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Cancer chemopreventive activity of serratane-type triterpenoids on two-stage mouse skin carcinogenesis
Cancer Letters 196 (2003) 121–126 www.elsevier.com/locate/canlet
Cancer chemopreventive activity of serratane-type triterpenoids on two-stage mouse skin carcinogenesis Reiko Tanakaa,*, Toshifumi Minamia, Yohei Ishikawaa, Shunyo Matsunagaa, Harukuni Tokudab, Hoyoku Nishinob a
Department of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan b Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan Received 10 January 2003; received in revised form 21 February 2003; accepted 3 March 2003
Abstract Eleven serratane-type triterpenoids isolated from the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. var. jezoensis (Pinaceae) and the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. var. hondoensis (Mayer) Rehder (Pinaceae) and three synthetic analogs were studied for their possible inhibitory effects on Epstein– Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). 21-Episerratenediol, serratenediol, diepiserratenediol, 3b-hydroxyserrat-14-en-21-one, 3a-methoxy-21b-hydroxyserrat-14-en-16-one, 3b-methoxyserrat-14-en-21b-yl acetate, 3a-methoxyserrat-14-en-21b-yl acetate and 3b-methoxyserrat-14-en-21a-yl acetate demonstrated strong inhibitory effects on the EBV-EA activation without showing any cytotoxicity, their effects being stronger than that of a representative control, oleanolic acid. Furthermore, 21-episerratenediol exhibited a remarkable inhibitory effect on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a ]anthracene as an initiator and TPA as a promoter. The result of the present investigation indicated that 21-episerratenediol might be valuable as a potent cancer chemopreventive agent. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Picea jezoensis (Sieb. et Zucc.) Carr. jezoensis; Picea jezoensis (Sieb. et Zucc.) Carr. hondoensis (Mayer); Pinaceae; Anti-tumor promoter; Epstein–Barr virus; Two-stage carcinogenesis; 21-Episerratenediol; Serratane; Triterpenoid; Structure –activity relationship
1. Introduction Cancer chemoprevention is regarded as one of the efficient strategies to prevent cancer [1]. In the course of a search for biologically active constituents from the leaves, bark and cones of coniferous trees, which had been treated as wastes in the forestry industry, we * Corresponding author. Tel.: þ81-72-690-1084; fax: þ 81-72690-1084. E-mail address: [email protected] (R. Tanaka).
previously found some compounds showing significant anti-tumor promoting activities in an in vivo two-stage mouse-skin carcinogenesis assay using 7,12-dimethyl-benz[a ] anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA): abiesenonic acid methyl, a chemical derivative of abieslactone isolated from the stem bark of Abies species (Pinaceae) [2], 15,16-bisnor-13-oxolabda-8(17),11Edien-19-oic acid [3] and 15-oxolabda-8(17),11Z,13Etrien-19-oic acid [4], diterpenoids isolated from the stem bark of Thuja standishii (Cupressaseae), and
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0304-3835(03)00214-3
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l3a,14a-epoxy-3b-methoxyserratan-21b-ol and 21ahydroxy-3b-methoxyserrat-14-en-29-al [5], 14b,15bepoxy-3b-methoxyserratan-21b-ol [6] triteroenoids from the bark of Picea jezoensis Carr. var. jezoensis (Pinaceae), and Picea jezoensis Carr. var. hondoensis (Mayer) Rehder (Pinaceae). In the course of our continuing study, we have focused on D13 and D14-serratane-type triterpenoids and their analogs, 21-episerratenediol (1) [7], serratenediol (2) [7], serrat-14-en-3,21-dione (3) [8], 3bmethoxyserrat-14-en-21-one (4) [9], 21a-methoxyserrat-13-en-3,15-dione (5) [8] from P. jezoensis Carr. var. jezoensis, and diepiserratenediol (6) [10], 21 b-hydroxyserrat- 14-en-3-one (7) [10], 3b-hydroxyserrat-14-en-21-one (8) [11], 3a-methoxyserrat-14en-21-one (9) [11], 3a-methoxy-21b-hydroxyserrat14-en-16-one (10) [12] and 29-nor-3b-methoxyserrat14-en-21-one (11) [13] from P. jezoensis Carr. var. hondoensis along with three synthetic derivatives, 3b-methoxyserrat-14-en-21b-yl acetate (12), 3amethoxyserrat-14-en-21b-yl acetate (13), and 3bmethoxyserrat-14-en-21a-yl acetate (14) (Fig. 1). This report deals with the results of in vitro and in vivo anti-tumor promoting activities of the above compounds (1 – 14). The assay methods employed were an in vitro assay estimating the inhibitory effect on Epstein – Barr virus antigen (EBV-EA) activation induced by TPA [14] and an in vivo two-stage mouseskin carcinogenesis assay using DMBA as an initiator and TPA as a promoter [15].
