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Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells
Toxicology in Vitro 28 (2014) 1359–1365
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Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells Zsuzsanna Rozmer a, Tímea Berki b, Gábor Maász c, Pál Perjési a,⇑ a
Department of Pharmaceutical Chemistry, University of Pécs, P.O. Box 99, H-7602 Pécs, Hungary Department of Immunology and Biotechnology, University of Pécs, P.O. Box 99, H-7602 Pécs, Hungary c Department of Biochemistry and Medical Chemistry, University of Pécs, P.O. Box 99, H-7602 Pécs, Hungary b
a r t i c l e
i n f o
Article history: Received 22 July 2013 Accepted 23 June 2014 Available online 8 July 2014 Keywords: Cyclic chalcone analogue Jurkat cells Glutathione ROS Chalcone-glutathione conjugate
a b s t r a c t Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids. Previous studies have demonstrated that chalcones and their conformationally rigid cyclic analogues have tumour cell cytotoxic and chemopreventive effects. It has been shown that equitoxic doses of the two cyclic chalcone analogues (E)-2-(40 -methoxybenzylidene)-(2) and (E)-2-(40 -methylbenzylidene)-1-benzosuberone (3) have different effect on cell cycle progress of the investigated Jurkat cells. It was also found that the compounds affect the cellular thiol status of the treated cells and show intrinsic (non-enzymecatalyzed) reactivity towards GSH under cell-free conditions. In order to gain new insights into the cytotoxic mechanism of the compounds, effects on the redox status and glutathione level of Jurkat cells were investigated. Detection of intracellular ROS level in Jurkat cells exposed to 2 and 3 was performed using the dichlorofluorescein-assay. Compound 2 did not influence ROS activity either on 1 or 4 h exposure; in contrast, chalcone 3 showed to reduce ROS level at both timepoints. The two compounds had different effects on cellular glutathione status as well. Compound 2 significantly increased the oxidized glutathione (GSSG) level showing an interference with the cellular antioxidant defence. On the contrary, chalcone 3 enhanced the reduced glutathione level, indicating enhanced cellular antioxidant activity. To investigate the chalcone–GSH conjugation reactions under cellular conditions, a combination of a RP-HPLC method with electrospray ionization mass spectrometry (ESI-MS) was performed. Chalcone–GSH adducts could not be observed either in the cell supernatant or the cell sediment after deproteinization. The investigations provide further details of dual – cytotoxic and chemopreventive – effects of the cyclic chalcone analogues. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The chemistry and biological activities of chalcones (1,3-diphenyl-2-propenones) (1) (Fig. 1) have been of interest for a long time. Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids (Harborne, 1986). The wide range of biological activities of both naturally occurring and synthetic analogues, among others cytotoxic, antitumor, antiinflammatory and chemopreventive properties are well documented in the literature (Dimmock et al., 1999a; Go et al., 2005; Nowakowska, 2007). Chalcones are a, b-unsaturated ketones and representatives of this class of compounds have demonstrated a preferential reactivity towards thiols in contrast to amino and hydroxyl groups (Dimmock et al., 1999a). Thus interactions with nucleic acids
⇑ Corresponding author. Tel./fax: +36 72 503 650/3538. E-mail address: [email protected] (P. Perjési). http://dx.doi.org/10.1016/j.tiv.2014.06.006 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.
may be absent which could eliminate the important genotoxic side effect which have been associated with certain anticancer drugs (Benvenuto et al., 1993). Several chalcones and cyclic chalcone analogues have been synthesized and their in vitro antineoplastic activity (IC50 values) has been investigated against several murine and human cancer cell lines. The primary aim of these studies was to get a better understanding of the mechanism as well as the effect of substitution and geometry of the enone moiety on activity of the compounds. It was found that cytotoxicity of the individual compounds greatly varied as a function of the ring size and the nature and position of substituents (Dimmock et al., 1999b, 2002). Among the compounds investigated (E)-2-(40 -methoxybenzylidene)-1-benzosuberone (2) (Fig. 1) showed the greatest average toxicity towards the investigated tumour cells, towards leukaemia cells (P388, L1210 and Jurkat cell) in particular (Dimmock et al., 1999b). Besides the documented tumour cytotoxic effects, some cyclic chalcone analogues were found to display emergent CYP1A
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Fig. 1. Chemical structures of chalcone (1) and the investigated compounds: (E)-2-(4-methoxybenzylidene)-1-benzosuberone (2) and (E)-2-(4-methylbenzylidene)-1benzosuberone (3).
inhibitor (Perjési et al., 2000; Monostory et al., 2003) and anti-angiogenic activities (Varinska et al., 2012). Accordingly, the compounds represent prototypes of molecules displaying both cytotoxic and chemopreventive effects. Previously we have investigated the effect of compound 2 and its methyl substituted analogue (3) (Fig. 1) on the cell cycle of Jurkat cells by flow cytometry (Rozmer et al., 2006; Pilatova et al., 2010). It was found that equitoxic doses of both compounds induced apoptosis, but 2 and 3 have different effect on cell cycle progress. Compound 2 showed to cause an immediate G1 lift and G2/M arrest, which was followed by cell death (apoptosis and/or necrosis) accompanied by formation of hyperdiploid cells. Such a remarkable effect of 3 on the G1 and G2 checkpoints could not be observed (Rozmer et al., 2006). Biologic effects of a, b-unsaturated carbonyl compounds, like chalcones, are frequently associated with the nucleophilic addition of cellular thiol groups onto the polarized carbon–carbon double bound (Michael-reaction) (Dimmock et al., 1999a; Go et al., 2005). Such a reaction can alter intracellular redox status, which can modulate events such as DNA synthesis, enzyme activation, selective gene expression and regulation of cell cycle (Powis et al., 1997). Several biological effects of chalcones, for example NQO1 (NADPH:quinone reductase) inducer effect (DinkovaKostova et al., 2001) anti-inflammatory effect (Jin et al., 2009) or GST (glutathione S-transferase) P1-1 inhibitory effect (Wang et al., 2009) associated with their Michael-type reactivity towards reduced glutathione (GSH). Previously we have investigated the mechanism of action on the total thiol content of the cells exposed to compounds 2 and 3 (Rozmer et al., 2006). Compound 2 reduced the total cellular thiol level both under nutrient-free and nutrient supplemented conditions. Under the latter conditions an increase of the total thiol level of the cells exposed to 3 could be observed. The different effect on the cellular thiol status might contribute to the different cytotoxicity of the chalcone analogues. Investigation of effects of 2 and other cyclic chalcone analogues on isolated rat liver mitochondria showed the compounds to deplete reduced glutathione and disrupt electron transfer chain and energetic mechanism. Increased generation of reactive oxygen species and covalent interaction with GSH were suggested as chemical events responsible for the observations resulted in on exposure of mitochondria to the investigated compounds (Perjési et al., 2008; Guzy et al., 2010). Incubation of 2 and 3 with reduced glutathione under cell-free conditions indicated spontaneous (non-enzyme catalyzed) conjugation (non-redox) reaction (Rozmer et al., 2006; Perjési et al., 2012). Investigation of antioxidant capacity of the compounds by monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose indicated that both compounds displayed hydroxyl radical scavenger activity (Rozmer et al., 2006; Perjési and Rozmer, 2011). As a continuation of our work towards a better understanding of the molecular mechanism taking part in the development of
