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Activation of oxidative stress response by hydroxyl substituted chalcones and cyclic chalcone analogues in mitochondria
FEBS Letters 584 (2010) 567–570
journal homepage: www.FEBSLetters.org
Activation of oxidative stress response by hydroxyl substituted chalcones and cyclic chalcone analogues in mitochondria J. Guzy a, J. Vašková-Kubálková a,*, Z. Rozmer b, K. Fodor b, M. Mareková a, M. Poškrobová a, P. Perjési b a b
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, P.J. Šafárik University, Košice, Slovak Republic Institute of Pharmaceutical Chemistry, Faculty of Medicine, University of Pécs, Pécs, Hungary
a r t i c l e
i n f o
Article history: Received 27 October 2009 Revised 19 November 2009 Accepted 20 November 2009 Available online 9 December 2009 Edited by Vladimir Skulachev Keywords: Cyclic chalcone analogue Mitochondria Respiration Glutathione Oxidative stress Hydroxyl radical
a b s t r a c t We investigated the effect of hydroxyl substituted chalcone (1a) and some chalcone analogues (1b– d) on isolated rat liver mitochondria to gain new insights into the cytotoxic mechanism of these compounds. We observed an inhibitory effect on phosphorylation and the partial uncoupling of compounds 1a and 1d. Increased radical generation and possible covalent interaction of the compounds with cellular thiols resulted in glutathione (GSH) depletion and modulation of the investigated mitochondrial activities. Disruption of interconnected mechanisms as electron transport chain and energetic metabolism, ROS production and insufficiency of antioxidant defensive system could lead to induction of cell death. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Chalcones are intermediary compounds of the biosynthetic pathway of a very large and widespread group of plant constituents known collectively as flavonoids [1]. Chalcones have been shown to possess antioxidant, oxygen scavenging, and anti-inflammatory properties in a variety of experimental systems [2,3]. Kohno et al. [4] found evidence that chalcone is converted to hydroxylated derivatives with estrogenic activity. Among the naturally occurring chalcones [5] and their synthetic analogues [6,7] several compounds displayed cytotoxic (cell growth inhibitor) activity towards cultured tumor cells. Chalcones are also effective in vivo as cell proliferating inhibitors, anti-tumor promoting and chemopreventive agents [5–7]. Chalcones, as a,bunsaturated carbonyl compounds, have a marked affinity for thiols in contrast to amines [6]. Since a number of clinically useful anticancer drugs have genotoxic effects due to their interaction with the amino groups of nucleic acids, chalcones may be devoid of this important side effect [6,7]. Recently we have investigated in vitro antineoplastic activity of several synthetic chalcones and cyclic chalcone analogues. The compounds were evaluated against P388, L1210, Molt 4/C8 and * Corresponding author. Address: Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, P.J. Šafárik University, Tr. SNP 1, 040 11 Košice, Slovak Republic. Fax: +421 55 6423849. E-mail address: [email protected] (J. Vašková-Kubálková).
CEM cells as well as against approximately 60 further human tumor cell lines from nine different neoplastic diseases [8–10]. Among the compounds investigated E-2-(40 -X-benzylidene)-1benzosuberones (Fig. 1, n = 3) had the greatest tumor toxicity. Structure–activity studies demonstrated that cytotoxicity of the cyclic chalcone analogues were influenced by the shape of the molecules [8–10]. Besides the documented tumor cytotoxic effects, some of the above cyclic chalcone analogues were found to display outstanding CYP1A inhibitor activity [11,12]. Their specific chemical structure renders them to be powerful inducers of the phase 2 family of detoxifying enzymes [13]. Accordingly, the compounds represent prototypes of molecules displaying both tumor cytotoxic and cytoprotective (chemopreventive) effects. The cytotoxic effect of a,b-unsaturated carbonyl compounds are frequently associated with their expected reactivity with the essential thiol groups in the living organisms [6,7,14,15]. Such a reaction can alter intracellular redox status (redox signaling), which can modulate processes such as DNA synthesis, enzyme activation, selective gene expression, and regulation of the cell cycle [16]. An important feature of the mitochondrial function of the cell is an interrelationship between three essential cellular processes: oxidative phosphorylation, generation of reactive oxygen species, and initiation of physiological cell death (apoptosis) via mitochondrial permeability transition pores [17]. Accordingly, modification of mitochondrial functions by xenobiotics may contribute to their cytotoxic and antineoplastic activities.
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.11.098
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J. Guzy et al. / FEBS Letters 584 (2010) 567–570
O
O
(CH2)n
OH
1a
n=1 n=2 n=3
OH
1b 1c 1d
Fig. 1. Chemical structure of the chalcones investigated.