2. Materials and methods 2.1. Chemicals The cell culture reagents, n-butyric acid and other reagents for the bioassay were purchased from Nacalai tesque, Inc. (Kyoto, Japan). TPA and DMBA were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Test products The natural serratane-type triterpenoids, 21-episerratenediol (1), serratenediol (2) [10], serrat-14-en3,21-dione (3), 3b-methoxyserrat-14-en-21-one (4), 21a-methoxyserrat-13-en-3,15-dione (5) were iso-
lated from the stem bark of Picea jezoensis Carr. var. jezoensis (Pinaceae), which was collected at Sapporo City, Japan. The isolation and characterization of compounds 1– 5 have been reported in the literature [7 – 9]. A voucher specimen was deposited at the Department of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences. The natural serratane-type triterpenoids, diepiserratenediol (6), 21b-hydroxyserrat-14-en-3-one (7), 3b-hydroxyserrat-14-en-21-one (8), 3a-methoxyserrat-14-en-21-one (9), 3a-methoxy-21b-hydroxyserrat-14-en-16-one (10) and 29-nor-3b-methoxyserrat14-en-21-one (11) were isolated from the stem bark of Picea jezoensis Carr. var. hondoensis (Pinaceae), which was collected at Gifu prefecture, Japan. The isolation and characterization of compounds 6 –11 have been reported [10 – 13]. A voucher specimen was deposited at the Department of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences. Three synthetic analogs, 3b-methoxyserrat-14-en21b-yl acetate (12), 3a methoxyserrat-14-en-21b-yl acetate (13), 3b-methoxyserrat-14-en-21a-yl acetate (14), were prepared as previously reported [7]. The chemical structures are shown in Fig. 1 and Table 1, respectively. Compounds 1 –14 had purities of over 99%.
2.3. Inhibition of EBV-EA activation assay The inhibition of EBV-EA activation was assayed according to Ito et al. [14]. The EBV-EA inhibiting activity of the test compounds were estimated on the basis of the percentage of the number of positive cells compared with that of a control without the test compound. The viability of the cells was assayed against treated cells using the Trypan-Blue staining method.
2.4. Two-stage mouse-skin carcinogenesis test Female ICR mice (6 weeks old) were used. Animals were divided into three experimental groups containing 15 mice each. The back of each mouse was shaved with surgical clippers, and the mice were painted with 390 nmol of DMBA in 0.1 ml acetone. One week after, the mice were treated topically
R. Tanaka et al. / Cancer Letters 196 (2003) 121–126
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Fig. 1. Structures of serratane triterpenoids from Picea jezoensis Carr. var. jezoensis and Picea jezoensis Carr. var. hondoensis and synthetic analogs.
with 1.7 nmol TPA in 0.1 ml acetone twice a week for 20 weeks. One hour before each treatment with TPA, the mice were treated with the 85 nmol of sample in 0.1 ml acetone, or 0.1 ml acetone alone, which served as a positive control. The incidence of papillomas was examined weekly over a period of 20 weeks.
