOMERASE I INHIBITION
Patrícia Mendes Jorge1, Iuri Marques de Oliveira1, Eduardo Cremonese Filippi Chiela1, Cassiana Macagnan Viau4, Jenifer Saffi4, Fabiana Horn1, Renato Moreira Rosa2, Temenouga Nikolova Guecheva1 and João Antonio Pêgas Henriques1,3
1
Departamento de Biofísica, Instituto de Biociências, Universidade Federal do Rio Grande do
Sul (UFRGS), Porto Alegre, RS, Brazil 2
Laboratório de Genética Toxicológica, Universidade Luterana do Brasil (ULBRA), Canoas,
RS, Brazil. 3
Instituto de Biotecnologia, Departamento de Ciências Biomédicas Universidade de Caxias
do Sul – UCS Caxias do Sul – RS, Brazil. 4
Departamento de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de
Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil.
Short title: Diphenyl ditelluride-induced topoisomerase I inhibition
(Received 11 February 2014; Accepted 18 August 2014)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bcpt.12315 This article is protected by copyright. All rights reserved.
Author for correspondence: Temenouga Nikolova Guecheva, Departamento de BiofísicaPrédio 43422- Laboratório 210, Campus do Vale – Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves 9500, Bairro Agronomia–CEP 91501-970, Porto Alegre, RS, Brazil (fax: +55 5133167003, e-mail: [email protected]).
Abstract: The diphenyl ditelluride (DPDT) is a prototype for the development of new biologically active molecules. In previous studies, DPDT showed an elevated cytotoxicity in Chinese hamster fibroblast (V79) cells but the mechanisms for reduction of cell viability still remain unknown. DPDT showed mutagenic properties by induction of frameshift mutations in bacterium Salmonella typhimurium and yeast Saccharomyces cerevisiae. This organotelluride also induced DNA strand breaks in V79 cells. In this work, we investigated the mechanism of DPDT cytotoxicity by evaluating the effects of this compound on cell cycle progression, apoptosis induction and topoisomerase I inhibition. Significant decrease of V79 cell viability following DPDT treatment was revealed by MTT assay. Morphological analysis showed induction of apoptosis and necrosis by DPDT in V79 cells. An increase of caspase 3/7 activity confirmed apoptosis induction. The cell cycle analysis showed an increase in the percentage of V79 cells in S-phase and sub-G1 phase. The yeast strain deficient in topoisomerase 1 (Top1p) showed higher tolerance to DPDT compared with the isogenic wildtype strain, suggesting that the interaction with this enzyme could be involved in DPDT toxicity. The sensitivity to DPDT found in top3∆ strain indicates that yeast topoisomerase 3 (Top3p) could participate in the repair of DNA lesions induced by the DPDT. We also demonstrated that DPDT inhibits human DNA topoisomerase I (Topo I) activity by DNA relaxation assay. Therefore, our results suggest that the DPDT-induced cell cycle arrest and reduction in cell viability could be attributed to interaction with topoisomerase I enzyme.
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Organotellurium (OT) compounds have been pointed out as promising and useful alternatives for numerous synthetic operations in organic synthesis, as seen in the increase of reports on OT chemistry appearing in the literature[1, 2]. In the last few decades, evidence has been accumulating that OT molecules are promising pharmacological agents. Several reports have been published showing immuno-modulatory, antioxidant, anti-cancer and antiinflammatory properties of OT compounds [3-5]. Despite the growing use of OT compounds in the chemical and biochemical fields, there has been little concern about their toxicity. Inorganic and OT compounds are highly toxic to some organisms [6, 7]. Thus, risks from occupational and environmental human exposure to these elements may be implied due to this use. Consequently, it is important to improve our understanding about the toxicological properties of organotellurides.
Diphenyl ditelluride (DPDT) (fig. 1) is a solid, non-volatile, hydrophobic, simple and stable OT compound used as an important and versatile intermediate in organic synthesis [8]. It is extremely toxic to rodents, causing marked neurotoxic effects in mice after acute or prolonged exposure [6, 9-11]. DPDT can also be teratogenic, causing various morphologic abnormalities in mice foetuses during development [12]. In addition, it produces renal and hepatic toxicity in rodents as well as hematological disorders in human beings [7, 13]. In contrast to these toxic effects, studies have demonstrated that DPDT in low concentrations is able to prevent oxidative stress induced by several oxidizing agents [14].
The toxicology of OT compounds has been studied mainly based on the toxicity observed in animal models, effects in tissues or by the inhibition of cellular growth [15]. However, the underlying mechanism of toxicity of tellurium (Te) compounds leading to cell
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death has been poorly investigated. Some studies have indicated that OT compounds can induce apoptosis and cell cycle arrest in cancer cells such as human promyelocytic (HL-60) cell line [3]. In this context, data from our laboratory showed that DPDT induced DNA double strands breaks (DSBs) in permanent lung fibroblast cell line derived from Chinese hamster (V79 cells). The observed DSBs formation could indicate the ability of this compound to intercalate into DNA and/or affect topoisomerase activity [16].
In this sense, the aim of present study was to evaluate DPDT-induced toxicity, its effect on cell cycle progression and mechanisms of cell death induced, as well as its possible interaction with DNA topoisomerase enzymes. To this end, two different test systems were employed: mammalian V79 cells and the yeast Saccharomyces cerevisiae. The DPDTinduced apoptosis/necrosis in V79 cells was evaluated by morphological analysis and determination of caspase 3/7 activity, whereas the cell cycle progression was evaluated by flow cytometry. The possible interaction with the yeast enzymes topoisomerase 1 (Top1p) and topoisomerase 3 (Top3p) was determined in S. cerevisiae strains proficient and deficient in these proteins. Interaction of DPDT with human topoisomerase I (Topo I) enzyme was confirmed in vitro by DNA relaxation assay.
Materials and Methods 1. Chemicals DPDT (CAS registry number 32294-60-3) was provided by Dr. Antônio Braga, Federal University of Santa Catarina, Florianopolis, Brazil. The chemical purity of DPDT (99.9%)
was
determined
by
gas
chromatography/high-performance
liquid
chromatography[17]. Yeast extract, bacto-peptone and bacto-agar were obtained from Difco Laboratories (Detroit, MI, USA). Dulbecco’s modified Eagle’s Medium (DMEM), foetal This article is protected by copyright. All rights reserved.
bovine serum (FBS), trypsin–ethylenediaminetetraacetic acid (EDTA), L-glutamine and antibiotics were purchased from Gibco BRL (Grand Island, NY, USA). L-histidine, Lthreonine, L-methionine, L-tryptophan, L-leucine, L-lysine, nitrogenous bases (adenine and uracil) were purchased from Sigma (St Louis, MO, USA). BCA protein assay reagent was purchased from Pierce (Rockford, IL, USA) and caspase 3/7 substrate was purchased from Peptide Institute (Osaka, Japan).
2. Assays in V79 cells V79 cell culture and treatments. V79 cells were cultured under standard conditions in DMEM supplemented with 10% heat-inactivated FBS, 0.2 mg/mL L-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were kept in tissue culture flasks at 37°C in a humidified atmosphere containing 5% CO2 in air and were harvested by treatment with 0.15% trypsin and 0.08% EDTA in PBS. Cells were seeded (3 × 106 cells) in 5 mL of complete medium in a 25-cm2 flask and grown for 48 hr up to 60–70% confluence before treatment with the test substance. DPDT was dissolved in DMSO and added to FBS-free medium to reach the different desired concentrations. The final DMSO concentration in the medium never exceeded 0.2%, and the control group was exposed to an equivalent concentration of solvent.
