Phenolic Compounds in Food and Their Effects on Health II

Chapter 30

Sarcophytol A and Its Analogs Cancer Preventive Activity 1

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H. Fujiki , M . Suganuma , K. Takagi , S. Nishiwaki , S. Yoshizawa , S. Okabe , J . Yatsunami , K. Frenkel , W. Troll , J . A. Marshall , and M . A. Tius 1

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Cancer Prevention Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan Department of Environmental Medicine, New York University Medical Center, New York, NY 10016 Department of Chemistry, University of South Carolina, Columbia, SC 29208 Department of Chemistry, University of Hawaii at Manoa, 2545 The Mall, Honolulu, HI 96822 2

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The aim of our research is screening for new cancer chemopreventive agents which inhibit tumor promotion in two-stage carcinogenesis experiments on mouse skin and studying their mechanisms of action. Sarcophytol A, isolated from the soft coral, Sarcophyton glaucum, inhibits the tumor promoting activities of okadaic acid as well as 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters on mouse skin. Based on these results, we extended the study to its analogs, (+)-α-2, 7, 11-cembratriene-4, 6,-diol (α-CBT), 3, 7, 11-trimethylcyclodeca-3E, 7E, 11E-triene-1-ol (Compound 1) and 2, 8, 12-trimethyldeca-1, 5Z, 7E, 11tetraene-4-ol (Compound 4). Compound 1 inhibited tumor promotion of okadaic acid on mouse skin more strongly than sarcophytol A or α-CBT. Compound 4 was less effective than sarcophytol A or α-CBT. A diet containing 0.05% sarcophytol A extended survival of A K R mice by 5 weeks. These mice die due to development of spontaneous thymic lymphoma. Distribution of H-sarcophytol A after a single oral admin istration was also studied. In addition, we briefly reviewed the inhibitory effects of sarcophytol A on chemical carcinogenesis in large bowel cancer and spontanenous tumor development in the mammary gland and liver in mice. It is worthwhile to investigate Compound 1, sarcophytol A and αCBT as promising cancer chemopreventive agents. 3

Sarcophytol A and Its Analogs In 1979, Kobayashi and his colleagues found significant amounts of cembrane-type diterpenes in the lipid extract of the soft coral, Sarcophvton glaucum. and characterized the structures of the four main compounds, based on the spectral data and degradative studies by ozonolysis (1). Sarcophytol A , with a molecular weight of 288, was one of the main compounds (Fig. 1). In 1985, Mizusaki and his 5

Current address: Gifu University School of Medicine, Gifu 500, Japan 0097-6156/92/0507-0380S06.00/0 © 1992 American Chemical Society

Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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FUJIKI ET AL.

Sarcophytol A & Its Analogs: Cancer Preventive Activity

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colleagues isolated two diastereoisomers of 2,7,1 l-cembratriene-4,6-diol (a- and βCBT) from the neutral fractions of cigarette smoke condensate (Fig. 1), that inhibited induction of early antigen of Epstein-Barr virus in lymphoblastoid Raji cells by 12-0tetradecanoylphorbol-13-acetate (TPA) (Fig. 2) and inhibited tumor promotion of TPA in a two-stage carcinogenesis experiment on mouse skin (2). Sarcophytol A is structurally similar to α-CBT (Fig. 1). In 1989, our group in Tokyo reported that sarcophytol A inhibits tumor promotion induced by teleocidin, one of the TPA-type tumor promoters on mouse skin (3). In 1989, J. A . Marshall and E. D . Robinson succeeded in the total synthesis of (+)-a-CBT from the achiral 17-membered ketone 3 by a sequence featuring asymmetric reduction, diastereoselective Wittig ring contraction and hydroxyl directed epoxidation (4). In 1990, Takayanagi et al. reported the stereoand enantioselective total synthesis of sarcophytol A (5). Tius et al. had already synthesized the other two analogs of sarcophytol A , Compound 1 and Compound 4 (Fig. 1). Compound 1 is 3,7, ll-trimethylcyclodeca-3E, 7E, llE-triene-l-ol, which differs from sarcophytol A in lacking the isopropyl group and one alkene group. Compound 4 is 2, 8,12-trimethyldeca-l, 5Z, 7E, 1 l-tetraene-4-ol, namely, an acyclic analog. In this experiment, sarcophytol A isolated from soft coral was used, and the other analogs used were chemically synthesized. 6