3. Results and discussion Compounds 1– 5 isolated from Picea jezoensis Carr. var. jezoensis and compounds 6– 11 isolated from Picea jezoensis Carr. var. hondoensis and synthetic derivatives 12 – 14 were evaluated for
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Table 1 Relative ratioa of EBV-EA activation with respect to positive control (100%) in the presence of compounds 1–14 Compounds
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Oleanolic acid a b c
% to control (% viability) 1000 mol ratio/TPAbc
500 mol ratio/TPAb
100 mol ratio/TPAb
10 mol ratio/TPAb
0 (70) 0 (70) 5.9 (70) 6.4 (70) 1.6 (70) 0 (70) 0 (70) 0 (70) 6.0 (70) 2.1 (60) 2.3 (70) 0 (70) 0 (70) 0 (70) 12.7 (70)
18.2 20.2 33.6 35.4 33.4 23.1 29.6 30.4 34.1 27.9 30.1 29.3 28.2 29.1 30.0
72.1 75.0 83.2 82.6 87.9 75.7 80.1 79.5 81.3 78.3 79.1 79.8 74.1 79.6 80.0
87.0 89.5 100.0 100.0 100.0 93.7 100.0 96.8 100.0 97.9 100.0 95.7 93.4 94.5 100.0
Values represent percentages relative to the positive control value (100%). TPA concentration was 20 ng/ml (32 pmol/ml). Values in parentheses are the viability percentages of Raji cells.
in vitro inhibitory activity against EBV-EA activation induced by TPA. All compounds exhibited dosedependent inhibitory activities, and the viability percentages of Raji cells treated with the test compounds (1 –14) were 70% at the highest concentration of 1000 mol ratio/TPA, suggesting that the cytotoxicities of all compounds were considerably moderate against in vitro cell lines (Table 1). As shown in Table 1, the inhibitory activities of 21-episerratenediol (1), serratenediol (2), diepiserratenediol (6), 3b-hydroxyserrat-14-en-21-one (8), 3a-methoxy-21b-hydroxyserrat-14-en-21b-ol (10), 3b-methoxyserrat-14-en-21b-yl acetate (12), 3amethoxyserrat-14-en-21b-yl acetate (13) and 3b-methoxyserrat-14-en-21a-yl acetate (14) were stronger at every concentration than that of oleanolic acid [16], known as a representive anti-tumor promoting agent, and compounds 1, 2 and 6 exhibited comparatively stronger inhibitory activities than others (8, 10, 12, 13, 14). The relative ratio of compound 1 with respect to TPA (100%) was 0, 18.2, 72.1 and 87.0% at the concentrations of 1000, 500, 100 and 10 mol ratio/ TPA, respectively (Table 1); meaning 100, 81.8, 27.9 and 13.0% inhibition of the EBV-EA activation by TPA, respectively, and compounds 2 and 6 showed 100, 79.8, 25.0 and 10.5%, and 100, 76.9, 24.3 and
6.3% inhibition of the EBV-EA activation by TPA, respectively, at concentrations of 1000, 500, 100 and 10 mol ratio/TPA. In conclusion, in studies of the inhibitory effects on EBV-EA activation of 11 natural and three synthetic serratane-type triterpenoids, compounds 1, 2 and 6 displayed the most potent activities. While compounds 3 – 5 and 9 were a little weaker than 1, 2, 6 and the positive control, oleanolic acid (70% inhibition at 500 mol ratio/TPA). It is interesting to note that the presence of two secondary OH groups at C-3 and C-21 were important to enhance the anti-tumor promoting activity. The inhibitory activities in the EBV-EA activation test of compounds 1, 2 and 6 were significantly stronger than those of 12 oleanene-type, five lupane-type, 23 