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Inhibitory effect of heliantriol C; a component of edible Chrysanthemum, on tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin
Phytomedicine, Vol. 5(3), pp. 215-218 © Gustav Fischer Verlag 1998
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I~~~~!~mml
Inhibitory effect of heliantriol C; a component of edible Chrysanthemum, on tumor promotion by 12-0-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin K. Yasukawa', T. Akihisa", Y. Kasahara', M. Ukiya'', K. Kumaki', T. Tamura", S. Yamanouchi! and M. Takido 1 lCollege of Pharmacy, Nihon University, Narashinodai Funabashi-shi, Chiba, japan 2College of Science and Technology, Nihon University, Kanda Surugadai, Chiyoda-ku, Tokyo, japan 3The Yamagata Prefectural Institute of Public Health, Tokamachi, Yamagata, japan
Summary Two hydroxy taraxastane-type triterpenes, faradiol and heliantriol C, have been isolated from the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino, the edible Chrysanthemum. These compounds showed strong inhibitory activity against 12-0-tetradecanoylphorbol-13-aetate (TPA)-induced inflammation in mice. At 0.2 umol/mouse, these compounds markedly inhibited the promoting effect of TPA (1 ug/mouse) on skin tumor formation followed by 7,12-dimethylbenz[a]anthracene (50 ug/mouse), Key words: Antitumor promoter, two-stage carcinogenesis, 12-0-tetradecanoylphorbol-13-acetate, heliantriol C, faradiol, taraxastane-type triterpene, Chrysanthemum Asteraceae.
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Introduction
Chrysanthemum is a bitter, aromatic herb that experimentally lowers fevers, soothes inflammation, dilates the coronary arteries (increasing blood flow to the heart), and inhibits the growth of pathogens. It is used in folkmisumia for hypertension, coronary artery disease, angina, feverish colds, and liver-related disorders. A methanol extract from the ligulate flowers of the edible chrysanthemum inhibited 12-0-tetradecanoylphorbol13-acetate (TPA)-induced inflammatory ear edema (our unpublished data). In our previous studies, two taraxastane-type triterpenes, taraxasterol from the flowers of Cynara cardunculus, cardoon, and faradiol from the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino, the edible Chrysanthemum, were demonstrated to inhibit the tumor-promoting activity of TPA in two-stage carcinogenesis in mouse skin, and in the group treated with 7,12dimethylbenz[a]anthracene (DMBA) plus TPA and fa-
radiol (2.0 umol/rnouse), no tumors appeared at week 20 (Yasukawa et aI., 1996b). Taraxastane-type di- and tri-hydroxytriterpenes inhibited TPA-induced inflammatory ear edema (Yasukawa et al., 1996c). Heliantriol C showed strong inhibitory activity against TPA-induced inflammation in mice; the 50% inhibitory dose of heliantriol C was 30 ug/ear similar to the 30 ug/ear dose of hydrocortisone (Yasukawa et al., 1996c). Heliantriol C had about ten times the activity of the other triterpene derivatives on TPA-induced .inflammation in mice (Yasukawa et a!., 1988; 1991; 1994b; 1995). Furthermore, in this study, topical application of faradiol (0.2 urnol/rnouse) and heliantriol C (0.2 umol/mouse) markedly inhibited the promotion of skin tumors induced by twice-weekly application of TPA following initiation by a single application of DMBA in mice. It is interestingto know that hydroxylated faradiol is more effective against skin carcinogenesis.
K. Yasukawa et al.
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Fig. 1. The chemical structures of faradiol and heliantriol C.
