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Constituents of Compositae plants
Cancer Letters 177 (2002) 7–12 www.elsevier.com/locate/canlet
Constituents of Compositae plants III. Anti-tumor promoting effects and cytotoxic activity against human cancer cell lines of triterpene diols and triols from edible chrysanthemum flowers Motohiko Ukiya a, Toshihiro Akihisa a,*, Harukuni Tokuda b, Hiroyuki Suzuki a, Teruo Mukainaka b, Eiichiro Ichiishi b, Ken Yasukawa c, Yoshimasa Kasahara d, Hoyoku Nishino b a
College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan b Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan c College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan d The Yamagata Prefectural Institute of Public Health, 1-6-6 Tokamachi, Yamagata 990-0031, Japan Received 19 July 2001; received in revised form 10 September 2001; accepted 10 September 2001
Abstract Fifteen pentacyclic triterpene diols and triols, consisting of: six taraxastanes, faradiol (1), heliantriol B0 (2), heliantriol C (3), 22a-methoxyfaradiol (4), arnidiol (5), and faradiol a-epoxide (6); five oleananes, maniladiol (7), erythrodiol (8), longispinogenin (9), coflodiol (10), and heliantriol A1 (11); two ursanes, brein (12) and uvaol (13); and two lupanes, calenduladiol (14) and heliantriol B2 (15), isolated from the non-saponifiable lipid fraction of the edible flower extract of chrysanthemum (Chrysanthemum morifolium) were evaluated for their inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, in Raji cells as a primary screening test for anti-tumorpromoters. All of the compounds tested showed inhibitory effects against EBV-EA activation with potencies either comparable with or stronger than that of glycyrrhetic acid, a known natural anti-tumor-promoter. Evaluation of the cytotoxic activity of six compounds, 1–3 and 5–7, against human cancer cell lines revealed that compound 5 possesses a wide range of cytotoxicity, with GI50 values (concentration that yields 50% growth) of mostly less than 6 mM. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chrysanthemum morifolium; Edible chrysanthemum flowers; Compositae; Triterpene diols and triols; Anti-tumor-promoter; Epstein–Barr virus early antigen; Cytotoxicity
1. Introduction Chrysanthemum morifolium Ramat. var. sinense Makino forma esculentum Makino (Japanese name: * Corresponding author. Fax: 181-3-3293-7572. E-mail address: [email protected] (T. Akihisa).
Ryouri-giku; Compositae) has been widely cultivated in the North-Eastern part of the Honshu Island of Japan as traditional edible flowers. Our recent study has demonstrated that the triterpene diols and triols isolated from the non-saponifiable lipid (NSL) fraction of the methanol extract of the edible chrysanthe-
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00769-8
8
M. Ukiya et al. / Cancer Letters 177 (2002) 7–12
mum ligulate flowers possess remarkable anti-inflammatory activity on 12-O-tetradecanoylphorbol-13acetate (TPA)-induced ear edema in mice [1]. Moreover, two taraxastane-type triterpenes, faradiol (1) and heliantriol C (3), isolated from the edible chrysanthemum flower extract, were revealed to markedly suppress the promoting effect of TPA on skin tumor formation in mice following initiation with 7,12dimethylbenz[a]anthracene (DMBA) [2]. Since the triterpene diols and triols from chrysanthemum flowers are expected to be potential anti-tumor-promoters (cancer chemopreventive agents) [3,4], we carried out a primary in vitro screening test of these compounds for their inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) induced by the tumor promoter, TPA, in Raji cells. In this paper, we report the inhibitory effects on EBV-EA activation of 15 compounds, 1–15, isolated from the chrysanthemum flower extract [1]. In addition, the cytotoxic activities of six compounds, 1–3 and 5–7, evaluated against human cancer cell lines are described.