The mitochondrion is the main metabolic sitewhere reactive oxygen species (ROS) are generated. Their administration to various xenobiotics may elevate ROS production [18]. The reactive oxygen species of primary biologic importance include superoxide (O 2 ), hydroxyl radicals (OH ) and hydrogen peroxide (H2O2). Cell damage occurs when there is an increased generation of reactive oxygen species or a relative shortage of antioxidant molecules [19]. Many of the stimuli that lead to apoptosis are associated with the generation of ROS [20]. As a continuation of our previous studies [15] focusing on the effect of chalcones on mitochondrial respiratory functions, in the present study the effect of four hydroxyl substituted chalcone (1a) and chalcone analogues (1b–d) on isolated rat liver mitochondria was investigated. Characterization of mitochondrial respiratory functions on enzyme activities as well as redox status of mitochondria administered to compounds of subcytotoxic doses was investigated. We studied the effect of the compounds on the reduced glutathione (GSH) content of the exposed mitochondria and characterized in vitro reactivity of the compounds with GSH. Since oxidation of hydroxyl-substituted xenobiotics could generate reactive oxygen species (ROS), the compounds could cause oxidative stress as a result of such a series of reactions [18]. To test this possibility, we have investigated time-dependence of in vitro antioxidant (hydroxyl radical scavenger) capacity of the compounds by means of the Fenton-reaction-initiated degradation of 2-deoxyribose [14]. 2. Materials and methods 4-Hydroxychalcone (1a) and its cyclic derivatives (1b–d) were synthesized and purified as described before [21]. Their structures were characterized by IR and NMR spectroscopy. The purity of the compounds was checked by thin layer chromatography (TLC) and gas chromatography (GC) methods. Other chemicals used were of the analytical grade available and, if otherwise not specified, purchased from Sigma–Aldrich or Serva (Heidelberg, Germany). Com-
Detected parameters (%)
80 *
60 40 20 0 1a
-20 -40
*
1c
*
1d
* *
*
-60 -80
1b * *
Hydroxychalcone derivatives state 4
state 3
RCR
Fig. 2. Effect of hydroxychalcone derivatives 1a–d on mitochondrial functions. (Results are calculated as the relative percentage of changes compared to the values of the control group). *Mean, significance P < 0.05.
pounds 1a–d were dissolved in dimethyl sulfoxide (DMSO) immediately before use. Male Wistar rats (Velaz, Praha, Czech Republic) weighing 200– 250 g were used. Adhering to procedures approved by the University of Košice Animal Care and Use Committee, the animals were sacrificed by cervical displacement and decapitation. Liver mitochondria were isolated from male Wistar rats using the method by Johnson and Lardy [22]. Mitochondrial protein was determined according Lowry after Hartree’s modification [23]. Polarographic measurement of oxygen consumption of isolated mitochondria was carried out with a Clark electrode (WTW oxi 325 (Germany)) at the experimental temperature of 25 °C. Measurements were carried out in respiratory medium (80 mM KCl, 300 mM KH2PO4, 300 mM K2HPO4, 15 mM TRIS–HCl, 6 mM MgCl2, 0.78 mM EDTA, pH 7.4) supplemented with 70 nmol of chalcone per mg of mitochondrial protein, 50 mM sodium succinate, 0.5 mM ADP and 20 mg/ml mitochondrial protein. The test compounds were added in dimethyl sulfoxide (DMSO), a dose that did not affect control rates of respiration. The respiratory rate is expressed as oxygen in nmol of oxygen consumed min1 mg1 of mitochondrial protein [24]. Respiratory control ratio (RCR) was determined as a ratio between oxygen consumption in state 3 (with addition of ADP) and state 4 (after exhaustion of ADP). The activity of glutathione reductase (E.C.1.6.4.2) was determined according to Carlberg and Mannervik [25], and glutathione peroxidase (E.C. 1.11.1.9) by the method of Flohe and Gunzler [26]. Reactivity of chalcones 1a–d with reduced glutathione (GSH) was studied by the method described before [15]. The reaction mixtures were kept in water bath (37 °C and 50 °C) and the progress of the reactions was followed by thin layer chromatography (TLC) on Kieselgel F254 plates. The developing solvent used was nbutanol:acetic acid:water (40:10:20, v/v %). Determination of reduced glutathione in mitochondria (GSH) was performed by the modified method of Abukhalaf et al. [27]. HPLC analyses were performed on LC-10ADVP liquid chromatograph (Schimadzu, Kyoto, Japan) equipped with an RF-10AXL fluorescence detector (Schimadzu). The elution gradient profile was as follows: 0 min: 0.5% ACN, 1–5 min: 10% ACN, 6–15 min.: 5% ACN. Investigation of the antioxidant capacity of chalcones 1a–d was performed as described before [14]. Quercetin (200 lM) was used as reference antioxidant. Each value is the average of three independent measurements. Results are presented as mean ± S.D. of at least three independent experiments. Statistical significance was determined by Student’s t-test with P < 0.05 level considered as significant.
3. Results The results of the present study demonstrate that the investigated compounds are able to modify the mitochondrial activity of the cell. Two of the selected compounds (1a and 1b) displayed an inhibitory effect on both the ADP-stimulated respiration (state 3) and the controlled respiration (state 4) (Table 1). The six-membered analogue 1c did not affect either mitochondrial function. On the contrary, compound 1d had no effect on state 3 respiration, however, increased the rate of oxygen consumption after ADP exhaustion (state 4) by 65.0% (Fig. 2). Respiratory control ratio (RCR) was significantly decreased by the compounds 1a, 1b and 1d by 27.1%, 27.1% and 40.5%, respectively (Table 1). Reactivity of the compounds towards cellular thiols was investigated under cell-free conditions. Thin layer chromatographic (TLC) analysis of 1:10 molar mixtures of chalcones and GSH incubated at pH 7.4 or pH 9.0 at 50 °C indicated the formation of
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J. Guzy et al. / FEBS Letters 584 (2010) 567–570 Table 1 Respiratory functions of mitochondria exposed to compounds 1a–d.
