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Correlation between reduction potentials and inhibitory effects on Epstein–Barr virus activation of poly-substituted anthraquinones
Cancer Letters 225 (2005) 193–198 www.elsevier.com/locate/canlet
Correlation between reduction potentials and inhibitory effects on Epstein–Barr virus activation of poly-substituted anthraquinones Junko Koyamaa,*, Izumi Moritaa, Norihiro Kobayashia, Toshiyuki Osakaib, Hoyoku Nishinoc, Harukuni Tokudac a
Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Higashinada, Kobe 658-8558, Japan b Department of Chemistry, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan c Department of Molecular Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan Received 4 November 2004; received in revised form 11 November 2004; accepted 11 November 2004
Abstract As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the reduction potentials of some poly-substituted anthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the reduction potentials and the inhibitory effects of the poly-substituted anthraquinones on Epstein–Barr virus early antigen activation. It has been further shown that the correlation can be enhanced by introducing log P as an additional parameter. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Epstein–Barr virus; Anthraquinone; Reduction potential; Cyclic voltammetry
1. Introduction Quinones (anthraquinones, naphthoquinones, and heteronaphthoquinones) being widely distributed in nature, are important naturally occurring pigments and known to demonstrate various physiological activities as antibiotics and anti-cancer agents. It has already been shown in previous papers that a number of naphthoquinones had been investigated in vitro * Corresponding author. Tel.: C81 78 441 7549; fax: C81 78 441 7550. E-mail address: [email protected] (J. Koyama).
anti-tumor promoting activity by determining the inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells [1,2]. We have also found inhibitory activities of the mono- and di-substituted anthraquinones and bianthraquinones on EBV-EA activation, and have studied their connections with the electronic properties of the anthraquinones [3,4]. In studies of the structure–activity relationships for drugs, the standard redox potential is an important parameter to determine the physiological activities. We employed cyclic voltammetry to determine
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.11.023
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J. Koyama et al. / Cancer Letters 225 (2005) 193–198
Fig. 1. Structures of anthraquinones.
the standard redox potentials of 9 anthraquinones, 9 naphthoquinones, and 19 azaanthraquinones at physiological pH 7.2, and found definite correlations between the standard redox potentials and the inhibitory effects (log IC50) on EBV-EA activation [5–8]. In the present study, we report the reduction potentials of 16 poly-substituted anthraquinones (Figs. 1 and 2) and the structure–activity relationship between their inhibitory effects and reduction potentials. Furthermore, we have calculated some electronic properties of the anthraquinones by the PM3 method using the CAChe MOPAC program [9], showing that the total energy could be used as another useful parameter to characterize the inhibitory effect on EBV-EA activation. It has also been found that the logarithm of the octanol–water partition coefficient (log P) [10] of the anthraquinones is one of the useful parameters for investigating their structure–activity relationship.
2. Material and methods 2.1. Reagents and materials Chrysophanol (1), emodin (2), obtusifolin (3), chryso-obtusin (4), obtusin (5), aurantio-obtusin (6), questin (7), and 1-hydroxy-8-methoxy-6-methylanthraquinone (8) were isolated from the seed of Cassia obtusifolia L. [11] and 1,7-dihydroxy-6,8-dimethoxy2-methylanthraquinone (9) from Galium spurium var. echinospermon Hayek [12]. Aloe-emodin (10) was isolated from the leaves of Aloe ferox Miller [13], and cassiamin C (11), cassiamin A (12), cassiamin B (13), 1,1 0 ,3,8,8 0 -pentahydroxy-3 0 ,6-dimethyl-[2,2 0 -bianthracene]-9,9 0 ,10,10 0 -tetrone (14) from Cassia siamea [14]. 1,1 0 ,8,8 0 -tetrahydroxy-3,3 0 -dihydroxymethyl-[2,2 0 -bianthracene]-9,9 0 ,10,10 0 -tetrone (15) and 1,1 0 ,3 0 ,8,8 0 -pentahydroxy-3-hydroxymethyl-6 0 methyl[2,2 0 -bianthracene]-9,9 0 ,10,10 0 -tetrone (16)
J. Koyama et al. / Cancer Letters 225 (2005) 193–198
195
control experiments with n-butyric acid (4 mM) plus TPA (32 pmol), in which EA induction was usually around 40%. 2.3. Electrochemical measurements
Fig. 2. Structures of bianthrquinones.
were derived from cascaroside A by Lemli’s method [15]. The tissue culture reagents, TPA, n-butyric acid and other reagents, were from Nacalai Tesque. The EBV-genome-carrying lymphoblastoid cells (Raji cells derived from Burkitt’s lymphoma) were cultured in RPMI 1640 medium (SIGMA, R8758), as described elsewhere [16]. Spontaneous activation of EBV-EA in our subline of Raji cells was less than 0.1%.
