Evaluation of skin cancer chemoprevention potential of sunscreen agents using the Epstein-Barr virus early antigen activation in vitro assay.

In our continuing search for novel cancer chemopreventive compounds of natural and synthetic origin, we have evaluated 14 commonly used ultraviolet (UV) sunscreen agents (designated UV-1 to UV-14) for their skin cancer chemoprevention potential. They belong to 8 different chemical categories: aminobenzoate (UV-5, UV-7, UV-8 and UV-14), benzophenone (UV-1, UV-2, UV-3 and UV-13), benzotriazole (UV-10), benzyloxyphenol (UV-9), cinnamate (UV-6), quinolone (UV-4), salicylate (UV-11) and xanthone (UV-12). In the in vitro assay employed, the sunscreens were assessed by their inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in human lymphoblastoid Raji cells. All sunscreens tested were found to exhibit anti-tumour promoting activity: listed in decreasing order, moderate (UV-11, UV-2, UV-7, UV-12, UV-3, UV-9 and UV-14) to weak (UV-1, UV-6, UV-8, UV-16, UV-5, UV-4 and UV-10) with octyl salicylate (UV-11) as the most potent and drometrizole (UV-10) as the least potent among the compounds evaluated. A plausible relationship between the antioxidant property of sunscreens and their ability to promote anti-tumour activity was noted. The results call for a comprehensive analysis of skin cancer chemoprevention potential of currently used UV sunscreen agents around the globe to identify those with the best clinical profile.

Cancer chemopreventive activity of odorine and odorinol from Aglaia odorata.

In the course of our continuing search for novel cancer chemo-preventive agents from natural sources, we have carried out a primary screening in vitro assay of the compounds isolated from Aglaia odorata. Consequently, aminopyrrolidine-diamides, odorine and odorinol, were obtained as active constituents. These compounds exhibited potent anti-carcinogenic effects in a two-stage carcinogenesis test of mouse skin induced by 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Further, both compounds showed remarkable inhibitory effects in two-stage mouse skin carcinogenesis models induced by nitric oxide (NO) donors such as (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR-1) or peroxynitrite as an initiator and TPA as a promoter. From these results, it was concluded that odorine and odorinol inhibited both the initiation and promotion stages of two-stage skin carcinogenesis.

Cancer chemoprevention by ginseng in mouse liver and other organs.

Oral administration of red ginseng extracts (1% in diet for 40 weeks) resulted in the significant suppression of spontaneous liver tumor formation in C3H/He male mice. Average number of tumors per mouse in control group was 1.06, while that in red ginseng extracts-treated group was 0.33 (p<0.05). Incidence of liver tumor development was also lower in red ginseng extracts-treated group, although the difference from control group was not statistically significant. Anti-carcinogenic activity of white ginseng extracts, besides red ginseng extracts, was also investigated. In the present study, the administration of white ginseng extracts was proven to suppress tumor promoter-induced phenomena in vitro and in vivo. It is of interest that oral administration of the extracts of Ren-Shen-Yang- Rong-Tang, a white ginseng-containing Chinese medicinal prescription, resulted in the suppression of skin tumor promotion by 12-o-tetradecanoylphorbol-13-acetate in 7,12-dimethylbenz[a]anthracene-initiated CD-1 mice. These results suggest the usefulness of ginseng in the field of cancer prevention.

Inhibition of epstein-barr virus early antigen activation promoted by 12-O-tetradecanoylphorbol-13-acetate by the non-steroidal anti-inflammatory drugs.

As part of our screening program for cancer inhibitory agents effective specifically in the promotion stage of cancer development, we have evaluated the possible inhibitory effects of 36 non-steroidal anti-inflammatory drugs (NSAIDs) on the Epstein-Barr virus early antigen (EBV-EA) activation which was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. All the drugs were observed to inhibit the EBV-EA activation at low doses with low toxicity. The two most active anti-tumor promoting agents were the arylacetic acid derivatives, etodolac and sulindac. We also report for the first time the activities of 14 new NSAIDs belonging to different classes as potential cancer chemopreventive agents. A structure-activity relationship study showed that among the salicylic acid derivative tested, the oxidation of the thiol group to dithiol derivatives results in the reduction of the activity. Introduction of amino group on the salicylic acid molecules also results in the reduction of activity in the EBV-EA assay. The results are of great interest in the development of NSAIDs as cancer chemopreventive agents, which halt cancer progression in multistage carcinogenesis, where successive activities are required to evolve into fully-fledged and metastatic cancer.

Inhibitory effect of flavonoids from citrus plants on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors.