Control 1a 1b 1c 1d *
O2 consumption in state 4 (nmol O2 min1 mg1p)
O2 consumption in state 3 (nmol O2 min1 mg1p)
RCR
21.84 ± 2.71 13.43 ± 2.44* 12.20 ± 2.24* 26.60 ± 3.25 36.05 ± 3.78*
53.53 ± 0.35 21.96 ± 2.14* 21.96 ± 2.14* 54.67 ± 3.84 52.86 ± 10.91
2.47 ± 0.32 1.80 ± 0.43* 1.80 ± 0.43* 2.06 ± 0.07 1.47 ± 0.34*
Mean, significance P < 0.05.
adducts that showed characteristics of both the aromatic and the peptide (GSH) moiety of them even after 2-h incubation. Similar adduct formation could be detected when the incubations were carried out at pH 9.0 at 37 °C. On the other hand, no adduct formation could be detected in incubations carried out at pH 7.4 at 37 °C. Determination of reduced glutathione (GSH) content of incubations free of adduct formation did not indicate in vitro redox reaction between the reactants. Determination of reduced glutathione (GSH) levels of mitochondria exposed to the compounds tested was performed by HPLC technique. On exposure of mitochondria to any of the tested compounds resulted in reduction of GSH levels compared to that of the DMSO-treated control experiments. Parallel to the reduction of the cellular GSH levels increased glutathione peroxidase (GPx) and glutathione reductase (GR) activities could be observed (Table 2). Antioxidant capacity and possible pro-oxidant action of the compounds were tested by measurement of time-dependence of Fenton-reaction-initiated degradation of 2-deoxyribose [14]. Fenton-reaction-initiated degradation of 2-deoxyribose may result from an attack of hydroxyl radicals (HO), which results in the formation of TBA-reactive substances with an absorption maximum of 532 nm. As it is shown in Table 2, all the compounds tested displayed antioxidant activity (HO scavenging) at the 20-, 120- and 240-min time points. 4. Discussion The mitochondrial electron transport chain is the source of constantly forming reactive oxygen species [28]. Exogenous sources could lead to increased or decreased production of reactive oxygen species and display a toxic or protective effect, respectively. Attention was paid to select and investigate 40 -hydroxychalcone (1a) and its cyclic analogues (1b–d) due to their possible redox properties that can manifest in an antioxidant/pro-oxidant effect. The results of the present study demonstrate that the investigated compounds are able to modify the energy-linked functions of the cells. In the selected concentrations 1a and 1b displayed an inhibitory effect on ADP-stimulated respiration (state 3) in 59.0% each (Table 1). A decrease in oxygen consumption in the presence of added ADP (state 3) is indicative of the inhibition of
mitochondrial electron transfer chain [29,30]. Since the compounds significantly reduced the controlled respiration (state 4) as well, the decreased respiratory control ration (RCR) suggested a phosphorylation inhibitory effect or partial uncoupling of the compounds (Table 1). This latter effect could be the result of the lipophilic weak acid character of the compounds [31,32]. On the other hand, compounds 1c and 1d, had no effect on state 3 respiration, however, it increased the rate of oxygen consumption (respiratory burst) after ADP exhaustion (state 4) by 21.8% and 67.1%, respectively. Respiratory control ration (RCR) was reduced by 16.6% and 40.5% in the case of 1c and 1d, respectively (Table 1). Since auto-oxidation of the compounds could not be responsible for the observed oxygen consumption, the consumed oxygen should be the substrate of the mitochondrial electron transfer of the mitochondrial activity [29]. Such an effect can increase the mitochondrial level of reactive oxygen species (ROS) that can initiate a series of biochemical events, involving cell death [18,33]. To keep reactive oxygen species production in balance, antioxidant defensive systems such as enzymatic removal of reactive oxygen species and application of potent radical scavengers are active. Several chalcones have been reported to display antioxidant activity [6,7]. On the other hand, a variety of chalcones were found to display antibacterial, antifungal, antiviral, and tumor cytotoxic activities [5–7]. There is also evidence that shows some chalcones to exhibit in vitro mutagenicity [6]. It is presumed that the toxic effects of the compounds might be due to their pro-oxidant activities [5–7]. To test this possibility we have investigated the in vitro antioxidant capacity of the compounds by means of the Fenton-reactioninitiated degradation of 2-deoxyribose. Fenton-reaction-initiated degradation of 2-deoxyribose is thought to be the result of attack of hydroxyl radicals (HO), which results in the formation of TBAreactive substances with an absorption maximum of 532 nm [15]. As shown in Table 2, all the compounds investigated displayed a continuous antioxidant (HO scavenging) activity at the investigated 20-, 120- and 240-min time points; i.e. the compounds reduced the formation of TBA-reactive deoxyribose degradation products. Glutathione peroxidase (GPx) has a well-established role in protecting cells against oxidative injury by giving adaptive response to oxidative stress conditions. GPx is non-specific for hydrogen peroxide and the lack of such substrate specificity results in a range of substrates from hydrogen peroxide to organic hydrogen peroxides [34]. An increase observed in GPx activities may be considered as a result of ROS production that can be accompanied by mitochondrial damage (Table 2). Free radical production is one of the pathogenic mechanisms leading to apoptosis [20]. Cytotoxic effect of a,b-unsaturated carbonyl compounds are frequently associated with the expected reactivity of the compounds with the essential thiol groups in the living organisms [6,7]. To study such reactivity, the compounds were allowed to react with reduced glutathione (GSH), the main soluble cellular thiol,
Table 2 Reduced glutathione (GSH) content, glutathione peroxidase (GPx), glutathione reductase (GR) activity of mitochondria and antioxidant capacity data of compounds 1a–d.
