Multi carotenoids report

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  • 1. Cancer and Metastasis Reviews 21: 257–264, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. Carotenoids in cancer chemoprevention Hoyoku Nishino1 , Michiaki Murakoshi1,3 , Tsunehiro Ii1 , Manabu Takemura1 , Masashi Kuchide1 , Motohiro Kanazawa2 , Xiao Yang Mou1 , Saeri Wada1 , Mitsuharu Masuda1 , Yasuhito Ohsaka1 , Shingo Yogosawa1 , Yoshiko Satomi1 and Kenji Jinno4 1 Department of Biochemistry, 2 Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto; 3 Research & Development Headquarters, Lion Corporation, Odawara, Kanagawa; 4 Internal Medicine, Shikoku Cancer Center, Matsuyama, Ehime, Japan Key words: natural carotenoids, multicarotenoids, cancer chemoprevention, bio-chemoprevention, functional foods Abstract Various natural carotenoids, besides β-carotene, were proven to have anticarcinogenic activity, and some of them showed more potent activity than β-carotene. Thus, these carotenoids (α-carotene, lutein, zeaxanthin, lycopene, β-cryptoxanthin, fucoxanthin, astaxanthin, capsanthin, crocetin and phytoene), as well as β-carotene, may be useful for cancer prevention. In the case of phytoene, the concept of ‘bio-chemoprevention’, which means biotechnologyassisted method for cancer chemoprevention, may be applicable. In fact, establishment of mammalian cells producing phytoene was succeeded by the introduction of crtB gene, which encodes phytoene synthase, and these cells were proven to acquire the resistance against carcinogenesis. Antioxidative phytoene-containing animal foods may be classified as a novel type of functional food, which has the preventive activity against carcinogenesis, as well as the ability to reduce the accumulation of oxidative damages, which are hazardous for human health. 1. Introduction Informations have been accumulated indicating that diets rich in vegetables and fruits can reduce the risk of a number of chronic diseases, including cancer, cardiovascular disease, diabetes and age-related macular degeneration. Various factors in plant foods, such as carotenoids, antioxidative vitamins, phenolic compounds, terpenoids, steroids, indoles and fibers, have been considered responsible for the risk reduction. Among them, carotenoids have been studied widely and proven to show diverse beneficial effects on human health. Initially, carotenoids in vegetables and fruits were suggested to serve as precursors of vitamin A as the active compounds. In this context, β-carotene has been studied most extensively, since β-carotene has the highest pro-vitamin A activity among carotenoids. However, Peto et al. [1] suggested that β-carotene could have a protective effect against cancer without converting to vitamin A. Then, carotenoids other than β-carotene may also contribute to protection of cancer and various diseases. Of more than 600 carotenoids identified up to date, about 40 carotenoids are found in our daily foods. However, as a result of selective uptake in digestive tract, only 14 carotenoids with some of their metabolites have been identified in human plasma and tissues. Thus, we decided to evaluate the biological activities of these carotenoids which are detectable in human body, and found that some of them showed more potent activity than β-carotene to suppress the process of carcinogenesis. In addition, antioxidative activity of α-carotene and lycopene was also proven to be higher than that of β-carotene. Therefore, various natural carotenoids, besides β-carotene, seem to be possible candidates as factors for anticarcinogenic functional foods. Here, at first, recent results of the evaluation for anticarcinogenic activity of various natural carotenoids are summarized. Some of the natural carotenoids, such as phytoene, are unstable when they are purified, and thus, it is very difficult to examine the biological activities of them. In such cases, stable production of these carotenoids in target cells may be