hopane-type and 21 cucurbitane-type triterpenoids from ferns of Adiantum, Dryopteris, Polypodium, Oleandra genera and rhizomes of Hemsleya panacis-scandens, H. canosiflora and the leaves and branches of Cowania mexicana reported by Konoshima et al. [17,18]. Based on the results obtained in vitro, we selected compound 1 to examine the effect on the in vivo twostage carcinogenesis test focusing on mouse skin papillomas induced by DMBA as an initiator and TPA as a promoter. During the in vivo assay,
R. Tanaka et al. / Cancer Letters 196 (2003) 121–126
the body-weight gains of the mice were not influenced by the treatment with the test compound and no toxic effects, such as lesional damages and inflammation (edema, erosion and ulcer) were observed on the areas of mouse skin topically treated with the test compound. Figs. 2 and 3 demonstrate the results of the papilloma formation in the skin of mice treated with compound 1. The papilloma-bearing mice in the positive control group treated with DMBA (390 nmol) and TPA (1.7 nmol, twice/week) appeared as early as week 6, and the percentages of the papilloma-bearers increased rapidly to reach 100% after week 10. On the other hand, the treatment with compound 1 (85 nmol) along with DMBA/TPA inhibited the formation of papillomas until week 11 and the first papilloma appeared as late as at week 12. The percentage of papilloma-bearers in the mice of this group was 46.6% over the period of week 20. As shown in Fig. 3, in the positive control group with DMBA/TPA, the number of papillomas formed per mouse increased rapidly after week 6 to reach 9.2 papillomas/mouse at week 20, whereas the mice treated with compound 1 bore only 2.0 papillomas even at week 20. These results suggested that the inhibitory effect of
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Fig. 3. Inhibition of TPA-induced tumor promotion by multiple application of 21-episerratenediol (1). All mice were initiated with DMBA (390 nmol) and promoted with 1.7 nmol of TPA, given twice weekly starting 1 week after initiation. Average number of papillomas per mouse. † control (TPA alone); A TPA þ 85 nmol of 1. aStatiscally different from the positive control (P , 0.01).
compound 1 on two-stage carcinogenesis test was stronger than that of glycyrretic acid [19]. Thus, 21episerratenediol (1) can be considered to become an appropriate lead compound to develop more potent agents with anti-tumor promoting activity for clinical use. The mechanism studied in this inhibitory activity is now in progress.
Acknowledgements The authors are grateful to Mr Kiyoshi Matsubara (Green Ace Co. Ltd, Hidaka town, Hokkaido, Japan) for the supply of the plant material. This study was supported by a Grant-in-Aid for High Technology from the Ministry of Education, Science, Sports and Culture, Japan.
Fig. 2. Inhibition of TPA-induced tumor promotion by multiple application of 21-episerratenediol (1). All mice were initiated with DMBA (390 nmol) and promoted with 1.7 nmol of TPA, given twice weekly starting 1 week after initiation. Percentage of mice bearing papillomas. † control (TPA alone); A TPA þ 85 nmol of 1. aStatiscally different from the positive control (P , 0.05).