2. Materials and methods 2.1. Test compounds Fifteen compounds: (1), faradiol (taraxast-20-ene3b,16b-diol); (2), heliantriol B0 (taraxast-20-ene3b,16b,28-triol); (3), heliantriol C (taraxast-20-ene3b,16b,22a-triol); (4), 22a-methoxyfaradiol (22amethoxytaraxast-20-ene-3b,16b-diol); (5), arnidiol (taraxast-20(30)-ene-3b,16b-diol); (6), faradiol aepoxide ((20R,21S)-20,21-epoxytaraxastane-3b,16bdiol); (7), maniladiol (olean-12-ene-3b,16b-diol); (8), erythrodiol (olean-12-ene-3b,28-diol); (9), longispinogenin (olean-12-ene-3b,16b,28-triol); (10), coflodiol (olean-13(18)-ene-3b,16b-diol); (11), heliantriol A1 (olean-13(18)-ene-3b,16b,28-triol); (12), brein (urs-12-ene-3b,16b-diol); (13), uvaol (urs-12-ene-3b,28-diol); (14), calenduladiol (lup20(29)-ene-3b,16b-diol); (15), and heliantriol B2 (lup-20(29)-ene-3b,16b,28-triol), tested in this study were isolated from the NSL fraction of the methanol
Fig. 1. Structures of the triterpene diols and triols described in this paper.
M. Ukiya et al. / Cancer Letters 177 (2002) 7–12
extract of the edible chrysanthemum ligulate flowers [1]. Glycyrrhetic acid (3b-hydroxy-11-oxoolean-12en-29-oic acid) [5,6] was used as a reference compound (Fig. 1). 2.2. In vitro EBV-EA activation experiment The inhibition of EBV-EA activation was assayed using Raji cells (EBV genome-carrying human lymphoblastoid cells; non-producer type), cultivated in 10% fetal bovine serum (FBS) RPMI-1640 medium (Nakalai Tesque, Kyoto, Japan). The indicator cells (Raji cells; 1 £ 10 6 cells/ml) were incubated in 1 ml of the medium containing 4 mM n-butyric acid as an inducer, 32 pM of TPA (20 ng/ml in dimethyl-sulfoxide (DMSO)), and a known amount (32, 16, 3.2, 0.32 nmol) of the test compound at 378C in a CO2 incubator. After 48 h, the cell suspensions were centrifuged at 1000 revs./min for 10 min, and the supernatant was removed. The activated cells were
9
stained with high-titer EBV-EA-positive sera from nasopharyngeal carcinoma patients, and the conventional indirect immunofluorescence technique was employed for detection. In each assay, at least 500 cells were counted and the experiments were repeated three times. The average extent of EA induction was determined and compared with that in positive control experiments in which the cells were treated with nbutyric acid plus TPA where the extent of EA induction was ordinarily more than around 40%. The viability of treated Raji cells was assayed by the TrypanBlue staining method [7,8]. 2.3. In vitro cytotoxicity assay There was a total of 60 human tumor cell lines derived from seven cancer types (lung, colon, melanoma, renal, ovarian, brain, and leukemia), against which compounds 1–3 and 5–7 were tested at a minimum of five concentrations at ten-fold dilutions, start-
Table 1 Relative ratio a of EBA-EA activation with respect to positive control b in the presence of triterpene diols from chrysanthemum flowers c Compound
Taraxastane-type Faradiol (1) Heliantriol B0 (2) Heliantriol C (3) 22a-Methoxyfaradiol (4) Arnidiol (5) Faradiol a-epoxide (6) Oleanane-type Maniladiol (7) Erythrodiol (8) Longispinogenin (9) Coflodiol (10) Heliantriol A1 (11) Ursane-type Brein (12) Uvaol (13) Lupane-type Calenduladiol (14) Heliantriol B2 (15) Reference compound Glycyrrhetic acid a b c d
Concentration (mol ratio/TPA) d 1000
500
100
10
6.9 (70) 0 (70) 3.3 (70) 2.5 (70) 9.1 (70) 12.4 (70)
42.4 (.80) 42.6 (.80) 39.0 (.80) 45.9 (.80) 49.2 (.80) 52.7 (.80)
78.8 (.80) 74.0 (.80) 74.1 (.80) 78.3 (.80) 81.0 (.80) 83.5 (.80)
100 (.80) 95.3 (.80) 100 (.80) 96.7 (.80) 100 (.80) 100 (.80)
0 (70) 0 (70) 0 (70) 5.4 (70) 0 (70)
40.0 (.80) 42.6 (.80) 38.8 (.80) 49.1 (.80) 39.0 (.80)
73.2 (.80) 74.8 (.80) 71.7 (.80) 76.2 (.80) 72.1 (.80)
94.6 (.80) 96.6 (.80) 92.4 (.80) 96.5 (.80) 93.0 (.80)
0 (70) 13.4 (70)
41.2 (.80) 57.2 (.80)
71.7 (.80) 82.3 (.80)
92.4 (.80) 100 (.80)
0 (70) 3.9 (70)
25.6 (.80) 32.5 (.80)
71.2 (.80) 71.0 (.80)
93.5 (.80) 93.1 (.80)
15.6 (.80)
54.3 (.80)
100 (.80)
100 (.80)
Values represent percentages relative to the positive control value. Positive control, 100%. Values in parentheses represent the viability percentage of Raji cells. The TPA concentration was 20 ng/ml (32 pmol/ml).