  • 2. 258 helpful for more accurate evaluation of their biological properties. In this context, we tried to develop the new method for the synthesis of phytoene in mammalian cells. And, establishment of mammalian cells producing phytoene was succeeded by the introduction of crtB gene, which encodes phytoene synthase. These cells were proven to acquire the resistance against carcinogenesis, as well as oxidative stress. Thus, usefulness of phytoene was proven. 2. Anti-carcinogenic activity of natural carotenoids Among the carotenoids, β-carotene has been expected to be the most promising candidate as cancer preventive agent. Thus, mainly β-carotene has been tested for cancer-preventive activity in interventional trials, i.e., two Linxian trials (Linxian 1 and Linxian 2), the Alpha-Tocopherol Beta-Carotene (ATBC) Cancer Prevention Study, the β-Carotene and Retinol Efficiency Trial (CARET), the Physicians’ Health Study (PHS) and the Skin Cancer Prevention Study (SCPS). In addition to these studies, we have recently completed the intervention trial with supplementation of the mixture of natural carotenoids (lycopene, β-carotene, α-carotene, and others) plus α-tocopherol, and the analysis of the results is now going on (Jinnno K, Nishino H, et al. 2002, unpublished information, see the Section 2.8. Multicarotenoids). 2.1. β-Carotene In the Linxian 1 study, a protective effect of supplemental β-carotene, vitamin E, and selenium was reported with regard to the incidence and mortality rates of gastric cancer when compared with untreated subjects. In the Linxian 2 study, the relative risk for cancer mortality was 0.97 in men and 0.92 in women (not significant). At the end of follow-up in ATBC cancer prevention study, 894 cases of lung cancer were reported. The numbers of lung cancer cases by intervention group were 204 in α-tocopherol, 242 in β-carotene, 240 in α-tocopherol, plus β-carotene, and 208 in placebo. The group receiving β-carotene had a 16% higher incidence of lung cancer than those not given β-carotene. The excess risk associated with β-carotene supplementation was concentrated mainly among people who currently smoked more than 20 cigarettes per day and who drank more than 11 g/day of ethanol. In the CARET, the relative risk of lung cancer incidence was 1.3 in the groups treated with β-carotene and retinal (p = 0.02), 1.4 in the current smoker group treated with β-carotene and retinal, and 1.4 in the asbestos-exposed group treated with β-carotene and retinal. In the PHS, no significant modification in risk was found. In the SCPS, the relative risk for skin cancer was 1.4 in the smoker group treated with β-carotene. 2.2. α-Carotene In recent studies, we found that α-carotene induced the G1-arrest in the process of cell cycle [2]. Since various agents which induce G1-arrest have been proven to have cancer preventive activity, we evaluated anticarcinogenic activity of α-carotene. α-Carotene showed higher activity than β-carotene to suppress the tumorigenesis in skin, lung, liver and colon [3,4]. In skin tumorigenesis experiment, two-stage mouse skin carcinogenesis model was used. Seven-week-old female ICR mice had their backs shaved with electric clipper. From 1 week after initiation by 100 µg of 7,12-dimethylbenz[a]anthracene (DMBA), 1.0 µg of 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied twice a week. α- or β-Carotene (200 nmol) was applied with each TPA application. The higher potency of α-carotene than β-carotene was observed. The percentage of tumor-bearing mice in the control group was 69%, whereas the percentages of tumor-bearing mice in the groups treated with α- and β-carotene were 25% and 31%, respectively. The average number of tumors per mouse in the control group was 3.7, whereas the α-carotene-treated group had 0.3 tumors per mouse (p < 0.01, Student’s t-test). β-Carotene treatment also decreased the average number of tumors per mouse (2.9 tumors per mouse), but the difference from the control group was not significant. The higher potency of α-carotene than β-carotene in the suppression of tumor promotion was confirmed by other two-stage carcinogenesis experiment, i.e., 4-nitroquinoline 1-oxide (4NQO)-initiated and glycerol-promoted ddY mouse lung carcinogenesis model. 4NQO (10 mg/kg body weight) was given by a single s.c. injection on the first experimental day. Glycerol (10% in drinking water) was given as tumor promoter from experimental week 5 to week 30 continuously. α- or β-Carotene (at the concentration of 0.05%) or vehicle as a control was mixed as
