Heterogeneity of ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin and in epidermal tumors

One of the earliest events after treatment of mouse skin with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is the induction of ornithine decarboxylase (ODC). Using an immunoperoxidase technique with a rabbit antiserum specific for ODC, the localization of cells containing high levels of ODC following TPA treatment was determined. CD-1 female mice treated with multiple topical applications of TPA and killed 4.5 h after the last TPA treatment exhibited a heterogeneous localization of ODC in this hyperplastic epidermis. The cells which exhibited intense immunostaining were found predominantly in the suprabasal cells lining the hair follicles. This specific ODC staining in cells surrounding hair follicles was inhibited by pretreatment of mice with either retinoic acid or cycloheximide 1 h before TPA treatment. The induction of ODC-specific staining after TPA treatment in hyperplastic mouse skin was transient, since no staining was observed 16 or 24 h after TPA treatment. In contrast, benign papillomas produced by two-stage tumorigenesis contained some cells demonstrating high levels of ODC a week after the last TPA application. These results indicate that both normal mouse epidermal cells as well as tumor tissue display cellular heterogeneity of ODC expression.     

Modulation of chrysarobin skin tumor promotion

The present study examined the effect of several prototypic inhibitors of phorbol ester skin tumor promotion on skin tumor promotion by chrysarobin, an anthrone tumor promoter. Retinoic acid (RA) inhibited skin tumor promotion by chrysarobin; however, the degree of inhibition was dependent on the treatment protocol. When RA (10 micrograms/mouse) was given 1 h after each twice-weekly application of chrysarobin (220 nmol/mouse), a marked inhibition of papilloma formation was observed (78%). In additional experiments, using a once-weekly application of chrysarobin, RA also inhibited skin tumor promotion but the magnitude of inhibition was less. Interestingly, RA (10 micrograms/mouse), given 1 or 6 h after the promoter, did not inhibit the induction of epidermal ornithine decarboxylase (ODC) activity induced by a single topical application of chrysarobin (220 nmol). Fluocinolone acetonide (1 microgram/mouse), given 5 min before each twice-weekly application of chrysarobin (220 nmol/mouse) effectively inhibited skin tumor promotion (88%). A 0.5 or 0.25% supplement of alpha-difluoromethylornithine (alpha-DFMO) in the drinking water inhibited the induction of epidermal ODC following chrysarobin (220 nmol/mouse) treatment by 85 or 70%, respectively. Supplements of both 0.25 and 0.5% of alpha-DFMO also led to a 50 and 61% inhibition, respectively, in the number of papillomas per mouse after 25 weeks of promotion with chrysarobin. Interestingly, 0.25% alpha-DFMO in the drinking water did not reduce the number of papillomas per mouse after 20 weeks of promotion with 1.7 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the number of papillomas per mouse that were greater than or equal to 4 mm in diameter was significantly reduced in both chrysarobin- and TPA-treated mice. The data indicate that RA, FA and alpha-DFMO may be general inhibitors of tumor promoter regardless of the chemical class of tumor promoter. The ability of these inhibitors of phorbol ester promotion to inhibit anthrone promotion indicates that some common biochemical pathways may exist for both classes of skin tumor promoters.     

Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis

Topical treatment of mouse skin with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) results in an array of biochemical alterations, one of the earliest being a more than 200-fold transient induction of epidermal ornithine decarboxylase (ODC) activity. There is an excellent correlation between the induction of epidermal ODC activity and changes in the level of immunoreactive ODC protein following a single TPA treatment to skin. Both ODC activity and protein levels peak at 4.5 h after TPA treatment and rapidly fall to basal levels by 24 h. Cycloheximide treatment of mice in which ODC had been previously induced by TPA indicated a similar rapid turnover of both ODC catalytic activity and protein levels. Northern blot analysis of polyadenylated RNA isolated from mouse epidermis after a single TPA treatment revealed the stimulation of one species of ODC mRNA of 2.0 kilobases with a maximum at 3.5 h declining by 16 h. The same-sized species of ODC mRNA was detected 4.5 h after multiple biweekly treatments with TPA as well as in mouse papillomas and carcinomas not treated with TPA for at least 1 week. Southern blot analysis of EcoRI or BamHI digests of DNA derived from mouse liver, papillomas, or carcinomas revealed no ODC gene amplification or rearrangement during neoplastic progression. These observations indicate that the induction of epidermal ODC activity following TPA treatment results in a transient increase in the steady state levels of ODC mRNA and in the rate of synthesis of ODC protein, in contrast to epidermal tumors where the levels of ODC mRNA and protein are constitutively elevated.     