HO
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Materials and methods
Chemicals
Faradiol and heliantriol C were isolated from the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino and were identified by chromatographic and spectroscopic comparison with authentic samples (Yasukawa et al., 1996c). DMBA and dimethylsulfoxide were purchased from Sigma Chemical (St Louis, Mo., U.S.A.); TPA was obtained from Chemicals for Cancer Research Inc. (Minneapolis, Minn., U.S.A.). Animals
Female ICR mice (7 weeks old) were obtained from the Japan SLC Inc. (Shizuoka, Japan). The animals were housed in an air-conditioned specific pathogen free room (22-23°C), with light from 8.00 to 20.00 h. Food and water were available ad libitum. Two-stage carcinogenesis experiment
The backs of mice (7 weeks old) were shaved with electric clippers. Initiation was accomplished by a single topical application of 50 ug DMBA. Promotion with 1 ug TPA, applied twice weekly, was begun 1 week after the initiation. Faradiol (0.2 umol) and heliantriol C (0.2 umol), or their vehicle, acetone-dimethylsulfoxide (9:1; 100 ul), were applied topically 30 min before each TPA treatment. DMBA and TPA were dissolved in acetone, and applied to the shaved area in a volume of 100 ul using a micropipette. The back of each animal was shaved once a week to remove hair. The number and diameter of skin tumors were measured every other week, and the experiment was continued for 20 weeks. Experimental and appropriate control groups each consisted of 15 mice.
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Results
Fig. 2A shows the time courses of skin tumor formation in the group treated with DMBA plus TPA, with or without faradiol and heliantriol C. The first tumor appeared at week 8 in the group treated with DMBA plus
TPA. In the group treated with DMBA plus TPA and faradiol and heliantriol C, the first tumor appeared at weeks 10 and 13, respectively. The proportion of tumor-bearing mice treated with DMBA plus TPA was 93% at week 20, whereas the proportions in the groups treated with DMBA plus TPA and faradiol, and heliantriol C were 40% and 20%, respectively. Figure 2B shows the average number of tumors per mouse. The group treated with DMBA plus TPA produced 8.6 tumors per mouse at week 20, whereas the group treated with DMBA plus TPA and faradiol and heliantriol C had 2.9 and 0.9 tumors per mouse, respectively. The treatment with faradiol and heliantriol C caused 66% and 90% reductions, respectively, in the average number of tumors per mouse at week 20.
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Discussion
A series of naturally occurring sterol and triterpene derivatives have been found to possess antitumor-promoting activities (Kaminaga et al., 1996; Kasahara et al., 1994; Yasukawa et al., 1988; 1991; 1994a; 1994b; 1995; 1996a; 1996b). The methanol extract of the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino, the edible flowers in Japan, has been reported to inhibit inflammatory ear edema induced by TPA, and various triterpenes were isolated from the unsaponifiable lipids of the methanol extract (Yasukawa et al., 1996b; 1996c). Heliantriol C had the strongest activity among these triterpenes, and faradiol consituted the main triterpene in the extract. In this study, we found that heliantriol C and faradiol markedly inhibited tumor promotion by TPA in DMBA-initiated mice. The inhibitory effect of 0.2 umol of these compounds corresponded to those of 2.0 umol of other sterols and triterpenes such as stigmasterol, ergosterol, ergosterol peroxide, lupeol, lupe01 acetate, betulin, betulinic acid, and karounidiol; therefore, these compounds had about ten times the activity of the other sterols and triterpenes on tumor promotion induced by TPA in mouse skin (Kasahara et al., 1994; Yasukawa et al., 1994a; 1994b; 1995; 1996a; 1996b). Heliantriol C was more effective than faradiol,
Inhibitory effect of heliantriol C on tumor promotion
217
B Fig. 2. Inhibitory effect of faradiol and heliA antriol C on the promotion of skin papillo10 r - - - - - - - - - - , 100 mas by TPA in DMBA-initiated mice. From 1 week after initiation by a single topical application of 50 ug of DMBA, 1 [-lg of TPA was applied twice weekly. Topical application of 0.2 urnol faradiol, heliantriol C and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). The treated groups were statistically o 6ooiOQCltoOctaOc>O-.....L..----l different from the control group by 15 20 10 5 20 10 15 5 o Student's t test (p <0.05 = heliantriol C treated group: after weeks 11-13, faradiol Weeks of promotion Weeks of promotion treated group: after weeks 11-16; p <0.01 = heliantriol C treated group: after week 14, faradiol treated group: after week 17-20). Heliantriol C treated group was statistically different from the faradiol treated group by Student's t test (p <0.05: after week 16-20). • = +TPA with vehicle alone; 0 = +TPA with heliantriol C; t::,. = +TPA with faradio!.