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M. Ukiya et al. / Cancer Letters 177 (2002) 7–12 24
ing from a high of 10 M. A 48 h continuous drug exposure protocol was used, and a sulforhodamine B protein assay was used to estimate cell viability or growth. Details of the assay procedure have been reported previously [9].
3. Results and discussion The triterpenes tested in this study were: six taraxastanes (1–6), five oleananes (7–11), two ursanes (12 and 13), and two lupanes (14 and 15), possessing either a 3b,16b-diol, 3b,28-diol, 3b,16b,22a-triol, or 3b,16b,28-triol system isolated from the NSL fraction of the edible flower extract of chrysanthemum [1]. Table 1 shows the inhibitory effects on an in vitro assay of TPA-induced EBV-EA activation in Raji cells of the 15 triterpene diols and triols and the reference substance, glycyrrhetic acid. All compounds evaluated showed an inhibitory effect on EBV activation at 1 £ 10 2 mol ratio, and exhibited significant activity at a high concentration (86.6– 100% inhibition at 1 £ 10 3 mol ratio) preserving high viability of Raji cells. The inhibitory effects of all compounds were almost equivalent to or stronger than that of glycyrrhetic acid, which is known to be a potent anti-tumor-promoter [4]. Among them, four oleananes (7–9 and 11), and one of each of the taraxastanes (2), ursanes (12), and lupanes (14), exhibited remarkably high inhibitory effects at a high concentration (100% inhibition at 1 £ 10 3 mol ratio). The inhibitory effects against EBV-EA activation have been demonstrated to closely parallel those against tumor promotion in vivo [6,10], and the triterpenes from chrysanthemum flowers are, therefore, suggested to be valuable anti-tumor-promoters (potential cancer chemopreventive agents). This was supported by our recent finding that faradiol (1) and heliantriol C (3) markedly suppressed the promoting effect of TPA on skin tumor formation in mice following initiation with DMBA [2]. The cytotoxic activities of six compounds, 1–3 and 5–7, were evaluated in the National Cancer Institute using 60 human cancer cell lines [9]. Remarkable cytotoxicity was observed for arnidiol (5), as shown in Table 2 with values for the concentration that yields 50% growth (GI50), the concentration at which no growth is observed (TGI), and the concentration at
Table 2 Cytotoxicity a of arnidiol (5) against human cancer cell lines in vitro in the National Cancer Institute Developmental Therapeutics Program b Panel/cell line Leukemia CCRF-CEM HL-60 (TB) K-562 MOLT-4 RPMT-8226 SR Non-small cell lung cancer A549/ATCC EKVX HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522 Colon cancer COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 CNS cancer SF-268 SF-295 SF-539 SNB-19 SNB-75 U251 Melanoma LOX IMVI MALME-3M M14 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62 Ovarian cancer IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 SK-OV-3 Renal cancer 786-0
GI50
TGI
LC50
5.92 0.465 ND 3.22 .100 .100
.100 31.1 .100 .100 .100 .100
.100 .100 .100 .100 .100 .100
1.93 3.09 1.70 ND 5.00 2.29 2.49 ND 1.72
4.00 7.99 3.33 ND 40.1 5.11 6.09 ND 4.22
8.32 .100 ND ND .100 .100 42.4 ND .100
4.23 ND 2.30 2.51 3.88 3.36 3.62
.100 ND 5.70 6.56 20.6 12.9 .100
.100 ND 41.8 94.6 .100 .100 .100
1.69 2.29 2.45 2.87 ND 1.78
3.56 6.28 6.29 8.44 ND 3.40
ND 47.0 98.5 .100 ND ND
1.86 3.27 5.87 6.17 4.60 6.46 9.53 7.13
3.47 15.7 29.9 26.4 23.5 25.3 31.7 26.6
6.48 77.4 .100 86.5 77.9 84.0 .100 83.9
1.80 1.77 1.98 4.58 1.78 3.01
3.31 3.56 45.9 22.0 3.39 41.9
6.10 ND .100 .100 ND .100
1.83
3.44
6.47
M. Ukiya et al. / Cancer Letters 177 (2002) 7–12 Table 2 (continued) Panel/cell line A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31 Prostate cancer PC-3 DU-145 Breast cancer MCF7 NCI/ADR-RES MDA-MB-231/ATCC HS 578T MDA-MB-435 MDA-N BT-549 T-47D a b
TGI
GI50 1.92 2.07 2.02 2.14 1.95 6.23 ND
3.99 4.50 3.71 5.06 4.26 31.8 ND
3.08 6.75
12.4 23.7
2.80 2.84 2.12 2.37 3.59 7.40 4.96 2.63
10.8 9.81 5.24 6.30 25.2 26.5 22.3 8.57
LC50 8.29 9.78 6.84 .100 ND .100 ND 77.2 76.3 77.8 .100 .100 .100 .100 83.2 98.7 .100
Cytotoxicity (mM). ND, not determined.