References [1] I. Berenblum, The mechanism of carcinogenesis, Cancer Res 1 (1941) 807 –814. [2] J. Takayasu, R. Tanaka, S. Matsunaga, H. Ueyama, H. Tokuda, T. Hasegawa, et al., Anti-tumor-promoting activity of
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[12] R. Tanaka, C. Mun, Y. Usami, S. Matsunaga, 3-Oxo-serratene triterpenoids from the stem bark of Picea jezoensis Can. hondoensis, Phytochemistry 35 (1994) 1517–1522. [13] R. Tanaka, K. Tsujimoto, S. Matsunaga, Two serratane triterpenes from the stem bark of Picea jezoensis var. hondoensis, Phytochemistry 52 (1999) 1581–1585. [14] Y. Ito, M. Kawanishi, T. Harayama, S. Takabayashi, Combined effect of the extracts from Croton tiglium, Euphorbia lathyris or Euphorbia tirucalli and n-butyrate on Epstein– Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells, Cancer Lett 12 (1981) 175–180. [15] H. Tokuda, K. Koshimizu, Inhibitory effects of ursolic acid and oleanolic acid on skin tumor promotion 12-O-tetradecanoylphorbol-13-acetate, Cancer Lett 33 (1986) 279 –285 [16] T. Konoshima, M. Takasaki, M. Kozuka, H. Tokuda, Studies on inhibitors on skin-tumor promotion I. Inhibitory effects of triterpenes from Euphorbia polyandra on Epstein–Barr virus activation, J Nat Prod 50 (1987) 1167–1170. [17] T. Konoshima, M. Takasaki, H. Tokuda, K. Masuda, Y. Arai, K. Shiojima, et al., Anti-tumor-promoting activities of triterpenoids from Ferns I, Biol Pharm Bull 19 (1996) 962 –965. [18] T. Konoshima, M. Takasaki, T. Tatsumoto, M. Kozuka, R. Kasai, O. Tanaka, et al., Inhibitory effects of cucurbitane triterpenoids on Epstein–Barr virus activation and two-stage carcinogenesis of skin tumors, Biol Pharm Bull 17 (1994) 668 –671. [19] H. Nishino, K. Yoshioka, A. Iwashima, H. Takizawa, S. Konishi, H. Okamoto, et al., Glycynetic acid inhibits tumorpromoting activity of teleocidin and 12-O-tetradecanoylphorbol-13-acetate in two-stage mouse skin carcinogenesis, Jpn J Cancer Res (Gann) 77 (1986) 33– 38.
Cancer chemopreventive activity of serratane-type triterpenoids on two-stage mouse skin carcinogenesis Reiko Tanakaa,*, Toshifumi Minamia, Yohei Ishikawaa, Shunyo Matsunagaa, Harukuni Tokudab, Hoyoku Nishinob a
Department of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan b Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan Received 10 January 2003; received in revised form 21 February 2003; accepted 3 March 2003
Abstract Eleven serratane-type triterpenoids isolated from the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. var. jezoensis (Pinaceae) and the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. var. hondoensis (Mayer) Rehder (Pinaceae) and three synthetic analogs were studied for their possible inhibitory effects on Epstein– Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). 21-Episerratenediol, serratenediol, diepiserratenediol, 3b-hydroxyserrat-14-en-21-one, 3a-methoxy-21b-hydroxyserrat-14-en-16-one, 3b-methoxyserrat-14-en-21b-yl acetate, 3a-methoxyserrat-14-en-21b-yl acetate and 3b-methoxyserrat-14-en-21a-yl acetate demonstrated strong inhibitory effects on the EBV-EA activation without showing any cytotoxicity, their effects being stronger than that of a representative control, oleanolic acid. Furthermore, 21-episerratenediol exhibited a remarkable inhibitory effect on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a ]anthracene as an initiator and TPA as a promoter. The result of the present investigation indicated that 21-episerratenediol might be valuable as a potent cancer chemopreventive agent. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Picea jezoensis (Sieb. et Zucc.) Carr. jezoensis; Picea jezoensis (Sieb. et Zucc.) Carr. hondoensis (Mayer); Pinaceae; Anti-tumor promoter; Epstein–Barr virus; Two-stage carcinogenesis; 21-Episerratenediol; Serratane; Triterpenoid; Structure –activity relationship
1. Introduction Cancer chemoprevention is regarded as one of the efficient strategies to prevent cancer [1]. In the course of a search for biologically active constituents from the leaves, bark and cones of coniferous trees, which had been treated as wastes in the forestry industry, we * Corresponding author. Tel.: þ81-72-690-1084; fax: þ 81-72690-1084. E-mail address: [email protected] (R. Tanaka).