biological activities of chalcones, we studied the effect of compounds 2 and 3 on redox status, investigating their effect on ROS production and on glutathione level (GSH and GSSG) in Jurkat cells. The previously developed reversed-phase high-performance liquid chromatographic (RP-HPLC) method coupled with electrospray ionization mass spectrometry (ESI-MS) detection (Perjési et al., 2012) was applied to investigate the chalcone–GSH reactions under cellular conditions. 2. Materials and methods 2.1. Materials The cyclic chalcone analogues 2 and 3 were synthesized and purified as described before (Dimmock et al., 1999b). Jurkat human T lymphocyte leukaemia cell line (Clone E6-1, ATCC TIB-152) was obtained from LGC Standards (Wesel, Germany). Cryopreservation and culturing of the cells were performed according to ATCC documentations. All the chemicals used were of the analytical grade available and, if otherwise not specified, purchased from Sigma– Aldrich Hungary (Budapest, Hungary). 2.2. Cell culture conditions and exposure Jurkat T lymphocytes were cultured under conventional conditions (37 °C humidified atmosphere of 95% air and 5% CO2 at 37 °C) in RPMI 1640 medium, pH 7.4 supplemented with 10% heat-inactivated foetal calf serum (FCS) (Gibco 110108-165) with addition of penicillin (100 U/ml) and streptomycin (100 lg/ml). Cells were grown until they reached 6–8 105 cells/ml density, refreshed with medium and transferred into new flask. Exponentially growing Jurkat cells (5 105 cells in 1.0 ml RPMI 1640 cell culture medium with 10% FCS) were exposed to the previously determined IC80 concentrations (Dimmock et al., 1999b) of 2 (5.6 lM) and 3 (23.3 lM) for 1 or 4 h. The compounds were added in 10 ll DMSO solution to the cells suspended in 1.0 ml medium. Control A cells were untreated; only vehicle, 10 ll DMSO, was added to the control B cells. Cell number was determined by exclusion of Trypan Blue dye and a haemocytometer (Tennant, 1964). 2.3. Measurement of intracellular reactive oxygen species (ROS) generation To detect production of intracellular ROS, oxidation-sensitive 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) was used (Frei et al., 2007; Aronis et al., 2003). After cells were incubated with 2 or 3 for 1 and 4 h and washed with PBS, they were treated with 10 lM DCFH-DA at 37 °C for 20 min in the dark, washed twice with PBS, collected by centrifugation and suspended in PBS. To detect the intensity of fluorescence of DCF each sample was analysed
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using a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA). At least 10.000 events were evaluated in each experiment. Data were collected and analysed by CellQuest (BD Biosciences) software program. The results show the change in the mean of fluorescent intensity compared to the untreated controls. Each value is the average of four independent experiments. Statistical evaluations were done by Student-t test (p < 0.05). 2.4. Determination of reduced and oxidized glutathione For determination of reduced and oxidized glutathione Jurkat T cells (2 106 cells in 5.0 ml RPMI 1640 cell culture medium with 10% FCS) were exposed to 2 and 3 for 4 h as described before (Section 2.2). After treatment cells were washed twice with cold 0.9% NaCl solution, suspended in 400 ll TRIS buffer (50 mM Tris–HCl, 0.1% SDS, pH 7.4) and lysed by three freeze–thaw cycles (freezing the cells for 30 min at 80 °C, followed by slow defrosting on room temperature). The cell lysates were centrifuged (5 min at 13,000g) and the clear supernatant was used for determination the protein concentration. The other part of the supernatant was stored in 80 °C for glutathione measurements. Each result is the average of four independent experiments. The protein concentration was determined by the BCA (bicinchoninic acid) assay (Smith et al., 1985), using bovine serum albumin as a standard. The absorbance was recorded in a Dynatech MR 7000 enzyme-immunoassay (ELISA) plate reader. Cell supernatant was mixed with 6% perchloric acid (210– 300 ll supernatant) and the mixture was centrifuged at 10,000g for 4 min to remove the precipitated protein. It was neutralised by 1 N KOH and centrifuged again at 3000g for 3 min to remove the salt precipitated. Determination of reduced (GSH) and oxidized (GSSG) glutathione content was performed by slight modification of Kand’ár et al. HPLC method coupled with fluorescence detection (Kand’ár et al., 2007). Briefly, GSH and GSSG content of the samples was derivatized with OPA (ortho phthalic aldehyde) and quantified by a reversed-phased high-performance liquid chromatography (HPLC). In the GSH assay, to 100 ll of the supernatant 1.0 ml of 0.1% EDTA in 0.1 M sodium hydrogen phosphate, pH 8.0, was added. To 20 ll portion of this mixture, 300 ll of 0.1% EDTA in 0.1 M sodium hydrogen phosphate and 20 ll of 0.1% OPA in methanol was added. The mixture was incubated at 25 °C for 15 min in dark and stored at 4 °C. In the GSSG assay 200 ll of the supernatant was incubated at 25 °C with 200 ll of 40 mM N-ethylmaleimide (NEM) for 25 min in dark to interact with GSH present in the sample. To this mixture 750 ll of 0.1 M NaOH was added. To 20 ll portion of this mixture, 300 ll of 0.1 M NaOH and 20 ll of 0.1% OPA in methanol was added. The mixture was incubated at 25 °C for 15 min in dark and stored at 4 °C. As reference freshly prepared GSH and GSSG containing standard series, dissolved in 50 mM Tris–HCl, 0.1% SDS, pH 7.4, (GSH: 10–100 lM; GSSG: 0–1 lM) was used. Effect of NEM to prevent interference of GSH at measurement of GSSG was checked using GSH standard. Standard solution of GSH was processed as well as samples for the determination of GSSG. HPLC analyses were performed with an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) integrated HPLC system equipped with a quaternary pump, a degasser, an autosampler and a fluorescent detector. Data were recorded and evaluated by use of Agilent Chem Station (Rev.A.10.02) software. The derivatives were measured on a Poroshell 120 EC-C18 (150 mm 4.1 mm i.d., particle size 2.7 lm) column with guard cartridge (TR-C-160-K1, ABLE&Jasco) at 37 °C. The mobile phase consisted of 15% methanol in 25 mM sodium hydrogen phosphate (v/v), pH 6.0. The flow rate was 0.4 ml/min. The injection volume was 10 ll. The fluorescence
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was monitored with an excitation wavelength of 340 nm and an emission wavelength of 425 nm. The concentration of GSH and GSSG in the samples was determined from the standard calibration curve. Each value is the average of five independent experiments. Statistical evaluations were done by Student-t test (p < 0.05). 2.5. HPLC–ES–MS study on chalcone–GSH conjugation For investigation of chalcone–GSH conjugation reactions under cellular conditions, Jurkat T cells (2 106 cells in 5.0 ml RPMI 1640 cell culture medium with 10% FCS) were exposed to 2 and 3 for 4 h as described above (Section 2.2). After treatment, cells were washed twice with ice cooled 0.9% NaCl solution, suspended in 600 ll TRIS buffer (50 mM Tris–HCl, 0.1% SDS, pH 7.4) and lysed in ice by sonication for 3 min. The cell lysates were centrifuged (at 13,000g for 5 min) and the separated supernatants and cell sediments were used for further investigations. The sedimented cells have been homogenized in 600 ll TRIS buffer and were explored by 6 10 s treatments with a high energy ultrasonicator, UIS250V (Hielsher Ultrasound Technology, Teltow, Germany) using ice cooling between the cycles. Samples were vortex mixed and centrifuged at 10,000g for 10 min. The supernatant and the explored cell samples were loaded in the Centrifugal Filter Devices (Amicon Ultra with 10 K cut off membrane, Merck-Millipore, Darmstadt, Germany) and centrifuged at 10,000g for 30 min. The filtrate was tested by MALDI and if protein was found in the sample, the experiment was repeated with a new filter. To the protein free filtrates 0.1% formic acid was added and the samples were subjected to HPLC–MS analysis. Analyses were performed with a complex Ultimate 3000 (Dionex, Sunnyvale, USA) micro HPLC system equipped with a quaternary pump, a degasser, and a Bruker micro-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Separations were performed on a Kinetex C-18 (150 mm 2.1 mm i.d., particle size 2.6 lm) column (Phenomenex, Torrance, USA). The flow rate was 200 mm3/ min; the injection volume was 1 mm3. Hystar 3.2 (Bruker Daltonics, Bremen, Germany) and Bruker micro-TOF control 1.3 software were used for controlling the instrument. Data acquisition and spectrum evaluations were performed by means of Brucker Data analysis 4.1 software. A binary gradient consisting of mobile phase A and B (A: methanol – 0.1% formic acid; B: water – 0.1% formic acid) was applied for the chromatographic separation. The HPLC analyses were performed using the following gradient profile: 40% A for 5 min, followed 10 min linear gradient to 90% and an isocratic period of 5 min. A Bruker microTOF mass spectrometer, equipped with an ESI source was used for mass detection. The ionization source was operated with an endplate potential of 3.8 kV in the positive ion mode. The following electrospray parameters were kept constant during the analysis: drying gas (N2) flow 8 dm3/min, drying gas temperature 210 °C, nebulizer pressure 20 psi. Spectra were acquired in the mass/charge ratio (m/z) range of 50–2000. 3. Results In order to investigate the effect of compounds 2 and 3 on the intracellular peroxide level in the Jurkat cells, oxidation-sensitive 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) was used (Frei et al., 2007; Aronis et al., 2003). This compound is a nonfluorescent, cell-diffusible dye. Intracellular esterases cleave the acetyl groups from the molecule to produce nonfluorescent 20 ,70 -dichlorofluorescein (DCFH). This is trapped inside the cell and in the presence of ROS, DCFH is subsequently modified to fluorescent DCF, which can be detected by flow-cytometry. The cells were incubated with
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Fig. 3. Effects of compounds 2 and 3 on the reduced (panel A) and the oxidized (panel B) glutathione content of the Jurkat cells after 4 h exposure. Control A are untreated cells, Control B are cells treated with 1% DMSO (vehicle). Experimental details are described in ‘‘Section 2’’. The values represent means ± standard error from five parallel experiments. Statistically significant difference from the untreated control, cells (Control A) (Student-t test, at the level of p < 0.05) is marked with *.
