Influence of diet and calorie restriction on the initiation and promotion of skin carcinogenesis in the SENCAR mouse model

Diets were restricted to 60% of the intake of the control mice by feeding less diet (total diet restriction, TDR) or by feeding fewer calories from fat and carbohydrate (calorie restriction, CR) during the initiation or promotion phases of skin tumorigenesis in female SENCAR mice. Skin cancer was initiated by topical treatment with 10 nmol of 7,12-dimethylbenzanthracene in acetone and promoted by twice weekly treatments with 12-O-tetradecanoylphorbol-13-acetate in acetone for 20 wk. Dietary restriction preceding and during 7,12-dimethylbenzanthracene treatment did not influence skin papilloma or carcinoma yield. Papilloma incidence and the number of papillomas per effective mouse were reduced in mice restricted by both TDR and CR protocols during and following promotion with 12-O-tetradecanoylphorbol-13-acetate. Papilloma size was reduced at experimental wk 16 and 20 in both TDR and CR groups fed these diet regimens during promotion. However, by wk 28 and 32, papilloma sizes were similar in the control and TDR groups, and smaller papillomas were observed only in the CR group. The average carcinoma latency was extended by 26% in the groups restricted during promotion, and incidence was reduced in both groups. The reduction, however, was statistically significant only in the CR group. Body weight gain was reduced during the times when dietary restriction was enforced, and in a short-term study, both restricted diet treatments reduced the percentage of carcass protein.     

Circadian rhythm of tumor promotion in the two-stage model of mouse tumorigenesis

The question of whether cancer risk is influenced by time-of-day exposure to potentially carcinogenic agents was approached in this study by exposing mouse skin to a single initiating dose of 7,12-dimethylbenz [-]anthracene, followed by a 12 week regime of bi-weekly skin treatments with the tumor promoter, 12-0-tetradecanoyl-phorbol acetate (TPA), given at four different circadian clock times (CCTs). Tumor incidence, average number of tumors per mouse and tumor size showed a dominant circadian component with an acrophase occurring at 23:00 h CCT. Pre-treatment with all trans-retinoic acid, prior to bi-weekly TPA promotion, reduced tumor incidence, average number and size of tumors per animal by greater than 80%, but did not suppress the underlying circadian rhythm of sensitivity to TPA-induced tumor formation.     

Dietary retinoids are essential for skin papilloma formation induced by either the two-stage or the complete tumorigenesis model in female SENCAR mice.

Our previous work has shown that dietary retinoic acid (RA) is necessary for skin tumor formation induced by the two-stage protocol with the initiator 7,12-dimethylbenz[a]anthracene (DMBA) and the promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) (De Luca et al., Cancer Res., 36 (1976) 2334-2339). Here we report that retinoids are required for tumorigenesis by the two-stage as well as by the complete tumorigenesis protocol. Mice were treated with a single dose of DMBA (20 micrograms), followed by 20 applications of TPA (2 micrograms), or by 20 applications of DMBA (25 micrograms for 2 weeks and 51 micrograms thereafter). Regardless of the tumor induction protocol, tumor formation was inhibited by vitamin A-deficiency, while RA (3 micrograms/g of diet) or retinyl palmitate (RP, 6 micrograms/g) supplementation permitted the appearance of tumors. In addition, in comparison to the purified diets and regardless of their RA levels, the non-purified Purina chow diet enhanced tumor yield especially in the two-stage tumorigenesis protocol. This effect was less striking in mice with tumors induced by the complete tumorigenesis protocol. In summary, dietary retinoids are essential for skin tumor formation induced either by the two-stage or the complete tumorigenesis protocol.     

Isoflavone genistein inhibits the initiation and promotion of two-stage skin carcinogenesis in mice