which only 50% of the cells are viable (LG50). It showed the activity with GI50 values less than 10 mM against all of the human cancer cells tested, with two exceptions for leukemia cells (RPMI-8226 and SR: GI50 . 100 mM) which were relatively insensitive to the compound. Compound 5 showed significant cytotoxicity, especially against a leukemia HL-60 cell with a GI50 0.47 mM. The cytotoxic activities of the other five compounds were not remarkable. Heliantriol C (3) (GI50 value, 10.5–23.3 mM) and faradiol a-epoxide (6) (GI50, 13.7–44.0 mM) exhibited some moderate cytotoxicity for all of the cells evaluated. Whereas faradiol (1) showed marked cytotoxicity for leukemia (CCRFCEM: GI50, 3.2 mM; K-582: GI50, 4.2 mM; SR: GI50, 3.5 mM) and non-small cell lung cancer (EKVX: GI50, 2.2 mM), it showed moderate activity for the other cells (GI50, 10.5–86.9 mM). Heliantriol B0 (2) for renal cancer (RXF 393: GI50, 7.1 mM) and breast cancer (MCF7: GI50, 8.4 mM), and maniladiol (7) for renal cancer (RFX 393: GI50, 7.3 mM) and breast cancer (T-47D: GI50, 9.8 mM) showed some marked activity, but the other cells were relatively insensitive for these compounds (GI50, 11.7–97.4 mM). In conclusion, the highly anti-inflammatory pentacyclic triterpene diols and triols from edible chry-
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santhemum flowers [1] possess high inhibitory effects against EBV-EA activation, suggesting that they could be useful as chemopreventive agents. In addition, arnidiol (5), among the six compounds evaluated, exhibited remarkable cytotoxic activity against the panels of NCI human cancer cell lines, which suggested this compound to be useful as an anticancer agent.
Acknowledgements The authors wish to thank Dr V.L. Narayanan, Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD, for performing the cytostatic and cytotoxic screening studies. This work was supported in part by a grant ‘Research and Development of Nanoscience’ from the Ministry of Education, Science, Sports and Culture to promote multi-disciplinary research project, and also supported in part by Grants-in-an Aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan.
References [1] M. Ukiya, T. Akihisa, K. Yasukawa, Y. Kasahara, Y. Kimura, K. Koike, T. Nikaido, M. Takido, Constituents of Compositae plants. 2. Triterpene diols and triols, and their 3-O-fatty acid esters from the edible chrysanthemum flower extract and their anti-inflammatory effects, J. Agric. Food Chem. 49 (2001) 3187–3197. [2] K. Yasukawa, T. Akihisa, Y. Kasahara, M. Ukiya, K. Kumaki, T. Tamura, S. Yamanouchi, M. Takido, 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 5 (1998) 215–218. [3] H. Tokuda, H. Ohigashi, K. Koshimizu, Y. Ito, Inhibitory effects of ursolic acid and oleanolic acid on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate, Cancer Lett. 33 (1986) 279–285. [4] T. Konoshima, M. Takasaki, E. Ichiishi, T. Murakami, H. Tokuda, H. Nishino, N.M. Duc, R. Kasai, K. Yamasaki, Cancer chemopreventive activity of majonoside-R2 from Vietnamese ginseng, Panax vietnamensis, Cancer Lett. 147 (1999) 11–16. [5] H. Ohigashi, H. Takamura, K. Koshimizu, H. Tokuda, Y. Ito, Search for possible antitumor promoters by inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced Epstein–Barr virus activation; ursolic acid and oleanolic acid from an
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anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L, Cancer Lett. 30 (1986) 143–151. [6] T. Konoshima, M. Takasaki, H. Tokuda, K. Masuda, Y. Arai, K. Shiojima, H. Ageta, Anti-tumor-promoting activities of triterpenoids from ferns. I, Biol. Pharm. Bull. 19 (1996) 962–965. [7] B. Diallo, M. Vanhaelen, R. Vanhaelen-Fastre´ , T. Konoshima, M. Kozuka, H. Tokuda, Studies on inhibitors of skin tumor promotion: inhibitory effects of triterpenes from Chlosperum tinctorium on Epstein–Barr virus activation, J. Nat. Prod. 52 (1989) 879–881.
