Cancer Prevention by Carotenoids

Mutation Research 402Ž. 1998 159±163

Cancer prevention by carotenoids

Hoyoku Nishino ) Department of Biochemistry, Kyoto Prefectural UniÕersity of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602, Japan

Received 13 May 1997; revised 30 June 1997; accepted 30 June 1997

Abstract

Epidemiological investigations have shown that cancer risk is inversely related to the consumption of green and yellow vegetables and fruits. Since b-carotene is present in abundance in these vegetables and fruits, it has been investigated extensively as a possible cancer-preventive agent. However, various carotenoids have also anti-carcinogenic activity. And we found that some of them, such as a-carotene, showed higher potency than b-carotene to suppress carcinogenesis in animal experiments. Thus, we have carried out more extended studies on the cancer-preventive activities of natural carotenoids. For example, lutein, zeaxanthin, lycopene, phytoene, fucoxanthin, peridinin and astaxanthin seem to be promising. In the present study, we confirmed the anti-carcinogenic activities of fucoxanthin and peridinin. Among natural carotenoids, phytoene has not been evaluated precisely since it becomes unstable when it is purified. Here, the cancer-preventive activity of phytoene was demonstrated using new a biotechnological method; i.e., establishment of mammalian cells producing phytoene was followed by the introduction of crtB gene which encodes phytoene synthase, and these cells were proven to acquire the resistance against transformation imposed by transfection of activated oncogenes. Further studies on various natural carotenoids, besides b-carotene, should be continued to get proper information regarding natural carotenoids in the field of cancer prevention. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: Cancer prevention; Natural carotenoid; Phytoene synthase gene

1. Introduction found that some carotenoids, such as a-carotene, showed higher potency than b-carotene to suppress Cancer chemoprevention is one of the promising carcinogenesis in animal experimentswx 2 . Therefore, methods for cancer control. Among the chemopre- we have extended the studies to evaluate cancer-preventive agents, b-carotene has been investigated ex- ventive activities of various natural carotenoids. tensivelywx 1 . However, other types of carotenoids FucoxanthinŽ. Fig. 1 is distributed on the earth as may also have anti-carcinogenic effect. In fact, we abundantly as b-carotene. Thus, it seems worthy to evaluate its biological activity. At first, we examined the free radical scavenging activity of fucoxanthin, since the anti-radical action of carotenoids has been ) Corresponding author. Tel.: q81-75-251-5316; fax: q81-75- suggested to play an important role in anti-mutagenic 213-2746. and anti-carcinogenic mechanism. We found that

Parasicyonis actinostoroides by modified procedures for the isolation and purification of fucoxanthin.

2.2. Electron spin resonance analysis for the effect of fucoxanthin on free radicals

The effect of fucoxanthin on free radicals, such as 12-doxyl-stearic acidŽ. 12-DS, 0.5 mM , 1,1-di- phenyl-2-picrylhydrazylŽ. DPPH, 0.5 mM , and the radical adduct of nitrosobenzene with linolenic acid radicalŽ NB-LP. wx 4 , which was produced by the Fig. 1. Structure of fucoxanthin and peridinin. incubation of linolenic acidŽ. 555 mM with ni- trosobenzeneŽ. 2.9 mM , was examined by the electron spin resonanceŽ. ESR method. Results were expressed as percent of control; the ESR signal fucoxanthin showed potent scavenging activity. Fur- intensity in control, which was obtained by the incu- thermore, fucoxanthin was proven to suppress liver bation of free radicals with dimethyl sulfoxide and skin carcinogenesis in vivo. Ž.DMSO , vehicle for fucoxanthin, is defined as 100%. PeridininŽ. Fig. 1 is also abundant in nature and resembles fucoxanthin in structure. Thus, we examined the anticarcinogenic activity of peridinin, com- 2.3. Carcinogenesis experiments paring with that of fucoxanthin. Some of the natural carotenoids, such as phy- The effect of fucoxanthin on spontaneous liver toene, are unstable when purified and thus their carcinogenesis in C3HrHe male mice was examined biological activities are very difficult to examine. In as described previouslywx 2 . such cases, stable production of these carotenoids in Two-stage mouse skin carcinogenesis experiments target cells may be helpful for a more accurate were carried out as described previouslywx 2 , or by a evaluation of their biological properties. In this con- modified procedure using two different kinds of text, we tried to develop the new method for the tumor promoters; i.e., 12-O-teradecanoylphorbol- synthesis of phytoene in animal cells. The establish- 13-acetateŽ. TPA, 1st-stage promoter and mezerein ment of mammalian cells producing phytoene was Ž.2nd-stage promoter . followed by the introduction of crtB gene which encodes phytoene synthase. These cells were proven to acquire resistance against transformation imposed 2.4. Establishment of phytoene-producing mam- by the transfection of activated oncogenes, and thus malian cells, and analysis of their properties the preventive activity of phytoene against carcinogenesis was confirmed. As the phytoene synthase encoding gene, crtB gene was cloned from Erwinia uredoÕora wx5 . Mam- malian expression plasmids, pCAcrtB, to transfer the crtB gene to mammalian cells, were constructed as 2. Materials and methods described previouslywx 6 . In the present study, possible resistance in phytoene-producing cells against oncogenic insult im- 2.1. Chemicals posed by transfection of the activated oncogenes was assessed. Plasmids with activated oncogenes were Fucoxanthin was isolated and purified as de- transfected to NIH3T3 cells with or without phy- scribed previouslywx 3 . Peridinin was prepared from toene production, and the rate of transformation fo- H. NishinorMutation Research 402() 1998 159±163 161