Fig. 2. Effects of 2 and 3 on intracellular ROS production in Jurkat cells (panel A). FACScan analysis of Jurkat cells incubated with 2 and 3 for 4 h and then stained with 10 lM DCFH-DA for 20 min. Control A are untreated cells (panel B). Relative fluorescent intensity (compared to Control A cells) of Control B cells (treated with DMSO 1%) and cells treated with 2 and 3. Further experimental details are described in ‘‘Section 2’’. The values represent means ± standard error from four parallel experiments. Statistically significant difference from the untreated control, cells (Control A) (Student-t test, at the level of p < 0.05) is marked with *.
1% DMSO, chalcones 2 (5.6 lM) or 3 (23.3 lM) for 1 and 4 h, respectively. The applied concentrations were previously determined IC80 concentrations (Dimmock et al., 1999b). ROS production level was evaluated using FACS analysis. As shown in Fig. 2A, ROS induction was markedly decreased in cells treated with 3; however, compound 2 did not influence ROS production compared to the untreated control cells. For comparison, the relative fluorescent intensity, expressed in per cent of the control cells, is illustrated in Fig. 2B. A significant decrease in the DCF fluorescence was observed in the Jurkat cells incubated with 3 both after 1 and 4 h exposure times (80.20 ± 10.35% and 71.35 ± 16.51% of control, respectively). Compound 2 did not affect the peroxide level in the Jurkat cells at any of the two timepoints. Effect of compounds 2 and 3 on reduced (GSH) and oxidized (GSSG) glutathione levels was investigated using a reversed phase HPLC method after derivatization with OPA (Kand’ár et al., 2007). Reduced glutathione reacts with OPA to form a stable, highly fluorescent tricyclic derivate at pH 8, while GSSG reacts with OPA only at pH 12 (Hissin and Hilf, 1976). While measurement of GSSG, the thiol function of GSH was chemically modified by N-ethylmaleimide to non-OPA-reactive derivative. The chromatogram of GSH standard solution (50 lM) treated with NEM (40 mM) and derivatized by OPA at pH 12 indicated that the used amount of NEM was
entirely sufficient to prevent interference of GSH while measuring GSSG. Concentration of GSH and GSSG in the samples was determined by means of standard curves. The results, normalized to protein concentration (nmol/mg protein), are represented in Fig. 3. Compound 2 was found not to influence the GSH level, but significantly increased the GSSG level in the cells (from 0.67 ± 0.26 nmol/mg protein to 1.15 ± 0.33 nmol/mg protein). Chalcone 3 enhanced the GSH level (from 35.3 ± 6.6 nmol/mg protein to 42.4 ± 7.2 nmol/mg protein), whereas did not markedly change the GSSG level, compared to the untreated (Control A) and DMSO-treated (Control B) cells. To investigate whether chalcone–GSH conjugation reactions undergoes under cellular conditions, analysis of cell supernatants and sediments by a RP-HPLC method coupled with ESI-MS was performed. The HPLC chromatograms of compound 2 from the properly prepared and investigated cell supernatant and cell sediment are shown in Fig. 4A-B. The selected ion chromatograms (Fig. 4A and B) and the ESI mass spectra of the analysed cell preparations, recorded in the 50–2000 Da range (Fig. 5A and B), give evidence of presence of the protonated molecule of 2 (m/ z = 279.2) both in cell supernatant and cell sediment. Presence of the diastereomeric GSH adducts of 2 (Figs. 4C and 5D) – formed in in vitro incubations of 2 and GSH under basic conditions (Perjési et al., 2012) – could not be observed. The results of similar investigations of compound 3 – not shown in the figures – were the same as those obtained with compound 2. Only the protonated molecule of compound 3 (m/z = 263.2) could be observed in the spectra of both the cell supernatant and the cell sediment of cells previously exposed to chalcone 3.
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Fig. 4. Selected ion chromatograms (m/z = 279.2) of cell supernatant (panel A) and of cell sediment (panel B) prepared from Jurkat cells exposed to compound 2 (5.6 lM) for 4 h, after deproteinization and centrifugation. Experimental details are described in ‘‘Section 2’’. Panel C: overlayed selected ion chromatograms (m/z = 279.2 and m/z = 586.2) of the reaction mixture of 2 (5 103 M) and GSH (5 102 M) after 300 min incubation time (pH 8.0; 50 °C).