[8] Y. Takaishi, K. Ujita, H. Tokuda, H. Nishino, A. Iwashima, T. Fujita, Inhibitory effects of dihydroagarofuran sesquiterpenes on Epstein–Barr virus activation, Cancer Lett. 65 (1992) 19– 26. [9] M.B. Boyd, K.D. Paul, Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen, Drug Dev. Res. 34 (1995) 91– 109. [10] M. Takasaki, T. Konoshima, H. Tokuda, K. Masuda, Y. Arai, K. Shiojima, H. Ageta, Anti-carcinogenic activity of Taraxacum plant. II, Biol. Pharm. Bull. 22 (1999) 606–610.
Constituents of Compositae plants III. Anti-tumor promoting effects and cytotoxic activity against human cancer cell lines of triterpene diols and triols from edible chrysanthemum flowers Motohiko Ukiya a, Toshihiro Akihisa a,*, Harukuni Tokuda b, Hiroyuki Suzuki a, Teruo Mukainaka b, Eiichiro Ichiishi b, Ken Yasukawa c, Yoshimasa Kasahara d, Hoyoku Nishino b a
College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan b Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan c College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan d The Yamagata Prefectural Institute of Public Health, 1-6-6 Tokamachi, Yamagata 990-0031, Japan Received 19 July 2001; received in revised form 10 September 2001; accepted 10 September 2001
Abstract Fifteen pentacyclic triterpene diols and triols, consisting of: six taraxastanes, faradiol (1), heliantriol B0 (2), heliantriol C (3), 22a-methoxyfaradiol (4), arnidiol (5), and faradiol a-epoxide (6); five oleananes, maniladiol (7), erythrodiol (8), longispinogenin (9), coflodiol (10), and heliantriol A1 (11); two ursanes, brein (12) and uvaol (13); and two lupanes, calenduladiol (14) and heliantriol B2 (15), isolated from the non-saponifiable lipid fraction of the edible flower extract of chrysanthemum (Chrysanthemum morifolium) were evaluated for their inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, in Raji cells as a primary screening test for anti-tumorpromoters. All of the compounds tested showed inhibitory effects against EBV-EA activation with potencies either comparable with or stronger than that of glycyrrhetic acid, a known natural anti-tumor-promoter. Evaluation of the cytotoxic activity of six compounds, 1–3 and 5–7, against human cancer cell lines revealed that compound 5 possesses a wide range of cytotoxicity, with GI50 values (concentration that yields 50% growth) of mostly less than 6 mM. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chrysanthemum morifolium; Edible chrysanthemum flowers; Compositae; Triterpene diols and triols; Anti-tumor-promoter; Epstein–Barr virus early antigen; Cytotoxicity
1. Introduction Chrysanthemum morifolium Ramat. var. sinense Makino forma esculentum Makino (Japanese name: * Corresponding author. Fax: 181-3-3293-7572. E-mail address: [email protected] (T. Akihisa).
Ryouri-giku; Compositae) has been widely cultivated in the North-Eastern part of the Honshu Island of Japan as traditional edible flowers. Our recent study has demonstrated that the triterpene diols and triols isolated from the non-saponifiable lipid (NSL) fraction of the methanol extract of the edible chrysanthe-
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00769-8
8
M. Ukiya et al. / Cancer Letters 177 (2002) 7–12
mum ligulate flowers possess remarkable anti-inflammatory activity on 12-O-tetradecanoylphorbol-13acetate (TPA)-induced ear edema in mice [1]. Moreover, two taraxastane-type triterpenes, faradiol (1) and heliantriol C (3), isolated from the edible chrysanthemum flower extract, were revealed to markedly suppress the promoting effect of TPA on skin tumor formation in mice following initiation with 7,12dimethylbenz[a]anthracene (DMBA) [2]. Since the triterpene diols and triols from chrysanthemum flowers are expected to be potential anti-tumor-promoters (cancer chemopreventive agents) [3,4], we carried out a primary in vitro screening test of these compounds for their inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) induced by the tumor promoter, TPA, in Raji cells. In this paper, we report the inhibitory effects on EBV-EA activation of 15 compounds, 1–15, isolated from the chrysanthemum flower extract [1]. In addition, the cytotoxic activities of six compounds, 1–3 and 5–7, evaluated against human cancer cell lines are described.