To search for possible anti-tumor promoters, thirteen flavones (1-13) obtained from the peel of Citrus plants were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation by a short-term in vitro assay. Of these flavones, 3,5,6,7,8,3',4'-heptamethoxyflavone (HPT) (13) exhibited significant inhibitory effects on the EBV-EA activation induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA). Further, compound 13 exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Cancer prevention by natural carotenoids.

Various natural carotenoids were proven to have anticarcinogenic activity. Epidemiological investigations have shown that cancer risk is inversely related to the consumption of green and yellow vegetables and fruits. Since beta-carotene is present in abundance in these vegetables and fruits, it has been investigated extensively as possible cancer preventive agent. However, various carotenoids which co-exist with beta-carotene in vegetables and fruits also have anti-carcinogenic activity. And some of them, such as alpha-carotene, showed higher potency than beta-carotene to suppress experimental carcinogenesis. Thus, we have carried out more extensive studies on cancer preventive activities of natural carotenoids in foods; i.e., lutein, lycopene, zeaxanthin and beta-cryptoxanthin. Analysis of the action mechanism of these natural carotenoids is now in progress, and some interesting results have already obtained; for example, beta-cryptoxanthin was suggested to stimulate the expression of RB gene, an anti-oncogene, and p73 gene, which is known as one of the p53-related genes. Based on these results, multi-carotenoids (mixture of natural carotenoids) seems to be of interest to evaluate its usefulness for practice in human cancer prevention.

Inhibitory effect of Epstein-Barr virus activation by Citrus fruits, a cancer chemopreventor.

To search useful compounds in Citrus fruit for cancer chemoprevention, we carried out a primary screening of extracts of fruit peels and seeds from 78 species of the genus Citrus and those from two Fortunella and one Poncirus species, which were closely related to the genus Citrus. These Citrus extracts inhibited the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) as a useful screening method for anti-tumor promoters. Our results indicated that Citrus containing substances may be inhibit susceptibility factors involved in the events leading to the development of cancer.

A new biflavonoid from Calophyllum panciflorum with antitumor-promoting activity.

A new biflavonoid named pancibiflavonol (1) was isolated from an EtOH extract of the stem bark of Calophyllum panciflorum, along with six known biflavonoids, and its structure was elucidated by spectroscopic methods. These biflavonoids all exhibited significant inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus early antigen activation in Raji cells.

Prevention of N-methylnitrosourea-induced colon carcinogenesis in F344 rats by lycopene and tomato juice rich in lycopene.

Epidemiological studies have suggested a protective effect of lycopene and lycopene-rich tomatoes against various cancers. Here, the inhibition of colon carcinogenesis by lycopene and tomato juice was investigated. Seven-week-old female F344/NSlc rats received an intrarectal dose of 2 mg (experiment I) or 4 mg (experiment II) of N-methylnitrosourea 3 times a week for 3 weeks, and had free access to one of 4 drinking fluids: plain water (control group), 17 ppm lycopene water solution (Ly group), and diluted tomato juice containing 17 ppm (Tj group) or 3.4 ppm (tj group) lycopene, throughout the experiments. The colon cancer incidence at week 35 was significantly lower in the Tj group, but not in the Ly group, than in the control group: 21% and 33% vs. 54%, in experiment I (24 rats in each group). It was significantly lower in the Tj group than in the tj and control groups, 40% vs. 72% and 84%, in experiment II (25 rats in each group). An appreciable amount of lycopene (0.02 microgram/g) was detected in the colon mucosa of rats in the Tj group, but not in the tj group. The results suggest that tomato juice rich in lycopene may have a protective effect against colon carcinogenesis.

Cancer chemopreventive activity of synthetic colorants used in foods, pharmaceuticals and cosmetic preparations.

In continuation with our studies to uncover cancer chemopreventive effects of non-toxic natural colorants and other products of biologic and synthetic origin, we tested several Food and Drug Administration-approved synthetic colorants for antitumor promoting potential by the in vitro Epstein-Barr virus early antigen activation in Raji cells in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Among 29 such colorants used in foods, pharmaceuticals and cosmetics and evaluated in vitro, six of the 10 most effective had an azo group. Three structurally unrelated colorants tested in this assay were also studied in vivo for chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced TPA-promoted mouse skin carcinogenesis. The results indicate that tartrazine, indigo carmine and erythrosine are potent inhibitors of skin tumor promotion in mice treated with DMBA and TPA.

Xanthones as inhibitors of Epstein-Barr virus activation.