which was almost comparable with that of the lanos-
tane-type triterpene acids, pachymic acid, 3-0-acetyl16a-hydroxytrametenolic acid and poricoic acid B on tumor promotion by TPA in two-stage carcinogenesis in mouse skin (Kaminaga et al., 1996). Thus faradiol (taraxast-Zu-ene-Jp.Lep-diol) showed a marked inhibitory effect and further hydroxylation of faradiol at C22 giving heliantriol C (taraxast-Zu-ene-Jp.Iep.Zzctriol) markedly enhanced the effect. There was a close relationship between the hydroxylation of triterpenes and the inhibitory effect. Our previous studies demonstrated that extracts from edible plants and fungi inhibit TPA-induced inflammation in mice, and sterols and triterpenes have been indentified by us as the active compounds separated from an edible mushroom Hypsizigus marrnoreus (Yasukawa et al., 1994a), safflower (Kasahara et al., 1994), an edible alga Chlorella vulgaris (Yasukawa et al., 1996a) and stevia (Yasukawa et al., 1993). Furthermore, these sterols and triterpenes have been shown to inhibit tumor promotion during twostage carcinogenesis in mouse skin. Other researchers have reported similar effects for triterpenes (Diallo et al., 1995; Konoshima et al., 1992; 1994; 1995; Nishino et al., 1986; 1988; Takayasu et al., 1990; Tokuda et al., 1986; 1991; Wrang et al., 1991; Yu et al., 1992; 1996). Many sterols and triterpenes, widely distributed in edible plants and fungi, inhibit the tumor-promoting activity of TPA in mouse skin, and this suggests that they may be of importance for cancer chemoprevention. Acknowledgements
This work was supported in part by an Interdisciplinary Joint Research Grant from Nihon University. We wish to thank Dr.
Tomio Takeuchi and Dr. Mieko Takeuchi, Institute of Microbial Chemistry, for their kind encouragements during the course of this study.
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References
Diallo, B., Vanhaelen-Fastre, R., Vanhaelen, M., Konoshima, T, Takasaki, M., Tokuda, H.: In vivo inhibitory effects of arjinolic acid derivatives on two-stage carcinogenesis in mouse skin. Phytother. Res. 9: 444-447,1995. Kaminaga, T., Yasukawa, Kanno, H., Tai, T, Nunoura, Y., Takido, M.: Inhibitory effects of lanostane-type triterpene acids, the components of Poria cocos, on tumor promotion by 12-0-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin. Oncology 53: 382-385, 1996. Kasahara, Y., Kumaki, K., Katagiri, 5., Yasukawa, K., Takido, M., Akihisa, T., Tamura, T.: Carthami Flos extract and its component, stigmasterol, inhibit tumor promotion in mouse skin two-stage carcinogenesis. Phytother. Res. 8: 327-331,1994. Konoshima, T., Kokumai, M., Kozuka, M., Tokuda, H., Nishino, H., Iwashima, A.: Antitumorpramoting activities of afromosin and soyasaponin I isolated from Wistaria brachybotrys. J. Nat. Prod. 55: 1776 -1778, 1992. Konoshima, T, Takasaki, M., Kozuka, M., Nagao, T., Okabe, H., Irino, N., Nakasumi, T, Tokuda, H., Nishino, H.: Inhibitory effects of cucurbitane triterpenoids on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumor. II. Bioi. Pharm. Bull. 18: 284-287,1995. Konoshima, T., Takasaki, M., Tatsumoto, T., Kozuka, M., Kasai, R., Takano, 0., Nie, R. L., Tokuda, H., Nishino, H., Iwashima, A.: Inhibitory effects of cucurbitane triterpenoids on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors. Bioi. Pharm. Bull. 17: 668-671, 1994. Konoshima, T., Takasaki, M., Tokuda, H., Masuda, K., Arai,