  • 3. 259 an emulsion into drinking water during the promotion stage. The average number of tumors per mouse in the control group was 4.1, whereas the α-carotenetreated group had 1.3 tumors per mouse (p < 0.001). β-Carotene treatment did not show any suppressive effect on the average number of tumors per mouse, but rather induced slight increase (4.9 tumors per mouse). In liver carcinogenesis experiment, spontaneous liver carcinogenesis model was used. Male C3H/He mice, which have a high incidence of spontaneous liver tumor development, were treated for 40 weeks with α- and β-carotene (at the concentration of 0.05%, mixed as an emulsion into drinking water) or vehicle as a control. The mean number of hepatomas was significantly decreased by α-carotene treatment as compared with that in the control group; the control group developed 6.3 tumors per mouse, whereas the α-carotene-treated group had 3.0 tumors per mouse (p < 0.001). On the other hand, the β-carotene-treated group did not show a significant difference from the control group, although a tendency toward a decrease was observed (4.7 tumors per mouse). A short-term experiment to evaluate the suppressive effect of α-carotene on colon carcinogenesis, effect on N-methylnitrosourea (MNU, three intrarectal administrations of 4 mg in week 1)-induced colonic aberrant crypt foci formation (ACF) was examined in Sprague– Dawley (SD) rats. α- or β-Carotene (6 mg, suspended in 0.2 ml of corn oil, intragastric gavage daily) or vehicle as control were adminstered during weeks 2 and 5. The mean number of colonic ACF in control group was 63, whereas α- or β-carotene-treated group had 42 (significantly lower than that in the control group: p < 0.05) and 56, respectively. Thus, the greater potency of α-carotene than β-carotene was again observed. 2.3. Lutein Lutein is dihydroxy-form of α-carotene, and distributed among variety of vegetables, such as kale, spinach and winter squash, and fruits, such as mango, papaya, peaches, prunes and oranges. Epidemiological study in Pacific Islands indicated that people with high intake of all three of β-carotene, α-carotene and lutein had the lowest risk of lung cancer [5]. Thus, the effect of lutein on lung carcinogenesis was examined. Lutein showed anti-tumor promoting activity in a two-stage carcinogenesis experiment in lung of ddY mice, initiated with 4NQO and promoted with glycerol. Lutein, 0.2 mg in 0.2 ml of mixture of olive oil and Tween 80 (49 : 1), was given by oral intubation three times a week during tumor promotion stage (25 weeks). Treatment with lutein showed a tendency of decrease of lung tumor formation; the control group developed 3.1 tumors per mouse, whereas the lutein-treated group had 2.2 tumors per mouse. The anti-tumor promoting activity of lutein was confirmed by another two-stage carcinogenesis experiment, i.e., it showed antitumor promoting activity in a two-stage carcinogenesis experiment in skin of ICR mice, initiated with DMBA and promoted with TPA and mezerein. At 1 week after the initiation by 100 µg of DMBA, TPA (10 nmol) was applied once, and then mezerein (3 nmol for 15 weeks, and 6 nmol for subsequent 15 weeks) twice a week. Lutein (1 µmol, molar ratio to TPA = 100) was applied twice (45 min before and 16 h after TPA application). At the experimental week 30, average number of tumors per mouse in the control group was 5.5, whereas the lutein-treated group had 1.9 tumors per mouse (p < 0.05). Lutein also inhibited the development of ACF in SD rat colon induced by MNU (three intrarectal administrations of 4 mg in week 1). Lutein (0.24 mg, suspended in 0.2 ml of corn oil, intragastric gavage daily) or vehicle as control were administrated during weeks 2–5. The mean number of colonic ACF in control group at week 5 was 69, whereas lutein-treated group had 40 (significantly lower than that in the control group: p < 0.05) [4]. 2.4. Zeaxanthin Zeaxanthin is dihydroxy-form of β-carotene, and distributed in our daily foods, such as corn and various vegetables. Since awareness of zeaxanthin as a beneficial carotenoid is achieved recently, available data for zeaxanthin are little. Recently, some features of zeaxanthin were elucidated. For example, zeaxanthin suppressed TPAinduced expression of early antigen of Epstein–Barr virus in Raji cells. TPA-enhanced 32 Pi-incorporation into phospholipids of cultured cells was also inhibited by zeaxanthin. Anti-carcinogenic activity of zeaxanthin in vivo was also examined. For example, it was found that spontaneous liver carcinogenesis in C3H/He male mice was suppressed by the treatment with zeaxanthin (at the concentration of 0.005%, mixed as an emulsion with drinking water).