Inhibition of Tumor Promotion Induced by Okadaic A c i d Okadaic acid is a polyether compound of a C38 fatty acid (Fig. 2), isolated from the black sponge, Halichondria okadai (6). and a new tumor promoter as strong as TPA on female CD-I mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA) (7). Okadaic acid acts differently on cells of mouse skin than TPA, namely, it inhibits the activities of protein phosphatases 1 and 2A, resulting in an increase of phosphoproteins, which is called the apparent activation of protein kinases (8) (Fig. 3). Since the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver, we think that the okadaic acid pathway is similar to the process of tumor promotion in human cancer development (9). In this experiment, therefore, we studied inhibition of tumor promotion of okadaic acid by sarcophytol A and its analogs. According to the two-stage carcinogenesis experiment, mouse skin was initiated with a single application of 100 μg D M B A followed by repeated applications of 1 (1.2 nmol) okadaic acid twice a week. Sarcophytol A and its analogs were applied 15 min before each application of okadaic acid. The amount of sarcophytol A and its analogs applied was 10 times (12 nmol) more than that of okadaic acid in this experiment (Table 1). Figure 4 shows percentages of tumor-bearing mice as well as average numbers of tumors per mouse. The control group treated with D M B A and okadaic acid resulted in 86.7% of mice with tumors and 4.7 average numbers of tumors per mouse in week 20 of tumor promotion. Table 1 summarizes the inhibition of tumor promotion of okadaic acid by sarcophytol A and its analogs. α-CBT inhibited tumor promotion of okadaic acid as effectively as sarcophytol A in week 20. Compound 4, an acyclic analog, was less effective than sarcophytol A , indicating that there is a cyclic structural requirement for inhibitory activity. The inhibitory activity of Compound 1 was exciting. The treatment with Compound 1 reduced the percentage of tumor-bearing mice from 86.7% to 26.7% and the average numbers of tumors per mouse from 4.7 to 0.3 in week 20 (Table 1). The absence of the isopropyl group and one alkene group in Compound 1 enhanced the inhibitory activity and was more effective than sarcophytol A and α-CBT. Compound 1 is a synthetic compound and is not naturally occurring. As for the mechanisms of action of sarcophytol A , we studied how sarcophytol A interacts with the tumor promoting process of okadaic acid. The


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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II

Compound 1

Fig. 1.

Compound 4

Structures of sarcophytol A and its analogs.

OCO(CH ) CH 2

12

Okadaic acid

TPA-type tumor promoter

Signal

Fig. 3. Mechanisms of action of the two different types of tumor promoters. Reproduced with permission from ref. 19. Copyright 1990 University of Nagoya Press.


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Table 1 Inhibition of Tumor Promotion of Okadaic Acid by Sarcophytol A and Its Analogs

Inhibitors

Amount

% of tumor-

Average numbers of

per

bearing mice

tumors per mouse

application

in week 20

in week 20

Control

86.7

4.7

Compound 4

2.8

73.3

2.7

α-CBT

3.7

46.7

2.0

Sarcophytol A

3.5

46.7

1.5

Compound 1

3.0

26.7

0.3

A g

B

ioo

X

c

X

CO CD

ο Ε

O

/ -

Ui

50

-

C 3

/

r i L

o

fj t i Y dp

D 4-

o

-oooodow

W 2

i c

-oooooWHX 20

0

20

Weeks of promotion

Fig. 4.

Inhibitory effects of sarcophytol A and its analogs on tumor promotion of okadaic acid. The groups treated with D M B A and okadaic acid (x); D M B A and okadaic acid plus Compound 4 ft, D M B A and okadaic acid plus α-CBT D M B A and okadaic acid plus sarcophytol A ft and D M B A and okadaic acid plus Compound 1 (Q.



384 3

specific H-okadaic acid binding to the particulate fraction of mouse skin was not inhibited by sarcophytol A at concentrations of up to 1 mM. Sarcophytol A also did not reverse the inhibition by okadaic acid of protein phosphatase 1 and 2A activities, whose enzymes are receptors of okadaic acid (10). Therefore, we think that sarco phytol A inhibits the process of tumor promotion after the binding of okadaic acid to protein phosphatases. Inhibition of Tumor Promotion of TPA-type Compounds As we previously reported, sarcophytol A inhibited tumor promotion by teleocidin (3). Sarcophytol A also inhibited tumor promotion by the TPA-types, TPA and aplysiatoxin (data not shown). We previously demonstrated that sarcophytol A inhibited H2O2 formation by TPA-activated human polymorphonuclear leukocytes (11). We extended the study to its analogs. Inhibitory potency of Compound 1 was much stronger than sarcophytol A . Therefore, the order of their inhibitory potency against H2O2 forma tion: Compound 1 > sarcophytol A >, probably, α-CBT > Compound 4 (data not shown), is comparable to that of their inhibitory activity of tumor promotion, although TPA rather than okadaic acid was used as a tumor promoter in the experiment We think Compound 1 might inhibit tumor promotion of the TPA-types more strongly than sarcophytol A . Inhibitory Effects of Spontaneous Thymic Lymphoma in A K R mice It is well known that A K R mice die due to compression of the trachea by enlarged thymic lymphoma within one year (12). To test the effects of sarcophytol A on development of spontaneous thymic lymphoma, female A K R mice were purchased from the Jackson Laboratory, Bar Harbor, M E , U.S.A. The control group (29 mice) was given a basal diet. The experimental group (29 mice) was given a diet containing 0.05% sarcophytol A from 7 weeks of age. Figure 5 shows percentages of survival of the two groups. The 50% mortality of the control group was reached at 33 weeks of age, whereas that of the experimental group was at 38 weeks of age. Thus, the survival of A K R mice was extended 5 weeks by sarcophytol A treatment, probably by inhibiting development of thymic lymphoma. It has been reported that a diet supplemented with choline and methionine extended the survival of A K R mice (13). 3