Cyclic voltammetry was performed by a conventional three-electrode system using a laboratoryconstructed microcomputer-controlled system, in which a potentiostat (Hokuto Denko, HA-301) was used for controlling the working electrode potential. A plastic-formed-carbon (PFC) electrode of a surface areaZ0.071 cm2 (BAS, PFCE-3), an Ag/AgCl (saturated NaCl) electrode, and a platinum coil electrode were used as the working, reference, and counter electrodes, respectively. Before recording each voltammogram, pretreatment of the working electrode was carried out as previously described [5,6]. The test solution was a 2:1 (v/v) phosphate buffer (pH 7.2)–ethanol solution containing 0.1 mM of a quinone derivative, and was degassed with prepurified N2 gas prior to the voltammetric measurements. The electrolytic cell was water-jacketed to maintain the temperature at 25G0.1 8C. Table 1 Inhibitory effects of poly-substituted anthraquinones on EBV-EA activation Compound
2.2. Procedure of EBV-EA activation The inhibition of the EBV-EA activation was assayed using Raji cells (virus non-producer), which were cultivated in 10% FBS RPMI 1640 medium. The indicator cells (Raji) (1!106 ml) were incubated at 37 8C for 48 h in 1 ml of the medium containing nbutyric acid (4 mM, inducer), 2 ml of TPA (20 ng/ml in DMSO), and a known amount of test compound dissolved in DMSO. Smears were made from the cell suspension. The activated cells were stained by hightiter EBV-EA positive sera from nasopharyngeal carcinoma (NPC) patients and detected by a conventional indirect immunofluorescence technique [17]. In each assay, at least 500 cells were counted, and the experiments were repeated three times. The average EA induction was compared with that of the positive
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% to control (% viability)
log IC50a
Molar ratio (to 32 pmol TPA) 1000
500
100
10
11.3 (60) 1.4 (70) 0 (60) 0 (70) 12.6 (60) 0 (60) 14.4 (60) 20.2 (70) 0 (70) 9.4 (70) 30.7 (70) 22.5 (70) 16.8 (70) 26.1 (70) 20.3 (70) 22.5 (70)
35.6 24.5 30.6 63.8 43.7 55.9 45.9 71.5 19.6 32.6 68.4 60.6 36.0 65.5 60.2 61.1
80.4 50.4 68.1 92.5 90.7 91.3 83.7 92.7 70.6 77.4 89.7 83.4 79.4 86.2 81.5 83.5
100 90.1 100 100 100 100 100 100 93.7 100 100 100 100 100 100 100
2.683 2.471 2.601 2.733 2.724 2.716 2.724 2.822 2.560 2.656 2.861 2.796 2.703 2.830 2.774 2.797
a The molar ratio of test compound to TPA giving 50% inhibition against a positive control (100%) was defined as IC50.
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When the voltage scan rate was increased (e.g. up to 200 mV sK1), the second cathodic peak accompanied with an anodic peak became larger than the first cathodic peak, and therefore the peak pair observed at more negative potentials should be considered as an adsorption wave due to the reduction–oxidation reactions of a quinone derivative adsorbed at the electrode surface. On the other hand, the first reduction peak was usually proportional to the square root of the scan rate, showing that the peak was limited mainly by diffusion of a quinone derivative. Unfortunately, however, the corresponding anodic
Fig. 3. Cyslic voltammograms of anthraquinone 4 and 16 at a PFC electrode in 2:1 (v/v) 0.1 M phosphate buffer (pH 7.2)–EtOH. Voltage scan rate: 20 mV sK1.