  • 4. 260 Anti-metastatic activity of zeaxanthin was also reported. Table 1. Effect of lycopene on tumorigenesis in mouse lung and liver Group Tumor-bearing mice (%) Average number of tumors per mouse Lung carcinogenesisc Control 15 +Lycopene 13 67 46 3.1 1.4a Liver carcinogenesisd Control 17 +Lycopene 13 88 39 7.7 0.9b 2.5. Lycopene Lycopene occurs in our diet, predominantly in tomatoes and tomato products. Recently, the exceptionally high singlet oxygen quenching ability of lycopene was found [6,7]. Epidemiological study in elderly Americans indicated that high tomato intake was associated with 50% reduction of mortality from cancers at all sites [8]. And the case-control study in Italy showed potential protection of high consumption of lycopene from tomatoes against cancers of digestive tract [9]. Inverse association between high intake of tomato products and prostate cancer risk was also reported [10]. Studies on anti-carcinogenic activity of lycopene in animal models were carried out in mammary gland, liver, lung, skin and colon [4,11]. The study in mice with a high rate of spontaneous mammary tumors showed that intake of lycopene delayed and reduced tumor growth. Lycopene showed anti-tumor promoting activity in a two-stage carcinogenesis experiment in lung of ddY mice, initiated with 4NQO and promoted with glycerol. Lycopene, 0.2 mg in 0.2 ml of mixture of olive oil and Tween 80 (49 : 1), was given by oral intubation three times a week during tumor promotion stage (25 weeks). Treatment with lycopene resulted in the significant decrease of lung tumor formation; the control group developed 3.1 tumors per mouse, whereas the lycopene-treated group had 1.4 tumors per mouse (p < 0.05) (Table 1). The anti-tumor promoting activity of lycopene was confirmed by another two-stage carcinogenesis experiment, i.e., it showed antitumor promoting activity in a two-stage carcinogenesis experiment in skin of ICR mice, initiated with DMBA and promoted with TPA. Lycopene (160 nmol, molar ratio to TPA = 100) was applied with each TPA application. At the experimental week 20, average number of tumors per mouse in the control group was 8.5, whereas the lycopenetreated group had 2.1 tumors per mouse (p < 0.05). Lycopene also inhibited the development of ACF in SD rat colon induced by MNU (three intrarectal administration of 4 mg in week 1). Lycopene (0.12 mg, suspended in 0.2 ml of corn oil, intragastric gavage daily) or vehicle as control were administered during weeks 2–5. The mean number of colonic ACF in Number of mice p < 0.05, b p < 0.05. Male ddY mice were used. 4NQO (10 mg/kg body weight), dissolved in a mixture of olive oil and cholesterol (20 : 1), was given by a single s.c. injection on the first experimental day. Glycerol (10% in drinkig water) was given as tumor promoter from experimental week 5 to week 30 continuously. Lycopene, 0.2 mg in 0.2 ml of mixture of olive oil and Tween 80 (49 : 1), was given by oral intubation three times a week during tumor promotion stage (25 weeks). d Male C3H/He mice at the age of 6 weeks were used. Lycopene, 0.005% in drinking water, was given during the whole period of experiment (40 weeks). a c control group at week 5 was 69, whereas lycopenetreated group had 34 (significantly lower than that in the control group: p < 0.05). Spontaneous liver carcinogenesis in C3H/He male mice was also suppressed. Treatment for 40 weeks with lycopene (at the concentration of 0.005%, mixed as an emulsion into drinking water) resulted in the significant decrease of liver tumor formation as shown in Table 1 (p < 0.005). 2.6. β-Cryptoxanthin β-Cryptoxanthin seems to be a promising carotenoid, since it showed the strongest inhibitory activity in the in vitro screening test, i.e., β-cryptoxanthin suppressed TPA-induced expression of early antigen of Epstein–Barr virus in Raji cells at the highest potency among carotenoids tested [12]. TPA-enhanced 32 Piincorporation into phospholipids of cultured cells was also inhibited by β-crypto-xanthin. β-Cryptoxanthin is distributed in our daily foodstuff, such as oranges, and is one of the major carotenoids detectable in human blood. Thus, it seems worthy to investigate more precisely. In this context, we further examined the anticarcinogenic activity in vivo. β-Cryptoxanthin showed anti-tumor promoting activity in a two-stage carcinogenesis experiment in
