Cytotechnology (2018) 70:1349–1362 https://doi.org/10.1007/s10616-018-0227-3(0123456789().,-volV)(0123456789().,-volV) Cytotoxic, tubulin-interfering and proapoptotic activities of 4 -methylthio-trans-stilbene derivatives, analogues of trans-resveratrol . . . Renata Mikstacka Małgorzata Zielinska-Przyjemska Zbigniew Dutkiewicz . . . Michał Cichocki Tomasz Stefanski Mariusz Kaczmarek Wanda Baer-Dubowska Received: 27 January 2017 / Accepted: 14 May 2018 / Published online: 28 May 2018 The Author(s) 2018 Abstract The aim of this study was to evaluate the MCF12A was observed, whereas no signiﬁcant dif- cytotoxicity of a series of seven 4 -methylthio-trans- ference was observed in cytotoxicity against A431 and stilbene derivatives against cancer cells: MCF7 and HaCaT. In the series of 4 -methylthio-trans-stilbenes, A431 in comparison with non-tumorigenic MCF12A 3,4,5-MTS and 2,4,5-MTS were the most promising and HaCaT cells. The mechanism of anti-proliferative compounds for further mechanistic studies. The activity of the most cytotoxic trans-resveratrol proapoptotic activity of 3,4,5-MTS and 2,4,5-MTS, analogs: 3,4,5-trimethoxy-4 -methylthio-trans-stil- estimated with the use of annexin-V/propidium iodide bene (3,4,5-MTS) and 2,4,5-trimethoxy-4 - assay, was comparable to that of trans-resveratrol. An methylthio-trans-stilbene (2,4,5-MTS) was analyzed analysis of cell cycle distribution showed a signiﬁcant and compared with the effect of trans-resveratrol. All increase in the percentage of apoptotic cells and G2/M the compounds that were studied exerted a stronger phase arrest in MCF7 and A431 as a result of treatment cytotoxic effect than trans-resveratrol did. MCF7 cells with 3,4,5-MTS, whereas trans-resveratrol tended to were the most sensitive to the cytotoxic effect of trans- increase the percentage of cells in S phase, particularly resveratrol analogs with IC in the range of in epithelial breast cells MCF12A and MCF7. Both 2.1–6.0 lM. Comparing the cytotoxicity of 3,4,5- trans-stilbene derivatives enhanced potently tubulin MTS and 2,4,5-MTS, a signiﬁcantly higher cytotoxic polymerization in a dose-dependent manner with activity of these compounds against MCF7 versus sulfur atom participating in the interactions with critical residues of the paclitaxel binding site of b- tubulin. Renata Mikstacka and Małgorzata Zielin ´ ska-Przyjemska contributed equally to this work. R. Mikstacka (&) ´ M. Zielinska-Przyjemska M. Cichocki Department of Inorganic and Analytical Chemistry, W. Baer-Dubowska Nicolaus Copernicus University, Ludwik Rydygier Department of Pharmaceutical Biochemistry, Poznan ´ Collegium Medicum, Dr A. Jurasza 2, 85-089 Bydgoszcz, University of Medical Sciences, Swie ˛cickiego 4, Poland 60-781 Poznan ´ , Poland e-mail: email@example.com; firstname.lastname@example.org M. Kaczmarek Department of Clinical Immunology, Poznan ´ University Z. Dutkiewicz T. Stefan ´ ski of Medical Sciences, Rokietnicka 5d, 60-806 Poznan ´ , Department of Chemical Technology of Drugs, Poznan ´ Poland University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan ´ , Poland 123 1350 Cytotechnology (2018) 70:1349–1362 Keywords Apoptosis Cytotoxicity Resveratrol (e.g. colchicine, vinblastine, vincristine, vinorelbine) Stilbenes Tubulin polymerization or by enhancing it (e.g. paclitaxel, docetaxel), driving the cells into apoptosis (Mollinedo and Gajate 2003; Singh et al. 2008; Nagle et al. 2012). Stilbenes, which are microtubule interfering agents (MIA), draw atten- Introduction tion in the context of the search for potent cancer remedies. Many natural MIAs have been identiﬁed Resveratrol (3,4 ,5-trihydroxy-trans-stilbene), a natu- recently, and still new classes of potential MIA are rally occurring phytoalexin, is the most extensively synthesized; among them, potent antitubulin agents studied stilbene derivative. This compound has been are selected (Kamal et al. 2015, 2016; Madadi et al. shown to exert several beneﬁcial effects, including 2015). cancer chemopreventive activity (Athar et al. 2009; Resveratrol has been shown to induce apoptosis Vang et al. 2011; Varoni et al. 2016; Jiang et al. 2017). and/or cell cycle arrest, among others in breast cancer Molecular mechanisms of anticancer properties of MCF7 cells (Su et al. 2013) and human epidermoid resveratrol concern three stages of carcinogenesis: carcinoma A431 cells (Ahmad et al. 2001). An initiation, promotion and progression. Resveratrol important issue related to the future application of exerts the suppressive activity on extracellular growth resveratrol in disease management is its low bioavail- factors, receptor tyrosine kinases and cytoplasmic ability due to its rapid metabolism, mainly sulphation tyrosine signalling pathways. It inﬂuences redox status and glucuronidation in mammals (Walle et al. 2004). of cells and demonstrates anti-inﬂammatory and anti- The problem that remains still unresolved is the dose estrogenic activities; and the inhibitory effect on