Cytotoxicity evaluation in V79 cells using MTT assay. V79 cells were seeded at 1×103 cells/well in DMEM/5% FBS in 96-well plates. They were then exposed to increasing concentrations (1, 5 and 10 µg/mL) of the DPDT, for 2 hr. After the treatment, the medium was replaced by drug-free medium with FBS. After 12 hr, the cell viability was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [18]. This assay measures the activity of cellular dehidrogenases (mainly from mitochondria) and, indirectly, the cell viability, even of the spontaneously detached cells in the culture medium. The
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method is based on the reduction of a tetrazolium bromide salt (MTT [3- (4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]). It provides a quantitative measure of the number of metabolically viable cells. The results were expressed as the percentage of cell viability in relation to the control.
Cell cycle analysis. The effects of DPDT on V79 cells cycle phase distribution was assessed using flow cytometry. Cells (3×105 cells per well) were cultured in triplicate in 24-well plates. Cells were treated with DPDT (1, 5, 10 µM) for 2 hr, and after that the medium was replaced by drug-free medium with FBS. After 12 hr, the cells were harvested, fixed with 70% ethanol (in PBS) and stained with PSSI solution (Triton X-100 0.1%, RNAse 0.5 mg/mL and Propidium Iodide 6 μM per sample, in PBS). Data acquisition and analysis were performed through flow cytometry using Guava EasyCyte 8HT Flow Cytometer (Millipore Corporation, MA, USA), and data from 10,000 cells were collected for each data file. Cell cycle analysis was performed with Guava Software.
Apoptosis Analysis. Apoptosis was evaluated by morphological analysis and enzymatic caspase 3/7 assay. For both assays, 5× 105 cells were treated with 1, 5, 10 μM of DPDT for 2 hr and analysed at 2, 4, 12 and 24 hr.
Morphological analysis was based on Matuo et al. (2009) [19]. After treatment, cells were harvested, centrifuged and resuspended in 20 μl of PBS buffer. A 2 μl aliquot of acridine orange (100 μg/mL−1) and ethidium bromide (100 μg/mL−1) were added in PBS buffer. Cells were analysed in fluorescence microscope with 600 × magnification.
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Protocols for conducting the apoptosis analysis by measuring the caspase 3/7 activity were described previous by Bastiani et al. (2005) [20]. The culture medium was removed after treatments; cells were washed with PBS and lysed with Triton X-100 0.2% in PBS for 10 min. on ice. After centrifugation at 12 000 rpm for 10 min., protein concentration in cell extracts was estimated using BCA Protein Assay Reagent. The 40-μg aliquot of protein was incubated with 20 μM caspase synthetic substrates in 100 mM HEPES-NaOH, pH 7.5, 10% sucrose, 0.1% CHAPS, 0.1 mg/mL−1 BSA and 10 mM DDT. The substrate tested was Ac– Asp–Glu–Val–Asp–MCA. Substrate hydrolysis was monitored for 1.5 hr at 370 nm excitation/460 nm emission in a microplate fluorescence reader (EnSpire Multimode Plate Readers - PerkinElmer Inc.); substrate hydrolysis was quantified by comparison with a standard curve for MCA substrates. Experiments were performed in triplicate.
3. Assays in yeast Strains and media for yeast assays. The S. cerevisiae strains used in this study are all isogenic derivatives of the wild-type (WT) strain BY4741. The relevant genotypes of S. cerevisiae strains used in this study are listed in table 1. Complete YPD medium containing 0.5% (w/v) yeast extract, 2% (w/v) bacto-peptone and 2% (w/v) glucose was used for routine growth of yeast cells (fermenting cells). For plating, the medium was solidified with 2% (w/v) bactoagar. Minimal medium (MM) containing 0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose and 2% (w/v) bacto-agar was supplemented with the appropriate amino acids. Synthetic complete medium (SynCo) was supplemented with 2 mg adenine, 2 mg arginine, 5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine, 2 mg of uracil, 2 mg of tryptophan and 24 mg of threonine per 100 mL MM.
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Yeast growth. Stationary phase cultures were obtained by the inoculation of S. cerevisiae strains in liquid YPD medium and growth for 48 hr at 30°C with aeration by shaking. After, cells were harvested by centrifugation (1 min/1500 g), washed twice in phosphate buffer (PBS 0.067 M pH 7.0) and the cell density was determined microscopically using a Neubauer counting chamber. The cultures contained 1–2 × 108 cells/mL with 2–3% budding cells. The cells were then resuspended in the same buffer at a final density of 1 × 108 cells mL-1 and further tests were carried out.
Cytotoxicity in yeast strains proficient and deficient in topoisomerases. The cytotoxicity of DPDT in WT and topoisomerase mutant yeast strains (top1∆ and top3∆) was determined by the drop test standard technique. Briefly, yeast cultures in early stationary growth phase (1 × 108 cells mL-1) were obtained after 2 days of growth in YPD media at 30°C. The yeast cultures were serially diluted (1:10 at each step) in phosphate buffer, and 10 µL of each suspension were plated on SynCo media supplemented with different concentrations of DPDT (1, 5 and 10 µM). Negative controls were obtained in media without DPDT. Plates were photographed after 48 hr of growth at 30°C.
4. DNA relaxation assay The inhibitory effects of DPDT (10 μM) on human Topo I were measured using Topo I Drug Screening Kit (TopoGEN, Inc). Supercoiled (pHOT-1) plasmid DNA (250 ng) was incubated with Topo I (4 units) at 37°C for 30 min. in relaxation buffer (10 mM Tris buffer pH 7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine and 5% glycerol) in the absence or presence of DPDT 10 μM (final 20 μL). 100 μM of campothecin (CPT) was used as positive control. The reaction was stopped by addition of 10% SDS (2 μL). Proteinase K (50 μg mL-1) was added and incubated at 37°C for 30 min. DNA samples were subjected to
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electrophoresis on a 1% agarose gel for 90 min. at room temperature, and visualized with 0.5 mg mL-1ethidium bromide.
Results Cytotoxic effects in V79 cells In order to evaluate the cytotoxicity of DPDT in V79 cells, subconfluent cultures of cells were incubated with various concentrations of the compound (0; 1; 5; 10 µM) for a period of 2 hr. The results of cell viability using the MTT assay are seen in fig. 2. Cells treated with DPDT showed significant reduction in cell viability at all concentrations tested when compared to control. The viability of the cells decreased further with an increase in the concentration.
Effect of DPDT on the cell cycle distribution The analysis of the cell cycle profile of V79 cells treated with DPDT (5 µM) showed that the fraction of cells in S-phase was significantly increased in treated cells as compared to control, whereas the fraction of G1 phase cells was decreased (fig. 3A). However, there are more cells in S-phase at 5 than at 10 µM. This fact can be due to significant increase in the percentage of sub-G1 cells after treatment at a higher concentration (10 µM) used, which is considered to indicate the frequency of apoptotic cells (fig. 3B).
Apoptosis induction To confirm if the cell death induction by DPDT can be related to apoptosis induction, we applied the morphological analysis using acridine orange and ethidium bromide staining. This analysis showed an increase of apoptotic cells starting at 2 hr for the higher concentrations used (5 and 10 µM). We also measured the caspase 3/7 activation and further This article is protected by copyright. All rights reserved.
performed a morphological analysis (after different treatment times). Our results showed that DPDT treatment induced apoptosis and necrosis (fig. 4 and table 2). Indeed, apoptosis induction was observed after only 4 hr of treatment with the OT compound, and the highest level of activation of the enzyme was at 12 hr (table 2 and fig. 4). The induction of necrosis was observed starting from 4-hr treatment at the highest concentration used (10 µM) (table 2).