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Y., Shiojima, K., Ageta, H .: Anti-tumo r-promoting activities of triterpenoids from ferns. I. Bioi. Pharm. Bull. 19: (962 - 965, 1996. Nish ino, H ., Nishino, A., Takayasu, J., Hasegawa, T., Iwashima, A., H irabayashi, K., Iwata, S., Shibata,S.: Inhibition of the tumor-promoting action of 12-0-tetradecanoylphorbol-13-acetate by some oleanane -type triter penoid compounds. Cancer Res. 48: 5210 -5215, 1988 . Nishino, H., Yoshioka, K., Iwashima, A., Takizawa, H., Konishi,S., Okamoto, H., Okabe, H., Shibata,S., Fujiki, H., Sugimura, T.: Glycyrrhet inic acid inhibits tumor-promoting 'a ctivity of .releocidin and 12-0-tetradecanoylphor bol-13, acetate. Jpn.]. Cancer Res. 77: 33 - 38, 1986 . Takayasu, J., Tanaka, R., Mats unaga,S., Ueyama, H., Tokuda, H., Hasegawa, T., Nishino, H. , Iwashima, A.: Anti-tumor-promoting activities of abieslactone, a natural tr iterpenoid isolated from several Abies genus. Cancer Lett. 53: 141-1 44,1990. Tokuda, H., Konos hima, T., Kozuka , M., Kimura, T.: Inhibition of 12-0-tetradecanoylphorbol-13-aceta te-promoted mouse skin papi llomas by saponins. On cology 48: 77 - 80, 1991. Tokud a, H. , Ohigashi, H ., Koshirn izu, K., Ito, Y.: Inhibitory effects of urso lic and oleano lic acid on skin tumor promotion by 12-0-tetradecano ylphorbol-13-acetate . Cancer Lett. 33: 279 -285, 1986. Wang, Z. Y. , Agarwa l, R., Zhou, Z. c., Bickers, D. R., Mukhtar, H.: Inhibition of mutagenicity in Salmone lla typhimurium and skin tumor initiating and tumor promoting activities in SENCAR mice by glycyrrhetinic acid: cornpar, ison of 18a-and 18j)-stereoisomers . Carcinogenesis 12: 187-192, 1991. Yasukawa, K., Aoki, T., Takido, M., Ikekawa, T., Saito, H., Matsuzawa, T.: Inhibitory effect of ergostero l isolated from the' edible mushroom Hypsizigus marmoreus on TPA-induced inflamma tory ear oedema and tumo ur promotion in mice. Pbytotber. Res. 8: 10-13, 1994a. Yasukawa, K., Akihisa, T., Kanno , H., Kamina ga, T., Izumida, M ., Sakoh, T., Tamura, T., Takido, M.: Inhibitory effect of sterols from Chlorella vulgaris on 12-0-tetradecanoylphorbol-13-acetate-induced inflammation and tumor pro m otion in mouse skin. Bioi. Pharm. Bull. 19: 573 -576, , 1996a. Yasukawa, K., Akihisa, T., Oinuma, H., Kaminaga , T., Kanno, H., Kasahara, Y., Tamura, T., Kumaki, K., Yarnanouchi, S., Takido, M.: Inhibitory effect of taraxastene-type rriterpenes on tumor promotion by 12-0-tetradecanoylphorbol-13-acetate in two -stage carcinogenesis in mouse skin. On cology 53: 341 -344, 1996b.