  • 5. 261 skin of ICR mice, initiated with DMBA and promoted with TPA. β-Cryptoxanthin (160 nmol, molar ratio to TPA = 1 : 100) was applied 1 h before each TPA application. At week 20 of promotion, the percentage of tumor-bearing mice in the control group was 64%, whereas the percentages of tumor-bearing mice in the group treated with β-cryptoxanthin were 29%. The average number of tumors per mouse in the control group was 2.7, whereas the β-crypto-xanthin-treated group had 1.6 tumors per mouse (p < 0.05). Effect of β-cryptoxanthin on colon carcinogenesis was also carried out. Four groups of F344 rats (n = 25 each) received an intrarectal dose of 2 mg MNU, 3 times a week for 5 weeks, and were fed the diet supplemented with or without β-cryptoxanthin (0.0025%). The colon cancer incidence at week 30 was significantly lower in the β-cryptoxanthin diet group (68%) than in the control group (96%). The tumor multiplicity was also lower in the β-crypto-xanthin-treated group (1.4 tumors per rat) than in the control group (1.7 tumors per rat), but statistically not significant. 2.7. Other carotenoids In addition to carotenoids mentioned above, fucoxanthin, astaxanthin, capsanthin, crocetin and phytoene seem to be a promising carotenoid, since these carotenoids showed the strong inhibitory activity in screening test. Fucoxanthin is distributed in our daily foodstuff, such as see weeds, and is one of the major carotenoids which distributed in marine organisms. Thus, it seems worthy to investigate more precisely. 2.8. Multicarotenoids It is now clear that various natural carotenoids are valuable to apply for cancer prevention. These carotenoids may be suitable in combinational use, as well as single use. In fact, we have recently found that multicarotenoids (i.e., mixture of various carotenoids, such as β-carotene, α-carotene, lutein, lycopene and so on) showed potent anti-carcinogenic activity. For example, administration of a prototype multicarotenoids preparation (Table 2) resulted in the suppression of lung tumor promotion, as shown in Table 3. Furthermore, we have recently proven that administration of natural multicarotenoids (mixture of lycopene, β-carotene, α-carotene, and other natural carotenoids) with α-tocopherol resulted in the significant suppression of liver tumor development in liver Table 2. Composition of multicarotenoids Carotenoids % β-carotene α-carotene Lutein Lycopene Zeaxanthin β-Cryptoxanthin 45.0 24.7 19.0 10.3 0.9 0.1 Total 100 Table 3. Effect of oral administration of multicarotenoids on the promotion of lung tumor formation by glycerol in 4NQOinitiated mice Group (n) Tumor-bearing mice (%) Average number of tumors per mouse Control +Multicarotenoids (15) (15) 73 27a 1.4 0.4b a p < 0.05, b p < 0.01. Multicarotenoids (2 mg in 0.2 ml of oil, i.e., 3 times per week) was given during the promoting period. cirrhosis patients (Jinno K, Nishino H et al. patent pending: #2002–022958, 2002.1.31., unpublished data). Thus, multicarotenoids seem to be promising for clinical use. By the way, it is important that the effectiveness or efficiency of multicarotenoids was different between individuals, i.e., responders and non-responders were founded in clinical trial. These difference may be explained by single nucleotide polymorphisms (SNPs). Thus, SNPs is now analyzing in responders and non-responders. 3. Production of phytoene in mammalian cells 3.1. Establishment of phytoene producing mammalian cells, and analysis of their properties Phytoene, which is detectable in human blood, was proven to suppress tumorigenesis in skin. And it was suggested that antioxidative activity of phytoene may play an important role in its action mechanism. In order to confirm the mechanism, more precise study should be carried out. However, phytoene becomes to be unstable when it is purified, and thus, is very difficult to examine the biological activities. Therefore, stable production of phytoene in target cells,