of resveratrol most suitable for effective cancer activation of procarcinogens catalysed by cytochrome preventive intervention. Mechanistic studies in cells P450 family 1 enzymes. Resveratrol inhibits cell in vitro have almost invariably used concentrations of -5 -4 proliferation by modulating the expression of cyclins resveratrol in the 10 –10 M range, which is much and cyclin-dependent kinases (CDKs) and CDK higher than those which can be achieved in humans. inhibitors (Varoni et al. 2016). The induction of Thus, new experimental paradigms need to be used to apoptosis is considered to be one of the most essential obtain information on pharmacological changes eli- among possible mechanisms of its chemopreventive cited by resveratrol when present at very low concen- and potentially chemotherapeutic activity. Resveratrol trations or when administered at dietary-relevant has been shown to stimulate an apoptotic pathway in doses (Scott et al. 2012). Recent multicenter studies various types of cells (Wang et al. 2010; Taguchi et al. indicated that the low doses of chemicals may act 2016). It was also shown that resveratrol has the efﬁciently, and this phenomenon is explained by potential to increase sensitivity to apoptotic stimuli by cumulative effect of chemicals acting on different suppressing the expression of anti-apoptotic mole- pathways and mechanisms (Goodson et al. 2015). cules in ovarian cancer cells (Taguchi et al. 2016). A natural analog of resveratrol, 3,4 ,5-trimethoxy- Apoptotic pathways are usually activated when the trans-stilbene which possesses substituents that could cell cycle cannot be restored (Moeller and Sheaff not be conjugated with sulphuric or glucuronic acid 2006). Moreover, cancerous cells exhibit high growth exerts a stronger proapoptotic effect in cancer cells rates because of cell cycle and apoptosis deregulation. (Wang et al. 2010). A strategy focused on discovering Thus, induction of cell cycle arrest is considered a and deﬁning novel analogs of resveratrol has been rational strategy to turn tumor cells into apoptotic introduced. The analogs should have the same struc- death (Schwarz and Shah 2005). tural backbone of resveratrol, with chemical modiﬁ- Tubulin monomers polymerize to form micro- cations resulting in superior efﬁcacy (Szekeres et al. tubules that are major components of the cytoskeleton. 2010; Chimento et al. 2016). Besides expected higher At the beginning of mitosis, the interphase micro- bioavailability, such new derivatives may be poten- tubular network forms a mitotic spindle that segre- tially used in combination with the parent compound gates the replicated chromosomes into daughter cells. or the other analogs. Such an approach when anti- Certain anti-cancer drugs cause suppression of micro- cancer drug combination are simultaneously targeting tubule dynamics either by inhibiting polymerization different pathways has been suggested as more 123 Cytotechnology (2018) 70:1349–1362 1351 effective, not only in therapy but also in chemopre- (RNase A) and 3-(4,5-dimethylthiazol-2-yl)-2,5- vention (Block et al. 2015). diphenyltetrazolium bromide (MTT) and all other Our earlier studies showed that the introduction of compounds were provided by Sigma-Aldrich Co. (St the methylthio-group into the stilbene core may Louis, MO, USA). inﬂuence the efﬁcacy and selectivity of the inhibitory Polymethoxy 4 -methylthio-trans-stilbenes were potency of these compounds toward the P450 synthesized as described previously (Mikstacka et al. isozymes (Mikstacka et al. 2012; Szaefer et al. 2012). Their structures are shown in Table 1. 2014), as well as enhance the expression of phase II enzymes controlled by Nrf2 transcription factor Cell culture and treatment in vivo and in vitro (Krajka-Kuz ´niak et al. 2014). These observations suggested that methylthio deriva- Spontaneously immortalized human keratinocyte tives of stilbenes may protect the cells, better than HaCaT cells were purchased from Cell Lines Service resveratrol does, against reactive electrophilic (CLS, Eppelheim, Germany). Human epidermoid metabolites of some carcinogens. On the other hand, carcinoma A431 cells were obtained from Deutsche other authors showed that substitution of the 4 oxygen Sammlung von Mikroorganismen und Zellkulturen with a less electronegative sulphur atom also reduced (DSMZ, Braunschweig, Germany). MCF7 (ECACC toxicity toward the HEK 293 cells, but enhanced the 86012803) and MCF12A (ECACC10782) cells were compound’s ability to activate human SIRT1 (Yang purchased from the European Collection of Cell et al. 2007). Activation of SIRT1 may contribute to Cultures (Salisbury, Wiltshire, UK). MCF12A cells reduced apoptosis and lead to cancer phenotype. In were cultured in 95% 1:1 mixture of DMEM and this regard, it was shown that SIRT1 activation is Ham’s F12 medium, 20 ng/mL human epidermal involved in benzo[a]pyrene induced lung tumorigen- growth factor, 500 ng/mL hydrocortisone and 5% esis (Lu et al. 