previously found some compounds showing significant anti-tumor promoting activities in an in vivo two-stage mouse-skin carcinogenesis assay using 7,12-dimethyl-benz[a ] anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA): abiesenonic acid methyl, a chemical derivative of abieslactone isolated from the stem bark of Abies species (Pinaceae) [2], 15,16-bisnor-13-oxolabda-8(17),11Edien-19-oic acid [3] and 15-oxolabda-8(17),11Z,13Etrien-19-oic acid [4], diterpenoids isolated from the stem bark of Thuja standishii (Cupressaseae), and
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0304-3835(03)00214-3
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l3a,14a-epoxy-3b-methoxyserratan-21b-ol and 21ahydroxy-3b-methoxyserrat-14-en-29-al [5], 14b,15bepoxy-3b-methoxyserratan-21b-ol [6] triteroenoids from the bark of Picea jezoensis Carr. var. jezoensis (Pinaceae), and Picea jezoensis Carr. var. hondoensis (Mayer) Rehder (Pinaceae). In the course of our continuing study, we have focused on D13 and D14-serratane-type triterpenoids and their analogs, 21-episerratenediol (1) [7], serratenediol (2) [7], serrat-14-en-3,21-dione (3) [8], 3bmethoxyserrat-14-en-21-one (4) [9], 21a-methoxyserrat-13-en-3,15-dione (5) [8] from P. jezoensis Carr. var. jezoensis, and diepiserratenediol (6) [10], 21 b-hydroxyserrat- 14-en-3-one (7) [10], 3b-hydroxyserrat-14-en-21-one (8) [11], 3a-methoxyserrat-14en-21-one (9) [11], 3a-methoxy-21b-hydroxyserrat14-en-16-one (10) [12] and 29-nor-3b-methoxyserrat14-en-21-one (11) [13] from P. jezoensis Carr. var. hondoensis along with three synthetic derivatives, 3b-methoxyserrat-14-en-21b-yl acetate (12), 3amethoxyserrat-14-en-21b-yl acetate (13), and 3bmethoxyserrat-14-en-21a-yl acetate (14) (Fig. 1). This report deals with the results of in vitro and in vivo anti-tumor promoting activities of the above compounds (1 – 14). The assay methods employed were an in vitro assay estimating the inhibitory effect on Epstein – Barr virus antigen (EBV-EA) activation induced by TPA [14] and an in vivo two-stage mouseskin carcinogenesis assay using DMBA as an initiator and TPA as a promoter [15].
2. Materials and methods 2.1. Chemicals The cell culture reagents, n-butyric acid and other reagents for the bioassay were purchased from Nacalai tesque, Inc. (Kyoto, Japan). TPA and DMBA were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Test products The natural serratane-type triterpenoids, 21-episerratenediol (1), serratenediol (2) [10], serrat-14-en3,21-dione (3), 3b-methoxyserrat-14-en-21-one (4), 21a-methoxyserrat-13-en-3,15-dione (5) were iso-
lated from the stem bark of Picea jezoensis Carr. var. jezoensis (Pinaceae), which was collected at Sapporo City, Japan. The isolation and characterization of compounds 1– 5 have been reported in the literature [7 – 9]. A voucher specimen was deposited at the Department of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences. The natural serratane-type triterpenoids, diepiserratenediol (6), 21b-hydroxyserrat-14-en-3-one (7), 3b-hydroxyserrat-14-en-21-one (8), 3a-methoxyserrat-14-en-21-one (9), 3a-methoxy-21b-hydroxyserrat-14-en-16-one (10) and 29-nor-3b-methoxyserrat14-en-21-one (11) were isolated from the stem bark of Picea jezoensis Carr. var. hondoensis (Pinaceae), which was collected at Gifu prefecture, Japan. The isolation and characterization of compounds 6 –11 have been reported [10 – 13]. A voucher specimen was deposited at the Department of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences. Three synthetic analogs, 3b-methoxyserrat-14-en21b-yl acetate (12), 3a methoxyserrat-14-en-21b-yl acetate (13), 3b-methoxyserrat-14-en-21a-yl acetate (14), were prepared as previously reported [7]. The chemical structures are shown in Fig. 1 and Table 1, respectively. Compounds 1 –14 had purities of over 99%.