  • 6. 262 which may be helpful for the evaluation of their biological properties, was tried. As phytoene synthase encoding gene, crtB, has already been cloned from Erwinia uredovora [13], we used it for the expression of the enzyme in animal cells. Mammalian expression plasmids, pCAcrtB, to transfer the crtB gene to mammalian cells, were constructed as follows. First, the sequence around the initiation codon of the crtB gene on the plasmid pCRT-B was modified by PCR using the primers to replace the original bacterial initiation codon TTG with CTCGAGCCACCATG, which is a composite of the typical mammalian initiation codon ATG preceded by the Kozak consensus sequence and a XhoI recognition site. The XhoI linker which harbors a cohesive end for the EcoRI site was ligated to the EcoRI site at the 3 -end of the crtB gene, and the 969-base pair (bp) XhoI fragment was cloned into the XhoI site of the expression vector pCAGGS. The resulting plasmid pCAcrtB drives the crtB gene by the CAG promoter (modified chicken b-actin promoter coupled with cytomegalovirus immediate early enhancer). In the pCAGGS vecter, a rabbit β-globin polyadenylation signal is provided just downstream of the XhoI cloning site. Plasmids were transfected either by electroporation or lipofection. For the gene transfer to NIH3T3 cells, which were cultured in Dulbecco’s modified minimum essential medium (DMEM) supplemented with 4 mM l-glutamin, 80 U/ml penicillin, 80 µg/ml streptomycin and 10% calf serum (CS), the parameter for electroporation using a Gene Pulser (BioRad) was set at 1500 V/25mF with a DNA concentration of 12.5–62.5 µg/ml. Lipofection was carried out using LIPOFECTAMINE (GIBCO BRL) according to the protocol supplied by the manufacturer. pCAcrtB or pCAGGS was cotransfected with the plasmid pKOneo, which harbors a neomycin resistance encoding gene (kindly provided by Dr. Douglas Hanahan, University of California, San Francisco). For Northern blot analysis, 20 µg of total RNA was loaded onto a 1.2% formaldehyde agarose gel, electrophoresed and transferred to a nitrocellulose filter (Nitroplus). The 969-bp XhoI fragment of the crtB gene as mentioned above was labeled with [32 P]dCTP by the random primer labeling method and used as a probe to hybridize the target RNA on the filter. NIH3T3 cells transfected with pCAcrtB showed the expression of a 1.5 kb mRNA from the crtB gene as a major transcript. That transcript was not present in the cells transfected with the vector alone (Table 4). Table 4. Expression of phytoene synthase mRNA and production of phytoene in mammalian cells by introduction of the crtB gene from Erwinia uredovora Transfection Phytoene synthase mRNA Phytoene (µg/107 cells) Electroporation Lipofection + + 4.4 1.5 Phytoene synthase mRNA was detected by Northern blot analysis, and phytoene in cell extracts was measured by HPLC. For analysis of phytoene by HPLC, the lipid fraction including phytoene was extracted from cells (107 –108 ). The sample was subjected to HPLC (column: 3.9 by 300 mm, Nova-pakHR, 6µ C18, Waters) at a flow rate of 1 ml/min. To detect phytoene, UV absorbance of the eluate at 286 nm was measured by a UV detector (JASCO875). Phytoene was detected as a major peak in HPLC profile in NIH3T3 cells transfected with pCAcrtB, but not in control cells (Table 4). Phytoene was identified by UV- and field desorption mass-spectra. Since lipid peroxidation is considered to play a critical role in tumorigenesis, and it was suggested that antioxidative activity of phytoene may play an important role in its mechanism