2015). horse serum. The other cell lines were cultured in In order to better characterize a potential anti- Dulbecco’s modiﬁed Eagle’s medium (DMEM) sup- carcinogenic activity of stilbene methylthio-deriva- plemented with 10% fetal bovine serum and 1% (v/v) tives, in this study the effect of the two most potent antibiotics solution. Cells were incubated at 37 Cin analogues of resveratrol: 3,4,5-trimethoxy-4 - an atmosphere consisting of 95% air and 5% CO in a methylthio-trans-stilbene (3,4,5-MTS) and 2,4,5-tri- humidiﬁed incubator until they reached 70% conﬂu- methoxy-4 -methylthio-trans-stilbene (2,4,5-MTS) on ency. 1 9 10 cells were seeded in 40 mm ø culture cell cycle viability, apoptosis and cell cycle distribu- dishes. After 24 h of preincubation in DMEM con- tion in breast and skin cancer cells, and their non- taining 5% of FBS, the cells were treated with tumorigenic counterparts was evaluated. Besides, the resveratrol or its analogs, and the incubation was effect of 3,4,5-MTS and 2,4,5-MTS on tubulin poly- continued for a subsequent 24 h to assess apoptosis or merization in vitro was investigated. Molecular dock- cell cycle distribution. Then, the cells were harvested. ing was employed to characterize the speciﬁcity of Control cells were treated with DMSO, at a concen- interactions of 3,4,5-MTS and 2,4,5-MTS with b- tration of less than 0.1%. tubulin. Cell viability assay Materials and methods The effect of resveratrol and 4 -methylthiostilbenes on cell viability was assessed with MTT assay according Chemicals to the standard protocol described earlier (Zielin ´ ska- Przyjemska et al. 2015). Brieﬂy, the cells were seeded Resveratrol (purity 99%), antibiotic solution (10,000 in 96-well plates at a density of 1 9 10 cells/well in units’ penicillin, 10 mg streptomycin and 25 lg 100 lL of growth medium. They were allowed to amphotericin B per mL), camptothecin, dimethyl attach overnight and either resveratrol or the appro- sulfoxide (DMSO), fetal bovine serum (FBS), Dul- priate analog was then added to the culture medium at becco’s Modiﬁed Eagle’s Medium (DMEM), Ham’s various concentrations (0–200 lM) for 48 h at 37 C. F12 mixture, propidium iodide (PI), ribonuclease A The cells were subsequently incubated with MTT 123 1352 Cytotechnology (2018) 70:1349–1362 (0.5 mg/mL) solution for another 4 h. The water ﬁxation of all cells and minimizing clumping. After a insoluble formazan crystals were solubilized in acidic 30 min incubation, the cells were washed twice in isopropanol before the measurement of absorbance PBS, and 250 lL of a solution containing 50 lg/mL using a microplate reader (TECAN Inﬁnite M200, TK PI, 100 lg/mL RNase A (Sigma, St. Louis, MO, USA) Biotech, Warsaw, Poland) at 540 and 690 nm. All of in PBS was added to the pellet and incubated for the experiments were repeated three times, with at 30 min at 37 C in the dark. The stained cells were least three measurements per assay. analyzed by FACSCanto Flow Cytometer (Becton– Dickinson). Camptothecin at a ﬁnal concentration of Apoptosis/necrosis determination: Annexin-V/ 50 nM was used as a positive control. Data analysis propidium iodide assay and acquisition were performed using FACS Diva software (Becton–Dickinson). Apoptosis and necrosis were detected using Annexin- V-FLUOS Staining Kit assay (Roche Diagnostics Tubulin polymerization assay GmbH, Mannheim, Germany), according to the man- ufacturer’s instruction. After have been treated with Tubulin polymerization was assessed with the use of test compounds for 24 h, the cells were transferred puriﬁed porcine tubulin purchased from Cytoskeleton (1 9 10 cells in 100 lL of the solution) into 5 mL Inc. (Denver, CO, USA) in accordance with a protocol culture tubes followed by the addition of 2 lLof recommended by the manufacturer. Tubulin was Annexin-V-Fluos and 2 lL PI. Camptothecin at a ﬁnal dissolved in a buffer containing: 80 mM PIPES pH concentration of 50 nM was used as a positive control. 6.9, 2 mM MgCl , 0.5 mM EGTA and 1 mM GTP, at Samples were gently mixed and incubated for 15 min a ﬁnal concentration of 3 mg/mL and placed in a at RT (25 C) in the dark. Fluorescence of cell surface 96-well plate (0.3 mg per well). The polymerization (AV) and DNA-bound PI markers was analyzed with reaction was started by increasing temperature from 4 ﬂow cytometry (Becton–Dickinson, San Jose, CA, to 37 C upon transfer of the reaction mixture to a pre- USA) at 488 nm excitation wavelength, emission warmed plate. The assembly of microtubules was 518 and 617 nm for AV and PI, respectively. monitored spectrophotometrically by measuring absorbance at 350 nm for 60 min, at a temperature TUNEL assay of 37 C. Paclitaxel was used as stabilizing positive control. TUNEL assay was applied to detect apoptotic cells using In Situ Cell Death Detection Kit (Roche Molecular docking Diagnostics, Indianapolis, IN, USA). Brieﬂy, after incubation with the test compounds for 24 h, the cells 3,4,5-MTS, 2,4,5-MTS and paclitaxel were