DPDT inhibit DNA topoisomerase I action Firstly, the effect of DTDT was evaluated using a drop test assay in mutant strains of S. cerevisiae defective in Top1p or Top3p (fig. 5). The yeast mutant deficient in Top1p activity (top1Δ) was more resistant to DPDT compared to the wild-type strain (BY4741) at all concentrations tested. These results indicate a possible interaction of DPDT with DNA or the enzyme, leading to inhibition of Top1p-action. Moreover, the strain without Top3p (top3Δ) was more sensitive to the DPDT. In addition, the effect of DPDT on Topo I activity was evaluated in a cell-free system. Purified human DNA Topo I was incubated with DPDT in the presence of supercoiled plasmid DNA. Relaxation of the plasmid was inhibited at concentration of 10 µM DPDT (fig. 6). The Topo I inhibitor CPT was used as positive control.
Discussion MTT assay was employed to evaluate the concentration range of DPDT-induced cytotoxicity in V79 cells (fig. 2). As expected, the cytotoxic threshold of DPDT was consistent with the results obtained by Degrandi et al. (2010) [16]. Recently, a significant decrease in cell viability was observed in human colon carcinoma (HT-29) treated at a
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concentration range of 62.5–1000 μM DPDT, and heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) cells, in MTT and luminescence assays [21].
In the present study, the mechanism of cell death upon exposure to DPDT was investigated by determination of apoptosis and/or necrosis induction. For this, we applied the enzymatic morphological analysis that showed dose-dependent induction of apoptosis and necrosis at all time periods studied (table 2). Sailer et al. (2003) [3] demonstrated a cell cycle specific (from S- and G2 phase) apoptosis induction in HL-60 cells after DPDT exposure. As caspases are important mediators of apoptosis [22], we determined apoptosis in DPDTtreated cells by the luminescence-based caspase 3/7 assay. An increase in 3/7 caspase activity was observed in all treatment concentrations (fig. 4). The decrease in the caspase activity after treatment at extended time periods was accompanied by the increase in the percentage of necrotic cells. Similarly, the study conducted by Vij and Hardej (2012) [21] showed an increase in caspase 3/7 and 9 activity in HT-29 and normal human colon (CCD-18Co) cells treated with DPDT (500–1000 μM). Caspase 3, -6 and -9 activation have also been demonstrated by Abondanza et al. (2008) [23] in HL60 cells when exposed to a novel OT compound RT-04. In addition, the study conducted by Yan et al. (2011) [24] demonstrated the endothelial toxicity of Cd-Te quantum dots, which area novel bio-imaging and drug delivery media, and related apoptosis induction preceded by cytochrome c release and activation of caspase 3 and 9. In contrast, the study conducted by Roy and Hardej (2011) [25] for DPDT treatment in rat hippocampal astrocytes did not demonstrate apoptosis induction in these cells but only the induction of necrosis. These discrepancies may be due to variation of glutathione (GSH) content in different cell types. Considering the ability of Te to bind to sulfhydryl groups, differences in the GSH levels might be at least partially responsible for the choice of either apoptosis or necrosis induction [25]. In this sense, the organochalcogens can
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inhibit the activity of mitochondrial complexes I and II by interaction of these compounds with
essential
cysteinyl
residues
of
mitochondrial
complexes
[26].
Thus,
the
organochalcogens should be considered as putative candidates for apoptotic cell death inducer via mitochondrial dysfunction caused by thiol oxidation is closely related to the, which may explain, at least in part, their action in apoptotic cell death induction [26-28].
We also observed an increase in the percentage of cells in sub-G1 phase, applying cell cycle analysis, that is the fraction considered indicative for the proportion of apoptotic cells over total. The cell cycle analysis also showed a significant increase in the fraction of S-phase in the treated cells when compared to control cells (fig. 3). Interestingly, it was found that the OT compound AS101 reduced tumour growth in a dose-dependent manner by inducing drag of the cell cycle in T cells in G2/M phase, and inducing apoptosis at the highest concentrations activating the caspases 3 and 9 [29]. Moreover, it was described that the compound AS101 may be involved in G2/M growth arrest and induced apoptosis in multiple myeloma [30]. Consistently, DPDT treatment induced a time-dependent increase in the number of apoptotic cells from the S and G2/M portions of the cell cycle in HL-60 cells [3]. This suggests that DPDT interferes either with DNA replication or with cell division processes.
Chemical compounds with planar topologies are often capable of intercalation between DNA bases [31]. Intercalating agent-induced genotoxicity manifests itself primarily as frameshift mutation in bacterial and yeast systems and as clastogenicity in mammalian systems. So, the results reported by Degrandi et al. (2010) [16] showing frameshift mutation induction by DPDT in Salmonella typhimurium and S. cerevisiae and an increase in DSBs in V79 cells, suggested intercalation activity and/or interaction with DNA topoisomerase enzymes. In order to determine the possible interaction of DPDT with Top1p and Top3p, we This article is protected by copyright. All rights reserved.
studied the response of S. cerevisiae mutants defective in these topoisomerase enzymes to treatment with this OT compound. The results of the survival assays demonstrated that DPDT treatment leads to pronounced tolerance in top1Δ (fig. 5). This observation indicates that DPDT could interact with the Top1p enzyme, when present in the cell, inducing DNA lesion responsible for the induced cell death. Consistently, DPDT also inhibited human Topo I activity in vitro (fig. 6). A key function of topoisomerase I enzyme is relaxation of DNA supercoiling ahead of the replication and transcription machinery, and during DNA repair and chromatin remodelling. Inhibitors of topoisomerase I block the second transesterification reaction, which prevents the DNA religation step and stabilizes the cleavage complex with topoisomerase I. Thus, transient reversible topoisomerase I-cleavage complex intermediates can be converted into irreversible DNA lesions upon collision with the DNA replication or transcription machinery. The DNA replication fork collision with the drug-stalled topoisomerase I-cleavage complex on the leading strand produces a replication-dependent DNA double-strand end, resulting in so-called “replication fork run-off” [32]. One of the possible causes for the increased resistance to DPDT of yeast strains deficient in Top1p, observed in our study, could be the impossibility to form stable complexes with DNA in the absence of this enzyme. This could prevent DSBs formation during replication and consequently, reduction of the toxic effect. Such replication induced DSBs trigger DNA damage response signalling, including activation of the protein kinases, γH2AX phosphorylation and p53 stabilization, activating DNA repair mechanisms or apoptosis induction [33-35]. Consistently, CPT, a known inhibitor of topoisomerase I, induced DNA damage that activates checkpoint kinases causing S- and G2-phase arrest [36]. The inhibition of topoisomerase I by DPDT (fig. 6) can explain the cell cycle arrest in S-phase observed in V79 cells (fig. 2).
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The absence of DNA Top1p in S. cerevisiae leads to a temporary delay in the extension of the short DNA chains [37]. This delay in chain elongation is also reflected in the rate of total DNA synthesis in the top1Δ, during the early S-phase [38]. However, if DPDT would exert its toxic effect through interaction with Top1p, absence of this enzyme in the top1Δ yeast strain could decrease the toxic effect induced by the treatment (fig. 5). This could explain the lower susceptibility of top1Δ mutant in relation to the WT strain observed in our experiment. In contrast, top3Δ mutant was more sensitive to DPDT (fig. 5) suggesting involvement of Top3p in repair of DPDT-induced lesions. In yeast S. cerevisiae the Top3p is a part of the Sgs1-Top3-Rmi1 complex, which in combination activity with Dna2 participates in the resection of DSBs with incompatible ends [39]. The end resection is initiated by the MRX complex and is an essential step of homologous recombination, which is one of the main pathways involved in repair of DPDT-induced DNA damage [16]. Moreover, the end processing can result in mutagenic deletions or insertions at the break site [39], which could explain the induction of frameshift mutations by DPDT [16].