Phytol is a novel tumor promoter on ICR mouse skin.

Phytol is a branched, long-chain aliphatic alcohol which has various biological effects. In this study, we examined phytol as a tumor promoter in a mouse skin initiation-promotion model, and compared its promotion activity with that of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Female ICR mice, 7 weeks of age, were initiated with 100 microg of 7,12-dimethylbenz(a)anthracene, and were then topically promoted twice a week for 16 weeks with 100 mg of phytol or with 2.5 microg of TPA. In this model 95% of animals treated with phytol developed skin tumors within 16 weeks. The average number of lesions per mouse treated with phytol was significantly lower than that in mice treated with TPA, and this significant difference continued up to 16 weeks after the end of promotion treatment. Characterization of hyperplasia 48 h after topical application of agents showed that epidermal thickness and vertical thickness following topical application of phytol were significantly increased compared with vehicle controls, but were significantly smaller than in animals treated with TPA. Ornithine decarboxylase (ODC) activity following topical application of phytol was increased in a dose-dependent manner and showed a weak, delayed induction (which was maximal 11-12 h after treatment) as compared with the case of TPA. The specific binding of [3H]phorbol-12,13-dibutyrate (PDBU) by JB6 cells was not inhibited by phytol at concentrations up to 1 mM. These results indicate that phytol has a weak tumor promoter activity compared to TPA and is a non-TPA-type tumor promoter in this model of mouse skin carcinogenesis.     

Phytol Is a Novel Tumor Promoter on ICR Mouse Skin

Phytol is a branched, long-chain aliphatic alcohol which has various biological effects. In this study, we examined phytol as a tumor promoter in a mouse skin initiation-promotion model, and compared its promotion activity with that of 12- O -tetradecanoyl phorbol-13-acetate (TPA). Female ICR mice, 7 weeks of age, were initiated with 100 μg of 7,12-dimethylbenz( a )anthracene, and were then topically promoted twice a week for 16 weeks with 100 mg of phytol or with 2.5 μg of TPA. In this model 95% of animals treated with phytol developed skin tumors within 16 weeks. The average number of lesions per mouse treated with phytol was significantly lower than that in mice treated with TPA, and this significant difference continued up to 16 weeks after the end of promotion treatment. Characterization of hyperplasia 48 h after topical application of agents showed that epidermal thickness and vertical thickness following topical application of phytol were significantly increased compared with vehicle controls, but were significantly smaller than in animals treated with TPA. Ornithine decarboxylase (ODC) activity following topical application of phytol was increased in a dose-dependent manner and showed a weak, delayed induction (which was maximal 11–12 h after treatment) as compared with the case of TPA. The specific binding of [³H]phorbol-12,13-dibutyrate (PDBU) by JB6 cells was not inhibited by phytol at concentrations up to 1 m M . These results indicate that phytol has a weak tumor promoter activity compared to TPA and is a non-TPA-type tumor promoter in this model of mouse skin carcinogenesis.     

Inhibition of the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate in mouse skin by sphingosine sulfate

We investigated the effect of sphingosine sulfate on the inductionof ODC (ornithine decarboxylase) activity by TPA (12-O-tetradecanoylphorbol-13-acetate)in mouse skin. When applied topically to the shaved skin ofSENCAR mice at dosages of 10–40 µunol per animal,30 min before the superficial application of 8.5 nmol of TPA,sphingosine sulfate dramatically inhibited the induction ofODC activity by the tumor promoter. Significant inhibition ofTPA-induced ODC activity was observed at 4, 6 and 8 h afterTPA treatment in separate studies. The results indicate thatsphingosine sulfate is an effective inhibitor of ODC inductionby TPA in mouse skin.     

Staurosporine, a potent protein kinase C inhibitor, augments phorbol ester-caused ornithine decarboxylase induction in mouse epidermis.

A single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin caused an induction of epidermal ornithine decarboxylase (ODC) activity. When mice were topically pretreated with staurosporine, a most potent protein kinase C inhibitor, 6-84 h prior to TPA treatment, TPA-caused ODC induction was markedly enhanced. The enhancement of TPA-caused ODC induction by staurosporine was most pronounced when the time interval between staurosporine and TPA treatment was 36 h. Staurosporine elicited this enhancing effect in a dose-related manner. Staurosporine by itself also induced epidermal ODC activity. But the activity induced was very slight and would not directly contribute to the enhancing effect of this compound. Although staurosporine markedly augmented TPA-caused ODC induction, staurosporine-caused ODC induction was not augmented by this compound. Other protein kinase C inhibitors, such as 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, sphingosine and palmitoylcarnitine did not mimic the enhancing effect of staurosporine. These results indicate that the enhancement of ODC induction by staurosporine is specific for the induction caused by TPA and that this enhancing effect is not related to the protein kinase C inhibitory action of staurosporine. TPA-caused epidermal ODC induction was inhibited by indomethacin, and this inhibition was reversed by prostaglandin E2 (PGE2). Staurosporine-caused ODC induction was also inhibited by indomethacin but the inhibition was not reversed by PGE2, indicating that the mechanism of staurosporine-caused ODC induction is different from that of TPA.     