docked to were detached with a 0.5% trypsin–EDTA solution the paclitaxel binding site of tubulin (PDB: 1JFF) and collected. Cell suspensions were ﬁxed with 4% (Lowe et al. 2001) by applying BIOVIA Discovery paraformaldehyde and permeabilized with 0.1% Tri- Studio 2016 (Dassault Systemes BIOVA 2016) ton X-100 in 0.1% sodium citrate. After the TUNEL CDOCKER procedure (Wu et al. 2003). Docking reaction mixture was added, cells were incubated for was performed with the use of CHARMm force ﬁeld 1 h at 37 C in a humidiﬁed chamber and samples with Momany-Rone charges for both the receptor and were analyzed by FACSCanto Flow Cytometer (Bec- ligands. The binding site was deﬁned around the ton–Dickinson). Camptothecin at a ﬁnal concentration centroid of the cocrystallized ligand (paclitaxel). The of 50 nM was used as a positive control. radius of the sphere was set to the value of 12 A. For each ligand, 20 initial conformations were generated Flow cytometry cell cycle analysis randomly using high-temperature grid-based molecu- lar dynamics, and then translated into the binding site. The cells harvested after a 24 h incubation with the Twenty orientations of each conformation were used test compounds were washed with 1 mL of PBS and for docking and further simulated annealing and ﬁxed with 70% ethanol. The ethanol was added minimization. Ten distinct binding complexes dropwise to the cell pellet while vortexing to ensure obtained by the docking search technique were 123 Cytotechnology (2018) 70:1349–1362 1353 subjected to energy ranking and protein–ligand inter- and 2,4,5-MTS exhibited higher cytotoxity against all actions analysis. cell lines tested when compared to the parent compound. Moreover, similarly to resveratrol, 2,4,5- Statistical analysis MTS reduced the viability of cancer cells (MCF7, A431) to a greater extent than their non-tumorigenic All results obtained in the experiments with cells counterpart cells (MCF12A and HaCaT). The 3,4,5- in vitro were presented as mean values ± SEM of three MTS exhibited the highest cytotoxicity toward MCF7 independent experiments. Differences were consid- cells, while IC of 2,4,5-MTS was lower than that of ered signiﬁcant for p values less than 0.05. The cell 3,4,5-MTS in MCF12A cells. The MCF7 cells were viability, cell cycle and apoptosis data were evaluated the most sensitive to the cytotoxic activity of all by analysis of variance (ANOVA) followed by studied stilbene derivatives with IC ranging between Dunnett’s post hoc test using GraphPad Prism Version 2.1 and 27.3 lM. 4.03 Sofware (San Diego, CA, USA). Based on the results o f viability assay, the concentrations of the tested compounds used in the further studies included values lower and higher than Results IC . 0 0 The effect of resveratrol and 4 -methylthio- The effect of resveratrol and 4 -methylthio- stilbenes on cell viability stilbenes on cell cycle distribution We determined IC values for a 48 h treatment of Flow cytometric cell cycle analysis was used in order human non-tumorigenic (MCF12A and HaCaT) and to assess the effect of resveratrol and 4 -methylthio- cancer derived (MCF-7 and A431) cell lines with stilbenes on cell cycle distribution (Fig. 1). The resveratrol and its analogs (Table 1). Only 3,4,5-MTS analysis of cell cycle distribution conﬁrmed the Table 1 The effect of resveratrol and 4 -methylthiostilbenes on cell viability (IC ; lM) Tested compounds IC (lM) MCF12A MCF7 HaCaT A431 Resveratrol (trans-3,4 ,5-trihydroxystilbene) 93.5 ± 12.1 24.6 ± 3.8 85.2 ± 29.2 49.4 ± 3.8 2-Methoxy-4 -methylthio-trans-stilbene n.d. 27.3 ± 12.4 106.4 ± 9.3 241.3 ± 10.0 3-Methoxy-4 -methylthio-trans-stilbene n.d. 6.6 ± 3.7 182.7 ± 38.7 141.3 ± 2.8 2,3-Dimethoxy-4 -methylthio-trans-stilbene n.d. 10.6 ± 6.1 51.8 ± 7.3 157.3 ± 3.0 2,5-Dimethoxy-4 -methylthio-trans-stilbene n.d. 10.2 ± 6.6 65.0 ± 24.5 47.1 ± 9.0 3,5-Dimethoxy-4 -methylthio-trans-stilbene n.d. 4.8 ± 3.4 25.4 ± 13.9 45.4 ± 7.2 3,4,5-Trimethoxy-4 -methylthio-trans-stilbene (3,4,5-MTS) 25.7 ± 4.3 2.1 ± 0.4 10.0 ± 4.6 19.3 ± 5.5 2,4,5-Trimethoxy-4 -methylthio-trans-stilbene (2,4,5-MTS) 10.0 ± 3.0 5.1 ± 1.4 50.9 ± 13.3 22.6 ± 7.2 n.d. not determined The structure of studied compounds is displayed with general formula, where R represents metoxy substituents linked to phenyl ring of stilbene 123 1354 Cytotechnology (2018) 70:1349–1362 The effect of resveratrol and 4 -methylthio- proapoptotic effect of resveratrol and methylthio- stilbenes, particularly in MCF7 and A431cells stilbenes on apoptosis induction (Fig. 1). The percentage of apoptotic cells in both cell lines increased in a dose dependent manner as a Proapoptotic activity of resveratrol and 4 -methylthio- stilbenes was estimated with the use of Annexin-V result of treatment with 3,4,5-MTS and 2,4,5-MTS. In normal immortalized cells (MCF12A and HaCaT), afﬁnity assay based on double staining. Phos- phatidylserine externalization occurs in early apop- this effect did not occur. Moreover, 3,4,5-MTS induced G2/M