Control 1a 1b 1c 1d Quercetine a *
GSH content of mitochondria (nmol/mgp)
GPx (U/ mgp)
GR (kat/kgp)
Antioxidant capacitya [20 min]
Antioxidant capacitya [120 min]
Antioxidant capacitya [240 min]
11.69 ± 2.56 9.19 ± 2.24 5.62 ± 2.05* 9.67 ± 1.83 6.86 ± 2.88* –
0.43 ± 0.12 0.76 ± 0.13* 0.62 ± 0.21 0.73 ± 0.22 0.69 ± 0.26 –
94.36 ± 12.30 147.50 ± 17.29* 159.72 ± 20.19* 186.00 ± 29.09* 124.62 ± 15.83* –
0.765 ± 0.020 0.708 ± 0.024 0.613 ± 0.008 0.645 ± 0.039 0.610 ± 0.064 0.291 ± 0.010
0.722 ± 0.020 0.637 ± 0.029 0.605 ± 0.018 0.620 ± 0.007 0.589 ± 0.036 0.315 ± 0.08
0.740 ± 0.020 0.693 ± 0.023 0.657 ± 0.020 0.668 ± 0.001 0.598 ± 0.049 0.315 ± 0.024
The antioxidant capacity was determined by degradation on deoxyribose with the initiation of Fenton reaction. Further experimental details are described in ‘‘Section 2”. Mean, significance P < 0.05.
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under cell-free conditions. According to the expectations, each compound showed intrinsic reactivity towards GSH at higher pH values (pH 9.0) and about physiological pH values (pH 7.4) at a higher temperature (50 °C). An analysis of GSH content of the adduct-free incubations did not indicate any measurable in vitro redox reactivity of the compounds towards GSH. In order to investigate whether the compounds can affect the cellular thiol status of the mitochondria, the reduced glutathione (GSH) level was determined. Under cellular conditions chalcones can modify the thiol status by enzyme-catalyzed conjugation and/or by oxidation of the reduced thiols to the respective disulfides [13,35]. On exposure of mitochondria to the compounds substantial reduction of the GSH level compared to the control could be detected in each case (Table 2). The observation could be the consequence of increased formation of ROS and a different glutathione-S-transferase-catalyzed reactivity of the compounds with GSH [35]. Simultaneously with the increased oxidative stress indicated by the increased GPx levels, increased glutathione reductase (GR) activity could also be observed (Table 2). In conclusion, it was demonstrated that compounds 1b and 1d displayed a pronounced effect on the modulation of mitochondrial respiratory functions. Phosphorylation inhibitory effect or partial uncoupling of the compounds resulted in stimulation of mitochondrial activity and increased formation of ROS. Increased formation of ROS and possible covalent interaction of the compounds with cellular thiols resulted in glutathione (GSH) depletion and inhibition or modulation of the investigated mitochondrial activities. The disruption of interconnected mechanisms of the electron transport chain and energetic metabolism in mitochondria, high production of ROS and insufficiency of the antioxidant defensive system could lead to the induction of cell death via the mitochondrial apoptotic pathway [20]. Acknowledgements This study was supported by VEGA grant 1/0624/08 from the Slovak Grant Agency for Science and by the Faculty of Medicine Research Fund, University of Pécs. The authors express special thanks to Zsuzsanna Moravecz and (University of Pécs) for her excellent technical assistance. References [1] Harborne, J.B. (1986) Nature, distribution, and function of plant flavonoids in: Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological and Structure–Activity Relationships (Cody, V., Middleton, E. Jr.Jr. and Harborne, J.B., Eds.), pp. 15–24, Alan R. Liss. Inc., New York. [2] Nakamura, Y., Watanabe, S., Miyake, N., Kohno, H. and Osawa, T. (2003) Dihydrochalcones: evaluation as novel radical scavenging antioxidants. J. Agric. Food Chem. 51, 3309–3312. [3] Alcaraz, M.J., Vicente, A.M., Araico, A., Dominiguez, J.N., Terencio, M.C. and Ferrandiz, M.L. (2004) Role of nuclear factor-jB and heme oxygenase-1 in the mechanism of action of anti-inflammatory chalcone derivate in RAW 264.7 cells. Br. J. Pharmacol. 142, 1191–1199. [4] Kohno, Y., Kitamura, S., Sanoh, S., Sugihara, K., Fujimoto, N. and Ohta, S. (2005) Metabolism of the a,b-unsaturated ketones, chalcone and trans-4-phenyl-3buten-2-one, by rat liver microsomes and estrogenic activity of the metabolites. Drug Metab. Depos. 33, 1115–1123. [5] Middleton Jr., E., Kandaswami, C. and Theoharides, T.C. (2000) The effect of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673–751. [6] Dimmock, J.R., Elias, D.W., Beazely, M.A. and Kandepu, N.M. (1999) Bioactivities of chalcones. Curr. Med. Chem. 6, 1125–1149. [7] Go, M.L., Wu, X. and Liu, X. (2005) Chalcones: an update on cytotoxic and chemopreventive properties. Curr. Med. Chem. 12, 483–499. [8] Dimmock, J.R., Kandepu, N.M., Nazarali, A.J., Kowalchuk, T.P., Motaganahalli, N., Quail, J.W., et al. (1999) Conformational and quantitative structure–activity relationship study of cytotoxic 2-arylidenebenzocycloalkanones. J. Med. Chem. 42, 1358–1366.