Inhibition of skin tumor promoter-caused induction of epidermal ornithine decarboxylase in SENCAR mice by polyphenolic fraction isolated from green tea and its individual epicatechin derivatives.

Green tea, next to water, is the most popular and commonly consumed beverage in the world, especially in eastern countries. In prior studies we have shown that the polyphenolic fraction isolated from green tea (GTP) exerts antigenotoxic effects in various mutagenicity test systems (Mutat. Res., 223: 273-285, 1989) and that its topical application or oral feeding in drinking water protects against polycyclic aromatic hydrocarbon-induced skin tumor initiation and complete carcinogenesis in SENCAR and BALB/c mice [Cancer Lett., 42: 7-12, 1988; Carcinogenesis (Lond.), 10: 411-415, 1989] and UV B radiation-induced photocarcinogenesis in SKH-1 hairless mice [Carcinogenesis (Lond.), 12: 1527-1530, 1991]. In the present study we assessed the effect of skin application of GTP to SENCAR mice on 12-O-tetradecanoylphorbol-13-acetate (TPA) and other skin tumor promoter-caused induction of epidermal ornithine decarboxylase (ODC) activity. Topical application of GTP to mouse skin inhibited TPA-induced epidermal ODC activity in a dose-dependent manner. The inhibitory effect of GTP was also dependent on the time of its application relative to TPA treatment. Maximum inhibitory effect was observed when GTP was applied 30 min prior to topical application of TPA. GTP application to animals also inhibited the induction of epidermal ODC activity caused by several structurally different mouse skin tumor promoters. In order to identify which of the specific epicatechin derivatives present in GTP is responsible for these inhibitory effects, they were isolated from GTP and evaluated for their inhibitory effects against TPA-caused induction of epidermal ODC activity. Among these, (-)epigallocatechin-3-gallate (EGCG), which was the major constituent present in GTP by weight, exerted the maximum inhibition. EGCG also showed greater inhibitory effects against TPA-caused induction of epidermal ODC activity when compared with several other naturally occurring polyphenols. The results of this study suggest that GTP, specifically its epicatechin derivative EGCG, could provide anti-tumor-promoting effects against a wide spectrum of skin tumor promoters.     

Cytotoxicity and absence of mutagenic activity of vomitoxin (4-deoxynivalenol) in a hepatocyte-mediated mutation assay with V79 Chinese hamster lung cells

The effects of naturally occurring sweetening agents, which inhibited the induction of Epstein-Barr virus-associated early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and related compounds on the induction of ornithine decarboxylase (ODC) by TPA is examined. Application of glycyrrhetinic acid or steviol to mouse skin 1 h before TPA treatment showed a remarkable decrease in TPA-induced ODC activity. Post-treatment with glycyrrhetinic acid or steviol 1 h after application of TPA also resulted in a considerable depression in the induction of ODC activity. Neither glycyrrhetinic acid nor steviol alone induced epidermal ODC activity. These results suggest that glycyrrhetinic acid and steviol interfere with the process of induction of epidermal ODC by TPA treatment of mouse skin. cis-Abienol, frullanolide and norambreinolide, which have a partially similar structure in the moiety with glycyrrhetinic acid or steviol, were tested. cis-Abienol and frullanolide showed an inhibitory effect when applied 1 h before TPA treatment, but norambreinolide was not effective. A relationship between suppression of ODC activity and inhibition of EBV-EA induction is discussed.     

Modulation of tissue and epidermal transglutaminases in mouse epidermal cells after treatment with 12-O-tetradecanoylphorbol-13-acetate and/or retinoic acid in vivo and in culture