Conclusions The present work demonstrated that DPDT promotes a reduction in cell viability by apoptosis induction, as shown by the increased caspase 3/7 activity and the cell cycle arrest in S-phase. According to our results, these events might well be a consequence of the putative interaction of DPDT with topoisomerase I, leading to the formation of stable complexes resulting in DSBs formation in proliferating cells, cell cycle arrest and cell death induction.
Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Bolsa de Pós Doutorado Júnior – PDJ – no 164160/2013-2), Fundação
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de Amparo à Pesquisa do Rio Grande do Sul - FAPERGS; Programa de Apoio a Núcleos de Excelência – PRONEX /FAPERGS /CNPq10/0044-3 and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior – CAPES.
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[14] Hassan W, Ibrahim M, Nogueira CW, Braga AL, Deobald AM, Mohammadzai IU, Rocha JB. Influence of pH on the reactivity of diphenyl ditelluride with thiols and antioxidant potential in rat brain. Chem Biol Interact 2009; 180: 47-53. [15] Sailer BL, Liles N, Dickerson S, Sumners S, Chasteen TG. Organotellurium compound toxicity in a promyelocytic cell line compared to non-tellurium-containing organic analog. Toxicology in vitro : an international journal published in association with BIBRA 2004; 18: 475-82. [16] Degrandi TH, de Oliveira IM, d'Almeida GS, Garcia CR, Villela IV, Guecheva TN, Rosa RM, Henriques JA. Evaluation of the cytotoxicity, genotoxicity and mutagenicity of diphenyl ditelluride in several biological models. Mutagenesis 2010; 25: 257-69. [17] Braga AL, Silveira CC, Reckziegel A, Menezes PH. Convenient Preparation of Alkynyl Selenides, Sulfides and Tellurides from Terminal Alkynes and Phenylchalcogenyl Halides in the Presence of Copper(I) Iodide. Tetrahedron Letters 1993; 34: 8041-2. [18] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63. [19] Matuo R, Sousa FG, Escargueil AE, Grivicich I, Garcia-Santos D, Chies JA, Saffi J, Larsen AK, Henriques JA. 5-Fluorouracil and its active metabolite FdUMP cause DNA damage in human SW620 colon adenocarcinoma cell line. Journal of applied toxicology : JAT 2009; 29: 308-16. [20] Bastiani M, Vidotto MC, Horn F. An avian pathogenic Escherichia coli isolate induces caspase 3/7 activation in J774 macrophages. FEMS Microbiol Lett 2005; 253: 133-40. [21] Vij P, Hardej D. Evaluation of tellurium toxicity in transformed and non-transformed human colon cells. Environ Toxicol Pharmacol 2012, 34: 768-82. [22] Lakhani SA, Masud A, Kuida K, Porter GA, Jr., Booth CJ, Mehal WZ, Inayat I, Flavell RA. Caspases 3 and 7: Key Mediators of Mitochondrial Events of Apoptosis. Science 2006; 311: 847-51. [23] Abondanza TS, Oliveira CR, Barbosa CM, Pereira FE, Cunha RL, Caires AC, Comasseto JV, Queiroz ML, Valadares MC, Bincoletto C. Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04. Food Chem Toxicol 2008; 46: 2540-5. [24] Yan M, Zhang Y, Xu K, Fu T, Qin H, Zheng X. An in vitro study of vascular endothelial toxicity of CdTe quantum dots. Toxicology 2011; 282: 94-103. [25] Roy S, Hardej D. Tellurium tetrachloride and diphenyl ditelluride cause cytotoxicity in rat hippocampal astrocytes. Food Chem Toxicol 2011; 49: 2564-74. [26] Puntel RL, Roos DH, Seeger RL, Rocha JB. Mitochondrial electron transfer chain complexes inhibition by different organochalcogens. Toxicology in vitro : an international journal published in association with BIBRA 2013; 27: 59-70. [27] Morin D, Zini R, Ligeret H, Neckameyer W, Labidalle S, Tillement JP. Dual effect of ebselen on mitochondrial permeability transition. Biochemical pharmacology 2003; 65: 164351. [28] Zhao R, Domann FE, Zhong W. Apoptosis induced by selenomethionine and methioninase is superoxide mediated and p53 dependent in human prostate cancer cells. Molecular cancer therapeutics 2006; 5: 3275-84. [29] Frei GM, Lebenthal I, Albeck M, Albeck A, Sredni B. Neutral and positively charged thiols synergize the effect of the immunomodulator AS101 as a growth inhibitor of Jurkat cells, by increasing its uptake. Biochemical pharmacology 2007; 74: 712-22. [30] Naor Y, Hayun M, Sredni B, Don J. Multiple signal transduction pathways are involved in G(2)/M growth arrest and apoptosis induced by the immunomodulator AS101 in multiple myeloma. Leuk Lymphoma 2013, 54: 160-6.
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[31] Snyder RD, Arnone MR. Putative identification of functional interactions between DNA intercalating agents and topoisomerase II using the V79 in vitro micronucleus assay. Mutat Res 2002; 503: 21-35. [32] Strumberg D, Pilon AA, Smith M, Hickey R, Malkas L, Pommier Y. Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'phosphorylated DNA double-strand breaks by replication runoff. Mol Cell Biol 2000; 20: 3977-87. [33] Sordet O, Khan QA, Kohn KW, Pommier Y. Apoptosis induced by topoisomerase inhibitors. Curr Med Chem Anticancer Agents 2003; 3: 271-90. [34] Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA, Pilch DR, Rogakou EP, Celeste A, Chen HT, Nussenzweig A, Aladjem MI, Bonner WM, Pommier Y. Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J Biol Chem 2003; 278: 20303-12. [35] Huang TH, Chen HC, Chou SM, Yang YC, Fan JR, Li TK. Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage. Cell Res 2010; 20: 1060-75. [36] Xiao Z, Chen Z, Gunasekera AH, Sowin TJ, Rosenberg SH, Fesik S, Zhang H. Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents. J Biol Chem 2003; 278: 21767-73. [37] Kim RA, Wang JC. Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae. Journal of Molecular Biology 1989; 208: 257-67. [38] Tomicic MT, Kaina B. Topoisomerase degradation, DSB repair, p53 and IAPs in cancer cell resistance to camptothecin-like topoisomerase I inhibitors. Biochim Biophys Acta 2012; 1835: 11-27. [39] Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Annu Rev Genet 2011; 45: 247-71.
Tables Table 1. Saccharomyces cerevisiae strains used in this study. Strains
Relevant genotypes
Sources
BY4741
wild-type, WT; MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0
EUROSCARF *
top1Δ
BY4741; with top1::kanMX4
EUROSCARF *
top3Δ
BY4741; with top3:: kanMX4
EUROSCARF *
∗European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF; Johan Wolfgang Goethe-University, Frankfurt, Germany). This article is protected by copyright. All rights reserved.
Table 2. Apoptosis and necrosis evaluation of DPDT in V79 cells by morphological assay DPDT (µM) a
Control 1 5 10
a
Control 1 5 10 a
2 hr 1.17 ± 0.76 1.67 ± 1.04 3.83 ± 1.04* 4.83 ± 1.53* 2 hr 1.33 ± 0.58 1.50 ± 0.87 1.83 ± 0.76 2.33 ± 0.76
Apoptosis (%)/treatment time 4 hr 12 hr 2.00 ± 0.50 4.00 ± 1.32 5.50 ± 1.00* 16.83 ± 3.79*** 8.83 ± 0.76*** 22.00 ± 2.50*** 9.83 ± 2.25*** 27.00 ± 2.29*** Necrosis (%)/treatment time 4 hr 12 hr 2.17 ± 0.76 3.50 ± 1.00 3.67 ± 1.26 11.33 ± 1.04* 3.83 ± 2.57 15.67 ± 3.75** 8.17 ± 1.76** 18.33 ± 5.13**
24 hr 4.50 ± 0.50 20.83 ± 3.33*** 25.50 ± 2.65*** 29.83 ± 3.21*** 24 hr 4.00 ± 1.00 12.00 ± 1.80* 28.00 ± 1.50*** 35.17 ± 5.01***
Negative control (solvent); Data are expressed as mean ± SD, n=3. *Data significantly different in relation to the
control group. *P < 0.05; **P < 0.01; ***P < 0.001/One-way ANOVA Tukey's Multiple Comparison Test.