phase arrest which was statistically totic stages of apoptosis as a result of lost membrane signiﬁcant in MCF12A, MCF7 and A431 cells. This integrity. Detection of phospholipid externalization effect was not observed for 2,4,5-MTS, whereas, in can be achieved by conjugation with the labeled MCF12A and MCF7 cells, 2,4,5-MTS- induced G0/ annexin-V (Annexin-V-Fluos), while, propidium G1 phase arrest was shown. Resveratrol at low iodide, the DNA binding dye enters only dead cells concentrations tended to increase the percentage of with a permeable membrane; thus this double staining discriminates viable cells from dead and/or apoptotic cells in S phase as compared with control, particularly in non-tumorigenic MCF-12 and cancer derived cells. As shown in Fig. 2, apoptosis was evident in all the MCF7 cells, while for higher concentrations the percentage of cells in G0/G1 was increasing with a tested cell lines as a result of treatment with resveratrol or its derivatives. In MCF12A, MCF7 and A431 cell simultaneous decrease of the fraction of cells in S phase. lines, resveratrol was an efﬁcient inducer of apoptosis, and this effect was dose dependent. In HaCaT cells, the effect of resveratrol and its derivatives was signiﬁcant. However, the amount of apoptotic cells did not change with an increasing concentration of the Fig. 1 Cell cycle distribution in MCF12A (a), MCF7 (b), ﬁnal concentration of 50 nM was used as a positive control. HaCaT (c) and A431 (d) cell lines after 24 h of incubation with Results of three independent experiments are presented as resveratrol, 3,4,5-MTS and 2,4,5-MTS followed by propidium mean ± SEM. *p \ 0.05, **p \ 0.01, ***p \ 0.001 compared iodide labeling and ﬂow cytometry analysis. Camptothecin at to untreated control using ANOVA followed by Dunnett’s test 123 Cytotechnology (2018) 70:1349–1362 1355 compounds studied. 3,4,5-MTS and 2,4,5-MTS in the statistically signiﬁcant increase in the number of range of concentrations used induced apoptosis in all TUNEL positive cells was observed only in HaCaT the cell lines. In MCF7 cells, the proapoptotic effect and A431 when higher concentrations of compounds was assessed for concentrations below 10 lM because were added. of a high cytotoxicity of 3,4,5-MTS and 2,4,5-MTS; 2.1 and 5.1 lM, respectively. In these cells, a signif- The effect of resveratrol and 4 -methylthio- icantly higher percentage of dead cells was observed stilbenes on tubulin polymerization in vitro as a result of treatment with 3,4,5-MTS and 2,4,5- MTS. The anti-proliferative activity of 3,4,5-MTS and 2,4,5- TUNEL assay was employed to estimate the MTS prompted us to investigate the effect of the proapoptotic properties of the studied compounds in studied compounds on microtubule organization. The the late stages of apoptosis. The results of TUNEL effect of 3,4,5-MTS and 2,4,5-MTS on tubulin poly- assay in the studied range of concentrations did not merization was determined in vitro and compared with show a signiﬁcant dose dependence (Fig. 3). How- paclitaxel used as a positive control. Both compounds ever, an increase in the number of TUNEL positive inﬂuenced tubulin polymerization in a concentration- cells in relation to the concentrations of the com- dependent manner (Fig. 4a, b). Absorbance measured pounds studied was partially observed in HaCaT and during the polymerization reaction expresses the mass A431 cells. The effect of resveratrol on the number of of polymer formed. Paclitaxel, 3,4,5-MTS and 2,4,5- TUNEL positive cells in relation to the control value MTS added to the reaction mixture at 10 lM concen- occurred in all the studied cell lines, with the highest tration increased the polymerized tubulin mass at the percentage of TUNEL positive cells observed in steady state level to 142.3, 132.9 and 123.2% of the MCF12A cells. The effect of 3,4,5-MTS and 2,4,5- control value, respectively (Table 2). However, pacli- MTS was noticeable in all cell lines, although a taxel signiﬁcantly more efﬁciently shortened the Fig. 2 Effect of resveratrol, 3,4,5-MTS and 2,4,5-MTS on AV?/PI- representing viable, dead and apoptotic cells, phosphatidylserine externalization and propidium iodide stain- respectively, were estimated. Camptothecin at ﬁnal concentra- ing in MCF12A (a), MCF7 (b), HaCaT (c) and A431 (d) cell tion of 50 nM was used as a positive control. Results of three lines after 24 h of incubation subjected to Annexin-V binding independent experiments are presented as mean ± SEM. and propidium iodide labeling followed by ﬂow cytometry *p \ 0.05, **p \ 0.01, ***p \ 0.001 compared to untreated analysis. The cell populations of AV-/PI-,AV?