Distribution of H-Sarcophytol A after O r a l Administration 3

A single oral administration of H-sarcophytol A (0.255 mCi/ml in sesame oil, specific activity of 12.2 Ci/mmol) was carried out by intubation. Two mice were sacrificed by decapitation 19 hours after housing in metabolic cages, and the tissues and feces were oxidized by combustion in a Packard Sample Oxidizer 306. The radioactivity of Η2θ produced by oxidization derived from H-sarcophytol A were measured by a liquid scintillation counter. Table 2 shows the distribution of radioactivity of H-sarcophytol A 19 hours after oral administration. The yield was 71%. Radioactivity was mainly found in the feces and urine. The 50% radioactivity extracted from the feces was found to be at the same Rf as that of sarcophytol A on thin-layer chromatography, whereas the other 50% migrated to different Rfs than that of the parent compound. Radio activity extracted from urine migrated differently than H-sarcophytol A on thin-layer chromatography (data not shown). In addition, our autoradiographical study of the 3

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385

Age ( weeks )

Fig. 5.

Inhibitory effects of sarcophytol A in the diet on development of spontaneous thymic lymphoma in female A K R mice. The groups treated with sarcophytol A (O) and control diet (·). Table 2 Distribution of Radioactivity 19 Hours After Oral Administration of 3

H-Sarcophytol A in Mice 7

Total 5.6 χ 10 dpm % of Radioactivity

Feces

53.60

Urine

16.10

liver

0.60

Stomach

0.30

Skin

0.30

Lung

0.09

Kidney

0.05

Spleen

0.01

Total yield

71.05



386 3

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distribution of H-sarcophytol A supported the evidence that most H-sarcophytol A was excreted during the first 24 hours. The rapid excretion observed strengthened our previous evidence that sarcophytol A is a compound with low toxicity potential (3). Additional Anticarcinogenic Activities We have previously reported anticarcinogenic activities of sarcophytol A in various organs. It is important to briefly review these anticarcinogenic effects. Dietary sarcophytol A inhibited the development of large bowel cancer induced by N-methylN-nitrosourea in female rats (14), spontaneous mammary tumors in female SHN mice (15) and spontaneous liver tumors in male C3H/HeN mice (16). However, sarcophytol A was not effective at inhibiting duodenal carcinogenesis by N-ethyl-N'nitro-N-nitrosoguanidine in male C57BL/6 mice. (Yamane, unpublished results). Recently, we have demonstrated that the inhibitory effect of sarcophytol A is enhanced by cotreatment with medroxyprogesterone acetate, an angiogenesis inhibitor, on tumor promotion by okadaic acid in mouse skin (17). As Moon and Mehta reported, combination chemoprevention approaches to various organs seems to be promising (18). Compound 1 indicates there is great potential in pursuing combination approaches to cancer chemoprevention. Acknowledgments This work was supported in part by Grants-in-Aid for cancer research from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan, and by grants from the Foundation for Promotion of Cancer Research, the Princess Takamatsu Cancer Research Fund, the Uehara Memorial Life Science Foundation and the Smoking Research Foundation. We thank Dr. T. Sugimura at National Cancer Center for his encouragement of the work and Dr. H . Takahara at the Smoking Research Foundation for his interest in the work. The authors (K. T., S. Ν. and J. Y.) thank the Foundation for Promotion of Cancer Research, Japan for support of their work at the National Cancer Center Research Institute, Tokyo. Racemic Compounds 1 and 4 were prepared by Jean M . Cullingham and Xue-quin Gu, University of Hawaii at Manoa. Literature Cited 1. Kobayashi, M.; Nakagawa, T.; Mitsuhashi, H. Chem. Pharm. Bull. (Tokyo) 1979, 27, 2382. 2. Saito, Y.; Takizawa, H.; Konishi, S.; Yoshida, D.; Mizusaki, S. Carcino genesis 1985, 6, 1189. 3. Fujiki, H.; Suganuma, M.; Suguri, H.; Yoshizawa, S.; Takagi, K.; Kobayashi, M. J. Cancer Res. Clin. Oncol 1989, 115, 25. 4. Marshall, J. Α.; Robinson, E. D.. Tetrahedron Lett. 1989, 30, 1055. 5. Takayanagi, H.; Kitano, Y.; Morinaka, Y. Tetrahedron Lett. 1990, 31, 3317. 6. Tachibana, Y.; Scheuer, P. J.; Tsukitani, Y.; Kikuchi, H.; Van Engen, D.; Clardy, J.; Gopichand, Y.; Schmitz, F. J. J. Am. Chem. Soc. 1981, 103, 2469. 7. Suganuma, M.; Fujiki, H.; Suguri, H.; Yoshizawa, S.; Hirota, M.; Nakayasu, M.; Ojika, M.; Wakamatsu, K.; Yamada, K.; Sugimura, T. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 1768.