Retinoic acid (RA) induces tissue transglutaminase (TGASE) and inhibits terminal differentiation induced either by calcium ion or by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in primary mouse epidermal cells in culture. The relevance of these effects on cultured cells to the antipromoting action of RA was investigated in female BALB/c and CD-1 mice in vivo. Tissue TGASE was distinguished from epidermal TGASE on the basis of different thermolability at pH 9 or elution from the anion exchanger Mono Q. After topical application of 3 to 5 micrograms (10 to 17 nmol) of RA to the shaved back skin, the specific activity of tissue TGASE increased up to 30-fold primarily in the basal cell fraction of Percoll-separated epidermal cells. Enzyme activity returned to basal levels by 7 days. Treatment with TPA (10 micrograms or 17 nmol/mouse) induced an increase in epidermal TGASE which reached a maximum at 12 h after application, primarily in suprabasal cells. RA applied 1 h before TPA caused no reduction of TPA-induced epidermal TGASE, but the increase in tissue TGASE due to RA was markedly inhibited by TPA. The effects of TPA and RA on TGASE activities in primary epidermal cells in culture were similar to those in vivo except that RA reduced the induction of epidermal TGASE by TPA. In culture the induction of epidermal TGASE by TPA was independent of Ca2+ concentration in the medium above 0.03 mM, but cornified envelope formation was markedly enhanced by Ca2+ above the level required for maintaining a basal cell population (0.03 to 0.05 mM). The TPA-induced formation of cornified envelope in the presence of elevated Ca2+ was completely inhibited by RA if cells were pretreated with RA for 24 h. Our results are consistent with RA causing a reprogramming of epidermal cells that alters their response to differentiation stimuli.     

Identification of epidermal cell subpopulations with increased ornithine decarboxylase activity following treatment of murine epidermis with 12-O-tetradecanoylphorbol-13-acetate

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.     

Effects of combined treatments with selenium, glutathione, and vitamin E on glutathione peroxidase activity, ornithine decarboxylase induction, and complete and multistage carcinogenesis in mouse skin

Several structurally different tumor promoters altered to various degrees both glutathione (GSH) peroxidase (EC 1.11.1.9) and ornithine decarboxylase (ODC, L-ornithine carboxy-lyase, EC 4.1.1.17) activities in mouse epidermis in vivo. At 5 h after their application to the skin, the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the stage 2 promoter mezerein were the most potent in inhibiting GSH peroxidase activity and inducing ODC activity. In comparison, the effects of anthralin, phorbol-12,13-didecanoate, benzoyl peroxide, H2O2, and phorbol-12,13-dibenzoate were much smaller, whereas the nontumor promoter phorbol, the hyperplastic agent ethyl phenylpropiolate, and the stage 1 promoter 4-O-methyl TPA did not alter GSH peroxidase and ODC activities. Various treatments including i.p. injections of 40 micrograms of Na2SeO3 and 100 mumol of GSH and/or topical applications of 40 mumol of D-alpha-tocopherol (vitamin E) 20 or 15 min, respectively, before tumor promoter treatment inhibited in an additive manner the effects of either TPA or mezerein on both GSH peroxidase activity and ODC induction. Moreover, these Na2SeO3, GSH, and/or vitamin E treatments inhibited in the same additive manner the tumor-promoting activity of TPA in the initiation-promotion protocol. However, when tested in the 2-stage promotion protocol with 4 doses of TPA followed by twice weekly applications of mezerein, Na2SeO3 plus vitamin E and GSH plus vitamin E treatments inhibited remarkably the tumor-promoting activity of mezerein but were ineffective in the first stage of promotion. The sequence and magnitude for the effects of 7,12-dimethylbenz[alpha]anthracene (DMBA) on GSH peroxidase and ODC activities were very different from those of the tumor promoters. In contrast with their antitumor-promoting activity, the treatments with Na2SeO3 plus vitamin E and GSH plus vitamin E failed to inhibit the carcinogenicity of a single large dose of DMBA and even enhanced the induction of skin tumors by repeated applications of subcarcinogenic doses of DMBA. These results suggest that the promoting component of DMBA carcinogenesis may be different from that of TPA. Moreover, the anticarcinogenicity of Na2SeO3, GSH, and vitamin E may be linked to their ability to facilitate or enhance the activity of the natural GSH-dependent antioxidant protective system of the epidermal cells during the later stages of skin tumor promotion.     

Epidermal cell proliferation and modulation of the protective potency of dexamethasone against phorbol ester-induced ornithine decarboxylase activity

Treating mouse skin with dexamethasone (DXME, 1 mumol) after a single TPA (3.25 nmol) application, inhibited both the dermal inflammatory reaction and the induction of epidermal ornithine decarboxylase (ODC) activity. At the hyperplastic stage, DXME was active against inflammation, though inhibited weakly the induction of ODC. In DXME-protected skin, the hyperplastic stage was delayed; unexpectedly, before that stage, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced strongly ODC activity in the epidermal cell layer. Provided that the proliferation process was induced, epidermal cells were increasingly sensitive toward TPA action; they may have been less dependent on inflammatory factors which may modulate the induction of ODC.     