Yasukawa, K., Akihisa, T., Oinuma, H., Kasahara, Y., Kimura, Y., Yamanouchi, 5., Kumaki, K., Tamura, T., Takido, M.: Inhibitory effect of di- and trih ydrox y triterpenes from the flowers of Compositae on 12-0-tetradecanoylphorbol13-acetate-induced inflammation in mice. BioI. Pharm. Bull. 19: 1329 -1331, 1996c. Yasukawa, K., Akihisa, T., Tamur a, T. Takido, M .: Inhibitory effect of karounidiol on 12-0-tetradecanoylphorbol-13acetate-induced tumor promotion. Bioi. Pharm. Bull. 17: 460-462,1994b. Yasukawa, K., Takido, M ., Matsumoto, T., Take uchi, M ., Nakagawa,S.: Sterol and trite rpene derivatives from plants inhibit the effect of a tumor promoter, and sitostero l and betulinic acid inhibit tumor formation in mouse skin twostage carcinogenesis. Oncology 48: 72 -76, 1991. Yasukawa, K., Takido, M. , Takeuchi, M. , Nakagawa,S.: Inhibitory effect of glycyrrhizin and caffeine on tw o-stage carcinogenes is in mice. Yakugaku Zasshi 108: 794 -796, 1988 . Yasukawa, K., Yamaguchi, A., Arita , ] ., Sakurai,S., Ikeda, A., Takido , M.: Inhibitory effect of edible plant extrac ts on 12-0-tetradecanoylphorbol -13-acetate-induced inflammator y ear oedema . Phytother. Res. 7: 185 -189, 1993. Yasukawa, K., Yu, S.-Y., Yamanouchi , 5., Takido, M., Akihisa, T., Tamura, T.: Some lupane-type tr iterpenes inhibit tumor promotion by 12-0-tetradecano ylphorbol-13-acetate in two-stage carcinogenes is in mouse skin. Phytomedicine 1: 309 -313, 1995. Yu, L., Ma, R., Wang, Y., Nishino, H., Taka yasu, j ., He, W., Chan g, M., Zhen , j., Liu, W., Fan,S.: Potent anti-tumorigenic effect of tubeimoside 1 isolated from the bulb of Bolbostemma paniculatum (Maxim) Franquet. Int. ]. Cancer 50: 635 - 638, 1992. Yu, L., Yu, T., Ma, R.: Inhibition of the tumor promoting action of 12-0-tetradecanoylphorbol-13-acetate by tubeimo side III isolated from Bolbostemma paniculatum. Carcinogenesis 16: 3045 - 304 8, 1996 .
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Address
K. Yasukawa, PhD, College of Pharmacy, Nihon University, 7-1, Narashinodai 7-chome, Funa bashi-shi, Chiba 274, Japan Tel.: +81474655440 Fax: +81 474 65 5487 e-mail:[email protected]
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Inhibitory effect of heliantriol C; a component of edible Chrysanthemum, on tumor promotion by 12-0-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin K. Yasukawa', T. Akihisa", Y. Kasahara', M. Ukiya'', K. Kumaki', T. Tamura", S. Yamanouchi! and M. Takido 1 lCollege of Pharmacy, Nihon University, Narashinodai Funabashi-shi, Chiba, japan 2College of Science and Technology, Nihon University, Kanda Surugadai, Chiyoda-ku, Tokyo, japan 3The Yamagata Prefectural Institute of Public Health, Tokamachi, Yamagata, japan
Summary Two hydroxy taraxastane-type triterpenes, faradiol and heliantriol C, have been isolated from the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino, the edible Chrysanthemum. These compounds showed strong inhibitory activity against 12-0-tetradecanoylphorbol-13-aetate (TPA)-induced inflammation in mice. At 0.2 umol/mouse, these compounds markedly inhibited the promoting effect of TPA (1 ug/mouse) on skin tumor formation followed by 7,12-dimethylbenz[a]anthracene (50 ug/mouse), Key words: Antitumor promoter, two-stage carcinogenesis, 12-0-tetradecanoylphorbol-13-acetate, heliantriol C, faradiol, taraxastane-type triterpene, Chrysanthemum Asteraceae.