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Activation of oxidative stress response by hydroxyl substituted chalcones and cyclic chalcone analogues in mitochondria J. Guzy a, J. Vašková-Kubálková a,*, Z. Rozmer b, K. Fodor b, M. Mareková a, M. Poškrobová a, P. Perjési b a b
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, P.J. Šafárik University, Košice, Slovak Republic Institute of Pharmaceutical Chemistry, Faculty of Medicine, University of Pécs, Pécs, Hungary
a r t i c l e
i n f o
Article history: Received 27 October 2009 Revised 19 November 2009 Accepted 20 November 2009 Available online 9 December 2009 Edited by Vladimir Skulachev Keywords: Cyclic chalcone analogue Mitochondria Respiration Glutathione Oxidative stress Hydroxyl radical
a b s t r a c t We investigated the effect of hydroxyl substituted chalcone (1a) and some chalcone analogues (1b– d) on isolated rat liver mitochondria to gain new insights into the cytotoxic mechanism of these compounds. We observed an inhibitory effect on phosphorylation and the partial uncoupling of compounds 1a and 1d. Increased radical generation and possible covalent interaction of the compounds with cellular thiols resulted in glutathione (GSH) depletion and modulation of the investigated mitochondrial activities. Disruption of interconnected mechanisms as electron transport chain and energetic metabolism, ROS production and insufficiency of antioxidant defensive system could lead to induction of cell death. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Chalcones are intermediary compounds of the biosynthetic pathway of a very large and widespread group of plant constituents known collectively as flavonoids [1]. Chalcones have been shown to possess antioxidant, oxygen scavenging, and anti-inflammatory properties in a variety of experimental systems [2,3]. Kohno et al. [4] found evidence that chalcone is converted to hydroxylated derivatives with estrogenic activity. Among the naturally occurring chalcones [5] and their synthetic analogues [6,7] several compounds displayed cytotoxic (cell growth inhibitor) activity towards cultured tumor cells. Chalcones are also effective in vivo as cell proliferating inhibitors, anti-tumor promoting and chemopreventive agents [5–7]. Chalcones, as a,bunsaturated carbonyl compounds, have a marked affinity for thiols in contrast to amines [6]. Since a number of clinically useful anticancer drugs have genotoxic effects due to their interaction with the amino groups of nucleic acids, chalcones may be devoid of this important side effect [6,7]. Recently we have investigated in vitro antineoplastic activity of several synthetic chalcones and cyclic chalcone analogues. The compounds were evaluated against P388, L1210, Molt 4/C8 and * Corresponding author. Address: Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, P.J. Šafárik University, Tr. SNP 1, 040 11 Košice, Slovak Republic. Fax: +421 55 6423849. E-mail address: [email protected] (J. Vašková-Kubálková).
CEM cells as well as against approximately 60 further human tumor cell lines from nine different neoplastic diseases [8–10]. Among the compounds investigated E-2-(40 -X-benzylidene)-1benzosuberones (Fig. 1, n = 3) had the greatest tumor toxicity. Structure–activity studies demonstrated that cytotoxicity of the cyclic chalcone analogues were influenced by the shape of the molecules [8–10]. Besides the documented tumor cytotoxic effects, some of the above cyclic chalcone analogues were found to display outstanding CYP1A inhibitor activity [11,12]. Their specific chemical structure renders them to be powerful inducers of the phase 2 family of detoxifying enzymes [13]. Accordingly, the compounds represent prototypes of molecules displaying both tumor cytotoxic and cytoprotective (chemopreventive) effects. The cytotoxic effect of a,b-unsaturated carbonyl compounds are frequently associated with their expected reactivity with the essential thiol groups in the living organisms [6,7,14,15]. Such a reaction can alter intracellular redox status (redox signaling), which can modulate processes such as DNA synthesis, enzyme activation, selective gene expression, and regulation of the cell cycle [16]. An important feature of the mitochondrial function of the cell is an interrelationship between three essential cellular processes: oxidative phosphorylation, generation of reactive oxygen species, and initiation of physiological cell death (apoptosis) via mitochondrial permeability transition pores [17]. Accordingly, modification of mitochondrial functions by xenobiotics may contribute to their cytotoxic and antineoplastic activities.
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.11.098
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O
O
(CH2)n
OH
1a
n=1 n=2 n=3
OH
1b 1c 1d
Fig. 1. Chemical structure of the chalcones investigated.