2.3. Inhibition of EBV-EA activation assay The inhibition of EBV-EA activation was assayed according to Ito et al. [14]. The EBV-EA inhibiting activity of the test compounds were estimated on the basis of the percentage of the number of positive cells compared with that of a control without the test compound. The viability of the cells was assayed against treated cells using the Trypan-Blue staining method.
2.4. Two-stage mouse-skin carcinogenesis test Female ICR mice (6 weeks old) were used. Animals were divided into three experimental groups containing 15 mice each. The back of each mouse was shaved with surgical clippers, and the mice were painted with 390 nmol of DMBA in 0.1 ml acetone. One week after, the mice were treated topically
R. Tanaka et al. / Cancer Letters 196 (2003) 121–126
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Fig. 1. Structures of serratane triterpenoids from Picea jezoensis Carr. var. jezoensis and Picea jezoensis Carr. var. hondoensis and synthetic analogs.
with 1.7 nmol TPA in 0.1 ml acetone twice a week for 20 weeks. One hour before each treatment with TPA, the mice were treated with the 85 nmol of sample in 0.1 ml acetone, or 0.1 ml acetone alone, which served as a positive control. The incidence of papillomas was examined weekly over a period of 20 weeks.
3. Results and discussion Compounds 1– 5 isolated from Picea jezoensis Carr. var. jezoensis and compounds 6– 11 isolated from Picea jezoensis Carr. var. hondoensis and synthetic derivatives 12 – 14 were evaluated for
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Table 1 Relative ratioa of EBV-EA activation with respect to positive control (100%) in the presence of compounds 1–14 Compounds
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Oleanolic acid a b c
% to control (% viability) 1000 mol ratio/TPAbc
500 mol ratio/TPAb
100 mol ratio/TPAb
10 mol ratio/TPAb
0 (70) 0 (70) 5.9 (70) 6.4 (70) 1.6 (70) 0 (70) 0 (70) 0 (70) 6.0 (70) 2.1 (60) 2.3 (70) 0 (70) 0 (70) 0 (70) 12.7 (70)
18.2 20.2 33.6 35.4 33.4 23.1 29.6 30.4 34.1 27.9 30.1 29.3 28.2 29.1 30.0
72.1 75.0 83.2 82.6 87.9 75.7 80.1 79.5 81.3 78.3 79.1 79.8 74.1 79.6 80.0
87.0 89.5 100.0 100.0 100.0 93.7 100.0 96.8 100.0 97.9 100.0 95.7 93.4 94.5 100.0
Values represent percentages relative to the positive control value (100%). TPA concentration was 20 ng/ml (32 pmol/ml). Values in parentheses are the viability percentages of Raji cells.
in vitro inhibitory activity against EBV-EA activation induced by TPA. All compounds exhibited dosedependent inhibitory activities, and the viability percentages of Raji cells treated with the test compounds (1 –14) were 70% at the highest concentration of 1000 mol ratio/TPA, suggesting that the cytotoxicities of all compounds were considerably moderate against in vitro cell lines (Table 1). As shown in Table 1, the inhibitory activities of 21-episerratenediol (1), serratenediol (2), diepiserratenediol (6), 3b-hydroxyserrat-14-en-21-one (8), 3a-methoxy-21b-hydroxyserrat-14-en-21b-ol (10), 3b-methoxyserrat-14-en-21b-yl acetate (12), 3amethoxyserrat-14-en-21b-yl acetate (13) and 3b-methoxyserrat-14-en-21a-yl acetate (14) were stronger at every concentration than that of oleanolic acid [16], known as a representive anti-tumor promoting agent, and compounds 1, 2 and 6 exhibited comparatively stronger inhibitory activities than others (8, 10, 12, 13, 14). The relative ratio of compound 1 with respect to TPA (100%) was 0, 18.2, 72.1 and 87.0% at the concentrations of 1000, 500, 100 and 10 mol ratio/ TPA, respectively (Table 1); meaning 100, 81.8, 27.9 and 13.0% inhibition of the EBV-EA activation by TPA, respectively, and compounds 2 and 6 showed 100, 79.8, 25.0 and 10.5%, and 100, 76.9, 24.3 and