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Introduction
Chrysanthemum is a bitter, aromatic herb that experimentally lowers fevers, soothes inflammation, dilates the coronary arteries (increasing blood flow to the heart), and inhibits the growth of pathogens. It is used in folkmisumia for hypertension, coronary artery disease, angina, feverish colds, and liver-related disorders. A methanol extract from the ligulate flowers of the edible chrysanthemum inhibited 12-0-tetradecanoylphorbol13-acetate (TPA)-induced inflammatory ear edema (our unpublished data). In our previous studies, two taraxastane-type triterpenes, taraxasterol from the flowers of Cynara cardunculus, cardoon, and faradiol from the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino, the edible Chrysanthemum, were demonstrated to inhibit the tumor-promoting activity of TPA in two-stage carcinogenesis in mouse skin, and in the group treated with 7,12dimethylbenz[a]anthracene (DMBA) plus TPA and fa-
radiol (2.0 umol/rnouse), no tumors appeared at week 20 (Yasukawa et aI., 1996b). Taraxastane-type di- and tri-hydroxytriterpenes inhibited TPA-induced inflammatory ear edema (Yasukawa et al., 1996c). Heliantriol C showed strong inhibitory activity against TPA-induced inflammation in mice; the 50% inhibitory dose of heliantriol C was 30 ug/ear similar to the 30 ug/ear dose of hydrocortisone (Yasukawa et al., 1996c). Heliantriol C had about ten times the activity of the other triterpene derivatives on TPA-induced .inflammation in mice (Yasukawa et a!., 1988; 1991; 1994b; 1995). Furthermore, in this study, topical application of faradiol (0.2 urnol/rnouse) and heliantriol C (0.2 umol/mouse) markedly inhibited the promotion of skin tumors induced by twice-weekly application of TPA following initiation by a single application of DMBA in mice. It is interestingto know that hydroxylated faradiol is more effective against skin carcinogenesis.
K. Yasukawa et al.
216
Fig. 1. The chemical structures of faradiol and heliantriol C.
HO
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Materials and methods
Chemicals
Faradiol and heliantriol C were isolated from the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino and were identified by chromatographic and spectroscopic comparison with authentic samples (Yasukawa et al., 1996c). DMBA and dimethylsulfoxide were purchased from Sigma Chemical (St Louis, Mo., U.S.A.); TPA was obtained from Chemicals for Cancer Research Inc. (Minneapolis, Minn., U.S.A.). Animals
Female ICR mice (7 weeks old) were obtained from the Japan SLC Inc. (Shizuoka, Japan). The animals were housed in an air-conditioned specific pathogen free room (22-23°C), with light from 8.00 to 20.00 h. Food and water were available ad libitum. Two-stage carcinogenesis experiment
The backs of mice (7 weeks old) were shaved with electric clippers. Initiation was accomplished by a single topical application of 50 ug DMBA. Promotion with 1 ug TPA, applied twice weekly, was begun 1 week after the initiation. Faradiol (0.2 umol) and heliantriol C (0.2 umol), or their vehicle, acetone-dimethylsulfoxide (9:1; 100 ul), were applied topically 30 min before each TPA treatment. DMBA and TPA were dissolved in acetone, and applied to the shaved area in a volume of 100 ul using a micropipette. The back of each animal was shaved once a week to remove hair. The number and diameter of skin tumors were measured every other week, and the experiment was continued for 20 weeks. Experimental and appropriate control groups each consisted of 15 mice.
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Results
Fig. 2A shows the time courses of skin tumor formation in the group treated with DMBA plus TPA, with or without faradiol and heliantriol C. The first tumor appeared at week 8 in the group treated with DMBA plus
TPA. In the group treated with DMBA plus TPA and faradiol and heliantriol C, the first tumor appeared at weeks 10 and 13, respectively. The proportion of tumor-bearing mice treated with DMBA plus TPA was 93% at week 20, whereas the proportions in the groups treated with DMBA plus TPA and faradiol, and heliantriol C were 40% and 20%, respectively. Figure 2B shows the average number of tumors per mouse. The group treated with DMBA plus TPA produced 8.6 tumors per mouse at week 20, whereas the group treated with DMBA plus TPA and faradiol and heliantriol C had 2.9 and 0.9 tumors per mouse, respectively. The treatment with faradiol and heliantriol C caused 66% and 90% reductions, respectively, in the average number of tumors per mouse at week 20.