4. Discussion Earlier we reported that equitoxic doses of compounds 2 (5.6 lM) and 3 (23.3 lM) induced apoptosis (accompanied by formation of hyperdiploid cells) and reduced the total cellular thiol level of Jurkat cells both under nutrient-free and nutrient supplemented conditions. Viability of the cells (determined by the Tripan Blue exclusion test) exposed to 2 and 3 at the 4 h timepoint was estimated about 80% in the case of both compounds (Rozmer et al., 2006). Under nutrient supplemented conditions (RPMI 1640 with 10% heat-inactivated FCS) a slight increase of the total thiol level of the cells exposed to 3 could be observed at the 4 h timepoint. In any other investigated timepoint (1 h or 8 h) both compounds reduced the total tiol level compared to those of the vehicle (DMSO)-treated controls. It was presumed that the more pronounced thioldepleting effect of 2 might contribute to its demonstrated tumour cell toxicity (Rozmer et al., 2006). Apoptosis is a tightly regulated form of cell death in which a cell effectively takes part in its own demise. There is a growing consensus that oxidative stress and the redox state of a cell plays a pivotal role in regulating apoptosis (Curtin et al., 2002). Several chalcones have been reported to cause oxidative stress which results cytotoxicity (Winter et al., 2010). On the other hand several chalcones have been described to demonstrate antioxidant activity (Dimmock et al., 1999a; Go et al., 2005) but it is presumed that the toxic effects of chalcones might be due to their pro-oxidant activities (Middleton et al., 2000; Dimmock et al., 1999a). To determine whether oxidative stress was involved in chalcone induced cell death, generation of reactive oxygen species (ROS) was evaluated using the DCFH-DA assay. The findings (Fig. 2) indicate that the compounds tested did not increase the intracellular ROS production under the experimental conditions; moreover compound 3 decreased the intracellular ROS level showing antioxidant activity. This latter result is in accordance with those of investigation of antioxidant capacity of the compounds by
monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose. In the hydroxyl radical initiated degradation test both compounds were found to display hydroxyl radical scavenger activity (Rozmer et al., 2006). Based on the present experimental results, hydroxyl radical scavenger activity of compound 3 is not associated with generation of ROS, which can be observed in the case of some aromatic antioxidants (Gregus and Klaassen, 2001; Prokai et al., 2003; Guzy et al., 2010) Different effect of the two compounds on intracellular ROS levels can be associated with the different reactivity of them towards GSH and other cellular thiols (Perjési et al., 2012; Dinkova-Kostova et al., 2001). The GSH system is one, but probably the most important cellular defence mechanism that exists in the cell. Glutathione acts as an ROS scavenger but also functions in the regulation of intracellular redox state (Curtin et al., 2002; Camera and Picardo, 2002). The ability of the cell to generate GSH (either by reduction of GSSG or de novo synthesis of GSH) is an important factor in the efficiency of that cell in managing oxidative stress. The exposure period used in the present experiments (4 h) was long enough for the cells to react to the possible change in the GSH levels (Gul et al., 2002). Under such conditions compound 3 was found to increase the reduced glutathione level of the cells (Fig. 3). Earlier, elevated cellular thiol level has been reported on exposure of Jurkat (Gul et al., 2002) and HeLa (Lóránd et al., 2001) cell lines to some electrophilic Mannich bases. Elevated GSH level of the Jurkat cells might have resulted from de novo synthesis in response to feedback control of GSH (Gul et al., 2002). In contrast, chalcone 2 induced an increase in GSSG content (Fig. 3), which can be evidence that this compound induces oxidative stress. This observation is in accordance with the previous results showing compound 2 to increase oxygen consumption without affecting state 3 activity of rat liver mitochondria (Kubálková et al., 2009). Such an effect can increase the mitochondrial level of reactive oxygen species (ROS) that can initiate a series of biochemical events, involving cell death (Chen et al., 2006; Sanz
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Fig. 5. ESI mass spectra of cell supernatant (A) and of cell sediment (B) prepared from Jurkat cells exposed to compound 2 (279.2 Da)(5.6 lM) for 4 h, after deproteinization and centrifugation. Experimental details are described in ‘‘Section 2’’. ESI mass spectra of 2 (C) and 2-GSH adduct (D) prepared from the reaction mixture of 2 (5 103 M) with GSH (5 102 M) after 300 min incubation time (pH 8.0; 50 °C) (m/z = 50–2000).
et al., 2006). GSSG is a physiological indicator of intracellular defence system activity against ROS, and it can be used to monitor oxidative stress even in vivo (Gitler et al., 1994; Hughes et al., 1990). Simultaneously, decrease in the GSH level could not be observed. This observation can be the result of increase in glutathione-reductase activity as an attempt to sustain GSH content and/or that of increased c-glutamyl-cysteine-synthetase (c-GCS) activity (Winter et al., 2010). Cytotoxic effect of a, b-unsaturated carbonyl compounds are frequently associated with expected reactivity of the compounds with the essential thiol groups in the living organism (Dimmock et al., 1999a; Go et al., 2005). Several other biological effects of chalcones have also been associated with their Michael-type reactivity towards reduced glutathione and other cellular thiols (Dinkova-Kostova et al., 2001). In our previous studies compounds 2 and 3 showed intrinsic reactivity towards GSH at higher pH (pH 9.0) and about physiological pH (pH 7.4) at a higher temperature
(Rozmer et al., 2006). The non-enzyme-catalyzed reaction of GSH and the chalcone analogues was investigated by RP-HPLC–MS (Perjési et al., 2012). The previously developed method provided the appropriate analytical tool to investigate the chalcone–GSH reactions in Jurkat cells. The compounds 2 and 3 could be detected even from cell supernatant and cell sediment after cell lysis and deproteinization, indicating that chalcones penetrate the cell membrane and affect intracellular biochemical events. However, chalcone–GSH adducts could not be observed either in the cell supernatants or the cell sediments (Figs. 4 and 5). This observation might be the consequence of reversible addition of GSH onto the chalcones’ polar carbon–carbon double bond, which can result in a retro-Michael-type decomposition of the adducts when the excess of GSH is reduced (Perjési et al., 2012). In conclusion, it was demonstrated that equitoxic doses of the two cyclic chalcone analogues 2 and 3 have different effect on glutathione level and redox status in Jurkat cells. For chalcone 2 the
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interference with the cellular antioxidant defence systems might contribute to its demonstrated tumour cell toxicity. Compound 3 decreased ROS production and increased cellular glutathione level, indicating antioxidant activity. Glutathione conjugates could not be detected either in cell supernatants and cell sediments. These results provide new insights into the dual – cytotoxic and chemopreventive – effects of the cyclic chalcone analogues. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version. Acknowledgements This study was supported by the University of Pécs, Faculty of Medicine Research Fund (PTE AOK KA 2012-2013/13-18 and PTE AOK-KA-2013/20). References Aronis, A., Melendez, J.A., Golan, O., Shilo, S., Dicter, N., Tirosh, O., 2003. Potentiation of FAS-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species. Cell Death Differ. 10, 335–344. Benvenuto, J.A., Connor, T.H., Monteith, D.K., Laidlaw, J.L., Adams, S.C., Matney, T.S., Theiss, J.C., 1993. Degradation and inactivation of antitumor drugs. J. Pharm. Sci. 82, 988–991. Camera, E., Picardo, M., 2002. Analytical methods to investigate glutathione and related compounds in biological and pathological processes. J. Chromatogr. B 781, 181–206. Chen, Q., Moghaddas, S., Hoppel, C.L., Lesnefsky, E.J., 2006. Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion. J. Pharmacol. Exp. Ther. 319, 1405– 1412. Curtin, J., Donovan, M., Cotter, T., 2002. Regulation and measurement of oxidative stress in apoptosis. J. Immunol. Methods 265, 49–72. Dimmock, J., Elias, D., Beazely, M., Kandepu, N., 1999a. Bioactivities of chalcones. Curr. Med. Chem. 6, 1125–1149. Dimmock, J.R., Kandepu, N.M., Nazarali, A.J., Kowalchuk, T.P., Motaganahalli, N., Quail, J.W., Mykytiuk, P.A., Audette, G.F., Prasad, L., Perjési, P., Allen, T.M., Santos, C.L., Szydlowski, J., De Clercq, E., Balzarini, J., 1999b. Conformational and quantitative structure-activity relationship study of cytotoxic 2arylidenebenzocycloalkanones. J. Med. Chem. 42, 1358–1366. Dimmock, J.R., Zello, G.A., Oloo, E.O., Quail, J.W., Kraatz, H.B., Perjési, P., Aradi, F., Takács-Novák, K., Allen, T.M., Santos, C.L., Balzarini, J., De Clercq, E., Stables, J.P., 2002. Correlations between cytotoxicity and topography of some 2arylidenebenzocycloalkanones determined by X-ray crystallography. J. Med. Chem. 45, 3103–3111. Dinkova-Kostova, A.T., Massiah, M.A., Bozak, R.E., Hicks, R.J., Talalay, P., 2001. Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc. Natl. Acad. Sci. 98, 3404–3409. Frei, G.M., Lebenthal, I., Albeck, M., Albeck, A., Sredni, B., 2007. Neutral and positively charged thiols synergize the effect on the immunomodulator AS101 as a growth inhibitor of Jurkat cells, by increasing its uptake. Biochem. Pharmacol. 74, 712–722. Gitler, C., Mogyoros, M., Kalef, E., 1994. Labeling of protein vicinal dithiols: role of protein-S2 to protein-(SH)2 conversion in metabolic regulation and oxidative stress. Methods Enzymol. 233, 403–415. Go, M., Wu, X., Liu, X., 2005. Chalcones: an update on cytotoxic and chemoprotective properties. Curr. Med. Chem. 12, 483–499. Gul, M., Gul, H., Hänninen, O., 2002. Effects of Mannich bases on cellular glutathione and related enzymes of Jurkat cells in culture conditions. Toxicol. in Vitro 16, 107–112. Guzy, J., Vasková-Kubálková, J., Rozmer, Zs., Fodor, K., Mareková, M., Proskrobová, M., Perjési, P., 2010. Activation of oxidative stress response by hydroxyl