2.4. Correlation coefficient The correlations of electrochemical and electronic parameters with EBV-EA activation of the anthraquinones were determined by using Pearson’s correlation coefficient.
3. Results and discussion Seven poly-substituted anthraquinones (3–9) were tested for their inhibitory activities using a short-term in vitro assay of the EBV-EA activation induced by TPA in Raji cells. The inhibitory effects of compounds 1, 2, and 10–16 were already reported [4]. Their inhibitory effects on activation of the EBV-EA and the log IC50 values are shown in Table 1. Anthraquinone monomers showed higher inhibitory effects than that of bianthraquinones except 8 and 13. Methylation of the phenolic hydroxyl group leads to a decrease of potency (1/8, 2/7, 6/5/4). Oxidation of phenyl ring enhances the activation (1/2, 8/3, 1/10). Cyclic voltammograms of some anthraquinone derivatives, which were obtained at 20 mV sK1, are shown in Fig. 3. As represented by the voltammogram for compound 4, poly-substituted anthraquinones except for compounds 11 and 16 had two cathodic (negative-current) peaks and one anodic (positive-current) peak. The cathodic and anodic peak potentials are summarized in Table 2.
Table 2 Epc, Epa values of poly-substituted anthraquinones Compound
EpcK1(V)
EpcK2 (V)
Epa(V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
K0.425 K0.470 K0.445 K0.403 K0.393 K0.385 K0.390 K0.378 K0.410 K0.405 K0.372 K0.386 K0.405 K0.410 K0.415 K0.405
K0.605 K0.652 K0.602 K0.584 K0.650 K0.665 K0.623 K0.558 K0.636 K0.591 ND* K0.600 K0.615 K0.628 K0.655 ND
K0.594 K0.258 K0.580 K0.554 K0.642 K0.270 K0.612 K0.548 K0.615 K0.580 ND K0.194 K0.240 K0.205 K0.230 ND
ND, not detected.
Fig. 4. Correlation of first reduction potential at pH 7.2 of anthraquinones with their EBV-EA activation. Plot of log IC50 against EpcK1.
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Table 3 log IC50 and Epc, Epa values of anthraquinones Compound
log IC50
EpcK1(V)
EpcK2 (V)
Epa(V)
Anthraquinone 1-Hydroxyanthraquinone 1,8-Dihydroxyanthraquinone 2,6-Dihydroxyanthraquinone 2-Methylanthraquinone 2-Ethylanthraquinone 1-Hydroxy-2-methylanthraquinone
3.037 2.770 2.778 2.875 2.643 2.684 2.704
K0.375 K0.390 K0.435 K0.400 K0.435 K0.405 K0.425
K0.595 K0.597 K0.600 K0.690 K0.582 K0.580 K0.638
K0.505 K0.579 K0.582 K0.615 K0.562 K0.544 K0.623
peak was not detected for all compounds tested, probably because of the instability of the reduction products in solution. Accordingly, we have employed the first cathodic peak potentials at 20 mV sK1 (EpcK1), which could be considered to reflect the ‘thermo-dynamic’ redox potentials, and examined their connections with the EBV-EA activation. In Fig. 4, the values of log IC50 are plotted versus the reduction potentials EpcK1 (in V). The plot shows that there is a certain correlation between the values of log IC50 and EpcK1. The log IC50 has been represented by a regression line log IC50 Z 4:034 C3:247 EpcK1 ðn Z 16; r Z 0:773Þ (1) where n and r are, respectively, the number of test compounds and the correlation coefficient. Thus, it has been revealed that the first reduction potential at pH 7.2 is an important parameter determining the EBV-EA activation of the poly-substituted anthraquinones. The more negative the EpcK1 of the anthraquinones, the stronger the anti-tumor promoting effect. This is the case for anthraquinone and some mono- and di-substituted anthraquinones (Table 3). By including these compounds in the