The mitochondrion is the main metabolic sitewhere reactive oxygen species (ROS) are generated. Their administration to various xenobiotics may elevate ROS production [18]. The reactive oxygen species of primary biologic importance include superoxide (O 2 ), hydroxyl radicals (OH ) and hydrogen peroxide (H2O2). Cell damage occurs when there is an increased generation of reactive oxygen species or a relative shortage of antioxidant molecules [19]. Many of the stimuli that lead to apoptosis are associated with the generation of ROS [20]. As a continuation of our previous studies [15] focusing on the effect of chalcones on mitochondrial respiratory functions, in the present study the effect of four hydroxyl substituted chalcone (1a) and chalcone analogues (1b–d) on isolated rat liver mitochondria was investigated. Characterization of mitochondrial respiratory functions on enzyme activities as well as redox status of mitochondria administered to compounds of subcytotoxic doses was investigated. We studied the effect of the compounds on the reduced glutathione (GSH) content of the exposed mitochondria and characterized in vitro reactivity of the compounds with GSH. Since oxidation of hydroxyl-substituted xenobiotics could generate reactive oxygen species (ROS), the compounds could cause oxidative stress as a result of such a series of reactions [18]. To test this possibility, we have investigated time-dependence of in vitro antioxidant (hydroxyl radical scavenger) capacity of the compounds by means of the Fenton-reaction-initiated degradation of 2-deoxyribose [14]. 2. Materials and methods 4-Hydroxychalcone (1a) and its cyclic derivatives (1b–d) were synthesized and purified as described before [21]. Their structures were characterized by IR and NMR spectroscopy. The purity of the compounds was checked by thin layer chromatography (TLC) and gas chromatography (GC) methods. Other chemicals used were of the analytical grade available and, if otherwise not specified, purchased from Sigma–Aldrich or Serva (Heidelberg, Germany). Com-
Detected parameters (%)
80 *
60 40 20 0 1a
-20 -40
*
1c
*
1d
* *
*
-60 -80
1b * *
Hydroxychalcone derivatives state 4
state 3
RCR
Fig. 2. Effect of hydroxychalcone derivatives 1a–d on mitochondrial functions. (Results are calculated as the relative percentage of changes compared to the values of the control group). *Mean, significance P < 0.05.
pounds 1a–d were dissolved in dimethyl sulfoxide (DMSO) immediately before use. Male Wistar rats (Velaz, Praha, Czech Republic) weighing 200– 250 g were used. Adhering to procedures approved by the University of Košice Animal Care and Use Committee, the animals were sacrificed by cervical displacement and decapitation. Liver mitochondria were isolated from male Wistar rats using the method by Johnson and Lardy [22]. Mitochondrial protein was determined according Lowry after Hartree’s modification [23]. Polarographic measurement of oxygen consumption of isolated mitochondria was carried out with a Clark electrode (WTW oxi 325 (Germany)) at the experimental temperature of 25 °C. Measurements were carried out in respiratory medium (80 mM KCl, 300 mM KH2PO4, 300 mM K2HPO4, 15 mM TRIS–HCl, 6 mM MgCl2, 0.78 mM EDTA, pH 7.4) supplemented with 70 nmol of chalcone per mg of mitochondrial protein, 50 mM sodium succinate, 0.5 mM ADP and 20 mg/ml mitochondrial protein. The test compounds were added in dimethyl sulfoxide (DMSO), a dose that did not affect control rates of respiration. The respiratory rate is expressed as oxygen in nmol of oxygen consumed min1 mg1 of mitochondrial protein [24]. Respiratory control ratio (RCR) was determined as a ratio between oxygen consumption in state 3 (with addition of ADP) and state 4 (after exhaustion of ADP). The activity of glutathione reductase (E.C.1.6.4.2) was determined according to Carlberg and Mannervik [25], and glutathione peroxidase (E.C. 1.11.1.9) by the method of Flohe and Gunzler [26]. Reactivity of chalcones 1a–d with reduced glutathione (GSH) was studied by the method described before [15]. The reaction mixtures were kept in water bath (37 °C and 50 °C) and the progress of the reactions was followed by thin layer chromatography (TLC) on Kieselgel F254 plates. The developing solvent used was nbutanol:acetic acid:water (40:10:20, v/v %). Determination of reduced glutathione in mitochondria (GSH) was performed by the modified method of Abukhalaf et al. [27]. HPLC analyses were performed on LC-10ADVP liquid chromatograph (Schimadzu, Kyoto, Japan) equipped with an RF-10AXL fluorescence detector (Schimadzu). The elution gradient profile was as follows: 0 min: 0.5% ACN, 1–5 min: 10% ACN, 6–15 min.: 5% ACN. Investigation of the antioxidant capacity of chalcones 1a–d was performed as described before [14]. Quercetin (200 lM) was used as reference antioxidant. Each value is the average of three independent measurements. Results are presented as mean ± S.D. of at least three independent experiments. Statistical significance was determined by Student’s t-test with P < 0.05 level considered as significant.
3. Results The results of the present study demonstrate that the investigated compounds are able to modify the mitochondrial activity of the cell. Two of the selected compounds (1a and 1b) displayed an inhibitory effect on both the ADP-stimulated respiration (state 3) and the controlled respiration (state 4) (Table 1). The six-membered analogue 1c did not affect either mitochondrial function. On the contrary, compound 1d had no effect on state 3 respiration, however, increased the rate of oxygen consumption after ADP exhaustion (state 4) by 65.0% (Fig. 2). Respiratory control ratio (RCR) was significantly decreased by the compounds 1a, 1b and 1d by 27.1%, 27.1% and 40.5%, respectively (Table 1). Reactivity of the compounds towards cellular thiols was investigated under cell-free conditions. Thin layer chromatographic (TLC) analysis of 1:10 molar mixtures of chalcones and GSH incubated at pH 7.4 or pH 9.0 at 50 °C indicated the formation of
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J. Guzy et al. / FEBS Letters 584 (2010) 567–570 Table 1 Respiratory functions of mitochondria exposed to compounds 1a–d.