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells Zsuzsanna Rozmer a, Tímea Berki b, Gábor Maász c, Pál Perjési a,⇑ a
Department of Pharmaceutical Chemistry, University of Pécs, P.O. Box 99, H-7602 Pécs, Hungary Department of Immunology and Biotechnology, University of Pécs, P.O. Box 99, H-7602 Pécs, Hungary c Department of Biochemistry and Medical Chemistry, University of Pécs, P.O. Box 99, H-7602 Pécs, Hungary b
a r t i c l e
i n f o
Article history: Received 22 July 2013 Accepted 23 June 2014 Available online 8 July 2014 Keywords: Cyclic chalcone analogue Jurkat cells Glutathione ROS Chalcone-glutathione conjugate
a b s t r a c t Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids. Previous studies have demonstrated that chalcones and their conformationally rigid cyclic analogues have tumour cell cytotoxic and chemopreventive effects. It has been shown that equitoxic doses of the two cyclic chalcone analogues (E)-2-(40 -methoxybenzylidene)-(2) and (E)-2-(40 -methylbenzylidene)-1-benzosuberone (3) have different effect on cell cycle progress of the investigated Jurkat cells. It was also found that the compounds affect the cellular thiol status of the treated cells and show intrinsic (non-enzymecatalyzed) reactivity towards GSH under cell-free conditions. In order to gain new insights into the cytotoxic mechanism of the compounds, effects on the redox status and glutathione level of Jurkat cells were investigated. Detection of intracellular ROS level in Jurkat cells exposed to 2 and 3 was performed using the dichlorofluorescein-assay. Compound 2 did not influence ROS activity either on 1 or 4 h exposure; in contrast, chalcone 3 showed to reduce ROS level at both timepoints. The two compounds had different effects on cellular glutathione status as well. Compound 2 significantly increased the oxidized glutathione (GSSG) level showing an interference with the cellular antioxidant defence. On the contrary, chalcone 3 enhanced the reduced glutathione level, indicating enhanced cellular antioxidant activity. To investigate the chalcone–GSH conjugation reactions under cellular conditions, a combination of a RP-HPLC method with electrospray ionization mass spectrometry (ESI-MS) was performed. Chalcone–GSH adducts could not be observed either in the cell supernatant or the cell sediment after deproteinization. The investigations provide further details of dual – cytotoxic and chemopreventive – effects of the cyclic chalcone analogues. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The chemistry and biological activities of chalcones (1,3-diphenyl-2-propenones) (1) (Fig. 1) have been of interest for a long time. Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids (Harborne, 1986). The wide range of biological activities of both naturally occurring and synthetic analogues, among others cytotoxic, antitumor, antiinflammatory and chemopreventive properties are well documented in the literature (Dimmock et al., 1999a; Go et al., 2005; Nowakowska, 2007). Chalcones are a, b-unsaturated ketones and representatives of this class of compounds have demonstrated a preferential reactivity towards thiols in contrast to amino and hydroxyl groups (Dimmock et al., 1999a). Thus interactions with nucleic acids
⇑ Corresponding author. Tel./fax: +36 72 503 650/3538. E-mail address: [email protected] (P. Perjési). http://dx.doi.org/10.1016/j.tiv.2014.06.006 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.
may be absent which could eliminate the important genotoxic side effect which have been associated with certain anticancer drugs (Benvenuto et al., 1993). Several chalcones and cyclic chalcone analogues have been synthesized and their in vitro antineoplastic activity (IC50 values) has been investigated against several murine and human cancer cell lines. The primary aim of these studies was to get a better understanding of the mechanism as well as the effect of substitution and geometry of the enone moiety on activity of the compounds. It was found that cytotoxicity of the individual compounds greatly varied as a function of the ring size and the nature and position of substituents (Dimmock et al., 1999b, 2002). Among the compounds investigated (E)-2-(40 -methoxybenzylidene)-1-benzosuberone (2) (Fig. 1) showed the greatest average toxicity towards the investigated tumour cells, towards leukaemia cells (P388, L1210 and Jurkat cell) in particular (Dimmock et al., 1999b). Besides the documented tumour cytotoxic effects, some cyclic chalcone analogues were found to display emergent CYP1A
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Fig. 1. Chemical structures of chalcone (1) and the investigated compounds: (E)-2-(4-methoxybenzylidene)-1-benzosuberone (2) and (E)-2-(4-methylbenzylidene)-1benzosuberone (3).
inhibitor (Perjési et al., 2000; Monostory et al., 2003) and anti-angiogenic activities (Varinska et al., 2012). Accordingly, the compounds represent prototypes of molecules displaying both cytotoxic and chemopreventive effects. Previously we have investigated the effect of compound 2 and its methyl substituted analogue (3) (Fig. 1) on the cell cycle of Jurkat cells by flow cytometry (Rozmer et al., 2006; Pilatova et al., 2010). It was found that equitoxic doses of both compounds induced apoptosis, but 2 and 3 have different effect on cell cycle progress. Compound 2 showed to cause an immediate G1 lift and G2/M arrest, which was followed by cell death (apoptosis and/or necrosis) accompanied by formation of hyperdiploid cells. Such a remarkable effect of 3 on the G1 and G2 checkpoints could not be observed (Rozmer et al., 2006). Biologic effects of a, b-unsaturated carbonyl compounds, like chalcones, are frequently associated with the nucleophilic addition of cellular thiol groups onto the polarized carbon–carbon double bound (Michael-reaction) (Dimmock et al., 1999a; Go et al., 2005). Such a reaction can alter intracellular redox status, which can modulate events such as DNA synthesis, enzyme activation, selective gene expression and regulation of cell cycle (Powis et al., 1997). Several biological effects of chalcones, for example NQO1 (NADPH:quinone reductase) inducer effect (DinkovaKostova et al., 2001) anti-inflammatory effect (Jin et al., 2009) or GST (glutathione S-transferase) P1-1 inhibitory effect (Wang et al., 2009) associated with their Michael-type reactivity towards reduced glutathione (GSH). Previously we have investigated the mechanism of action on the total thiol content of the cells exposed to compounds 2 and 3 (Rozmer et al., 2006). Compound 2 reduced the total cellular thiol level both under nutrient-free and nutrient supplemented conditions. Under the latter conditions an increase of the total thiol level of the cells exposed to 3 could be observed. The different effect on the cellular thiol status might contribute to the different cytotoxicity of the chalcone analogues. Investigation of effects of 2 and other cyclic chalcone analogues on isolated rat liver mitochondria showed the compounds to deplete reduced glutathione and disrupt electron transfer chain and energetic mechanism. Increased generation of reactive oxygen species and covalent interaction with GSH were suggested as chemical events responsible for the observations resulted in on exposure of mitochondria to the investigated compounds (Perjési et al., 2008; Guzy et al., 2010). Incubation of 2 and 3 with reduced glutathione under cell-free conditions indicated spontaneous (non-enzyme catalyzed) conjugation (non-redox) reaction (Rozmer et al., 2006; Perjési et al., 2012). Investigation of antioxidant capacity of the compounds by monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose indicated that both compounds displayed hydroxyl radical scavenger activity (Rozmer et al., 2006; Perjési and Rozmer, 2011). As a continuation of our work towards a better understanding of the molecular mechanism taking part in the development of