Legends for figures
Fig. 1. Chemical structure of diphenyl ditelluride (DPDT).
Fig. 2. Dose-dependent cytotoxicity of DPDT in V79 cells as determined by MTT reduction method (% of negative control). Cells were treated with 1; 5 and 10 µM DPDT for 2 hr. Negative control (solvent) was included; Data are expressed as mean ± SD, n=4. *Data significantly different in relation to the control group ***P < 0.001/One-way ANOVA Tukey's Multiple Comparison Test.
Fig. 3. A -Effect of DPDT treatment on the cell cycle distribution. V79 cells were treated for 2 hr with 1; 5 and 10 µM DPDT. Values are the relative numbers of cells in the sub-G1/G1, S and G2/M phases of cell cycle. B -Each column represents the mean of sub-G1 cells of four independent experiments performed in triplicate, and the lines indicate the standard error
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means. Data were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test. *Significantly different from the control group *P < 0.05; **P < 0.01;***P < 0.001.
Fig. 4. Apoptosis evaluation by caspase 3/7 activation in V79 cells treated with DPDT. The quantity of pMol MCA released per µg of protein after treatment with:() negative control; () 1 μM; () 5 μM; () 10 μM of DPDT; and () positive control. Treatments were performed for: (a) 2 hr; (b) 4 hr; (c) 12 hr; and (d) 24 hr. Data are representative of at least three experiments performed in duplicate. The mean ± SD of Vmax for each curve is indicated on the right of the curve.
Fig 5. Sensitivity of yeast strains BY4741 (WT), top1∆ and top3∆ to DPDT treatment. Cells were diluted between 108 and 103 cells mL-1, and 10µL of each dilution were plated on SC solid medium without DPDT (A) or containing 1 µM (B), 5 µM (C), and 10 µM (D) DPDT.
Fig 6. Inhibition of topoisomerase I-mediated plasmid relaxation in the presence of DPDT. Line 1 of agarose gel contains marker DNA - relaxed and supercoiled pHOT1. CPT served as positive control (Line 2). The supercoiled DNA was incubated with Topo I in the presence of DPDT at the indicated concentration (Line 3). In the negative control, Topo I enzyme was incubated with the vehicle used for substance dilution (Line 4). The DNA was analysed by electrophoresis using a 1% agarose gel. The gel was stained with ethidium bromide and photographed under UV light.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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DIPHENYL DITELLURIDE-INDUCED CELL CYCLE ARREST AND APOPTOSIS: A RELATION WITH TOPOISOMERASE I INHIBITION
Patrícia Mendes Jorge1, Iuri Marques de Oliveira1, Eduardo Cremonese Filippi Chiela1, Cassiana Macagnan Viau4, Jenifer Saffi4, Fabiana Horn1, Renato Moreira Rosa2, Temenouga Nikolova Guecheva1 and João Antonio Pêgas Henriques1,3
1
Departamento de Biofísica, Instituto de Biociências, Universidade Federal do Rio Grande do
Sul (UFRGS), Porto Alegre, RS, Brazil 2
Laboratório de Genética Toxicológica, Universidade Luterana do Brasil (ULBRA), Canoas,
RS, Brazil. 3
Instituto de Biotecnologia, Departamento de Ciências Biomédicas Universidade de Caxias
do Sul – UCS Caxias do Sul – RS, Brazil. 4
Departamento de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de
Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil.
Short title: Diphenyl ditelluride-induced topoisomerase I inhibition
(Received 11 February 2014; Accepted 18 August 2014)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bcpt.12315 This article is protected by copyright. All rights reserved.
Author for correspondence: Temenouga Nikolova Guecheva, Departamento de BiofísicaPrédio 43422- Laboratório 210, Campus do Vale – Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves 9500, Bairro Agronomia–CEP 91501-970, Porto Alegre, RS, Brazil (fax: +55 5133167003, e-mail: [email protected]).
Abstract: The diphenyl ditelluride (DPDT) is a prototype for the development of new biologically active molecules. In previous studies, DPDT showed an elevated cytotoxicity in Chinese hamster fibroblast (V79) cells but the mechanisms for reduction of cell viability still remain unknown. DPDT showed mutagenic properties by induction of frameshift mutations in bacterium Salmonella typhimurium and yeast Saccharomyces cerevisiae. This organotelluride also induced DNA strand breaks in V79 cells. In this work, we investigated the mechanism of DPDT cytotoxicity by evaluating the effects of this compound on cell cycle progression, apoptosis induction and topoisomerase I inhibition. Significant decrease of V79 cell viability following DPDT treatment was revealed by MTT assay. Morphological analysis showed induction of apoptosis and necrosis by DPDT in V79 cells. An increase of caspase 3/7 activity confirmed apoptosis induction. The cell cycle analysis showed an increase in the percentage of V79 cells in S-phase and sub-G1 phase. The yeast strain deficient in topoisomerase 1 (Top1p) showed higher tolerance to DPDT compared with the isogenic wildtype strain, suggesting that the interaction with this enzyme could be involved in DPDT toxicity. The sensitivity to DPDT found in top3∆ strain indicates that yeast topoisomerase 3 (Top3p) could participate in the repair of DNA lesions induced by the DPDT. We also demonstrated that DPDT inhibits human DNA topoisomerase I (Topo I) activity by DNA relaxation assay. Therefore, our results suggest that the DPDT-induced cell cycle arrest and reduction in cell viability could be attributed to interaction with topoisomerase I enzyme.
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Organotellurium (OT) compounds have been pointed out as promising and useful alternatives for numerous synthetic operations in organic synthesis, as seen in the increase of reports on OT chemistry appearing in the literature[1, 2]. In the last few decades, evidence has been accumulating that OT molecules are promising pharmacological agents. Several reports have been published showing immuno-modulatory, antioxidant, anti-cancer and antiinflammatory properties of OT compounds [3-5]. Despite the growing use of OT compounds in the chemical and biochemical fields, there has been little concern about their toxicity. Inorganic and OT compounds are highly toxic to some organisms [6, 7]. Thus, risks from occupational and environmental human exposure to these elements may be implied due to this use. Consequently, it is important to improve our understanding about the toxicological properties of organotellurides.
Diphenyl ditelluride (DPDT) (fig. 1) is a solid, non-volatile, hydrophobic, simple and stable OT compound used as an important and versatile intermediate in organic synthesis [8]. It is extremely toxic to rodents, causing marked neurotoxic effects in mice after acute or prolonged exposure [6, 9-11]. DPDT can also be teratogenic, causing various morphologic abnormalities in mice foetuses during development [12]. In addition, it produces renal and hepatic toxicity in rodents as well as hematological disorders in human beings [7, 13]. In contrast to these toxic effects, studies have demonstrated that DPDT in low concentrations is able to prevent oxidative stress induced by several oxidizing agents [14].
The toxicology of OT compounds has been studied mainly based on the toxicity observed in animal models, effects in tissues or by the inhibition of cellular growth [15]. However, the underlying mechanism of toxicity of tellurium (Te) compounds leading to cell
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death has been poorly investigated. Some studies have indicated that OT compounds can induce apoptosis and cell cycle arrest in cancer cells such as human promyelocytic (HL-60) cell line [3]. In this context, data from our laboratory showed that DPDT induced DNA double strands breaks (DSBs) in permanent lung fibroblast cell line derived from Chinese hamster (V79 cells). The observed DSBs formation could indicate the ability of this compound to intercalate into DNA and/or affect topoisomerase activity [16].