regression analysis, we have ascertained some definite correlation between log IC50 and EpcK1 for the 23 anthraquinones, the regression equation being log IC50Z4.135C3.436 EpcK1 (nZ23, rZ0.703). Furthermore, we examined the correlation of log IC50 with the electronic properties of the polysubstituted anthraquinones. Table 4 shows the electronic properties including LUMO energy, total energy, and steric energy. Among these electronic properties, the LUMO energy shows no correlation with log IC50. However, the total energy demonstrates
some definite correlation with log IC50. As also shown in Table 4, there is a very small correlation of the steric energy with log IC50. In studies of the structure– activity relationships, log P has so far been used as a useful parameter representative of molecular hydrophobicity. The log P values have been calculated for the anthraquinone compounds using the atom typing scheme of Ghose and Crippen [10]. A small correlation of log P and log IC50 was then obtained. The lower log P value of the compounds leads to more potent activity. Thus, we introduced log P as an additional parameter, and performed a regression analysis. Then, it has been found that the log IC50 can Table 4 Electronic properties and log P values of poly-substituted anthraquinones Compound
LUMO
Steric energy (kcal/mole)
Total energy (hartree)
log P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 ra(with IC50)
K1.673 K1.642 K1.382 K1.164 K1.416 K1.336 K1.392 K1.377 K1.458 K1.792 K1.703 K1.700 K1.687 K1.700 K1.948 K1.879 0.187
K21.65 K23.96 K18.31 K7.77 K11.08 K14.98 K20.48 K18.14 K18.55 K25.30 K54.74 K62.27 K62.35 K60.19 K63.83 K70.44 0.497
K139.01 K151.00 25.00 K158.35 K204.22 K215.20 K223.37 K146.14 K150.06 K151.20 K276.65 K288.88 K301.11 K288.88 K301.00 K301.05 0.611
2.358 2.073 2.105 1.631 1.600 1.568 2.105 2.389 1.852 1.355 4.353 4.069 3.785 4.069 2.348 3.066 0.561
a
r, correlation coefficient.
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be expressed by the following equation much better than Eq. (1): log IC50Z3.761C2.831 EpcK1C0.041 log P ðn Z 16; r Z 0:860Þ
(2)
In conclusion, the first reduction potential of polysubstituted anthraquinones determined at the physiological pH 7.2 is a quite useful parameter for the estimation of the inhibitory effects on EBV-EA activation. The log P value would also be useful as an additional parameter.
Acknowledgements This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan, and also from the National Cancer Institute (CA 17625), USA.
References [1] T. Konoshima, M. Kozuka, J. Koyama, T. Okatani, K. Tagahara, H. Tokuda, Studies on inhibitors of skin tumor promotion, VI. Inhibitory effects of quinones on Epstein–Barr virus activation, J. Nat. Prod. 52 (1989) 987–995. [2] G.J. Kapadia, V. Balasubramanian, H. Tokuda, T. Konoshima, M. Takasaki, J. Koyama, et al., Anti-tumor promoting effects of naphthoquinone derivatives on short term Epstein–Barr early antigen activation assay and in mouse skin carcinogenesis, Cancer Lett. 113 (1997) 47–53. [3] K. Tagahara, J. Koyama, T. Ogura, T. Konoshima, M. Kozuka, H. Tokuda, et al., Electronic properties and inhibitory effects on Epstein–Barr virus activation of mono- and di-substituted anthraquinones, Chem. Express 7 (1992) 557–560. [4] J. Koyama, I. Morita, K. Tagahara, M. Ogata, T. Mukainaka, H. Tokuda, H. Nishino, Inhibitory effects of anthraquinones and bianthraquinones on Epstein–Barr virus activation, Cancer Lett. 170 (2001) 15–18.