2. Materials and methods 2.1. Test compounds Fifteen compounds: (1), faradiol (taraxast-20-ene3b,16b-diol); (2), heliantriol B0 (taraxast-20-ene3b,16b,28-triol); (3), heliantriol C (taraxast-20-ene3b,16b,22a-triol); (4), 22a-methoxyfaradiol (22amethoxytaraxast-20-ene-3b,16b-diol); (5), arnidiol (taraxast-20(30)-ene-3b,16b-diol); (6), faradiol aepoxide ((20R,21S)-20,21-epoxytaraxastane-3b,16bdiol); (7), maniladiol (olean-12-ene-3b,16b-diol); (8), erythrodiol (olean-12-ene-3b,28-diol); (9), longispinogenin (olean-12-ene-3b,16b,28-triol); (10), coflodiol (olean-13(18)-ene-3b,16b-diol); (11), heliantriol A1 (olean-13(18)-ene-3b,16b,28-triol); (12), brein (urs-12-ene-3b,16b-diol); (13), uvaol (urs-12-ene-3b,28-diol); (14), calenduladiol (lup20(29)-ene-3b,16b-diol); (15), and heliantriol B2 (lup-20(29)-ene-3b,16b,28-triol), tested in this study were isolated from the NSL fraction of the methanol
Fig. 1. Structures of the triterpene diols and triols described in this paper.
M. Ukiya et al. / Cancer Letters 177 (2002) 7–12
extract of the edible chrysanthemum ligulate flowers [1]. Glycyrrhetic acid (3b-hydroxy-11-oxoolean-12en-29-oic acid) [5,6] was used as a reference compound (Fig. 1). 2.2. In vitro EBV-EA activation experiment The inhibition of EBV-EA activation was assayed using Raji cells (EBV genome-carrying human lymphoblastoid cells; non-producer type), cultivated in 10% fetal bovine serum (FBS) RPMI-1640 medium (Nakalai Tesque, Kyoto, Japan). The indicator cells (Raji cells; 1 £ 10 6 cells/ml) were incubated in 1 ml of the medium containing 4 mM n-butyric acid as an inducer, 32 pM of TPA (20 ng/ml in dimethyl-sulfoxide (DMSO)), and a known amount (32, 16, 3.2, 0.32 nmol) of the test compound at 378C in a CO2 incubator. After 48 h, the cell suspensions were centrifuged at 1000 revs./min for 10 min, and the supernatant was removed. The activated cells were
9
stained with high-titer EBV-EA-positive sera from nasopharyngeal carcinoma patients, and the conventional indirect immunofluorescence technique was employed for detection. In each assay, at least 500 cells were counted and the experiments were repeated three times. The average extent of EA induction was determined and compared with that in positive control experiments in which the cells were treated with nbutyric acid plus TPA where the extent of EA induction was ordinarily more than around 40%. The viability of treated Raji cells was assayed by the TrypanBlue staining method [7,8]. 2.3. In vitro cytotoxicity assay There was a total of 60 human tumor cell lines derived from seven cancer types (lung, colon, melanoma, renal, ovarian, brain, and leukemia), against which compounds 1–3 and 5–7 were tested at a minimum of five concentrations at ten-fold dilutions, start-
Table 1 Relative ratio a of EBA-EA activation with respect to positive control b in the presence of triterpene diols from chrysanthemum flowers c Compound
Taraxastane-type Faradiol (1) Heliantriol B0 (2) Heliantriol C (3) 22a-Methoxyfaradiol (4) Arnidiol (5) Faradiol a-epoxide (6) Oleanane-type Maniladiol (7) Erythrodiol (8) Longispinogenin (9) Coflodiol (10) Heliantriol A1 (11) Ursane-type Brein (12) Uvaol (13) Lupane-type Calenduladiol (14) Heliantriol B2 (15) Reference compound Glycyrrhetic acid a b c d
Concentration (mol ratio/TPA) d 1000
500
100
10
6.9 (70) 0 (70) 3.3 (70) 2.5 (70) 9.1 (70) 12.4 (70)
42.4 (.80) 42.6 (.80) 39.0 (.80) 45.9 (.80) 49.2 (.80) 52.7 (.80)
78.8 (.80) 74.0 (.80) 74.1 (.80) 78.3 (.80) 81.0 (.80) 83.5 (.80)