Isoflavone genistein is a specific inhibitor of protein tyrosine kinase (PTK) and has been shown to have a variety of anticancer activities in cultured cells and animal models. We report here that genistein significantly inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-promoted skin tumorigenesis in a two-stage carcinogenesis model. In an initiation study, 10 micromol genistein was applied daily to female SENCAR mouse skin for 1 week, followed by initiation with 10 nmol DMBA. Mice were then treated with twice weekly 4 microg TPA. Genistein was shown to reduce tumor incidence and multiplicity in DMBA-initiated skin tumors by approximately 20 (P < 0.05) and 50% (P < 0.01), respectively. Two promotion studies were conducted using CD-1 and SENCAR mice. In experiment 1, CD-1 mice were initiated with 100 nmol DMBA and followed by a twice weekly regimen of 1 and 5 micromol genistein/4 microg TPA. In experiment 2, SENCAR mice were initiated with 10 nmol DMBA and followed by a regimen of 5, 10 and 20 micromol genistein/2 microg TPA. Both studies consistently showed that genistein substantially inhibited TPA-promoted skin tumorigenesis by reducing the tumor multiplicity by approximately 60 and 75%, respectively (P < 0.01). However, the tumor incidence appeared to be less affected. Mechanistic studies showed that genistein inhibited DMBA-induced bulky DNA adduct formation and substantially suppressed TPA-stimulated H2O2 and inflammatory responses in mouse skin by >60% (P < 0.01). In contrast, genistein only exhibited a moderate inhibition of TPA-induced ornithine decarboxylase activity (P > 0.05). Our results suggest that genistein exerts its anti- initiational and anti-promotional effects on skin carcinogenesis probably through blockage of DNA adduct formation and inhibition of oxidative and inflammatory events in vivo.      

Dietary fat versus caloric content in initiation and promotion of 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats

Enhancement of mammary tumor formation by dietary fat may be mediated via increased caloric intake. Three experiments were performed to study this relationship in 7,12-dimethyl-benz(a)anthracene (DMBA)-treated female Sprague-Dawley rats: (a) high- or low-fat isocaloric diets were fed in a crossover design; (b) low-fat, high-calorie and high-fat, low-calorie diets were fed in a crossover design; (c) pair-fed rats were restricted to 60% of the calories of controls with ad libitum access to food beginning 10 days after DMBA administration. The pair-fed rats received daily 60% of calories, the same level of fiber, and 115% more fat than did rats fed ad libitum. Tumor yield but not tumor incidence was greater in rats fed high-fat rather than low-fat isocaloric diets prior to initiation of tumorigenesis. A low-fat, high-calorie diet led to more tumor incidence and yield than was associated with feeding of a high-fat, low-calorie diet. Caloric restriction (although with concomitant intake of more fat) led to complete inhibition of tumor formation. These results indicate that both high-fat and high-calorie diets exhibit cocarginogenic, not merely promotional, properties. Caloric intake may be a greater determinant than dietary fat of a tumor-enhancing regimen. Finally, restriction of caloric intake during promotion markedly suppresses tumor formation, despite the increased fat content of the restricted diet, suggesting a permissive role for calories in tumor formation. The possibility remains that alterations in levels of other dietary components could also have contributed to the observed effects.     

On the persistence of tumor initiation in two-stage carcinogenesis on mouse skin

The persistence of the initiated state during two-step car-cinogenesisin mouse epidermis is a generally accepted phenomenon, however,conflicting results exist with regard to the degree of irreversibiltyrelative to the age of the animals. Several factors such asage-dependent alterations in the response of the epidermis tothe promoter and skin damage following the initiation step havebeen proposed to account for the observed discrepancies. Inthe present investigation we have tried to circumvent skin-damagingeffects of topically applied 7,12-dimethylbenz[a]anthraceneby intragastric administration of the drug. Tumor productionby topical promotion with 12-O-tetradecanoylphorbol-13-acetatewas subsequently determined in 600 female NMRI mice using intervalsof 4, 8, 16, 24, 32 and 40 weeks between initiation and promotion.Independent of the delay between the initiating and promotingstep, we observed a similar time course and extent of tumorproduction in the different experimental groups. This indirectlyproves that the promoting capacity of 12-O-tetradecanoylphorbol-13-acetateis age-independent and that during aging no substantial lossof initiated cells occurs in mouse epidermis.     

Antipromotional activity of dietary N-(4-hydroxyphenyl)retinamide in two-stage skin tumorigenesis in CD-1 and SENCAR mice

The activity of the synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR), as a promoter and as an inhibitor of tumor promotion in mouse skin was investigated using CD-1 and SENCAR mice. Dietary administration of 4-HPR inhibited skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in both mouse strains, although protective activity was observed only at high TPA doses. Dietary 4-HPR had no promoting activity in mice receiving initiation and no TPA promotion. These data suggest that retinoid promotion of skin tumorigenesis may be specific to retinoic acid, and is not necessarily characteristic of the entire chemical class.     