In this sense, the aim of present study was to evaluate DPDT-induced toxicity, its effect on cell cycle progression and mechanisms of cell death induced, as well as its possible interaction with DNA topoisomerase enzymes. To this end, two different test systems were employed: mammalian V79 cells and the yeast Saccharomyces cerevisiae. The DPDTinduced apoptosis/necrosis in V79 cells was evaluated by morphological analysis and determination of caspase 3/7 activity, whereas the cell cycle progression was evaluated by flow cytometry. The possible interaction with the yeast enzymes topoisomerase 1 (Top1p) and topoisomerase 3 (Top3p) was determined in S. cerevisiae strains proficient and deficient in these proteins. Interaction of DPDT with human topoisomerase I (Topo I) enzyme was confirmed in vitro by DNA relaxation assay.
Materials and Methods 1. Chemicals DPDT (CAS registry number 32294-60-3) was provided by Dr. Antônio Braga, Federal University of Santa Catarina, Florianopolis, Brazil. The chemical purity of DPDT (99.9%)
was
determined
by
gas
chromatography/high-performance
liquid
chromatography[17]. Yeast extract, bacto-peptone and bacto-agar were obtained from Difco Laboratories (Detroit, MI, USA). Dulbecco’s modified Eagle’s Medium (DMEM), foetal This article is protected by copyright. All rights reserved.
bovine serum (FBS), trypsin–ethylenediaminetetraacetic acid (EDTA), L-glutamine and antibiotics were purchased from Gibco BRL (Grand Island, NY, USA). L-histidine, Lthreonine, L-methionine, L-tryptophan, L-leucine, L-lysine, nitrogenous bases (adenine and uracil) were purchased from Sigma (St Louis, MO, USA). BCA protein assay reagent was purchased from Pierce (Rockford, IL, USA) and caspase 3/7 substrate was purchased from Peptide Institute (Osaka, Japan).
2. Assays in V79 cells V79 cell culture and treatments. V79 cells were cultured under standard conditions in DMEM supplemented with 10% heat-inactivated FBS, 0.2 mg/mL L-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were kept in tissue culture flasks at 37°C in a humidified atmosphere containing 5% CO2 in air and were harvested by treatment with 0.15% trypsin and 0.08% EDTA in PBS. Cells were seeded (3 × 106 cells) in 5 mL of complete medium in a 25-cm2 flask and grown for 48 hr up to 60–70% confluence before treatment with the test substance. DPDT was dissolved in DMSO and added to FBS-free medium to reach the different desired concentrations. The final DMSO concentration in the medium never exceeded 0.2%, and the control group was exposed to an equivalent concentration of solvent.
Cytotoxicity evaluation in V79 cells using MTT assay. V79 cells were seeded at 1×103 cells/well in DMEM/5% FBS in 96-well plates. They were then exposed to increasing concentrations (1, 5 and 10 µg/mL) of the DPDT, for 2 hr. After the treatment, the medium was replaced by drug-free medium with FBS. After 12 hr, the cell viability was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [18]. This assay measures the activity of cellular dehidrogenases (mainly from mitochondria) and, indirectly, the cell viability, even of the spontaneously detached cells in the culture medium. The
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method is based on the reduction of a tetrazolium bromide salt (MTT [3- (4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]). It provides a quantitative measure of the number of metabolically viable cells. The results were expressed as the percentage of cell viability in relation to the control.
Cell cycle analysis. The effects of DPDT on V79 cells cycle phase distribution was assessed using flow cytometry. Cells (3×105 cells per well) were cultured in triplicate in 24-well plates. Cells were treated with DPDT (1, 5, 10 µM) for 2 hr, and after that the medium was replaced by drug-free medium with FBS. After 12 hr, the cells were harvested, fixed with 70% ethanol (in PBS) and stained with PSSI solution (Triton X-100 0.1%, RNAse 0.5 mg/mL and Propidium Iodide 6 μM per sample, in PBS). Data acquisition and analysis were performed through flow cytometry using Guava EasyCyte 8HT Flow Cytometer (Millipore Corporation, MA, USA), and data from 10,000 cells were collected for each data file. Cell cycle analysis was performed with Guava Software.
Apoptosis Analysis. Apoptosis was evaluated by morphological analysis and enzymatic caspase 3/7 assay. For both assays, 5× 105 cells were treated with 1, 5, 10 μM of DPDT for 2 hr and analysed at 2, 4, 12 and 24 hr.
Morphological analysis was based on Matuo et al. (2009) [19]. After treatment, cells were harvested, centrifuged and resuspended in 20 μl of PBS buffer. A 2 μl aliquot of acridine orange (100 μg/mL−1) and ethidium bromide (100 μg/mL−1) were added in PBS buffer. Cells were analysed in fluorescence microscope with 600 × magnification.
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Protocols for conducting the apoptosis analysis by measuring the caspase 3/7 activity were described previous by Bastiani et al. (2005) [20]. The culture medium was removed after treatments; cells were washed with PBS and lysed with Triton X-100 0.2% in PBS for 10 min. on ice. After centrifugation at 12 000 rpm for 10 min., protein concentration in cell extracts was estimated using BCA Protein Assay Reagent. The 40-μg aliquot of protein was incubated with 20 μM caspase synthetic substrates in 100 mM HEPES-NaOH, pH 7.5, 10% sucrose, 0.1% CHAPS, 0.1 mg/mL−1 BSA and 10 mM DDT. The substrate tested was Ac– Asp–Glu–Val–Asp–MCA. Substrate hydrolysis was monitored for 1.5 hr at 370 nm excitation/460 nm emission in a microplate fluorescence reader (EnSpire Multimode Plate Readers - PerkinElmer Inc.); substrate hydrolysis was quantified by comparison with a standard curve for MCA substrates. Experiments were performed in triplicate.
3. Assays in yeast Strains and media for yeast assays. The S. cerevisiae strains used in this study are all isogenic derivatives of the wild-type (WT) strain BY4741. The relevant genotypes of S. cerevisiae strains used in this study are listed in table 1. Complete YPD medium containing 0.5% (w/v) yeast extract, 2% (w/v) bacto-peptone and 2% (w/v) glucose was used for routine growth of yeast cells (fermenting cells). For plating, the medium was solidified with 2% (w/v) bactoagar. Minimal medium (MM) containing 0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose and 2% (w/v) bacto-agar was supplemented with the appropriate amino acids. Synthetic complete medium (SynCo) was supplemented with 2 mg adenine, 2 mg arginine, 5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine, 2 mg of uracil, 2 mg of tryptophan and 24 mg of threonine per 100 mL MM.
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Yeast growth. Stationary phase cultures were obtained by the inoculation of S. cerevisiae strains in liquid YPD medium and growth for 48 hr at 30°C with aeration by shaking. After, cells were harvested by centrifugation (1 min/1500 g), washed twice in phosphate buffer (PBS 0.067 M pH 7.0) and the cell density was determined microscopically using a Neubauer counting chamber. The cultures contained 1–2 × 108 cells/mL with 2–3% budding cells. The cells were then resuspended in the same buffer at a final density of 1 × 108 cells mL-1 and further tests were carried out.
Cytotoxicity in yeast strains proficient and deficient in topoisomerases. The cytotoxicity of DPDT in WT and topoisomerase mutant yeast strains (top1∆ and top3∆) was determined by the drop test standard technique. Briefly, yeast cultures in early stationary growth phase (1 × 108 cells mL-1) were obtained after 2 days of growth in YPD media at 30°C. The yeast cultures were serially diluted (1:10 at each step) in phosphate buffer, and 10 µL of each suspension were plated on SynCo media supplemented with different concentrations of DPDT (1, 5 and 10 µM). Negative controls were obtained in media without DPDT. Plates were photographed after 48 hr of growth at 30°C.