[5] J. Koyama, K. Tagahara, T. Osakai, Y. Tsujino, S. Tsurumi, H. Nishino, H. Tokuda, Inhibitory effects on Epstein–Barr virus activation of anthraquinones: correlation with redox potentials, Cancer Lett. 115 (1997) 179–183. [6] J. Koyama, I. Morita, K. Tagahara, T. Osakai, H. Hotta, M.X. Yang, et al., Correlation with redox potentials and inhibitory effects on Epstein–Barr virus activation of azaanthraquinones, Chem. Pharm. Bull. 49 (2001) 1214–1216. [7] J. Koyama, I. Morita, N. Kobayashi, T. Osakai, H. Hotta, J. Takayasu, et al., Correlation of redox potentials and inhibitory effects on Epstein–Barr virus activation of naphthoquinones, Cancer Lett. 201 (2003) 25–30. [8] J. Koyama, I. Morita, N. Kobayashi, T. Osakai, H. Hotta, J. Takayasu, et al., Correlation of redox potentials and inhibitory effects on Epstein–Barr virus activation of naphthoquinones, Cancer Lett. 212 (2004) 1–6. [9] J.J.P. Stewart, Optimization of parameters for semi-empirical methods. I. Method (CAChe scientific, Computer-aided chemistry from the Oxford Molecular Group), J. Comput. Chem. 10 (1989) 209–220. [10] A.K. Ghose, A. Pritchett, G.M. Crippen, Atomic physicochemical parameters for three dimensional structure directed quantitative structure–activity relationships III: modeling hydrophobic interactions, J. Comput. Chem. 9 (1988) 80–90. [11] M. Takido, Studies on the constituents of the seeds of Cassia obtusifolia L. II. The structure of obtusin, Chem. Pharm. Bull. 8 (1960) 246–251. [12] J. Koyama, T. Ogura, K. Tagahara, Anthraquinones from Galium spurium, Phytochemistry 33 (1993) 1540–1542. [13] J. Koyama, T. Ogura, K. Tagahara, Naphtho[2,3- c]furan-4,9dione and its derivatives from Aloe ferox, Phytochemistry 37 (1994) 1147–1148. [14] J. Koyama, I. Morita, K. Tagahara, M. Aqil, Bianthraquinones from Cassia siamea, Phytochemistry 56 (2001) 849–851. [15] P. Witte, J. Cuveele, J. Lemli, Bicascarosides in fluid extract of Cascara, Planta Med. 57 (1991) 440–443. [16] Y. Ito, S. Yanase, J. Fujita, T. Harayama, M. Takashima, H. Imanaka, A short-term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein–Barr virus, Cancer Lett. 13 (1981) 29–37. [17] G. Henle, W. Henle, Immunofluorescence in cells derived from Brukitt’s lymphoma, J. Bacteriol. 91 (1966) 1248–1256.
Correlation between reduction potentials and inhibitory effects on Epstein–Barr virus activation of poly-substituted anthraquinones Junko Koyamaa,*, Izumi Moritaa, Norihiro Kobayashia, Toshiyuki Osakaib, Hoyoku Nishinoc, Harukuni Tokudac a
Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Higashinada, Kobe 658-8558, Japan b Department of Chemistry, Faculty of Science, Kobe University, Nada, Kobe 657-8501, Japan c Department of Molecular Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan Received 4 November 2004; received in revised form 11 November 2004; accepted 11 November 2004
Abstract As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the reduction potentials of some poly-substituted anthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the reduction potentials and the inhibitory effects of the poly-substituted anthraquinones on Epstein–Barr virus early antigen activation. It has been further shown that the correlation can be enhanced by introducing log P as an additional parameter. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Epstein–Barr virus; Anthraquinone; Reduction potential; Cyclic voltammetry
1. Introduction Quinones (anthraquinones, naphthoquinones, and heteronaphthoquinones) being widely distributed in nature, are important naturally occurring pigments and known to demonstrate various physiological activities as antibiotics and anti-cancer agents. It has already been shown in previous papers that a number of naphthoquinones had been investigated in vitro * Corresponding author. Tel.: C81 78 441 7549; fax: C81 78 441 7550. E-mail address: [email protected] (J. Koyama).
anti-tumor promoting activity by determining the inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells [1,2]. We have also found inhibitory activities of the mono- and di-substituted anthraquinones and bianthraquinones on EBV-EA activation, and have studied their connections with the electronic properties of the anthraquinones [3,4]. In studies of the structure–activity relationships for drugs, the standard redox potential is an important parameter to determine the physiological activities. We employed cyclic voltammetry to determine
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.11.023
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J. Koyama et al. / Cancer Letters 225 (2005) 193–198
Fig. 1. Structures of anthraquinones.
the standard redox potentials of 9 anthraquinones, 9 naphthoquinones, and 19 azaanthraquinones at physiological pH 7.2, and found definite correlations between the standard redox potentials and the inhibitory effects (log IC50) on EBV-EA activation [5–8]. In the present study, we report the reduction potentials of 16 poly-substituted anthraquinones (Figs. 1 and 2) and the structure–activity relationship between their inhibitory effects and reduction potentials. Furthermore, we have calculated some electronic properties of the anthraquinones by the PM3 method using the CAChe MOPAC program [9], showing that the total energy could be used as another useful parameter to characterize the inhibitory effect on EBV-EA activation. It has also been found that the logarithm of the octanol–water partition coefficient (log P) [10] of the anthraquinones is one of the useful parameters for investigating their structure–activity relationship.