Initiating activity of eight pyrolysates of carbohydrates in a two-stage mouse skin tumorigenesis model

Tumor initiating potential was tested for eight pyrolysates of carbohydrates in a two-stage mouse skin carcinogenesis model using 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. The pyrolysates were levoglucosan (LG-I), levoglucosenone (LG-II), furfural (FF), 5-(hydroxymethyl)-2-furfural (HMF), glyoxal (GL), methylglyoxal (MGL), 3-deoxy-D-glucosone (DG) and thiazolidine (TZ). The total initiating doses were 200 mumol for LG-I and DG, 25 mumol for LG-II and 500 mumol for FF, HMF, GL, MGL and TZ. 7,12-Dimethylbenz[a]anthracene (DMBA) was used as a positive control agent applied to a total dose of 100 micrograms. All compounds were topically administered to the dorsal skin twice weekly for 5 weeks with or without TPA treatment for the following 47 weeks. In conjunction with promotion TZ induced skin tumors in 40% of the mice (average 0.65 tumors/mouse), FF in 25% (0.40/mouse), LG-I in 25% (0.35/mouse) and LG-II, HMF, DG and GL in 10-20% (0.11-0.25/mouse) respectively whereas DMBA induced skin tumors in 100% of the animals (6.7/mouse). MGL did not induce any tumors during the experiment and no tumors appeared in any of the groups treated with test chemicals alone. As assessed by Fisher's exact test, tumor incidences were significant in the TZ (0.01 less than P less than 0.05) and DMBA (P less than 0.01) groups as compared with the dimethylsulfoxide (DMSO) followed by TPA groups (5%, 0.05/mouse). Statistical analyses with Peto's trend test revealed significant tumor development in the LG-I (P less than 0.01), LG-II (0.05 less than P less than 0.01), FF (0.01 less than P less than 0.05) and TZ (P less than 0.01) followed by TPA groups (compared with DMSO + TPA). The results indicate that LG-I, LG-II, FF and TZ potentially possess tumor initiating activity, while HMF, GL, MGL and DG do not, as judged by the statistical analysis based on the incidences and development of the skin papillomas and/or carcinomas. A positive correlation between tumor initiating potential and clastogenic activity based on calculated ID50 (50% initiating dose) and published CD20 (20% clastogenic dose) values was evident for LG-II, FF and TZ, while LG-I was an exception.     

On the persistence of tumour initiation and the acceleration of tumour progression in mouse skin tumorigenesis

Evidence has been obtained for the reversibility of initiation of carcinogenesis as evoked by 100 μ 7,12-dimethylbenz (a)anthracene (DMBA) applied to the skin of Swiss mice. Mice exposed to twice-weekly applications of a phorbol ester, TPA, for 15 weeks developed multiple papillomas when treatment was started 3 weeks after tumour initiation with DMBA, but very few when the interval was 50 weeks. This finding is not necessarily at variance with the postulate of the irreversibility of formation of “latent tumour cells” by subcarcinogenic doses of DMBA. Intraperitoneal injections of urethane increased the risk of development of malignant skin tumours by mice bearing multiple papillomas as a result of previous treatment with 100 μMg DMBA and TPA as compared with intraperitoneal injections of distilled water. This finding may allow a more clear-cut experimental definition of the stages in the process of tumour progression in mouse skin tumorigenesis.     

Guggulsterone modulates MAPK and NF-kappaB pathways and inhibits skin tumorigenesis in SENCAR mice

Guggulsterone (GUG), a resin of the Commiphora mukul tree, has been used in ayurvedic medicine for centuries to treat a variety of ailments. Recent studies have suggested that GUG may also possess anticancer effects. In the present study, we show that GUG possesses antitumor-promoting effects in SENCAR mouse skin tumorigenesis model. We first determined the effect of topical application of GUG to mice against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of GUG (1.6 micromol per mouse) 30 min prior to TPA (3.2 nmol per mouse) application onto the skin of mice afforded significant inhibition against TPA-mediated increase in skin edema and hyperplasia. Topical application of GUG was also found to result in substantial inhibition against TPA-induced epidermal (i) ornithine decarboxylase (ODC) activity; (ii) ODC, cyclooxygenase-2 and inducible nitric oxide synthase protein expressions; (iii) phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinases and p38; (iv) activation of NF-kappaB/p65 and IKK alpha/beta and (v) phosphorylation and degradation of I kappaB alpha. We next assessed the effect of topically applied GUG on TPA-induced skin tumor promotion in 7,12-dimethyl benz[a]anthracene-initiated mice. Compared with non-GUG-pretreated mice, animals pretreated with GUG showed significantly reduced tumor incidence, lower tumor body burden and a significant delay in the latency period for tumor appearance from 5 to 11 weeks. These results provide the first evidence that GUG possesses anti-skin tumor-promoting effects in SENCAR mice and inhibits conventional as well as novel biomarkers of tumor promotion. In summary, GUG could be useful for delaying tumor growth in humans.     