4. DNA relaxation assay The inhibitory effects of DPDT (10 μM) on human Topo I were measured using Topo I Drug Screening Kit (TopoGEN, Inc). Supercoiled (pHOT-1) plasmid DNA (250 ng) was incubated with Topo I (4 units) at 37°C for 30 min. in relaxation buffer (10 mM Tris buffer pH 7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine and 5% glycerol) in the absence or presence of DPDT 10 μM (final 20 μL). 100 μM of campothecin (CPT) was used as positive control. The reaction was stopped by addition of 10% SDS (2 μL). Proteinase K (50 μg mL-1) was added and incubated at 37°C for 30 min. DNA samples were subjected to
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electrophoresis on a 1% agarose gel for 90 min. at room temperature, and visualized with 0.5 mg mL-1ethidium bromide.
Results Cytotoxic effects in V79 cells In order to evaluate the cytotoxicity of DPDT in V79 cells, subconfluent cultures of cells were incubated with various concentrations of the compound (0; 1; 5; 10 µM) for a period of 2 hr. The results of cell viability using the MTT assay are seen in fig. 2. Cells treated with DPDT showed significant reduction in cell viability at all concentrations tested when compared to control. The viability of the cells decreased further with an increase in the concentration.
Effect of DPDT on the cell cycle distribution The analysis of the cell cycle profile of V79 cells treated with DPDT (5 µM) showed that the fraction of cells in S-phase was significantly increased in treated cells as compared to control, whereas the fraction of G1 phase cells was decreased (fig. 3A). However, there are more cells in S-phase at 5 than at 10 µM. This fact can be due to significant increase in the percentage of sub-G1 cells after treatment at a higher concentration (10 µM) used, which is considered to indicate the frequency of apoptotic cells (fig. 3B).
Apoptosis induction To confirm if the cell death induction by DPDT can be related to apoptosis induction, we applied the morphological analysis using acridine orange and ethidium bromide staining. This analysis showed an increase of apoptotic cells starting at 2 hr for the higher concentrations used (5 and 10 µM). We also measured the caspase 3/7 activation and further This article is protected by copyright. All rights reserved.
performed a morphological analysis (after different treatment times). Our results showed that DPDT treatment induced apoptosis and necrosis (fig. 4 and table 2). Indeed, apoptosis induction was observed after only 4 hr of treatment with the OT compound, and the highest level of activation of the enzyme was at 12 hr (table 2 and fig. 4). The induction of necrosis was observed starting from 4-hr treatment at the highest concentration used (10 µM) (table 2).
DPDT inhibit DNA topoisomerase I action Firstly, the effect of DTDT was evaluated using a drop test assay in mutant strains of S. cerevisiae defective in Top1p or Top3p (fig. 5). The yeast mutant deficient in Top1p activity (top1Δ) was more resistant to DPDT compared to the wild-type strain (BY4741) at all concentrations tested. These results indicate a possible interaction of DPDT with DNA or the enzyme, leading to inhibition of Top1p-action. Moreover, the strain without Top3p (top3Δ) was more sensitive to the DPDT. In addition, the effect of DPDT on Topo I activity was evaluated in a cell-free system. Purified human DNA Topo I was incubated with DPDT in the presence of supercoiled plasmid DNA. Relaxation of the plasmid was inhibited at concentration of 10 µM DPDT (fig. 6). The Topo I inhibitor CPT was used as positive control.
Discussion MTT assay was employed to evaluate the concentration range of DPDT-induced cytotoxicity in V79 cells (fig. 2). As expected, the cytotoxic threshold of DPDT was consistent with the results obtained by Degrandi et al. (2010) [16]. Recently, a significant decrease in cell viability was observed in human colon carcinoma (HT-29) treated at a
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concentration range of 62.5–1000 μM DPDT, and heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) cells, in MTT and luminescence assays [21].
In the present study, the mechanism of cell death upon exposure to DPDT was investigated by determination of apoptosis and/or necrosis induction. For this, we applied the enzymatic morphological analysis that showed dose-dependent induction of apoptosis and necrosis at all time periods studied (table 2). Sailer et al. (2003) [3] demonstrated a cell cycle specific (from S- and G2 phase) apoptosis induction in HL-60 cells after DPDT exposure. As caspases are important mediators of apoptosis [22], we determined apoptosis in DPDTtreated cells by the luminescence-based caspase 3/7 assay. An increase in 3/7 caspase activity was observed in all treatment concentrations (fig. 4). The decrease in the caspase activity after treatment at extended time periods was accompanied by the increase in the percentage of necrotic cells. Similarly, the study conducted by Vij and Hardej (2012) [21] showed an increase in caspase 3/7 and 9 activity in HT-29 and normal human colon (CCD-18Co) cells treated with DPDT (500–1000 μM). Caspase 3, -6 and -9 activation have also been demonstrated by Abondanza et al. (2008) [23] in HL60 cells when exposed to a novel OT compound RT-04. In addition, the study conducted by Yan et al. (2011) [24] demonstrated the endothelial toxicity of Cd-Te quantum dots, which area novel bio-imaging and drug delivery media, and related apoptosis induction preceded by cytochrome c release and activation of caspase 3 and 9. In contrast, the study conducted by Roy and Hardej (2011) [25] for DPDT treatment in rat hippocampal astrocytes did not demonstrate apoptosis induction in these cells but only the induction of necrosis. These discrepancies may be due to variation of glutathione (GSH) content in different cell types. Considering the ability of Te to bind to sulfhydryl groups, differences in the GSH levels might be at least partially responsible for the choice of either apoptosis or necrosis induction [25]. In this sense, the organochalcogens can
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inhibit the activity of mitochondrial complexes I and II by interaction of these compounds with
essential
cysteinyl
residues
of
mitochondrial
complexes
[26].
Thus,
the
organochalcogens should be considered as putative candidates for apoptotic cell death inducer via mitochondrial dysfunction caused by thiol oxidation is closely related to the, which may explain, at least in part, their action in apoptotic cell death induction [26-28].
We also observed an increase in the percentage of cells in sub-G1 phase, applying cell cycle analysis, that is the fraction considered indicative for the proportion of apoptotic cells over total. The cell cycle analysis also showed a significant increase in the fraction of S-phase in the treated cells when compared to control cells (fig. 3). Interestingly, it was found that the OT compound AS101 reduced tumour growth in a dose-dependent manner by inducing drag of the cell cycle in T cells in G2/M phase, and inducing apoptosis at the highest concentrations activating the caspases 3 and 9 [29]. Moreover, it was described that the compound AS101 may be involved in G2/M growth arrest and induced apoptosis in multiple myeloma [30]. Consistently, DPDT treatment induced a time-dependent increase in the number of apoptotic cells from the S and G2/M portions of the cell cycle in HL-60 cells [3]. This suggests that DPDT interferes either with DNA replication or with cell division processes.