biological activities of chalcones, we studied the effect of compounds 2 and 3 on redox status, investigating their effect on ROS production and on glutathione level (GSH and GSSG) in Jurkat cells. The previously developed reversed-phase high-performance liquid chromatographic (RP-HPLC) method coupled with electrospray ionization mass spectrometry (ESI-MS) detection (Perjési et al., 2012) was applied to investigate the chalcone–GSH reactions under cellular conditions. 2. Materials and methods 2.1. Materials The cyclic chalcone analogues 2 and 3 were synthesized and purified as described before (Dimmock et al., 1999b). Jurkat human T lymphocyte leukaemia cell line (Clone E6-1, ATCC TIB-152) was obtained from LGC Standards (Wesel, Germany). Cryopreservation and culturing of the cells were performed according to ATCC documentations. All the chemicals used were of the analytical grade available and, if otherwise not specified, purchased from Sigma– Aldrich Hungary (Budapest, Hungary). 2.2. Cell culture conditions and exposure Jurkat T lymphocytes were cultured under conventional conditions (37 °C humidified atmosphere of 95% air and 5% CO2 at 37 °C) in RPMI 1640 medium, pH 7.4 supplemented with 10% heat-inactivated foetal calf serum (FCS) (Gibco 110108-165) with addition of penicillin (100 U/ml) and streptomycin (100 lg/ml). Cells were grown until they reached 6–8 105 cells/ml density, refreshed with medium and transferred into new flask. Exponentially growing Jurkat cells (5 105 cells in 1.0 ml RPMI 1640 cell culture medium with 10% FCS) were exposed to the previously determined IC80 concentrations (Dimmock et al., 1999b) of 2 (5.6 lM) and 3 (23.3 lM) for 1 or 4 h. The compounds were added in 10 ll DMSO solution to the cells suspended in 1.0 ml medium. Control A cells were untreated; only vehicle, 10 ll DMSO, was added to the control B cells. Cell number was determined by exclusion of Trypan Blue dye and a haemocytometer (Tennant, 1964). 2.3. Measurement of intracellular reactive oxygen species (ROS) generation To detect production of intracellular ROS, oxidation-sensitive 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) was used (Frei et al., 2007; Aronis et al., 2003). After cells were incubated with 2 or 3 for 1 and 4 h and washed with PBS, they were treated with 10 lM DCFH-DA at 37 °C for 20 min in the dark, washed twice with PBS, collected by centrifugation and suspended in PBS. To detect the intensity of fluorescence of DCF each sample was analysed
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using a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA). At least 10.000 events were evaluated in each experiment. Data were collected and analysed by CellQuest (BD Biosciences) software program. The results show the change in the mean of fluorescent intensity compared to the untreated controls. Each value is the average of four independent experiments. Statistical evaluations were done by Student-t test (p < 0.05). 2.4. Determination of reduced and oxidized glutathione For determination of reduced and oxidized glutathione Jurkat T cells (2 106 cells in 5.0 ml RPMI 1640 cell culture medium with 10% FCS) were exposed to 2 and 3 for 4 h as described before (Section 2.2). After treatment cells were washed twice with cold 0.9% NaCl solution, suspended in 400 ll TRIS buffer (50 mM Tris–HCl, 0.1% SDS, pH 7.4) and lysed by three freeze–thaw cycles (freezing the cells for 30 min at 80 °C, followed by slow defrosting on room temperature). The cell lysates were centrifuged (5 min at 13,000g) and the clear supernatant was used for determination the protein concentration. The other part of the supernatant was stored in 80 °C for glutathione measurements. Each result is the average of four independent experiments. The protein concentration was determined by the BCA (bicinchoninic acid) assay (Smith et al., 1985), using bovine serum albumin as a standard. The absorbance was recorded in a Dynatech MR 7000 enzyme-immunoassay (ELISA) plate reader. Cell supernatant was mixed with 6% perchloric acid (210– 300 ll supernatant) and the mixture was centrifuged at 10,000g for 4 min to remove the precipitated protein. It was neutralised by 1 N KOH and centrifuged again at 3000g for 3 min to remove the salt precipitated. Determination of reduced (GSH) and oxidized (GSSG) glutathione content was performed by slight modification of Kand’ár et al. HPLC method coupled with fluorescence detection (Kand’ár et al., 2007). Briefly, GSH and GSSG content of the samples was derivatized with OPA (ortho phthalic aldehyde) and quantified by a reversed-phased high-performance liquid chromatography (HPLC). In the GSH assay, to 100 ll of the supernatant 1.0 ml of 0.1% EDTA in 0.1 M sodium hydrogen phosphate, pH 8.0, was added. To 20 ll portion of this mixture, 300 ll of 0.1% EDTA in 0.1 M sodium hydrogen phosphate and 20 ll of 0.1% OPA in methanol was added. The mixture was incubated at 25 °C for 15 min in dark and stored at 4 °C. In the GSSG assay 200 ll of the supernatant was incubated at 25 °C with 200 ll of 40 mM N-ethylmaleimide (NEM) for 25 min in dark to interact with GSH present in the sample. To this mixture 750 ll of 0.1 M NaOH was added. To 20 ll portion of this mixture, 300 ll of 0.1 M NaOH and 20 ll of 0.1% OPA in methanol was added. The mixture was incubated at 25 °C for 15 min in dark and stored at 4 °C. As reference freshly prepared GSH and GSSG containing standard series, dissolved in 50 mM Tris–HCl, 0.1% SDS, pH 7.4, (GSH: 10–100 lM; GSSG: 0–1 lM) was used. Effect of NEM to prevent interference of GSH at measurement of GSSG was checked using GSH standard. Standard solution of GSH was processed as well as samples for the determination of GSSG. HPLC analyses were performed with an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) integrated HPLC system equipped with a quaternary pump, a degasser, an autosampler and a fluorescent detector. Data were recorded and evaluated by use of Agilent Chem Station (Rev.A.10.02) software. The derivatives were measured on a Poroshell 120 EC-C18 (150 mm 4.1 mm i.d., particle size 2.7 lm) column with guard cartridge (TR-C-160-K1, ABLE&Jasco) at 37 °C. The mobile phase consisted of 15% methanol in 25 mM sodium hydrogen phosphate (v/v), pH 6.0. The flow rate was 0.4 ml/min. The injection volume was 10 ll. The fluorescence
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was monitored with an excitation wavelength of 340 nm and an emission wavelength of 425 nm. The concentration of GSH and GSSG in the samples was determined from the standard calibration curve. Each value is the average of five independent experiments. Statistical evaluations were done by Student-t test (p < 0.05). 2.5. HPLC–ES–MS study on chalcone–GSH conjugation For investigation of chalcone–GSH conjugation reactions under cellular conditions, Jurkat T cells (2 106 cells in 5.0 ml RPMI 1640 cell culture medium with 10% FCS) were exposed to 2 and 3 for 4 h as described above (Section 2.2). After treatment, cells were washed twice with ice cooled 0.9% NaCl solution, suspended in 600 ll TRIS buffer (50 mM Tris–HCl, 0.1% SDS, pH 7.4) and lysed in ice by sonication for 3 min. The cell lysates were centrifuged (at 13,000g for 5 min) and the separated supernatants and cell sediments were used for further investigations. The sedimented cells have been homogenized in 600 ll TRIS buffer and were explored by 6 10 s treatments with a high energy ultrasonicator, UIS250V (Hielsher Ultrasound Technology, Teltow, Germany) using ice cooling between the cycles. Samples were vortex mixed and centrifuged at 10,000g for 10 min. The supernatant and the explored cell samples were loaded in the Centrifugal Filter Devices (Amicon Ultra with 10 K cut off membrane, Merck-Millipore, Darmstadt, Germany) and centrifuged at 10,000g for 30 min. The filtrate was tested by MALDI and if protein was found in the sample, the experiment was repeated with a new filter. To the protein free filtrates 0.1% formic acid was added and the samples were subjected to HPLC–MS analysis. Analyses were performed with a complex Ultimate 3000 (Dionex, Sunnyvale, USA) micro HPLC system equipped with a quaternary pump, a degasser, and a Bruker micro-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Separations were performed on a Kinetex C-18 (150 mm 2.1 mm i.d., particle size 2.6 lm) column (Phenomenex, Torrance, USA). The flow rate was 200 mm3/ min; the injection volume was 1 mm3. Hystar 3.2 (Bruker Daltonics, Bremen, Germany) and Bruker micro-TOF control 1.3 software were used for controlling the instrument. Data acquisition and spectrum evaluations were performed by means of Brucker Data analysis 4.1 software. A binary gradient consisting of mobile phase A and B (A: methanol – 0.1% formic acid; B: water – 0.1% formic acid) was applied for the chromatographic separation. The HPLC analyses were performed using the following gradient profile: 40% A for 5 min, followed 10 min linear gradient to 90% and an isocratic period of 5 min. A Bruker microTOF mass spectrometer, equipped with an ESI source was used for mass detection. The ionization source was operated with an endplate potential of 3.8 kV in the positive ion mode. The following electrospray parameters were kept constant during the analysis: drying gas (N2) flow 8 dm3/min, drying gas temperature 210 °C, nebulizer pressure 20 psi. Spectra were acquired in the mass/charge ratio (m/z) range of 50–2000. 3. Results In order to investigate the effect of compounds 2 and 3 on the intracellular peroxide level in the Jurkat cells, oxidation-sensitive 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) was used (Frei et al., 2007; Aronis et al., 2003). This compound is a nonfluorescent, cell-diffusible dye. Intracellular esterases cleave the acetyl groups from the molecule to produce nonfluorescent 20 ,70 -dichlorofluorescein (DCFH). This is trapped inside the cell and in the presence of ROS, DCFH is subsequently modified to fluorescent DCF, which can be detected by flow-cytometry. The cells were incubated with
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Fig. 3. Effects of compounds 2 and 3 on the reduced (panel A) and the oxidized (panel B) glutathione content of the Jurkat cells after 4 h exposure. Control A are untreated cells, Control B are cells treated with 1% DMSO (vehicle). Experimental details are described in ‘‘Section 2’’. The values represent means ± standard error from five parallel experiments. Statistically significant difference from the untreated control, cells (Control A) (Student-t test, at the level of p < 0.05) is marked with *.