Characterization of skin tumor promotion and progression by chrysarobin in SENCAR mice

The characteristics of the skin tumor promotion response with anthrone derivatives has been further examined in SENCAR mice. Chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) was an effective skin tumor promoter when applied twice weekly with dose-dependent increases in both papillomas and squamous cell carcinomas between 25 and 100 nmol/mouse. A similar dose-response relationship for papilloma and carcinoma formation was observed when chrysarobin was applied once weekly. Interestingly, chrysarobin was approximately twice as active as a skin tumor promoter when applied once weekly versus twice weekly. Doses of 25,100, and 220 nmol/mouse gave maximal papilloma responses of 2.90, 8.15, and 9.38 versus 0.73, 4.70, and 5.42 papillomas/mouse, respectively, in mice initiated with 25 nmol 7,12-dimethylbenz(a)anthracene. Thus, unlike 12-O-tetradecanoylphorbol-13-acetate (TPA), where a twice weekly application frequency is optimal, application of anthrone promoters such as chrysarobin once weekly is a more optimal frequency for papilloma development. Chrysarobin was also a much more effective skin tumor promoter when the start of promotion was delayed by an additional 10 weeks. Thus, groups of mice initiated with 10 nmol 7,12-dimethylbenz(a)anthracene and having promotion started in either the 3rd or the 13th week after initiation had maximal responses of 5.6 or 11.0 papillomas/mouse, respectively. In addition, the rate of papilloma development was faster in the delayed promotion group. The progression of papillomas to carcinomas was examined in all chrysarobin-treated groups and compared with three groups of mice treated with 3.4 nmol TPA. After 60 weeks of promotion, the anthrone promoter-treated groups had carcinoma:papilloma ratios 2.5 to 5.0 times higher than the TPA-treated groups. This was due primarily to the fact that similar carcinoma responses were observed in both anthrone- and TPA-treated mice at optimal promoting doses whereas the papilloma responses were significantly lower in the former groups. The data suggest that anthrone derivatives are very efficient tumor promoters. The results are further discussed in terms of mechanisms of skin tumor promotion.     

Further studies on the influence of initiation dose on papilloma growth and progression during two-stage carcinogenesis in SENCAR mice

The present study was designed to further evaluate the growthand progression of papillomas to squamous cell carcinomas (SCCs)in groups of animals receiving initiating doses of 7,12-dimethylbenz[a]anthracene(DMBA) producing relatively low papilloma yields following longterm promotion (60 weeks) with 12-O-tetradecanoylphorbol-13-acetate(TPA). For comparison, groups of animals were initiated withvarious doses of DMBA and then promoted with mezerein (MEZ),benzoyl peroxide (BzPo) and chrysarobin (CHRY). Following initiation,groups of female SENCAR mice received the following promoterdoses: TPA (1.0 or 2.0 µg per mouse); MEZ (2.0 µgper mouse); BzPo (20.0 mg per mouse); and CHRY (52.8 µgper mouse). The maximum papilloma to SCC conversion ratio obtainedwith TPA in the current study was 0.32. This value was in therange of maximum conversion ratios obtained with the other compounds:MEZ, 0.40; CHRY, 0.32 and BzPo, 0.19. In general, the highestpapilloma to SCC conversion ratios observed with TPA as thepromoter were obtained in groups that received the lowest dosesof DMBA and had relatively low papilloma burdens. A comparisonof papilloma to SCC conversion in groups of mice promoted withTPA, MEZ or CHRY and having similar papilloma yields, revealedvery similar conversion ratios. Comparison of the BzPo groupwith a similar papilloma yield indicated that the conversionratio was slightly lower with this promoter. The present resultsindicate that in mice promoted with TPA and having relativelylow papilloma numbers, a larger proportion of these papillomasprogress to SCCs during continued promoter treatment. Furthermore,the results suggest that papillomas behave similarly in theirability to progress to SCCs regardless of the promoter usedwhen comparing groups of mice with similar tumor numbers. Thedata are discussed in terms of possible mechanisms for the observedresults.     