Chemical compounds with planar topologies are often capable of intercalation between DNA bases [31]. Intercalating agent-induced genotoxicity manifests itself primarily as frameshift mutation in bacterial and yeast systems and as clastogenicity in mammalian systems. So, the results reported by Degrandi et al. (2010) [16] showing frameshift mutation induction by DPDT in Salmonella typhimurium and S. cerevisiae and an increase in DSBs in V79 cells, suggested intercalation activity and/or interaction with DNA topoisomerase enzymes. In order to determine the possible interaction of DPDT with Top1p and Top3p, we This article is protected by copyright. All rights reserved.
studied the response of S. cerevisiae mutants defective in these topoisomerase enzymes to treatment with this OT compound. The results of the survival assays demonstrated that DPDT treatment leads to pronounced tolerance in top1Δ (fig. 5). This observation indicates that DPDT could interact with the Top1p enzyme, when present in the cell, inducing DNA lesion responsible for the induced cell death. Consistently, DPDT also inhibited human Topo I activity in vitro (fig. 6). A key function of topoisomerase I enzyme is relaxation of DNA supercoiling ahead of the replication and transcription machinery, and during DNA repair and chromatin remodelling. Inhibitors of topoisomerase I block the second transesterification reaction, which prevents the DNA religation step and stabilizes the cleavage complex with topoisomerase I. Thus, transient reversible topoisomerase I-cleavage complex intermediates can be converted into irreversible DNA lesions upon collision with the DNA replication or transcription machinery. The DNA replication fork collision with the drug-stalled topoisomerase I-cleavage complex on the leading strand produces a replication-dependent DNA double-strand end, resulting in so-called “replication fork run-off” [32]. One of the possible causes for the increased resistance to DPDT of yeast strains deficient in Top1p, observed in our study, could be the impossibility to form stable complexes with DNA in the absence of this enzyme. This could prevent DSBs formation during replication and consequently, reduction of the toxic effect. Such replication induced DSBs trigger DNA damage response signalling, including activation of the protein kinases, γH2AX phosphorylation and p53 stabilization, activating DNA repair mechanisms or apoptosis induction [33-35]. Consistently, CPT, a known inhibitor of topoisomerase I, induced DNA damage that activates checkpoint kinases causing S- and G2-phase arrest [36]. The inhibition of topoisomerase I by DPDT (fig. 6) can explain the cell cycle arrest in S-phase observed in V79 cells (fig. 2).
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The absence of DNA Top1p in S. cerevisiae leads to a temporary delay in the extension of the short DNA chains [37]. This delay in chain elongation is also reflected in the rate of total DNA synthesis in the top1Δ, during the early S-phase [38]. However, if DPDT would exert its toxic effect through interaction with Top1p, absence of this enzyme in the top1Δ yeast strain could decrease the toxic effect induced by the treatment (fig. 5). This could explain the lower susceptibility of top1Δ mutant in relation to the WT strain observed in our experiment. In contrast, top3Δ mutant was more sensitive to DPDT (fig. 5) suggesting involvement of Top3p in repair of DPDT-induced lesions. In yeast S. cerevisiae the Top3p is a part of the Sgs1-Top3-Rmi1 complex, which in combination activity with Dna2 participates in the resection of DSBs with incompatible ends [39]. The end resection is initiated by the MRX complex and is an essential step of homologous recombination, which is one of the main pathways involved in repair of DPDT-induced DNA damage [16]. Moreover, the end processing can result in mutagenic deletions or insertions at the break site [39], which could explain the induction of frameshift mutations by DPDT [16].
Conclusions The present work demonstrated that DPDT promotes a reduction in cell viability by apoptosis induction, as shown by the increased caspase 3/7 activity and the cell cycle arrest in S-phase. According to our results, these events might well be a consequence of the putative interaction of DPDT with topoisomerase I, leading to the formation of stable complexes resulting in DSBs formation in proliferating cells, cell cycle arrest and cell death induction.
Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Bolsa de Pós Doutorado Júnior – PDJ – no 164160/2013-2), Fundação
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de Amparo à Pesquisa do Rio Grande do Sul - FAPERGS; Programa de Apoio a Núcleos de Excelência – PRONEX /FAPERGS /CNPq10/0044-3 and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior – CAPES.
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Tables Table 1. Saccharomyces cerevisiae strains used in this study. Strains
Relevant genotypes
Sources
BY4741
wild-type, WT; MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0
EUROSCARF *
top1Δ
BY4741; with top1::kanMX4
EUROSCARF *
top3Δ
BY4741; with top3:: kanMX4
EUROSCARF *
∗European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF; Johan Wolfgang Goethe-University, Frankfurt, Germany). This article is protected by copyright. All rights reserved.
Table 2. Apoptosis and necrosis evaluation of DPDT in V79 cells by morphological assay DPDT (µM) a
Control 1 5 10
a
Control 1 5 10 a
2 hr 1.17 ± 0.76 1.67 ± 1.04 3.83 ± 1.04* 4.83 ± 1.53* 2 hr 1.33 ± 0.58 1.50 ± 0.87 1.83 ± 0.76 2.33 ± 0.76
Apoptosis (%)/treatment time 4 hr 12 hr 2.00 ± 0.50 4.00 ± 1.32 5.50 ± 1.00* 16.83 ± 3.79*** 8.83 ± 0.76*** 22.00 ± 2.50*** 9.83 ± 2.25*** 27.00 ± 2.29*** Necrosis (%)/treatment time 4 hr 12 hr 2.17 ± 0.76 3.50 ± 1.00 3.67 ± 1.26 11.33 ± 1.04* 3.83 ± 2.57 15.67 ± 3.75** 8.17 ± 1.76** 18.33 ± 5.13**
24 hr 4.50 ± 0.50 20.83 ± 3.33*** 25.50 ± 2.65*** 29.83 ± 3.21*** 24 hr 4.00 ± 1.00 12.00 ± 1.80* 28.00 ± 1.50*** 35.17 ± 5.01***
Negative control (solvent); Data are expressed as mean ± SD, n=3. *Data significantly different in relation to the
control group. *P < 0.05; **P < 0.01; ***P < 0.001/One-way ANOVA Tukey's Multiple Comparison Test.
Legends for figures
Fig. 1. Chemical structure of diphenyl ditelluride (DPDT).
Fig. 2. Dose-dependent cytotoxicity of DPDT in V79 cells as determined by MTT reduction method (% of negative control). Cells were treated with 1; 5 and 10 µM DPDT for 2 hr. Negative control (solvent) was included; Data are expressed as mean ± SD, n=4. *Data significantly different in relation to the control group ***P < 0.001/One-way ANOVA Tukey's Multiple Comparison Test.
Fig. 3. A -Effect of DPDT treatment on the cell cycle distribution. V79 cells were treated for 2 hr with 1; 5 and 10 µM DPDT. Values are the relative numbers of cells in the sub-G1/G1, S and G2/M phases of cell cycle. B -Each column represents the mean of sub-G1 cells of four independent experiments performed in triplicate, and the lines indicate the standard error
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means. Data were analyzed by one-way ANOVA, followed by Tukey’s post-hoc test. *Significantly different from the control group *P < 0.05; **P < 0.01;***P < 0.001.
Fig. 4. Apoptosis evaluation by caspase 3/7 activation in V79 cells treated with DPDT. The quantity of pMol MCA released per µg of protein after treatment with:() negative control; () 1 μM; () 5 μM; () 10 μM of DPDT; and () positive control. Treatments were performed for: (a) 2 hr; (b) 4 hr; (c) 12 hr; and (d) 24 hr. Data are representative of at least three experiments performed in duplicate. The mean ± SD of Vmax for each curve is indicated on the right of the curve.
Fig 5. Sensitivity of yeast strains BY4741 (WT), top1∆ and top3∆ to DPDT treatment. Cells were diluted between 108 and 103 cells mL-1, and 10µL of each dilution were plated on SC solid medium without DPDT (A) or containing 1 µM (B), 5 µM (C), and 10 µM (D) DPDT.
Fig 6. Inhibition of topoisomerase I-mediated plasmid relaxation in the presence of DPDT. Line 1 of agarose gel contains marker DNA - relaxed and supercoiled pHOT1. CPT served as positive control (Line 2). The supercoiled DNA was incubated with Topo I in the presence of DPDT at the indicated concentration (Line 3). In the negative control, Topo I enzyme was incubated with the vehicle used for substance dilution (Line 4). The DNA was analysed by electrophoresis using a 1% agarose gel. The gel was stained with ethidium bromide and photographed under UV light.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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