To search for possible antitumor promoters, we carried out a primary screening of 20 xanthones isolated from plants of the Guttiferae family, using their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Some of these xanthones, namely 1,3,7-trihydroxy-2-(3-methyl-2-butenyl)xanthone (8), dulxanthone-B (10) and latisxanthone-C (15), were observed to significantly inhibit the EBV-EA activation. The investigation indicated that 8, 10 and 15 might be valuable antitumor promoters.

Anti-tumor promoting activity of Dryopteris phlorophenone derivatives.

As a part of screening studies for cancer chemopreventive agents (anti-tumor promoters) 33 Dryopteris phlorophenone derivatives have been evaluated. The compounds tested comprised of monomeric acylphloroglucinols (e.g. desaspidinol, aspidinol) as well as dimeric (e.g. aspidin, desaspidin), trimeric (e.g. filixic acids), and tetrameric (e.g. dryocrassin) phlorophenone, wherein hexacyclic rings are bound together by a methylene bridge. These compounds were examined for their in vitro anti-tumor promoting effect on Epstein-Barr virus antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The two dimeric compounds aspidin and desaspidin, which were found to be the most active among the tested phlorophenones, were also examined in vivo on two stage mouse skin carcinogenesis, and found to show significant inhibitory effect on 7,12-dimethylbenz[alpha]anthracene (DMBA)-TPA tumor promotion.

Structures of new coumarins and antitumor-promoting activity of coumarins from Angelica edulis.

From the fruits of Angelica edulis Miyabe (Umbelliferae), three new angular furanocoumarins, edulisin III (1), edulisin IV (2), and edulisin V (3), were isolated along with three known coumarins, 2'(S), 3'(R)-3'-isobutyryloxy-4'-acetoxy-2',3'-dihydrooroselol (4), edultin (5), and 2'(S),3'(R)-3'-senecioyloxy-4'-acetoxy-2',3'-dihydrooroselol (6), respectively. The structures of 1 and 2 were established to be 2'(S),3'(R)-3'-(2-methylbutyryloxy)-4'-acetoxy-2',3'-dihydrooro selol and 2'-(S),3'(R)-3'-propyryloxy-4'-acetoxy-2',3'-dihydrooroselol by chemical studies and spectral analyses. Coumarin 3 was proved to be 3'-(2-methylbutyryl-oxy)-4'-angeloyloxy-2',3'-dihydrooroselol++ + by chemical and spectral analyses and H-C COLOC. Coumarins 1-6 were examined for the effects on tumor-promotor induced phenomena in vitro. Among these coumarins, 3 showed the most potent inhibitory activity on 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated 32Pi incorporation into phospholipids of cultured cells.

Potent preventive action of alpha-carotene against carcinogenesis: spontaneous liver carcinogenesis and promoting stage of lung and skin carcinogenesis in mice are suppressed more effectively by alpha-carotene than by beta-carotene.

Although beta-carotene has been considered to be a key cancer preventive agent in green and yellow vegetables, other types of carotenoids, such as alpha-carotene, may also contribute to anticarcinogenic action, since these carotenoids usually coexist with beta-carotene and are detectable in human blood and tissues. In this study, we compared the inhibitory effect of natural alpha-carotene, obtained from palm oil, with that of beta-carotene on spontaneous liver carcinogenesis in C3H/He male mice. The mean number of hepatomas per mouse was significantly decreased by alpha-carotene supplementation (per os administration in drinking water at a concentration of 0.05%, ad libitum) as compared with that in the control group (P < 0.001, Student's t test). On the other hand, beta-carotene, at the same dose as alpha-carotene, did not show any such significant difference from the control group. Furthermore, we also compared the antitumor-promoting activity of alpha-carotene with that of beta-carotene against two-stage mouse lung carcinogenesis (initiator, 4-nitroquinoline 1-oxide; promoter, glycerol). alpha-Carotene, but not beta-carotene, reduced the number of lung tumors per mouse to about 30% of that in the control group (P < 0.001, Student's t test). The higher potency of the antitumor-promoting action of alpha-carotene compared to beta-carotene was confirmed in other experimental systems; e.g., alpha-carotene was also found to have a stronger effect than beta-carotene in suppressing the promoting activity of 12-O-tetradecanoylphorbol-13-acetate on skin carcinogenesis in 7,12-dimethylbenz[a]anthracene-initiated mice. These results suggest that not only beta-carotene, but also other types of carotenoids, such as alpha-carotene, may play an important role in cancer prevention.

Anti-neoplastic effect of halocynthiaxanthin, a metabolite of fucoxanthin.