2. Material and methods 2.1. Reagents and materials Chrysophanol (1), emodin (2), obtusifolin (3), chryso-obtusin (4), obtusin (5), aurantio-obtusin (6), questin (7), and 1-hydroxy-8-methoxy-6-methylanthraquinone (8) were isolated from the seed of Cassia obtusifolia L. [11] and 1,7-dihydroxy-6,8-dimethoxy2-methylanthraquinone (9) from Galium spurium var. echinospermon Hayek [12]. Aloe-emodin (10) was isolated from the leaves of Aloe ferox Miller [13], and cassiamin C (11), cassiamin A (12), cassiamin B (13), 1,1 0 ,3,8,8 0 -pentahydroxy-3 0 ,6-dimethyl-[2,2 0 -bianthracene]-9,9 0 ,10,10 0 -tetrone (14) from Cassia siamea [14]. 1,1 0 ,8,8 0 -tetrahydroxy-3,3 0 -dihydroxymethyl-[2,2 0 -bianthracene]-9,9 0 ,10,10 0 -tetrone (15) and 1,1 0 ,3 0 ,8,8 0 -pentahydroxy-3-hydroxymethyl-6 0 methyl[2,2 0 -bianthracene]-9,9 0 ,10,10 0 -tetrone (16)
J. Koyama et al. / Cancer Letters 225 (2005) 193–198
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control experiments with n-butyric acid (4 mM) plus TPA (32 pmol), in which EA induction was usually around 40%. 2.3. Electrochemical measurements
Fig. 2. Structures of bianthrquinones.
were derived from cascaroside A by Lemli’s method [15]. The tissue culture reagents, TPA, n-butyric acid and other reagents, were from Nacalai Tesque. The EBV-genome-carrying lymphoblastoid cells (Raji cells derived from Burkitt’s lymphoma) were cultured in RPMI 1640 medium (SIGMA, R8758), as described elsewhere [16]. Spontaneous activation of EBV-EA in our subline of Raji cells was less than 0.1%.
Cyclic voltammetry was performed by a conventional three-electrode system using a laboratoryconstructed microcomputer-controlled system, in which a potentiostat (Hokuto Denko, HA-301) was used for controlling the working electrode potential. A plastic-formed-carbon (PFC) electrode of a surface areaZ0.071 cm2 (BAS, PFCE-3), an Ag/AgCl (saturated NaCl) electrode, and a platinum coil electrode were used as the working, reference, and counter electrodes, respectively. Before recording each voltammogram, pretreatment of the working electrode was carried out as previously described [5,6]. The test solution was a 2:1 (v/v) phosphate buffer (pH 7.2)–ethanol solution containing 0.1 mM of a quinone derivative, and was degassed with prepurified N2 gas prior to the voltammetric measurements. The electrolytic cell was water-jacketed to maintain the temperature at 25G0.1 8C. Table 1 Inhibitory effects of poly-substituted anthraquinones on EBV-EA activation Compound
2.2. Procedure of EBV-EA activation The inhibition of the EBV-EA activation was assayed using Raji cells (virus non-producer), which were cultivated in 10% FBS RPMI 1640 medium. The indicator cells (Raji) (1!106 ml) were incubated at 37 8C for 48 h in 1 ml of the medium containing nbutyric acid (4 mM, inducer), 2 ml of TPA (20 ng/ml in DMSO), and a known amount of test compound dissolved in DMSO. Smears were made from the cell suspension. The activated cells were stained by hightiter EBV-EA positive sera from nasopharyngeal carcinoma (NPC) patients and detected by a conventional indirect immunofluorescence technique [17]. In each assay, at least 500 cells were counted, and the experiments were repeated three times. The average EA induction was compared with that of the positive
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
% to control (% viability)
log IC50a
Molar ratio (to 32 pmol TPA) 1000
500
100
10
11.3 (60) 1.4 (70) 0 (60) 0 (70) 12.6 (60) 0 (60) 14.4 (60) 20.2 (70) 0 (70) 9.4 (70) 30.7 (70) 22.5 (70) 16.8 (70) 26.1 (70) 20.3 (70) 22.5 (70)
35.6 24.5 30.6 63.8 43.7 55.9 45.9 71.5 19.6 32.6 68.4 60.6 36.0 65.5 60.2 61.1
80.4 50.4 68.1 92.5 90.7 91.3 83.7 92.7 70.6 77.4 89.7 83.4 79.4 86.2 81.5 83.5
100 90.1 100 100 100 100 100 100 93.7 100 100 100 100 100 100 100
2.683 2.471 2.601 2.733 2.724 2.716 2.724 2.822 2.560 2.656 2.861 2.796 2.703 2.830 2.774 2.797
a The molar ratio of test compound to TPA giving 50% inhibition against a positive control (100%) was defined as IC50.