6.3% inhibition of the EBV-EA activation by TPA, respectively, at concentrations of 1000, 500, 100 and 10 mol ratio/TPA. In conclusion, in studies of the inhibitory effects on EBV-EA activation of 11 natural and three synthetic serratane-type triterpenoids, compounds 1, 2 and 6 displayed the most potent activities. While compounds 3 – 5 and 9 were a little weaker than 1, 2, 6 and the positive control, oleanolic acid (70% inhibition at 500 mol ratio/TPA). It is interesting to note that the presence of two secondary OH groups at C-3 and C-21 were important to enhance the anti-tumor promoting activity. The inhibitory activities in the EBV-EA activation test of compounds 1, 2 and 6 were significantly stronger than those of 12 oleanene-type, five lupane-type, 23 hopane-type and 21 cucurbitane-type triterpenoids from ferns of Adiantum, Dryopteris, Polypodium, Oleandra genera and rhizomes of Hemsleya panacis-scandens, H. canosiflora and the leaves and branches of Cowania mexicana reported by Konoshima et al. [17,18]. Based on the results obtained in vitro, we selected compound 1 to examine the effect on the in vivo twostage carcinogenesis test focusing on mouse skin papillomas induced by DMBA as an initiator and TPA as a promoter. During the in vivo assay,
R. Tanaka et al. / Cancer Letters 196 (2003) 121–126
the body-weight gains of the mice were not influenced by the treatment with the test compound and no toxic effects, such as lesional damages and inflammation (edema, erosion and ulcer) were observed on the areas of mouse skin topically treated with the test compound. Figs. 2 and 3 demonstrate the results of the papilloma formation in the skin of mice treated with compound 1. The papilloma-bearing mice in the positive control group treated with DMBA (390 nmol) and TPA (1.7 nmol, twice/week) appeared as early as week 6, and the percentages of the papilloma-bearers increased rapidly to reach 100% after week 10. On the other hand, the treatment with compound 1 (85 nmol) along with DMBA/TPA inhibited the formation of papillomas until week 11 and the first papilloma appeared as late as at week 12. The percentage of papilloma-bearers in the mice of this group was 46.6% over the period of week 20. As shown in Fig. 3, in the positive control group with DMBA/TPA, the number of papillomas formed per mouse increased rapidly after week 6 to reach 9.2 papillomas/mouse at week 20, whereas the mice treated with compound 1 bore only 2.0 papillomas even at week 20. These results suggested that the inhibitory effect of
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Fig. 3. Inhibition of TPA-induced tumor promotion by multiple application of 21-episerratenediol (1). All mice were initiated with DMBA (390 nmol) and promoted with 1.7 nmol of TPA, given twice weekly starting 1 week after initiation. Average number of papillomas per mouse. † control (TPA alone); A TPA þ 85 nmol of 1. aStatiscally different from the positive control (P , 0.01).
compound 1 on two-stage carcinogenesis test was stronger than that of glycyrretic acid [19]. Thus, 21episerratenediol (1) can be considered to become an appropriate lead compound to develop more potent agents with anti-tumor promoting activity for clinical use. The mechanism studied in this inhibitory activity is now in progress.
Acknowledgements The authors are grateful to Mr Kiyoshi Matsubara (Green Ace Co. Ltd, Hidaka town, Hokkaido, Japan) for the supply of the plant material. This study was supported by a Grant-in-Aid for High Technology from the Ministry of Education, Science, Sports and Culture, Japan.
Fig. 2. Inhibition of TPA-induced tumor promotion by multiple application of 21-episerratenediol (1). All mice were initiated with DMBA (390 nmol) and promoted with 1.7 nmol of TPA, given twice weekly starting 1 week after initiation. Percentage of mice bearing papillomas. † control (TPA alone); A TPA þ 85 nmol of 1. aStatiscally different from the positive control (P , 0.05).
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