We have reported that fucoxanthin, a natural carotenoid, inhibited the growth of human neuroblastoma GOTO cells. In the present study, we show that a metabolite of fucoxanthin, halocynthiaxanthin, which is isolated from sea squirt Halocynthia roretzi, has a more potent inhibitory effect. Halocynthiaxanthin (5 micrograms/ml) caused complete suppression of GOTO cell proliferation, whereas fucoxanthin reduced the growth rate by only 88.8% compared with the control, at day 2 after the drug treatment. Furthermore, halocynthiaxanthin also inhibited the growth of other human malignant tumor cells. Thus halocynthiaxanthin seems to be a promising anti-neoplastic agent.

Potent anti-tumorigenic effect of tubeimoside 1 isolated from the bulb of Bolbostemma paniculatum (Maxim) Franquet.

Tubeimoside 1, one of the new triterpenoid saponins from the bulb of Bolbostemma paniculatum (Maxim) Franquet, had an anti-inflammatory effect on mouse ear edema induced by arachidonic acid and 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, a potent anti-tumorigenic effect of tubeimoside 1 was observed in 2-stage carcinogenesis of mouse skin after oral administration as well as topical application. Thus, tubeimoside 1 appears to be a promising agent for cancer chemoprevention.

Inhibitory effects of licochalcone A isolated from Glycyrrhiza inflata root on inflammatory ear edema and tumour promotion in mice.

Licochalcone A, 3-a,a-dimethylallyl-4,4'-dihydroxy-6-methoxychalcone, from the root of Glycyrrhiza inflata Beta (Leguminosae) (Xin-jiang liquorice) showed anti-inflammatory action towards mouse ear edema induced by arachidonic acid (AA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) by topical application. Anti-tumour promoting action of licochalcone A was also observed in vivo for mouse skin papilloma initiated by dimethylbenz[a]anthracene (DMBA) and promoted by TPA. It inhibited in vitro 32Pi-incorporation to phospholipids in HeLa cells promoted by TPA. A competitive interaction of licochalcone A with the TPA-receptors in the cell membrane has been suggested.

Anti-tumor-promotion by principles obtained from Angelica keiskei.

Potent anti-tumor promoter activity has been found in the nonpolar extracts of the root of "Ashita-Ba", Angelica keiskei Koidz. (Umbelliferae), which is eaten as a vegetable in Japan. From this active fraction, two angular furanocoumarins, archangelicin (1) and 8(S),9(R)-9-angeloyloxy-8,9-dihydrooroselol (2), three linear furanocoumarins, psoralen (3), bergapten (4) and xanthotoxin (5), and three chalcones, 4-hydroxyderricin (6), xanthoangelol (7) and a novel chalcone named ashitaba-chalcone (8), were isolated. Among these compounds, two angular type furanocoumarins, 1 and 2, and three chalcones, 6-8, suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32Pi-incorporation into phospholipids of cultured cells, whereas coumarins 3-5 were less effective. In addition, chalcones 6 and 7 were proved to have anti-tumor-promoting activity in mouse skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) plus TPA. Since chalcones 6 and 7 showed calmodulin-interacting property, both chalcones may reveal anti-tumor-promoting activity via the modulation of calmodulin involved systems. These chalcones may be useful to develop the effective method for cancer prevention.

Anti-tumor-promoting activity of derivatives of abieslactone, a natural triterpenoid isolated from several Abies genus.

Abiesenonic acid methyl ester (AVB-I acid methyl ester), a triterpenoid compound prepared from abieslactone, suppressed tumor promoter-induced phenomena in vitro and in vivo; i.e., AVB-I acid methyl ester inhibited 12-o-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32Pi-incorporation into phospholipids of cultured cells and the promoting action of TPA on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene.

Studies on the anti-tumor-promoting activity of naturally occurring substances. IV. Pd-II [(+)anomalin, (+)praeruptorin B], a seselin-type coumarin, inhibits the promotion of skin tumor formation by 12-O-tetradecanoylphorbol-13-acetate in 7,12-dimethylbenz[a]anthracene-initiated mice.

Since Pd-II [(+)anomalin, (+)praeruptorin B], a seselin-type coumarin, was found to inhibit tumor promoter induced phenomenon in vitro, the effect of Pd-II on the in vivo tumor-promoting action of 12-O-tetradecanoylphorbol-13-acetate (TPA) in 7,12-dimethylbenz[a]anthracene-initiated mouse skin was investigated. Pd-II, applied 40 min before the TPA treatment, at a dose of 10 mumol/painting, completely suppressed tumor formation up to 20 weeks of tumor promotion, without any toxicity. Besides Pd-II, various anti-tumor-promoter coumarins were found in the traditional Chinese medicine Qian-Hu, from which Pd-II was obtained. These coumarins may be useful for the development of an effective method to prevent cancer.