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When the voltage scan rate was increased (e.g. up to 200 mV sK1), the second cathodic peak accompanied with an anodic peak became larger than the first cathodic peak, and therefore the peak pair observed at more negative potentials should be considered as an adsorption wave due to the reduction–oxidation reactions of a quinone derivative adsorbed at the electrode surface. On the other hand, the first reduction peak was usually proportional to the square root of the scan rate, showing that the peak was limited mainly by diffusion of a quinone derivative. Unfortunately, however, the corresponding anodic
Fig. 3. Cyslic voltammograms of anthraquinone 4 and 16 at a PFC electrode in 2:1 (v/v) 0.1 M phosphate buffer (pH 7.2)–EtOH. Voltage scan rate: 20 mV sK1.
2.4. Correlation coefficient The correlations of electrochemical and electronic parameters with EBV-EA activation of the anthraquinones were determined by using Pearson’s correlation coefficient.
3. Results and discussion Seven poly-substituted anthraquinones (3–9) were tested for their inhibitory activities using a short-term in vitro assay of the EBV-EA activation induced by TPA in Raji cells. The inhibitory effects of compounds 1, 2, and 10–16 were already reported [4]. Their inhibitory effects on activation of the EBV-EA and the log IC50 values are shown in Table 1. Anthraquinone monomers showed higher inhibitory effects than that of bianthraquinones except 8 and 13. Methylation of the phenolic hydroxyl group leads to a decrease of potency (1/8, 2/7, 6/5/4). Oxidation of phenyl ring enhances the activation (1/2, 8/3, 1/10). Cyclic voltammograms of some anthraquinone derivatives, which were obtained at 20 mV sK1, are shown in Fig. 3. As represented by the voltammogram for compound 4, poly-substituted anthraquinones except for compounds 11 and 16 had two cathodic (negative-current) peaks and one anodic (positive-current) peak. The cathodic and anodic peak potentials are summarized in Table 2.
Table 2 Epc, Epa values of poly-substituted anthraquinones Compound
EpcK1(V)
EpcK2 (V)
Epa(V)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
K0.425 K0.470 K0.445 K0.403 K0.393 K0.385 K0.390 K0.378 K0.410 K0.405 K0.372 K0.386 K0.405 K0.410 K0.415 K0.405
K0.605 K0.652 K0.602 K0.584 K0.650 K0.665 K0.623 K0.558 K0.636 K0.591 ND* K0.600 K0.615 K0.628 K0.655 ND
K0.594 K0.258 K0.580 K0.554 K0.642 K0.270 K0.612 K0.548 K0.615 K0.580 ND K0.194 K0.240 K0.205 K0.230 ND
ND, not detected.
Fig. 4. Correlation of first reduction potential at pH 7.2 of anthraquinones with their EBV-EA activation. Plot of log IC50 against EpcK1.