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8. Sassa, T.; Richter, W. W.; Uda, N.; Suganuma, M.; Suguri, H.; Yoshizawa, S.; Hirota, M.; Fujiki, H. Biochem. Biophys. Res. Commun. 1989, 159, 939. 9. Fujiki, H.; Suganuma, M.; Nishiwaki, S.; Yoshizawa, S.; Yatsunami, J.; Matsushima, R.; Furuya, H.; Okabe, S.; Matsunaga, S.; Sugimura, T. In Relevance of Animal Studies to Evaluate Human Cancer Risk; D'Amato, R., Slaga, T. J., Farland, W., Henry, C., Eds.; John Wiley & Sons, Inc. New York, Ν. Y. in press. 10. Nishiwaki, S.; Fujiki, H.; Suganuma, M.; Ojika, M.; Yamada, K.; Sugimura, T. Biochem. Biophys. Res. Commun. 1990, 170, 1359. 11. Frenkel, K.; Zhong, Z.; Rashid, K.; Fujiki, H. In Anticarcinogenesis and Radiation Protection: Strategies in ProtectionfromRadiation and Cancer; Nygaard, O. F., Ed.; Plenum Press, New York, in press. 12. Gross, L. In Mouse Leukemia in Oncogenic Viruses, Vol 1; Pergamon Press, New York, 1983, p. 305. 13. Wainfan, E.; Dizik, M.; Kelkenny, M.; O'Callaghan, J. P. Carcinogenesis 1990, 11, 361. 14. Narisawa, T.; Takahashi, M.; Niwa, M.; Fukaura, Y.; Fujiki, H. Cancer Res. 1989, 49, 3287. 15. Fujiki, H.; Suganuma, M.; Suguri, H.; Takagi, K.; Yoshizawa, S.; Ootsuyama, Α.; Tanooka, H.; Okuda, T.; Kobayashi, M.; Sugimura, T. In Antimutagenesis and Anticarcinogenesis Mechanisms II; Kuroda, K., Shankel, D. M., Waters, M. D., Eds.; Plenum Press, New York, London, 1990, p. 205. 16. Yamauchi, O.; Omori, M.; Ninomiya, M.; Okuno, M.; Moriwaki, H.; Suganuma, M.; Fujiki, H.; Muto, Y. Jpn. J. Cancer Res. 1991, 82, 1234. 17. Suganuma, M.; Yoshizawa, S.; Yatsunami, J.; Nishiwaki, S.; Furuya, H.; Okabe, S.; Nishiwaki-Matsushima, R.; Frenkel, K.; Troll, W.; Verma, A. K.; Fujiki, H. In Antimutagenesis and Anticarcinogenesis Mechanisms III; Bronzetti, G., De Flora, S., Shankel, D. M., Waters, M. D., Eds.; Plenum Press, New York, London, in press. 18. Moon, R. C.; Mehta, R. G. In Antimutagenesis and Anticarcinogenesis Mechanisms II; Kuroda, K., Shankel, D. M., Waters, M. D., Eds.; Plenum Press, New York, London, 1990, p. 213. 19. Fujika, H. et al. In Epidemiology and Prevention of Cancer; Sasaki, R. and Aoki, K., Eds. University of Nagoya Press: Nagoya, Japan; 1990, pp36-41. RECEIVED

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Phenolic Compounds in Food and Their Effects on Health II

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