100 (.80) 95.3 (.80) 100 (.80) 96.7 (.80) 100 (.80) 100 (.80)
0 (70) 0 (70) 0 (70) 5.4 (70) 0 (70)
40.0 (.80) 42.6 (.80) 38.8 (.80) 49.1 (.80) 39.0 (.80)
73.2 (.80) 74.8 (.80) 71.7 (.80) 76.2 (.80) 72.1 (.80)
94.6 (.80) 96.6 (.80) 92.4 (.80) 96.5 (.80) 93.0 (.80)
0 (70) 13.4 (70)
41.2 (.80) 57.2 (.80)
71.7 (.80) 82.3 (.80)
92.4 (.80) 100 (.80)
0 (70) 3.9 (70)
25.6 (.80) 32.5 (.80)
71.2 (.80) 71.0 (.80)
93.5 (.80) 93.1 (.80)
15.6 (.80)
54.3 (.80)
100 (.80)
100 (.80)
Values represent percentages relative to the positive control value. Positive control, 100%. Values in parentheses represent the viability percentage of Raji cells. The TPA concentration was 20 ng/ml (32 pmol/ml).
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M. Ukiya et al. / Cancer Letters 177 (2002) 7–12 24
ing from a high of 10 M. A 48 h continuous drug exposure protocol was used, and a sulforhodamine B protein assay was used to estimate cell viability or growth. Details of the assay procedure have been reported previously [9].
3. Results and discussion The triterpenes tested in this study were: six taraxastanes (1–6), five oleananes (7–11), two ursanes (12 and 13), and two lupanes (14 and 15), possessing either a 3b,16b-diol, 3b,28-diol, 3b,16b,22a-triol, or 3b,16b,28-triol system isolated from the NSL fraction of the edible flower extract of chrysanthemum [1]. Table 1 shows the inhibitory effects on an in vitro assay of TPA-induced EBV-EA activation in Raji cells of the 15 triterpene diols and triols and the reference substance, glycyrrhetic acid. All compounds evaluated showed an inhibitory effect on EBV activation at 1 £ 10 2 mol ratio, and exhibited significant activity at a high concentration (86.6– 100% inhibition at 1 £ 10 3 mol ratio) preserving high viability of Raji cells. The inhibitory effects of all compounds were almost equivalent to or stronger than that of glycyrrhetic acid, which is known to be a potent anti-tumor-promoter [4]. Among them, four oleananes (7–9 and 11), and one of each of the taraxastanes (2), ursanes (12), and lupanes (14), exhibited remarkably high inhibitory effects at a high concentration (100% inhibition at 1 £ 10 3 mol ratio). The inhibitory effects against EBV-EA activation have been demonstrated to closely parallel those against tumor promotion in vivo [6,10], and the triterpenes from chrysanthemum flowers are, therefore, suggested to be valuable anti-tumor-promoters (potential cancer chemopreventive agents). This was supported by our recent finding that faradiol (1) and heliantriol C (3) markedly suppressed the promoting effect of TPA on skin tumor formation in mice following initiation with DMBA [2]. The cytotoxic activities of six compounds, 1–3 and 5–7, were evaluated in the National Cancer Institute using 60 human cancer cell lines [9]. Remarkable cytotoxicity was observed for arnidiol (5), as shown in Table 2 with values for the concentration that yields 50% growth (GI50), the concentration at which no growth is observed (TGI), and the concentration at
Table 2 Cytotoxicity a of arnidiol (5) against human cancer cell lines in vitro in the National Cancer Institute Developmental Therapeutics Program b Panel/cell line Leukemia CCRF-CEM HL-60 (TB) K-562 MOLT-4 RPMT-8226 SR Non-small cell lung cancer A549/ATCC EKVX HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522 Colon cancer COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 CNS cancer SF-268 SF-295 SF-539 SNB-19 SNB-75 U251 Melanoma LOX IMVI MALME-3M M14 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62 Ovarian cancer IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 SK-OV-3 Renal cancer 786-0