Control 1a 1b 1c 1d *
O2 consumption in state 4 (nmol O2 min1 mg1p)
O2 consumption in state 3 (nmol O2 min1 mg1p)
RCR
21.84 ± 2.71 13.43 ± 2.44* 12.20 ± 2.24* 26.60 ± 3.25 36.05 ± 3.78*
53.53 ± 0.35 21.96 ± 2.14* 21.96 ± 2.14* 54.67 ± 3.84 52.86 ± 10.91
2.47 ± 0.32 1.80 ± 0.43* 1.80 ± 0.43* 2.06 ± 0.07 1.47 ± 0.34*
Mean, significance P < 0.05.
adducts that showed characteristics of both the aromatic and the peptide (GSH) moiety of them even after 2-h incubation. Similar adduct formation could be detected when the incubations were carried out at pH 9.0 at 37 °C. On the other hand, no adduct formation could be detected in incubations carried out at pH 7.4 at 37 °C. Determination of reduced glutathione (GSH) content of incubations free of adduct formation did not indicate in vitro redox reaction between the reactants. Determination of reduced glutathione (GSH) levels of mitochondria exposed to the compounds tested was performed by HPLC technique. On exposure of mitochondria to any of the tested compounds resulted in reduction of GSH levels compared to that of the DMSO-treated control experiments. Parallel to the reduction of the cellular GSH levels increased glutathione peroxidase (GPx) and glutathione reductase (GR) activities could be observed (Table 2). Antioxidant capacity and possible pro-oxidant action of the compounds were tested by measurement of time-dependence of Fenton-reaction-initiated degradation of 2-deoxyribose [14]. Fenton-reaction-initiated degradation of 2-deoxyribose may result from an attack of hydroxyl radicals (HO), which results in the formation of TBA-reactive substances with an absorption maximum of 532 nm. As it is shown in Table 2, all the compounds tested displayed antioxidant activity (HO scavenging) at the 20-, 120- and 240-min time points. 4. Discussion The mitochondrial electron transport chain is the source of constantly forming reactive oxygen species [28]. Exogenous sources could lead to increased or decreased production of reactive oxygen species and display a toxic or protective effect, respectively. Attention was paid to select and investigate 40 -hydroxychalcone (1a) and its cyclic analogues (1b–d) due to their possible redox properties that can manifest in an antioxidant/pro-oxidant effect. The results of the present study demonstrate that the investigated compounds are able to modify the energy-linked functions of the cells. In the selected concentrations 1a and 1b displayed an inhibitory effect on ADP-stimulated respiration (state 3) in 59.0% each (Table 1). A decrease in oxygen consumption in the presence of added ADP (state 3) is indicative of the inhibition of
mitochondrial electron transfer chain [29,30]. Since the compounds significantly reduced the controlled respiration (state 4) as well, the decreased respiratory control ration (RCR) suggested a phosphorylation inhibitory effect or partial uncoupling of the compounds (Table 1). This latter effect could be the result of the lipophilic weak acid character of the compounds [31,32]. On the other hand, compounds 1c and 1d, had no effect on state 3 respiration, however, it increased the rate of oxygen consumption (respiratory burst) after ADP exhaustion (state 4) by 21.8% and 67.1%, respectively. Respiratory control ration (RCR) was reduced by 16.6% and 40.5% in the case of 1c and 1d, respectively (Table 1). Since auto-oxidation of the compounds could not be responsible for the observed oxygen consumption, the consumed oxygen should be the substrate of the mitochondrial electron transfer of the mitochondrial activity [29]. Such an effect can increase the mitochondrial level of reactive oxygen species (ROS) that can initiate a series of biochemical events, involving cell death [18,33]. To keep reactive oxygen species production in balance, antioxidant defensive systems such as enzymatic removal of reactive oxygen species and application of potent radical scavengers are active. Several chalcones have been reported to display antioxidant activity [6,7]. On the other hand, a variety of chalcones were found to display antibacterial, antifungal, antiviral, and tumor cytotoxic activities [5–7]. There is also evidence that shows some chalcones to exhibit in vitro mutagenicity [6]. It is presumed that the toxic effects of the compounds might be due to their pro-oxidant activities [5–7]. To test this possibility we have investigated the in vitro antioxidant capacity of the compounds by means of the Fenton-reactioninitiated degradation of 2-deoxyribose. Fenton-reaction-initiated degradation of 2-deoxyribose is thought to be the result of attack of hydroxyl radicals (HO), which results in the formation of TBAreactive substances with an absorption maximum of 532 nm [15]. As shown in Table 2, all the compounds investigated displayed a continuous antioxidant (HO scavenging) activity at the investigated 20-, 120- and 240-min time points; i.e. the compounds reduced the formation of TBA-reactive deoxyribose degradation products. Glutathione peroxidase (GPx) has a well-established role in protecting cells against oxidative injury by giving adaptive response to oxidative stress conditions. GPx is non-specific for hydrogen peroxide and the lack of such substrate specificity results in a range of substrates from hydrogen peroxide to organic hydrogen peroxides [34]. An increase observed in GPx activities may be considered as a result of ROS production that can be accompanied by mitochondrial damage (Table 2). Free radical production is one of the pathogenic mechanisms leading to apoptosis [20]. Cytotoxic effect of a,b-unsaturated carbonyl compounds are frequently associated with the expected reactivity of the compounds with the essential thiol groups in the living organisms [6,7]. To study such reactivity, the compounds were allowed to react with reduced glutathione (GSH), the main soluble cellular thiol,
Table 2 Reduced glutathione (GSH) content, glutathione peroxidase (GPx), glutathione reductase (GR) activity of mitochondria and antioxidant capacity data of compounds 1a–d.