/PI? and control using ANOVA followed by Dunnett’s test 123 1356 Cytotechnology (2018) 70:1349–1362 Fig. 3 DNA fragmentation in MCF12A (a), MCF7 (b), HaCaT Camptothecin at ﬁnal concentration of 50 nM was used as a (c) and A431 (d) cell lines assessed by TUNEL test using positive control. Results of three independent experiments are terminal transferase and ﬂow cytometry analysis after 24 h of presented as mean ± SEM. *p \ 0.05, **p \ 0.01 compared to incubation with resveratrol, 3,4,5-MTS and 2,4,5-MTS. untreated control using ANOVA followed by Dunnett’s test nucleation phase and increased the initial velocity of hydrogen bond acceptor and also enables an attractive the polymerization (Fig. 4a, b). nonbonded SX (X=O) interaction with Thr276 of b- tubulin. CDOCKER interaction energies calculated Molecular docking for 3,4,5-MTS and 2,4,5-MTS were –31.4 and – 33.1 kcal/mol, respectively. The representative poses We used a molecular docking approach to elucidate of docked compounds in the paclitaxel binding site are whether the inﬂuence of the analyzed molecules on shown in Fig. 5. tubulin polymerization is due to a speciﬁc interaction with paclitaxel binding site of b-tubulin. 3,4,5-MTS and 2,4,5-MTS were successfully docked to the Discussion appropriate binding site of tubulin (PDB: 1JFF) by means of the CDOCKER procedure. The predicted The cell-cycle dysregulation and inhibition of apop- binding modes for both molecules were very similar; tosis are critical in the growth and development of they bind to the domain localized between helix H7 neoplasms. Thus, these two events, along with inter- and M-loop. As shown in Fig. 5, the analyzed ligands ference with microtubule assembly/disassembly, interact with Pro360 and Leu371 (p-alkyl interac- became attractive targets toward cancer chemopre- tions), Ala233 (weak hydrogen bond), Arg369 (amide vention and therapy (Athar et al. 2009; Vang et al. p-stacked interaction) and form a conventional hydro- 2011). In this study, we investigated the cytotoxic gen bond with Thr276. However, 3,4,5-MTS has a effect of resveratrol and its methylthio-derivatives slightly different orientation, which allows for addi- with the use of cell lines derived from breast tional contact with Asp26 and speciﬁc S…O interac- epithelium, non-tumorigenic MCF12A and tumori- tion with the carbonyl oxygen atom of Thr276. The genic MCF7 and two cell lines derived from human sulfur atom in methylthio substituent acts as a skin epidermis, spontaneously immortalized HaCaT 123 Cytotechnology (2018) 70:1349–1362 1357 2,4,5-MTS, we found a signiﬁcantly higher cytotoxic activity of these compounds against MCF7 versus MCF12A, whereas no signiﬁcant difference in cyto- toxicity against A431 and HaCaT was observed. This effect was in agreement with the results of Annexin-V binding assay, which demonstrated a signiﬁcant increase in the number of dead MCF7 cells in comparison with non-tumorigenic MCF12A cells, while in case of HaCaT and A431 cells a negligible difference occurred. In conclusion, the hypothesis that 3,4,5-MTS and 2,4,5-MTS are selectively proapop- totic against tumorigenic cells was proven in human epithelial-like mammary cells, but failed in relation to human normal and malignant keratinocytes. Our results indicate a proapoptotic effect of resveratrol in all studied cells with signiﬁcant dose- dependent increase in the number of apoptotic cells in tumorigenic MCF7 and A431cells. Earlier studies in A431 cells have shown that resveratrol treatment resulted in cell growth inhibition, G0/G1-phase cell cycle arrest and induction of apoptosis (Ahmad et al. 2001; Madan et al. 2008). Thus, our study basically conﬁrmed these observations. Moreover, the proapop- totic effects were observed at doses lower than that usually studied for resveratrol in in vitro experiments Fig. 4 Effect of 3,4,5-MTS (a) and 2,4,5-MTS (b) on tubulin (Scott et al. 2012). However, as assessed by ﬂow polymerization. Puriﬁed tubulin protein (cytoskeleton) in PIPES cytometry, in MCF12A, MCF7 and A431 cells, buffer containing 1 mM GTP were incubated at 37 C in the resveratrol blocked the cell cycle in S-phase. Our absence (control) and presence of studied compounds. Turbidity results conﬁrm the cell cycle block at S-phase and was measured at 340 nm in 2 min intervals during 60 min. As a positive standard 10 lM paclitaxel was used. Each point apoptosis induction in MCF7 cells reported earlier represents an average of two independent samples (Pozo-Guisado et al. 2002) and the particular sensi- tivity of MCF7 line to cytotoxic effect of resveratrol and epidermoid carcinoma A431 cells. The results of (Li et al. 2006). screening studies prompted us to investigate the In contrast to A431 cell line, normally differenti- proapoptotic activity of two methoxy derivatives of ated non-tumorigenic epithelial HaCaT cells treated 4 -methylthio-trans-stilbene: 3,4,5-MTS and 2,4,5- with resveratrol were arrested in G0/G1-phase. There- MTS in comparison with the effect of trans-resvera- fore, it is possible that in HaCaT cells resveratrol trol. By comparing the cytotoxicity of 3,4,5-MTS and upregulates p53, leading to G0/G1 phase extension. Table 2 The effect of 3,4,5-MTS, 2,4,5-MTS and paclitaxel on tubulin polymerization expressed as a percent of control absorbance at steady state level