of anticarcinogenic action, the level of phospholipid peroxidation induced by oxidative stress in cells transfected with pCAcrtB or vector alone was compared. Oxidative stress was imposed by culturing the cells in a Fe3+ /adenosine 5 -diphosphate (ADP) containing medium (374 mM iron (III) chloride, 10 mM ADP dissolved in DMEM) for 4 h. The cells were then washed three times with a Ca2+ and Mg2+ -free phosphate buffered saline (PBS(−)), harvested by scraping, washed once with PBS(−), suspended in 1 ml of PBS(−) and freeze-thawed once. The lipid fraction was extracted from the disrupted cells twice with 6 ml of chloroform/methanol (2 : 1). The chloroform layer was collected, dried with sodium sulfate. The sample was evaporated, and its residue was dissolved in a small volume of HPLC solvent (2-propanol : n-hexane : methanol : H2 O = 7 : 5 : 1 : 1) and then subjected to chemiluminescence-HPLC (CL-HPLC). The lipid was separated with the column (Finepack SIL NH2-5250 mm × 4.6 mm i.d. JASCO) by eluting with the HPLC solvent (see above) at a flow rate of 1 ml/min at 35◦ C. Post column chemiluminescent reaction was carried out in the mixture of 10 µg/ml cytochrome c and 2 µg/ml luminol in a borate buffer (pH 10.0) at a flow rate of 1.1 ml/min. To detect lipids, UV absorbance of
  • 7. 263 the eluate at 210 nm was measured by a UV-8011 detector (TOSOH), and chemiluminescence was detected with a CLD-110 detector (Tohoku Electric Ind.). The phospholipid hydroperoxidation level in the cells transfected with pCAcrtB and confirmed to produce phytoene by HPLC, was lower than that in the cells transfected with vector alone (Table 5). Thus, anti-oxidative activity of phytoene in animal cells was confirmed. It is of interest to test the effect of the endogenous synthesis of phytoene on the malignant transformation process which is newly triggered in noncancerous cells. Thus, the study was carried out on the NIH3T3 cells producing phytoene for its possible resistance against oncogenic insult imposed by transfection of the activated H-ras oncogene. Plasmids with activated H-ras gene was transfected to NIH3T3 cells with or without phytoene production, and the rate of transformed focus formation in 100 mm diameter dishes was compared. As the results, it was proven that the rate of transformed focus formation induced by the transfection of activated H-ras oncogene was lower in the phytoene producing cells than in control cells (Table 6). This type of experimental method may be possible to apply for the evaluation of anti-carcinogenic and/or anti-oxidative activity of other phytochemicals, since the cloning of genes for synthesis of various kinds of substances in vegetables and fruits, has already been accomplished. It is particularly useful to evaluate biological activity of unstable phytochemicals, such as phytoene and other unstable carotenoids. Table 5. Reduction of oxidative stress induced lipid hydroperoxidation levels in cells producing phytoene Transfected plasmid PCOOH + PEOOH/PC + PE Vector crtB 4.6 2.5 (% Inhibition) (46) PCOOH: phosphatidylcholine hydroperoxide; PEOOH: phosphatidylethanolamine hydroperoxide; PC: phosphatidylcholine; PE: phosphatidylethanolamine. Table 6. Suppression of transformed focus formation induced by activated H-ras gene in cells producing phytoene Oncogene Number of transformed foci Control ras-1 (pNCO102) ras-2 (pNCO602) +crtB 47 80 22 15 3.2. Bio-chemoprevention Valuable chemopreventive substances, including carotenoids, may be produced in wide variety of foods by means of bio-technology; this kind of new concept may be named as ‘bio-chemoprevention’. As a prototype experiment, phytoene synthesis in animal cells is demonstrated as described above. Since phytoene produced in animal cells was proven to prevent oxidative damage of cellular lipids, it may become a valuable factor in animal foods to reduce the formation of oxidized oils, which may be carcinogenic and hazardous for health, as