Further characterization of skin tumor promotion and progression by mezerein in SENCAR mice

This study evaluated the skin tumor-promoting activity of mezerein in SENCAR mice. The effect of initiation dose of 7,12-dimethylbenz(a)anthracene (DMBA) on tumor promotion by mezerein was examined. Excellent dose-response relationships were observed for initiation with DMBA at 0.2-20 micrograms per mouse with mezerein as a complete promoter. None of the mezerein-only promotion groups had papilloma responses similar to those of the corresponding groups receiving two-stage promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) followed by mezerein, even when a 40-micrograms initiating dose of DMBA was used. The effect delaying promotion with mezerein for 10 weeks was also examined in mice initiated with either 0.2, 2, 20, or 40 micrograms of DMBA per mouse. The 10-week delay led to a slight increase in the number of papillomas per mouse in some but not all treatment groups. Again, none of the delayed-mezerein-treatment groups had papilloma responses similar to those of the corresponding two-stage promotion (TPA-mezerein) groups at any corresponding initiating dose of DMBA. Finally, the progression of papillomas to carcinomas during promotion with mezerein was examined in groups of mice initiated with either 2 or 20 micrograms of DMBA. Higher ratios of carcinomas to papillomas were observed in mice promoted with mezerein than in mice receiving TPA promotion or two-stage promotion (TPA-mezerein). However, the presence of two to four times more papillomas in some mezerein-treated groups did not lead to greater numbers of carcinomas than in the groups with fewer papillomas. The data do not support the idea that spontaneous stage I promotion can be induced by delaying mezerein treatment for 10 weeks. Furthermore, the data suggest that the higher ratio of carcinomas to papillomas observed with mezerein promotion may be a function of the lower tumor burdens obtained after promotion with this compound rather than a specific property of the chemical.     

Influences of sphingosine on two-stage skin tumorigenesis in Sencar mice.

Sphingosine (SPH) was studied as an inhibitor of skin tumor promotion in skin cancer initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Two tumorigenesis studies were conducted using female Sencar mice treated with 10 nmol DMBA in 0.2 ml acetone at 8 weeks of age and promoted beginning 1 week later with 3.2 nmol TPA applied twice per week. In the high-dose study, 10 mumol SPH was applied 30 min before each TPA treatment. The low-dose study used 0.5, 0.05, or 0.01 mumol treatments with SPH 30 min before TPA. In the high-dose study SPH treatment alone following initiation by DMBA, and SPH treatment preceding TPA in DMBA-initiated mice accelerated the development of papillomas in comparison with the DMBA/TPA-treated group. The low-dose experiment showed no consistent alteration of tumorigenesis by SPH in the DMBA/TPA-treated groups, and low doses of SPH following DMBA did not promote skin tumorigenesis. SPH treatment did not alter body weight in either experiment.     

Initiation, promotion and complete carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine or ethylnitrosourea in the Sencar mouse skin tumorigenesis model

Five doses of either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ethylnitrosourea (ENU) were tested as complete carcinogens, tumor initiators and tumor promoters in the SENCAR skin tumorigenesis model. As tumor initiators, MNNG-induced papillomas at all doses tested, while ENU was active from 10-40 mumol. As complete carcinogens, MNNG from doses of 0.5-5.0 mumol and ENU from doses of 10 mumol-40 mumol were potent inducers of both papillomas and carcinomas indicating that these agents are active as both tumor initiators and tumor promoters.     