Fig. 2. Effects of 2 and 3 on intracellular ROS production in Jurkat cells (panel A). FACScan analysis of Jurkat cells incubated with 2 and 3 for 4 h and then stained with 10 lM DCFH-DA for 20 min. Control A are untreated cells (panel B). Relative fluorescent intensity (compared to Control A cells) of Control B cells (treated with DMSO 1%) and cells treated with 2 and 3. Further experimental details are described in ‘‘Section 2’’. The values represent means ± standard error from four parallel experiments. Statistically significant difference from the untreated control, cells (Control A) (Student-t test, at the level of p < 0.05) is marked with *.
1% DMSO, chalcones 2 (5.6 lM) or 3 (23.3 lM) for 1 and 4 h, respectively. The applied concentrations were previously determined IC80 concentrations (Dimmock et al., 1999b). ROS production level was evaluated using FACS analysis. As shown in Fig. 2A, ROS induction was markedly decreased in cells treated with 3; however, compound 2 did not influence ROS production compared to the untreated control cells. For comparison, the relative fluorescent intensity, expressed in per cent of the control cells, is illustrated in Fig. 2B. A significant decrease in the DCF fluorescence was observed in the Jurkat cells incubated with 3 both after 1 and 4 h exposure times (80.20 ± 10.35% and 71.35 ± 16.51% of control, respectively). Compound 2 did not affect the peroxide level in the Jurkat cells at any of the two timepoints. Effect of compounds 2 and 3 on reduced (GSH) and oxidized (GSSG) glutathione levels was investigated using a reversed phase HPLC method after derivatization with OPA (Kand’ár et al., 2007). Reduced glutathione reacts with OPA to form a stable, highly fluorescent tricyclic derivate at pH 8, while GSSG reacts with OPA only at pH 12 (Hissin and Hilf, 1976). While measurement of GSSG, the thiol function of GSH was chemically modified by N-ethylmaleimide to non-OPA-reactive derivative. The chromatogram of GSH standard solution (50 lM) treated with NEM (40 mM) and derivatized by OPA at pH 12 indicated that the used amount of NEM was
entirely sufficient to prevent interference of GSH while measuring GSSG. Concentration of GSH and GSSG in the samples was determined by means of standard curves. The results, normalized to protein concentration (nmol/mg protein), are represented in Fig. 3. Compound 2 was found not to influence the GSH level, but significantly increased the GSSG level in the cells (from 0.67 ± 0.26 nmol/mg protein to 1.15 ± 0.33 nmol/mg protein). Chalcone 3 enhanced the GSH level (from 35.3 ± 6.6 nmol/mg protein to 42.4 ± 7.2 nmol/mg protein), whereas did not markedly change the GSSG level, compared to the untreated (Control A) and DMSO-treated (Control B) cells. To investigate whether chalcone–GSH conjugation reactions undergoes under cellular conditions, analysis of cell supernatants and sediments by a RP-HPLC method coupled with ESI-MS was performed. The HPLC chromatograms of compound 2 from the properly prepared and investigated cell supernatant and cell sediment are shown in Fig. 4A-B. The selected ion chromatograms (Fig. 4A and B) and the ESI mass spectra of the analysed cell preparations, recorded in the 50–2000 Da range (Fig. 5A and B), give evidence of presence of the protonated molecule of 2 (m/ z = 279.2) both in cell supernatant and cell sediment. Presence of the diastereomeric GSH adducts of 2 (Figs. 4C and 5D) – formed in in vitro incubations of 2 and GSH under basic conditions (Perjési et al., 2012) – could not be observed. The results of similar investigations of compound 3 – not shown in the figures – were the same as those obtained with compound 2. Only the protonated molecule of compound 3 (m/z = 263.2) could be observed in the spectra of both the cell supernatant and the cell sediment of cells previously exposed to chalcone 3.
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Fig. 4. Selected ion chromatograms (m/z = 279.2) of cell supernatant (panel A) and of cell sediment (panel B) prepared from Jurkat cells exposed to compound 2 (5.6 lM) for 4 h, after deproteinization and centrifugation. Experimental details are described in ‘‘Section 2’’. Panel C: overlayed selected ion chromatograms (m/z = 279.2 and m/z = 586.2) of the reaction mixture of 2 (5 103 M) and GSH (5 102 M) after 300 min incubation time (pH 8.0; 50 °C).
4. Discussion Earlier we reported that equitoxic doses of compounds 2 (5.6 lM) and 3 (23.3 lM) induced apoptosis (accompanied by formation of hyperdiploid cells) and reduced the total cellular thiol level of Jurkat cells both under nutrient-free and nutrient supplemented conditions. Viability of the cells (determined by the Tripan Blue exclusion test) exposed to 2 and 3 at the 4 h timepoint was estimated about 80% in the case of both compounds (Rozmer et al., 2006). Under nutrient supplemented conditions (RPMI 1640 with 10% heat-inactivated FCS) a slight increase of the total thiol level of the cells exposed to 3 could be observed at the 4 h timepoint. In any other investigated timepoint (1 h or 8 h) both compounds reduced the total tiol level compared to those of the vehicle (DMSO)-treated controls. It was presumed that the more pronounced thioldepleting effect of 2 might contribute to its demonstrated tumour cell toxicity (Rozmer et al., 2006). Apoptosis is a tightly regulated form of cell death in which a cell effectively takes part in its own demise. There is a growing consensus that oxidative stress and the redox state of a cell plays a pivotal role in regulating apoptosis (Curtin et al., 2002). Several chalcones have been reported to cause oxidative stress which results cytotoxicity (Winter et al., 2010). On the other hand several chalcones have been described to demonstrate antioxidant activity (Dimmock et al., 1999a; Go et al., 2005) but it is presumed that the toxic effects of chalcones might be due to their pro-oxidant activities (Middleton et al., 2000; Dimmock et al., 1999a). To determine whether oxidative stress was involved in chalcone induced cell death, generation of reactive oxygen species (ROS) was evaluated using the DCFH-DA assay. The findings (Fig. 2) indicate that the compounds tested did not increase the intracellular ROS production under the experimental conditions; moreover compound 3 decreased the intracellular ROS level showing antioxidant activity. This latter result is in accordance with those of investigation of antioxidant capacity of the compounds by
monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose. In the hydroxyl radical initiated degradation test both compounds were found to display hydroxyl radical scavenger activity (Rozmer et al., 2006). Based on the present experimental results, hydroxyl radical scavenger activity of compound 3 is not associated with generation of ROS, which can be observed in the case of some aromatic antioxidants (Gregus and Klaassen, 2001; Prokai et al., 2003; Guzy et al., 2010) Different effect of the two compounds on intracellular ROS levels can be associated with the different reactivity of them towards GSH and other cellular thiols (Perjési et al., 2012; Dinkova-Kostova et al., 2001). The GSH system is one, but probably the most important cellular defence mechanism that exists in the cell. Glutathione acts as an ROS scavenger but also functions in the regulation of intracellular redox state (Curtin et al., 2002; Camera and Picardo, 2002). The ability of the cell to generate GSH (either by reduction of GSSG or de novo synthesis of GSH) is an important factor in the efficiency of that cell in managing oxidative stress. The exposure period used in the present experiments (4 h) was long enough for the cells to react to the possible change in the GSH levels (Gul et al., 2002). Under such conditions compound 3 was found to increase the reduced glutathione level of the cells (Fig. 3). Earlier, elevated cellular thiol level has been reported on exposure of Jurkat (Gul et al., 2002) and HeLa (Lóránd et al., 2001) cell lines to some electrophilic Mannich bases. Elevated GSH level of the Jurkat cells might have resulted from de novo synthesis in response to feedback control of GSH (Gul et al., 2002). In contrast, chalcone 2 induced an increase in GSSG content (Fig. 3), which can be evidence that this compound induces oxidative stress. This observation is in accordance with the previous results showing compound 2 to increase oxygen consumption without affecting state 3 activity of rat liver mitochondria (Kubálková et al., 2009). Such an effect can increase the mitochondrial level of reactive oxygen species (ROS) that can initiate a series of biochemical events, involving cell death (Chen et al., 2006; Sanz
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Fig. 5. ESI mass spectra of cell supernatant (A) and of cell sediment (B) prepared from Jurkat cells exposed to compound 2 (279.2 Da)(5.6 lM) for 4 h, after deproteinization and centrifugation. Experimental details are described in ‘‘Section 2’’. ESI mass spectra of 2 (C) and 2-GSH adduct (D) prepared from the reaction mixture of 2 (5 103 M) with GSH (5 102 M) after 300 min incubation time (pH 8.0; 50 °C) (m/z = 50–2000).