GI50
TGI
LC50
5.92 0.465 ND 3.22 .100 .100
.100 31.1 .100 .100 .100 .100
.100 .100 .100 .100 .100 .100
1.93 3.09 1.70 ND 5.00 2.29 2.49 ND 1.72
4.00 7.99 3.33 ND 40.1 5.11 6.09 ND 4.22
8.32 .100 ND ND .100 .100 42.4 ND .100
4.23 ND 2.30 2.51 3.88 3.36 3.62
.100 ND 5.70 6.56 20.6 12.9 .100
.100 ND 41.8 94.6 .100 .100 .100
1.69 2.29 2.45 2.87 ND 1.78
3.56 6.28 6.29 8.44 ND 3.40
ND 47.0 98.5 .100 ND ND
1.86 3.27 5.87 6.17 4.60 6.46 9.53 7.13
3.47 15.7 29.9 26.4 23.5 25.3 31.7 26.6
6.48 77.4 .100 86.5 77.9 84.0 .100 83.9
1.80 1.77 1.98 4.58 1.78 3.01
3.31 3.56 45.9 22.0 3.39 41.9
6.10 ND .100 .100 ND .100
1.83
3.44
6.47
M. Ukiya et al. / Cancer Letters 177 (2002) 7–12 Table 2 (continued) Panel/cell line A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31 Prostate cancer PC-3 DU-145 Breast cancer MCF7 NCI/ADR-RES MDA-MB-231/ATCC HS 578T MDA-MB-435 MDA-N BT-549 T-47D a b
TGI
GI50 1.92 2.07 2.02 2.14 1.95 6.23 ND
3.99 4.50 3.71 5.06 4.26 31.8 ND
3.08 6.75
12.4 23.7
2.80 2.84 2.12 2.37 3.59 7.40 4.96 2.63
10.8 9.81 5.24 6.30 25.2 26.5 22.3 8.57
LC50 8.29 9.78 6.84 .100 ND .100 ND 77.2 76.3 77.8 .100 .100 .100 .100 83.2 98.7 .100
Cytotoxicity (mM). ND, not determined.
which only 50% of the cells are viable (LG50). It showed the activity with GI50 values less than 10 mM against all of the human cancer cells tested, with two exceptions for leukemia cells (RPMI-8226 and SR: GI50 . 100 mM) which were relatively insensitive to the compound. Compound 5 showed significant cytotoxicity, especially against a leukemia HL-60 cell with a GI50 0.47 mM. The cytotoxic activities of the other five compounds were not remarkable. Heliantriol C (3) (GI50 value, 10.5–23.3 mM) and faradiol a-epoxide (6) (GI50, 13.7–44.0 mM) exhibited some moderate cytotoxicity for all of the cells evaluated. Whereas faradiol (1) showed marked cytotoxicity for leukemia (CCRFCEM: GI50, 3.2 mM; K-582: GI50, 4.2 mM; SR: GI50, 3.5 mM) and non-small cell lung cancer (EKVX: GI50, 2.2 mM), it showed moderate activity for the other cells (GI50, 10.5–86.9 mM). Heliantriol B0 (2) for renal cancer (RXF 393: GI50, 7.1 mM) and breast cancer (MCF7: GI50, 8.4 mM), and maniladiol (7) for renal cancer (RFX 393: GI50, 7.3 mM) and breast cancer (T-47D: GI50, 9.8 mM) showed some marked activity, but the other cells were relatively insensitive for these compounds (GI50, 11.7–97.4 mM). In conclusion, the highly anti-inflammatory pentacyclic triterpene diols and triols from edible chry-
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santhemum flowers [1] possess high inhibitory effects against EBV-EA activation, suggesting that they could be useful as chemopreventive agents. In addition, arnidiol (5), among the six compounds evaluated, exhibited remarkable cytotoxic activity against the panels of NCI human cancer cell lines, which suggested this compound to be useful as an anticancer agent.
Acknowledgements The authors wish to thank Dr V.L. Narayanan, Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD, for performing the cytostatic and cytotoxic screening studies. This work was supported in part by a grant ‘Research and Development of Nanoscience’ from the Ministry of Education, Science, Sports and Culture to promote multi-disciplinary research project, and also supported in part by Grants-in-an Aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health and Welfare, Japan.
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