Mechanisms of initiation and promotion in mouse epidermis

Studies performed on mouse skin have indicated that chemical carcinogenesis can be subdivided into two distinct stages--initiation and promotion. Initiation results from exposure to a classical mutagenic carcinogen and is irreversible even after a single exposure. The permanently altered initiated cell and its progeny may never form a tumour or be in any way recognizable in the target tissue. Exposure to tumour promoters permits the expression of the neoplastic change in initiated cells, and tumours develop. In contrast to initiators, promoters must be given repeatedly to be effective; individual exposures are reversible. Studies in mouse skin cell cultures have provided new insights into the changes associated with initiation and promotion. Some carcinogen-treated cells and cells derived from initiated mouse skin are resistant to signals for terminal differentiation and can proliferate under conditions in which normal cells are obliged to cease proliferation and begin their maturation programme. This change is essential for a tumour cell, since it provides it with the ability to grow away from a basement membrane attachment site. In cultured epidermal cells, tumour promoters are capable of stimulating selectively the growth of certain cells, including initiated cells, while inducing simultaneously terminal differentiation in other epidermal cells. The induction of terminal differentiation may be mediated by the phorbol ester receptor. The net effect of these responses to promoters is the clonal expansion of cells stimulated to proliferate. In this way, promoters are capable of increasing the clone size of initiated cells and producing benign tumours. In-vivo studies have indicated that promoters are incapable of accelerating the conversion of benign to malignant tumours. However, exposure of papilloma-bearing mice to genotoxic initiating carcinogens accelerates malignant conversion markedly. Thus, initiation and promotion appear to be relevant to the formation of preneoplastic lesions, but further genetic damage may be required for the carcinogenic event.     

SENCAR mouse skin tumors produced by promotion alone have A to G mutations in codon 61 of the c-rasHa gene

SENCAR mice, developed by selective breeding for high susceptibilityto skin carcinogenesis by initiation with 7, 12-dimethylbenz[a]anthraceneand promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA),form squamous papillomas in     

Potent suppressive effect of a Japanese edible seaweed, Enteromorpha prolifera (Sujiao-nori) on initiation and promotion phases of chemically induced mouse skin tumorigenesis.

Potent antigenotoxic and anti-tumor promoting activities of a Japanese edible seaweed, Enteromorpha prolifera (Sujiao-nori in Japanese) were previously identified using an in vitro cell culture experiment (Y. Okai, K. Higashi-Okai, S. Nakamura, Y. Yano, S. Otani, Cancer Lett. 87 (1994) 25–32). However, in vivo anti-carcinogenic activity of this seaweed has not been elucidated until now. In the present study, the anticarcinogenic activity of E. prolifera was analyzed using an initiation and promotion model experiment of mouse skin tumorigenesis caused by 7,12-dimethylbenz[a]anthracene (initiator) and 12-O-tetradecanoylphorbol-13-acetate (promoter). (1) Application of the extract of E. prolifera prior to the treatment with a tumor initiator or promoter caused a significant suppression against skin tumorigenesis, and the combined application of the extract prior to both treatments with initiator and promoter exhibited much stronger suppression against the same skin tumorigenesis. (2) As a possible active principle for the anticarcinogenic activity of the extract, we propose a chlorophyll-related compound, pheophytin-a, which has been recently identified in the extract of this alga as an antigenotoxic substance (Y. Okai, K. Higashi-Okai, J. Sci. Food Agric. 74 (1997) 531–535), and showed significant suppressive effects in the same tumorigenesis experiment. These results suggest that E. prolifera has a potent suppressive activity against chemically induced mouse skin tumorigenesis through the suppression at the initiation and promotion phases, and that pheophytin-a might be partially associated with the in vivo anticarcinogenic activity.     

Keratin modifications in epidermis, papillomas, and carcinomas during two-stage carcinogenesis in the SENCAR mouse

To elucidate the role of keratin modification in tumor promotion, we investigated the keratin polypeptide patterns of mouse epidermis, papillomas, and carcinomas throughout an initiation-promotion experiment. The epidermal keratin modifications induced by repetitive 12-O-tetradecanoylphorbol-13-acetate treatments in both initiated and noninitiated mouse skin were essentially identical to those observed with a single 12-O-tetradecanoylphorbol-13-acetate application. These changes were even more pronounced in epidermal papillomas. In addition, the keratins of the papillomas displayed greater charge heterogeneity, particularly among the high-molecular-weight keratins (Mr 60,000 to 62,000). As the experiment progressed, there appeared to be a selective loss of one group of high-molecular-weight keratins (Mr 62,000) in some of the papillomas. Interestingly, the carcinomas that appeared at this time had significant reduction in both groups of high-molecular-weight keratins. In fact, the keratin profiles of carcinomas were very similar to the patterns observed in basal cells after a single 12-O-tetradecanoylphorbol-13-acetate treatment of adult epidermis. This may indicate that the program of keratin expression of a carcinoma becomes permanently fixed at a basal cell pattern. Changes in keratin patterns may serve as a biochemical marker of malignant progression in mouse epidermis.