Concentration (lM) Steady state level [% of control] 3,4,5-MTS 2,4,5-MTS Paclitaxel 5 113.4 ± 3.6 106.3 ± 4.2 – 10 132.9 ± 17.1 123.2 ± 4.5 142.3 ± 9.2 The results are mean of two independent experiments performed in duplicate ± SEM 123 1358 Cytotechnology (2018) 70:1349–1362 Fig. 5 Representative binding modes of 3,4,5-MTS (a) and dashed line). For 3,4,5-MTS sulfur atom enables additional 2,4,5-MTS (b). Both trans-stilbene derivatives bind between attractive nonbonded SO interaction with carbonyl oxygen helix H7 and M-loop forming hydrogen bond with Thr276 (blue atom of Thr276 (orange dashed line). (Color ﬁgure online) Our results are consistent with the recent studies (Chu In contrast to the parent compound, its methylthio et al. 2017) which demonstrated the arrest in G1 stage derivative 3,4,5-MTS exhibited a more speciﬁc effect of cell cycle in HaCaT cells by epigallocatechin toward both MCF7 and A431 cancer cell lines, gallate. showing the dose-dependent increase of the percent- A431 cells have a nonfunctional (mutated) TP53 age of apoptotic cells and causing G2/M phase cycle gene. In response to DNA damage, protein p53 arrest. Although such tendency was observed also in triggers a variety of cell-regulatory events to limit normal immortalized cells, this effect was more the proliferation of damaged cells (Amaral et al. signiﬁcant in cancer cells, particularly at the highest 2010). However, in some cell lines apoptosis induced dose. Moreover, similarly to paclitaxel, 3,4,5-MTS by resveratrol was evidently p53 independent (Pozo- derivative promoted microtubule polymerization Guisado et al. 2002; Yuan et al. 2015; Mahyar-Roemer in vitro. It can be assumed, therefore, that by et al. 2001). The studies of Gogada et al. (2011) interacting with tubulin this compound dysregulates demonstrated that resveratrol is an inducer of Bax- mitotic spindle formation and induces mitotic arrest mediated caspase activation, cytochrome c release and and apoptosis. These results indicate that the pattern of apoptosis in cancer cells, which lack functional p53. 3,4,5-trimethoxy phenyl may be particularly impor- Taking into account many biological activities of tant for cell cytotoxicity and mechanisms involved in resveratrol it is presumed that resveratrol activates it. Such an assumption is further supported by the fact different pathways and ﬁnally the experimental obser- that DMU-212 (3,4,5,4 -trans-tetramethoxystilbene) vations may be a consequence of the resveratrol possessing 4 –methoxy group, extensively studied impact on multiple proapoptotic targets. However, the over the last 10 years, is considered to be the most molecular mechanism of resveratrol-induced apopto- promising polymethoxy-trans-stilbene derivative (Ci- sis may be different in cancer cells representing chocki et al. 2014). In this regard, it was shown that different malignancies, additionally displaying differ- DMU-212 exerts a stronger anti-proliferative and pro- ent redox status (Benitez et al. 2007; Bresgen et al. apoptotic effect than resveratrol in ovarian cancer cells 2010; Hecht et al. 2016). Moreover, the ERa status of in vitro (Piotrowska et al. 2012). Similar effects were studied cells may be important in studies of cell also found in other cell systems including human speciﬁc effect of chemopreventive agents on cell breast cancer cells HCA7 and MCF7 (Ma et al. 2008; proliferation (Chin et al. 2015). Sale et al. 2005; Androutsopoulos et al. 2011). In the 123 Cytotechnology (2018) 70:1349–1362 1359 latter cells, DMU-212 exhibited submicromolar tox- 34.6 kcal/mol, respectively, whereas for the paclitaxel icity after a 96 h exposure, while 3,4,5-MTS inhibited molecule the interaction energy was – 71.8 kcal/mol. a proliferation of MCF7 cells with IC of 2.07 lM It must be noted that, owing to its size and complex after a 48 h exposure to the test compound. structure, the molecule of paclitaxel may bind more As shown in Annexin-V assay, 3,4,5-MTS and tightly in comparison with planar molecules of 2,4,5-MTS in the range of the concentrations used stilbenes. induced apoptosis in all the studied cell lines. How- With the use of molecular docking, we conﬁrmed ever, these results were not fully supported by TUNEL the results of our experimental studies, demonstrating assay which determines the number of sites of free 3 - the stabilizing effect of 3,4,5-MTS and 2,4,5-MTS on OH DNA ends yielded during the apoptotic process. In tubulin polymerization, although the effect of the the studied range of concentrations, a signiﬁcant dose studied stilbenes was weaker than that observed for dependence for 3,4,5-MTS and 2,4,5-MTS was not paclitaxel. Molecular docking revealed interactions of demonstrated. However, an increase in the number of the studied derivatives with critical residues of the TUNEL positive cells in relation to several concen- paclitaxel binding site: Pro360, Leu371, Asp26, trations of the studied compounds was observed in Arg369, Ala233 and Thr276. It