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Discussion
A series of naturally occurring sterol and triterpene derivatives have been found to possess antitumor-promoting activities (Kaminaga et al., 1996; Kasahara et al., 1994; Yasukawa et al., 1988; 1991; 1994a; 1994b; 1995; 1996a; 1996b). The methanol extract of the ligulate flowers of Chrysanthemum morifolium Ramat. var. sinense Makino fa. esculentum Makino, the edible flowers in Japan, has been reported to inhibit inflammatory ear edema induced by TPA, and various triterpenes were isolated from the unsaponifiable lipids of the methanol extract (Yasukawa et al., 1996b; 1996c). Heliantriol C had the strongest activity among these triterpenes, and faradiol consituted the main triterpene in the extract. In this study, we found that heliantriol C and faradiol markedly inhibited tumor promotion by TPA in DMBA-initiated mice. The inhibitory effect of 0.2 umol of these compounds corresponded to those of 2.0 umol of other sterols and triterpenes such as stigmasterol, ergosterol, ergosterol peroxide, lupeol, lupe01 acetate, betulin, betulinic acid, and karounidiol; therefore, these compounds had about ten times the activity of the other sterols and triterpenes on tumor promotion induced by TPA in mouse skin (Kasahara et al., 1994; Yasukawa et al., 1994a; 1994b; 1995; 1996a; 1996b). Heliantriol C was more effective than faradiol,
Inhibitory effect of heliantriol C on tumor promotion
217
B Fig. 2. Inhibitory effect of faradiol and heliA antriol C on the promotion of skin papillo10 r - - - - - - - - - - , 100 mas by TPA in DMBA-initiated mice. From 1 week after initiation by a single topical application of 50 ug of DMBA, 1 [-lg of TPA was applied twice weekly. Topical application of 0.2 urnol faradiol, heliantriol C and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). The treated groups were statistically o 6ooiOQCltoOctaOc>O-.....L..----l different from the control group by 15 20 10 5 20 10 15 5 o Student's t test (p <0.05 = heliantriol C treated group: after weeks 11-13, faradiol Weeks of promotion Weeks of promotion treated group: after weeks 11-16; p <0.01 = heliantriol C treated group: after week 14, faradiol treated group: after week 17-20). Heliantriol C treated group was statistically different from the faradiol treated group by Student's t test (p <0.05: after week 16-20). • = +TPA with vehicle alone; 0 = +TPA with heliantriol C; t::,. = +TPA with faradio!.
which was almost comparable with that of the lanos-
tane-type triterpene acids, pachymic acid, 3-0-acetyl16a-hydroxytrametenolic acid and poricoic acid B on tumor promotion by TPA in two-stage carcinogenesis in mouse skin (Kaminaga et al., 1996). Thus faradiol (taraxast-Zu-ene-Jp.Lep-diol) showed a marked inhibitory effect and further hydroxylation of faradiol at C22 giving heliantriol C (taraxast-Zu-ene-Jp.Iep.Zzctriol) markedly enhanced the effect. There was a close relationship between the hydroxylation of triterpenes and the inhibitory effect. Our previous studies demonstrated that extracts from edible plants and fungi inhibit TPA-induced inflammation in mice, and sterols and triterpenes have been indentified by us as the active compounds separated from an edible mushroom Hypsizigus marrnoreus (Yasukawa et al., 1994a), safflower (Kasahara et al., 1994), an edible alga Chlorella vulgaris (Yasukawa et al., 1996a) and stevia (Yasukawa et al., 1993). Furthermore, these sterols and triterpenes have been shown to inhibit tumor promotion during twostage carcinogenesis in mouse skin. Other researchers have reported similar effects for triterpenes (Diallo et al., 1995; Konoshima et al., 1992; 1994; 1995; Nishino et al., 1986; 1988; Takayasu et al., 1990; Tokuda et al., 1986; 1991; Wrang et al., 1991; Yu et al., 1992; 1996). Many sterols and triterpenes, widely distributed in edible plants and fungi, inhibit the tumor-promoting activity of TPA in mouse skin, and this suggests that they may be of importance for cancer chemoprevention. Acknowledgements
This work was supported in part by an Interdisciplinary Joint Research Grant from Nihon University. We wish to thank Dr.
Tomio Takeuchi and Dr. Mieko Takeuchi, Institute of Microbial Chemistry, for their kind encouragements during the course of this study.
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References
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Address
K. Yasukawa, PhD, College of Pharmacy, Nihon University, 7-1, Narashinodai 7-chome, Funa bashi-shi, Chiba 274, Japan Tel.: +81474655440 Fax: +81 474 65 5487 e-mail:[email protected]