J. Koyama et al. / Cancer Letters 225 (2005) 193–198
197
Table 3 log IC50 and Epc, Epa values of anthraquinones Compound
log IC50
EpcK1(V)
EpcK2 (V)
Epa(V)
Anthraquinone 1-Hydroxyanthraquinone 1,8-Dihydroxyanthraquinone 2,6-Dihydroxyanthraquinone 2-Methylanthraquinone 2-Ethylanthraquinone 1-Hydroxy-2-methylanthraquinone
3.037 2.770 2.778 2.875 2.643 2.684 2.704
K0.375 K0.390 K0.435 K0.400 K0.435 K0.405 K0.425
K0.595 K0.597 K0.600 K0.690 K0.582 K0.580 K0.638
K0.505 K0.579 K0.582 K0.615 K0.562 K0.544 K0.623
peak was not detected for all compounds tested, probably because of the instability of the reduction products in solution. Accordingly, we have employed the first cathodic peak potentials at 20 mV sK1 (EpcK1), which could be considered to reflect the ‘thermo-dynamic’ redox potentials, and examined their connections with the EBV-EA activation. In Fig. 4, the values of log IC50 are plotted versus the reduction potentials EpcK1 (in V). The plot shows that there is a certain correlation between the values of log IC50 and EpcK1. The log IC50 has been represented by a regression line log IC50 Z 4:034 C3:247 EpcK1 ðn Z 16; r Z 0:773Þ (1) where n and r are, respectively, the number of test compounds and the correlation coefficient. Thus, it has been revealed that the first reduction potential at pH 7.2 is an important parameter determining the EBV-EA activation of the poly-substituted anthraquinones. The more negative the EpcK1 of the anthraquinones, the stronger the anti-tumor promoting effect. This is the case for anthraquinone and some mono- and di-substituted anthraquinones (Table 3). By including these compounds in the regression analysis, we have ascertained some definite correlation between log IC50 and EpcK1 for the 23 anthraquinones, the regression equation being log IC50Z4.135C3.436 EpcK1 (nZ23, rZ0.703). Furthermore, we examined the correlation of log IC50 with the electronic properties of the polysubstituted anthraquinones. Table 4 shows the electronic properties including LUMO energy, total energy, and steric energy. Among these electronic properties, the LUMO energy shows no correlation with log IC50. However, the total energy demonstrates
some definite correlation with log IC50. As also shown in Table 4, there is a very small correlation of the steric energy with log IC50. In studies of the structure– activity relationships, log P has so far been used as a useful parameter representative of molecular hydrophobicity. The log P values have been calculated for the anthraquinone compounds using the atom typing scheme of Ghose and Crippen [10]. A small correlation of log P and log IC50 was then obtained. The lower log P value of the compounds leads to more potent activity. Thus, we introduced log P as an additional parameter, and performed a regression analysis. Then, it has been found that the log IC50 can Table 4 Electronic properties and log P values of poly-substituted anthraquinones Compound
LUMO
Steric energy (kcal/mole)
Total energy (hartree)
log P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 ra(with IC50)
K1.673 K1.642 K1.382 K1.164 K1.416 K1.336 K1.392 K1.377 K1.458 K1.792 K1.703 K1.700 K1.687 K1.700 K1.948 K1.879 0.187
K21.65 K23.96 K18.31 K7.77 K11.08 K14.98 K20.48 K18.14 K18.55 K25.30 K54.74 K62.27 K62.35 K60.19 K63.83 K70.44 0.497
K139.01 K151.00 25.00 K158.35 K204.22 K215.20 K223.37 K146.14 K150.06 K151.20 K276.65 K288.88 K301.11 K288.88 K301.00 K301.05 0.611
2.358 2.073 2.105 1.631 1.600 1.568 2.105 2.389 1.852 1.355 4.353 4.069 3.785 4.069 2.348 3.066 0.561
a
r, correlation coefficient.
198
J. Koyama et al. / Cancer Letters 225 (2005) 193–198
be expressed by the following equation much better than Eq. (1): log IC50Z3.761C2.831 EpcK1C0.041 log P ðn Z 16; r Z 0:860Þ
(2)
In conclusion, the first reduction potential of polysubstituted anthraquinones determined at the physiological pH 7.2 is a quite useful parameter for the estimation of the inhibitory effects on EBV-EA activation. The log P value would also be useful as an additional parameter.
Acknowledgements This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan, and also from the National Cancer Institute (CA 17625), USA.
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