et al., 2006). GSSG is a physiological indicator of intracellular defence system activity against ROS, and it can be used to monitor oxidative stress even in vivo (Gitler et al., 1994; Hughes et al., 1990). Simultaneously, decrease in the GSH level could not be observed. This observation can be the result of increase in glutathione-reductase activity as an attempt to sustain GSH content and/or that of increased c-glutamyl-cysteine-synthetase (c-GCS) activity (Winter et al., 2010). Cytotoxic effect of a, b-unsaturated carbonyl compounds are frequently associated with expected reactivity of the compounds with the essential thiol groups in the living organism (Dimmock et al., 1999a; Go et al., 2005). Several other biological effects of chalcones have also been associated with their Michael-type reactivity towards reduced glutathione and other cellular thiols (Dinkova-Kostova et al., 2001). In our previous studies compounds 2 and 3 showed intrinsic reactivity towards GSH at higher pH (pH 9.0) and about physiological pH (pH 7.4) at a higher temperature
(Rozmer et al., 2006). The non-enzyme-catalyzed reaction of GSH and the chalcone analogues was investigated by RP-HPLC–MS (Perjési et al., 2012). The previously developed method provided the appropriate analytical tool to investigate the chalcone–GSH reactions in Jurkat cells. The compounds 2 and 3 could be detected even from cell supernatant and cell sediment after cell lysis and deproteinization, indicating that chalcones penetrate the cell membrane and affect intracellular biochemical events. However, chalcone–GSH adducts could not be observed either in the cell supernatants or the cell sediments (Figs. 4 and 5). This observation might be the consequence of reversible addition of GSH onto the chalcones’ polar carbon–carbon double bond, which can result in a retro-Michael-type decomposition of the adducts when the excess of GSH is reduced (Perjési et al., 2012). In conclusion, it was demonstrated that equitoxic doses of the two cyclic chalcone analogues 2 and 3 have different effect on glutathione level and redox status in Jurkat cells. For chalcone 2 the
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interference with the cellular antioxidant defence systems might contribute to its demonstrated tumour cell toxicity. Compound 3 decreased ROS production and increased cellular glutathione level, indicating antioxidant activity. Glutathione conjugates could not be detected either in cell supernatants and cell sediments. These results provide new insights into the dual – cytotoxic and chemopreventive – effects of the cyclic chalcone analogues. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version. Acknowledgements This study was supported by the University of Pécs, Faculty of Medicine Research Fund (PTE AOK KA 2012-2013/13-18 and PTE AOK-KA-2013/20). References Aronis, A., Melendez, J.A., Golan, O., Shilo, S., Dicter, N., Tirosh, O., 2003. Potentiation of FAS-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species. Cell Death Differ. 10, 335–344. Benvenuto, J.A., Connor, T.H., Monteith, D.K., Laidlaw, J.L., Adams, S.C., Matney, T.S., Theiss, J.C., 1993. Degradation and inactivation of antitumor drugs. J. Pharm. Sci. 82, 988–991. Camera, E., Picardo, M., 2002. Analytical methods to investigate glutathione and related compounds in biological and pathological processes. J. Chromatogr. B 781, 181–206. Chen, Q., Moghaddas, S., Hoppel, C.L., Lesnefsky, E.J., 2006. Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion. J. Pharmacol. Exp. Ther. 319, 1405– 1412. Curtin, J., Donovan, M., Cotter, T., 2002. Regulation and measurement of oxidative stress in apoptosis. J. Immunol. Methods 265, 49–72. Dimmock, J., Elias, D., Beazely, M., Kandepu, N., 1999a. Bioactivities of chalcones. Curr. Med. Chem. 6, 1125–1149. Dimmock, J.R., Kandepu, N.M., Nazarali, A.J., Kowalchuk, T.P., Motaganahalli, N., Quail, J.W., Mykytiuk, P.A., Audette, G.F., Prasad, L., Perjési, P., Allen, T.M., Santos, C.L., Szydlowski, J., De Clercq, E., Balzarini, J., 1999b. Conformational and quantitative structure-activity relationship study of cytotoxic 2arylidenebenzocycloalkanones. J. Med. Chem. 42, 1358–1366. Dimmock, J.R., Zello, G.A., Oloo, E.O., Quail, J.W., Kraatz, H.B., Perjési, P., Aradi, F., Takács-Novák, K., Allen, T.M., Santos, C.L., Balzarini, J., De Clercq, E., Stables, J.P., 2002. Correlations between cytotoxicity and topography of some 2arylidenebenzocycloalkanones determined by X-ray crystallography. J. Med. Chem. 45, 3103–3111. Dinkova-Kostova, A.T., Massiah, M.A., Bozak, R.E., Hicks, R.J., Talalay, P., 2001. Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc. Natl. Acad. Sci. 98, 3404–3409. Frei, G.M., Lebenthal, I., Albeck, M., Albeck, A., Sredni, B., 2007. Neutral and positively charged thiols synergize the effect on the immunomodulator AS101 as a growth inhibitor of Jurkat cells, by increasing its uptake. Biochem. Pharmacol. 74, 712–722. Gitler, C., Mogyoros, M., Kalef, E., 1994. Labeling of protein vicinal dithiols: role of protein-S2 to protein-(SH)2 conversion in metabolic regulation and oxidative stress. Methods Enzymol. 233, 403–415. Go, M., Wu, X., Liu, X., 2005. Chalcones: an update on cytotoxic and chemoprotective properties. Curr. Med. Chem. 12, 483–499. Gul, M., Gul, H., Hänninen, O., 2002. Effects of Mannich bases on cellular glutathione and related enzymes of Jurkat cells in culture conditions. Toxicol. in Vitro 16, 107–112. Guzy, J., Vasková-Kubálková, J., Rozmer, Zs., Fodor, K., Mareková, M., Proskrobová, M., Perjési, P., 2010. Activation of oxidative stress response by hydroxyl
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