well as to keep freshness, resulting in the maintenance of safety and good quality of foods. Furtheremore, phytoene-containing foods are valuable to prevent cancer, since phytoene is known as an anticarcinogenic substance. Recently, we have succeeded to produce phytoene in mice (Satomi Y, Nishino H et al. 2002, unpublished data), and now analyze their properties. In the next step, we are planning to produce phytoene in pigs and cows. 4. Conclusions Various natural carotenoids were proven to have anticarcinogenic activity, and seem to be useful for cancer control in human. Some of them may also be applicable for bio-chemoprevention project. 5. Key unanswered questions Action mechanism of natural carotenoids should be elucidated. Assessment of usefulness of these natural carotenoids in human cancer control project should be carried out more extensively. Taylor made-cancer prevention, including counterplan for non-responders to carotenoids, should be developed. For this purpose, SNPs analysis and comparison of SNPs between responders and nonresponders should be carried out systematically. In any case, establishment of the most effective and safe combination recipes of natural carotenoids for each individual person is the most important problem. Acknowledgements This work was supported in part by grants from the Program for Promotion of Basic Research Activities
  • 8. 264 for Innovative Biosciences (ProBRAIN), the Ministry of Agriculture, Forestry, and Fisheries, the Ministry of Health and Welfare, the Ministry of Education, Science and Culture, and Institute of Free Radical Control (IFRC), Japan. The study was carried out in collaboration with research groups of Kyoto Prefectural University of Medicine, Akita University College of Allied Medical Science, Kyoto Pharmaceutical University, Food Research Institute, Fruit Tree Research Station, National Cancer Center Research Institute, Shikoku Cancer Center, Lion Co., Dainippon Ink & Chemicals, Inc., Kagome Co., Kirin Brewery Co., Koyo Mercantile Co., Japan, Dr. Frederick Khachik, Department of Chemistry and Biochemistry, University of Maryland, USA, and Dr. Zohar Nir, LycoRed Natural Products Industries, Ltd., Israel. Authors are grateful to Dr. Takashi Sugimura, Emeritus President, National Cancer Center, Japan, for his kind encouragement during this study. References 1. Peto R, Doll R, Buckley JD, Sporn MB: Nature 290: 201–208, 1981 2. Murakoshi M, Takayasu J, Kimura O, Kohmura E, Nishino H, Iwashima A, Okuzumi J, Sakai T, Sugimoto T, Imanishi J, Iwasaki R: J Natl Cancer Inst 81: 1649–1652, 1989 3. Murakoshi M, Nishino H, Satomi Y, Takayasu J, Hasegawa T, Tokuda H, Iwashima A, Okuzumi J, Okabe H, Kitano H, Iwasaki R: Cancer Res 52: 6583–6587, 1992 4. Narisawa T, Fukaura Y, Hasebe M, Ito M, Aizawa R, Murakoshi M, Uemura S, Khachik F, Nishino H: Cancer Lett 107: 137–142, 1996 5. Le Marchand L, Hankin JH, Kolonel LN, Beecher GR, Wilkens LR, Zhao LP: Cancer Epidemiol Biomarkers Prev 2: 183–187, 1993 6. Stahl W, Sies H: Ann NY Acad Sci 691: 10–19, 1993 7. Ukai N, Lu Y, Etoh H, Yagi A, Ina K, Oshima S, Ojima F, Sakamoto H, Ishiguro Y: Biosci Biotech Biochem 58: 1718–1719, 1994 8. Colditz GA, Branch LG, Lipnick RJ: Am J Clin Nutr 41: 32–36, 1985 9. Franceschi S, Bidoli E, La Veccia C, Talamini R, D’Avanzo B, Negri E: Int J Cancer 59: 181–184, 1994 10. Giovannucci E, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA, Willett WC: J Natl Cancer Inst 87: 1767–1776, 1995 11. Nagasawa K, Mitamura T, Sakamoto S, Yamamoto K: Anticancer Res 15: 1173–1178, 1995 12. Tsushima M, Maoka T, Katsuyama M, Kozuka M, Matsuno T, Tokuda H, Nishino H, Iwashima A: Biol Pharm Bull 18: 227–233, 1995 13. Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa Y, Nakamura K, Harashima K: J Bacteriol 172: 6704–6712, 1990 Address for offprints: Hoyoku Nishino, Department of Biochemistry, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602-8566, Japan; e-mail: hnishno@basic.kpum.ac.jp