Table 1 Effect of fucoxanthin on free radicals Free radicals % of controlŽ. Inhibition % 12-DS 34%Ž. 66% DPPHa 72%Ž. 28% NB-LP 43%Ž. 57%

ESR signal intensity of free radicals was measured after the incubation with fucoxanthin or its vehicleŽ. DMSO as control, for 12 hŽ or 1 ha . . Final concentration of fucoxanthin was 0.5 mM for 12-DS and DPPH, or 2.9 mM for NB-LP. cus formation in 100-mm diameter dishes was compared.

Fig. 2. Effect of peridinin and fucoxanthin on skin-tumor formation in female ICR mice, initiated with DMBA and promoted with 3. Results TPA and mezerein. At 1 week after initiation by DMBAŽ 100 mg.Ž , TPA 10 nmol . was applied once, and then mezerein Ž 3 nmol 3.1. Inhibitory effect of fucoxanthin on free radicals for 15 weeks, and 6 nmol for the subsequent 15 weeks. was applied twice a week. Peridinin or fucoxanthinŽ 1 mmol, molar As shown in Table 1, fucoxanthin scavenged or- ratio to TPA s100.Ž was applied twice 45 min before and 16 h . ` v ganic free radicals, such as 12-DS, DPPH, and NB-LP. after TPA application . , control group; , peridinin-treated group; ', fucoxanthin-treated group. More than 50% inhibition in the ESR signal intensi- ties of 12-DS and NB-LP was observed after the incubation with fucoxanthin for 12 h. In the case of DPPH, some precipitation was observed after the Fucoxanthin also showed antitumor-promoting ac- incubation for 12 h. Thus, analysis was performed tivity in a two-stage carcinogenesis experiment in the after 1 h, and 28% inhibition was observed by skin of ICR mice, initiated with dimethyl- wx treatment with fucoxanthin. benz a anthraceneŽ DMBA, 100 mg, single application.Ž and promoted with TPA 1.6 nmol, twice a 3.2. Anti-carcinogenic actiÕity of fucoxanthin and week from 1 week after the initiation. . Tumor devel- peridinin opment was completely suppressed by fucoxanthin Žat the dose of 0.6 mmol, molar ratio to TPAs380, Fucoxanthin was proven to suppress liver tumori- applied simultaneously with TPA. up to week 20 of genesis in C3HrHe male miceŽ. Table 2 . promotionŽ. Table 2 .

Table 2 Effect of fucoxanthin on mouse liver and skin carcinogenesis Group Number of mice Tumor-bearing miceŽ. % Average number of tumors per mouse ()ALiÕer carcinogenesis Control 15 100 5.93) qFucoxanthin 15 86.7 3.07 )

()B Skin carcinogenesis Control 15 53.3 2.20 qFucoxanthin 15 0 0

Ž.A Spontaneous liver carcinogenesis model in C3HrHe male mice was used. FucoxanthinŽ. 0.001% in drinking water was given during the whole period of experimentŽ. 44 weeks . ) p-0.05. Ž.B Two-stage skin carcinogenesis model in ICR mice Ž initiated with DMBA and promoted with TPA . was used. Fucoxanthin Ž at the dose of 0.6 mmol, molar ratio to TPAs380. was applied simultaneously with TPA for 20 weeks. 162 H. NishinorMutation Research 402() 1998 159±163