is noteworthy that MCF12A, HaCaT and A431 cells. Nonetheless, in for 3,4,5-MTS the interaction of the 4 -methylthio compliance with Annexin-V assay, the effect of group with Thr276 consisted of a hydrogen bond of resveratrol on the number of TUNEL positive cells sulfur with the hydroxyl group of Thr and an attractive was statistically signiﬁcant in all the cell lines studied. nonbonded SO interaction with the carbonyl oxygen The effect of stilbenes on the process of tubulin atom (Fig. 5a). polymerization depends on the cis or trans structure of According to the studies by Ma and coworkers compounds. Polymethoxy derivatives of trans-stil- (2008), resveratrol does not inﬂuence tubulin poly- bene are found to enhance tubulin polymerization, merization, while the effect of DMU-212 on micro- contrary to cis-stilbenes which exhibit a microtubule- tubule polymerization at the dose of 2.5 lM was inhibiting effect (Kingston 2009; Tron et al. 2006; comparable to that of paclitaxel at the dose of 10 lM. Mikstacka et al. 2013). More recently, Scherzberg Thus, the contribution of the tubulin-interfering et al. (2015) observed the inhibitory effect of cis-3,4,5- activity of polymethoxy trans-stilbene derivatives in trimethoxystilbene on tubulin polymerization with the their cytotoxic action might be important and charac- immunoﬂuorescence staining method, while trans- teristic of this group of anticancer agents. However, resveratrol did not affect the process. Their results apoptotic cell death induced by tubulin-interfering indicate that methylation of the hydroxyl groups is a agents may occur also via a signaling pathway critical modiﬁcation inﬂuencing the antitubulin effect. independent of microtubules and G2/M arrest. More- The opposed effect of cis and trans isomers on over, in our study Annexin-V/PI assay showed in microtubule dynamics relies on different sites of their MCF7 cells a signiﬁcant increase in the percentage of interaction with ab-tubulin heterodimers; cis-stilbenes necrotic cells as a result of treatment with 3,4,5-MTS. link preferentially to the colchicine binding site, which This effect might result partly from the fact that these is located at the interface between subunits of the cells do not express casapase-3, which is crucial for tubulin dimer (Ravelli et al. 2004), while trans- the induction of apoptosis. The lack of caspase 3 may stilbenes demonstrate afﬁnity to the paclitaxel binding affect apoptotic response in MCF7. However, these site. This site is located in a deep hydrophobic pocket cells are still sensitive to cell death induction (Janicke on the b-tubulin (Nogales et al. 1995). However, the 2009), and their apoptotic death may proceed via a effect of trans-stilbenes on tubulin polymerization sequential activation of caspases 9, 7 and 6 (Liang might be dependent on factors other than a speciﬁc et al. 2001). The contribution of tubulin polymeriza- binding of compounds to tubulin. That is why we tion impairment in the mechanism of cytotoxic action employed molecular docking in order to evaluate the of 3,4,5-MTS should be investigated further. putative interactions between the studied stilbenes with tubulin. In fact, 3,4,5-MTS and 2,4,5-MTS interacted with amino acid residues of the paclitaxel binding site with interaction energies – 33.1 and – 123 1360 Cytotechnology (2018) 70:1349–1362 Conclusion effects of the metabolic products of the resveratrol ana- logue, DMU-212: structural requirements for potency. 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J Biomed Sci 24:55 Acknowledgements This study was supported by Poznan ´ Cichocki M, Baer-Dubowska W, Wierzchowski M, Murias M, University of Medical Sciences, Grants Nos. 502-01-03302403- Jodynis-Liebert J (2014) 3,4,5,4 -trans-tetramethoxystil- 02524 and 502-01-03313427-08870. bene (DMU-212) modulates the activation of NF-jB, AP- 1, and STAT3 transcription factors in rat liver carcino- Compliance with ethical standards genesis induced by initiation-promotion regimen. Mol Cell Biochem 391:27–35 Conﬂict of interest The authors declare that they have no Dassault Syste `mes BIOVIA (2016) Discovery studio modeling conﬂict interests. environment, release 2016. Dassault Syste `mes, San Diego, CA Open Access This article is distributed under the terms of the Gogada R, Prabhu V, Amadori M, Scott R, Hashmi S, Chandra Creative Commons Attribution 4.0 International License (http:// D (2011) Resveratrol induces p53-independent, X-linked creativecommons.org/licenses/by/4.0/), which permits unre- inhibitor of apoptosis protein (XIAP)-mediated Bax pro- stricted use, distribution, and reproduction in any medium, tein oligomerization on mitochondria to initiate cyto- provided you give appropriate credit to the original chrome c release and caspase activation. J Biol Chem author(s) and the source, provide a link to the Creative Com- 286:28749–28760 mons license, and indicate if changes were made. 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