Control 1a 1b 1c 1d Quercetine a *
GSH content of mitochondria (nmol/mgp)
GPx (U/ mgp)
GR (kat/kgp)
Antioxidant capacitya [20 min]
Antioxidant capacitya [120 min]
Antioxidant capacitya [240 min]
11.69 ± 2.56 9.19 ± 2.24 5.62 ± 2.05* 9.67 ± 1.83 6.86 ± 2.88* –
0.43 ± 0.12 0.76 ± 0.13* 0.62 ± 0.21 0.73 ± 0.22 0.69 ± 0.26 –
94.36 ± 12.30 147.50 ± 17.29* 159.72 ± 20.19* 186.00 ± 29.09* 124.62 ± 15.83* –
0.765 ± 0.020 0.708 ± 0.024 0.613 ± 0.008 0.645 ± 0.039 0.610 ± 0.064 0.291 ± 0.010
0.722 ± 0.020 0.637 ± 0.029 0.605 ± 0.018 0.620 ± 0.007 0.589 ± 0.036 0.315 ± 0.08
0.740 ± 0.020 0.693 ± 0.023 0.657 ± 0.020 0.668 ± 0.001 0.598 ± 0.049 0.315 ± 0.024
The antioxidant capacity was determined by degradation on deoxyribose with the initiation of Fenton reaction. Further experimental details are described in ‘‘Section 2”. Mean, significance P < 0.05.
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under cell-free conditions. According to the expectations, each compound showed intrinsic reactivity towards GSH at higher pH values (pH 9.0) and about physiological pH values (pH 7.4) at a higher temperature (50 °C). An analysis of GSH content of the adduct-free incubations did not indicate any measurable in vitro redox reactivity of the compounds towards GSH. In order to investigate whether the compounds can affect the cellular thiol status of the mitochondria, the reduced glutathione (GSH) level was determined. Under cellular conditions chalcones can modify the thiol status by enzyme-catalyzed conjugation and/or by oxidation of the reduced thiols to the respective disulfides [13,35]. On exposure of mitochondria to the compounds substantial reduction of the GSH level compared to the control could be detected in each case (Table 2). The observation could be the consequence of increased formation of ROS and a different glutathione-S-transferase-catalyzed reactivity of the compounds with GSH [35]. Simultaneously with the increased oxidative stress indicated by the increased GPx levels, increased glutathione reductase (GR) activity could also be observed (Table 2). In conclusion, it was demonstrated that compounds 1b and 1d displayed a pronounced effect on the modulation of mitochondrial respiratory functions. Phosphorylation inhibitory effect or partial uncoupling of the compounds resulted in stimulation of mitochondrial activity and increased formation of ROS. Increased formation of ROS and possible covalent interaction of the compounds with cellular thiols resulted in glutathione (GSH) depletion and inhibition or modulation of the investigated mitochondrial activities. The disruption of interconnected mechanisms of the electron transport chain and energetic metabolism in mitochondria, high production of ROS and insufficiency of the antioxidant defensive system could lead to the induction of cell death via the mitochondrial apoptotic pathway [20]. Acknowledgements This study was supported by VEGA grant 1/0624/08 from the Slovak Grant Agency for Science and by the Faculty of Medicine Research Fund, University of Pécs. The authors express special thanks to Zsuzsanna Moravecz and (University of Pécs) for her excellent technical assistance. References [1] Harborne, J.B. (1986) Nature, distribution, and function of plant flavonoids in: Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological and Structure–Activity Relationships (Cody, V., Middleton, E. Jr.Jr. and Harborne, J.B., Eds.), pp. 15–24, Alan R. Liss. Inc., New York. [2] Nakamura, Y., Watanabe, S., Miyake, N., Kohno, H. and Osawa, T. (2003) Dihydrochalcones: evaluation as novel radical scavenging antioxidants. J. Agric. Food Chem. 51, 3309–3312. [3] Alcaraz, M.J., Vicente, A.M., Araico, A., Dominiguez, J.N., Terencio, M.C. and Ferrandiz, M.L. (2004) Role of nuclear factor-jB and heme oxygenase-1 in the mechanism of action of anti-inflammatory chalcone derivate in RAW 264.7 cells. Br. J. Pharmacol. 142, 1191–1199. [4] Kohno, Y., Kitamura, S., Sanoh, S., Sugihara, K., Fujimoto, N. and Ohta, S. (2005) Metabolism of the a,b-unsaturated ketones, chalcone and trans-4-phenyl-3buten-2-one, by rat liver microsomes and estrogenic activity of the metabolites. Drug Metab. Depos. 33, 1115–1123. [5] Middleton Jr., E., Kandaswami, C. and Theoharides, T.C. (2000) The effect of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52, 673–751. [6] Dimmock, J.R., Elias, D.W., Beazely, M.A. and Kandepu, N.M. (1999) Bioactivities of chalcones. Curr. Med. Chem. 6, 1125–1149. [7] Go, M.L., Wu, X. and Liu, X. (2005) Chalcones: an update on cytotoxic and chemopreventive properties. Curr. Med. Chem. 12, 483–499. [8] Dimmock, J.R., Kandepu, N.M., Nazarali, A.J., Kowalchuk, T.P., Motaganahalli, N., Quail, J.W., et al. (1999) Conformational and quantitative structure–activity relationship study of cytotoxic 2-arylidenebenzocycloalkanones. J. Med. Chem. 42, 1358–1366.
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