Table 3 tivity in vitro, in addition to the carotenoids men- Suppression of transformation focus formation induced by onco- tioned above. Among them, b-cryptoxanthin and genes in phytoene-producing cells lactucaxanthin showed potent activity. Thus, we are Oncogene Number of transformed foci now planning to evaluate their anticarcinogenic ac- Control qcrtB tivity in vivo. In any case, it appears that more ras Ž.pNCO602 79.5 15.0 precise investigation of biological activity not only hst Ž.pKOHST1-6 90.5 54.5 of b-carotene, but also of other kinds of carotenoids, is important for understanding their significance in cancer prevention. Phytoene is present in various vegetables and The antitumor promoting activity of peridinine fruits, such as tomatoes and oranges. Phytoene has was examined in a two-stage carcinogenesis in the been suggested to have anti-oxidative activity. This skin of ICR mice, initiated with DMBAŽ 100 mg, hypothesis was confirmed in our previous studywx 6 ; single application.Ž and promoted with TPA 10 nmol, the level of phospholipid hydroperoxidation induced single application.Ž and mezerein 3 nmol for 15 by the Fe3qrADP system was significantly reduced weeks, and 6 nmol for the subsequent 15 weeks, in cells producing phytoene. Thus, mutagenesis via twice a week.Ž . Peridinin 1 mmol, molar ratio to oxidative stress may also be reduced in cells that TPAs100, applied twice; i.e., 45 min before and 16 produce phytoene. h after TPA application. suppressed the development In the present study, phytoene-producing cells of skin tumor, and its potency was proven to be were proven to acquire the resistance against trans- higher than that of fucoxanthinŽ. Fig. 2 . formation imposed by transfection of oncogenes. This type of experimental method may possibly be 3.3. Resistance against oncogenic insult imposed by applied for the evaluation of the anticarcinogenic transfection of the actiÕated oncogenes in activity of other phytochemicals, since the cloning of phytoene-producing cells genes for the synthesis of various kinds of sub- stances in vegetables and fruits has already been The rate of oncogene-induced transformation in accomplished. It is particularly useful to evaluate the cells transfected with pCAcrtB or vector alone was biological activity of unstable phytochemicals. compared. As a result, it was proven that the rate of transformation focus formation induced by the transfection of activated oncogenes was lower in the Acknowledgements phytoene-producing cells than in control cellsŽ Table 3.. This work was supported in part by grants from the Program for Promotion of Basic Research Activi- ties for Innovative Biosciences, the Organization for 4. Discussion Drug ADR Relief, R&D Promotion and Product Review, the Ministry of Health and WelfareŽ the The anti-carcinogenic activity of fucoxanthin and 2nd-term Comprehensive 10-year Strategy for Can- peridinin was confirmed in this study. Peridinin cer Control. , the Ministry of Education, Science and showed higher activity than fucoxanthin, and buteno- Culture, SRF, and the Plant Science Research Foun- lide ring structure in peridinin may contribute to dation, Faculty of Agriculture, Kyoto University, potentiate anti-carcinogenic activity. In this context, Japan. The study was carried out in collaboration we are now investigating various butenolide com- with research groups of Kyoto Prefectural University pounds, and some of them have been found to have of Medicine, Kagoshima University, National Cancer potent antitumor promoter activityŽ unpublished Center Research Institute, Tokyo Metropolitan Insti- data. . tute of Medical Science, Kirin Brewery, Nippon Recently, we have found that various kinds of Suisan Kaisha, and Electric Power Development, natural carotenoids showed antitumor promoter ac- Japan. H. NishinorMutation Research 402() 1998 159±163 163

References wx4 H. Iwahashi, C.E. Parker, R.P. Mason, K.B. Tomer, Radical adducts of nitrosobenzene and 2-methyl-2-nitrosopropane with wx1 R. Peto, R. Doll, J.D. Buckley, M.B. Sporn, Can dietary 12,13- epoxylinolenic acid radical, and 14,15- Ž. beta-carotene materially reduce human cancer rates?, Nature epoxyarachidonic acid radical, Biochem. J. 276 1991 447± Ž. 453. 290 1981 201±208. wx wx2 M. Murakoshi, H. Nishino, Y. Satomi, J. Takayasu, T. 5 N. Misawa, M. Nakagawa, K. Kobayashi, S. Yamano, Y. Hasegawa, H. Tokuda, A. Iwashima, J. Okuzumi, H. Okabe, Izawa, K. Nakamura, K. Harashima, Elucidation of the Er- H. Kitano, R. Iwasaki, Potent preventive action of a-carotene winia uredoÕora carotenoid biosynthetic pathway by func- against carcinogenesis: spontaneous liver carcinogenesis and tional analysis of gene products expressed in Escherichia coli, J. Bacteriol. 172Ž. 1990 6704±6712. promoting stage of lung and skin carcinogenesis in mice are wx suppressed more effectively by a-carotene than by b-carotene, 6 Y. Satomi, T. Yoshida, K. Aoki, N. Misawa, M. Masuda, M. Cancer Res. 52Ž. 1992 6583±6587. Murakoshi, N. Takasuka, T. Sugimura, H. Nishino, Production wx3 J. Okuzumi, H. Nishino, M. Murakoshi, A. Iwashima, Y. of phytoene, an oxidative stress protective carotenoid, in Tanaka, T. Yamane, Y. Fujita, T. Takahashi, Inhibitory effects mammalian cells by introduction of phytoene synthase gene of fucoxanthin, a natural carotenoid, on N-myc expression and crtB isolated from bacterium Erwinia uredoÕora, Proc. Jpn. Ž. cell cycle progression in human malignant tumor cells, Cancer Acad. Ser. B 71 1995 236±240. Lett. 55Ž. 1990 75±81.