Xanthorrhizol inhibits 12-O-tetradecanoylphorbol-13-acetate-induced acute inflammation and two-stage mouse skin carcinogenesis by blocking the expression of ornithine decarboxylase, cyclooxygenase-2 and inducible nitric oxide synthase through mitogen-activated protein kinases and/or the nuclear factor-kappa B

Xanthorrhizol is an active component isolated from Curcuma xanthorrhiza Roxb. (Zingiberaceae) that is traditionally used in Indonesia for medicinal purposes. In the present study, we found that the topical application of xanthorrhizol before 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment significantly inhibits TPA-induced mouse ear edema and TPA-induced tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated ICR mouse skin. The topical application of xanthorrhizol following the induction of papillomas with TPA-induced hyperplasia and dysplasia also reduced tumor multiplicity and incidence in DMBA-initiated mouse skin. To further elucidate the molecular mechanisms underlying the antitumor-promoting activity of xanthorrhizol, its effect on the TPA-induced expression of ornithine decarboxylase (ODC), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and the upstream signaling molecules controlling these proteins were explored in mouse skin. The pre-treatment with xanthorrhizol inhibited the expression of ODC, iNOS and COX-2 proteins and nuclear factor-kappaB (NF-kappaB) activation in both mouse skin with TPA-induced acute inflammation and DMBA-initiated mouse skin promoted by TPA for 19 weeks. When mouse skin was treated after TPA-induced production of papillomas, xanthorrhizol remarkably suppressed the expression of ODC, iNOS and COX-2 and inhibited the activation of NF-kappaB. Furthermore, western blot analysis showed that xanthorrhizol suppressed the activation of extracellular signal-regulated protein kinase, p38, c-Jun-N-terminal kinase and Akt in mice after topical application for 6 weeks following the induction of papillomas. Taken together, the present study demonstrates that xanthorrhizol not only delays or inhibits tumor formation, but also reverses the carcinogenic process at pre-malignant stages by reducing the protein levels of ODC, iNOS and COX-2 regulated by the NF-kappaB, mitogen-activated protein kinases and/or Akt.     

Anti-tumor promoting action of phthalic acid mono-n-butyl ester cupric salt, a biomimetic superoxide dismutase

Skin tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by a concurrent and topical application of phthalic acid mono-n-butyl ester cupric salt (PAMBCu) in CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene. PAMBCu inhibited TPA-caused epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. skin inflammation. However, neither PAMBCu nor superoxide dismutase (SOD) inhibited TPA-caused ODC induction in primary cultured mouse epidermal cells. 7-Bromomethylbenz[a]anthracene (BrMBA) is known to be a non-TPA type of tumor promoting agent. Epidermal ODC induction and inflammation caused by BrMBA were not inhibited by a concurrent application of PAMBCu. When mice were topically treated twice with PAMBCu, i.e. concurrently with and 7 h after BrMBA treatment, BrMBA-caused ODC induction was markedly suppressed. The same dose regimen of PAMBCu, however, failed to inhibit tumor promotion and inflammation caused by BrMBA. PAMBCu showed SOD-mimetic activity in superoxide generating systems, i.e. xanthine-xanthine oxidase reaction and TPA-stimulated polymorphonuclear leukocytes (PMN). Mono-n-butyl phthalate, which lacks SOD-mimetic activity, failed to inhibit TPA-caused ODC induction and skin inflammation. Therefore, inhibition by PAMBCu of TPA-caused tumor promotion, epidermal ODC induction and inflammation may be attributable to its SOD-mimetic activity. The results also support the contention that a superoxide anion of non-epidermal cell origin, such as PMN and macrophages, plays a role (probably some enhancing role) in in vivo ODC induction and tumor promotion caused by TPA. Failure of PAMBCu to inhibit BrMBA-caused tumor promotion suggests that superoxide anion generation is not involved in the tumor promoting action of this agent and that the anti-tumor promoting action of PAMBCu is dependent on the nature of the tumor promoting agents.     

Inhibition of tumor promoter-induced ornithine decarboxylase activity by tannic acid and other polyphenols in mouse epidermis in vivo

Naturally occurring plant phenols with antimutagenic and anticarcinogenic activities were tested for their abilities to inhibit the ornithine decarboxylase (ODC) response linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical applications of tannic acid (TA) inhibit remarkably and in a dose-dependent manner TPA-induced ODC activity in mouse epidermis in vivo. This inhibitory effect of TA is dependent on the time of its administration relative to TPA. The induction of epidermal ODC activity by 8.5 nmol of TPA is inhibited maximally when 20 mumol of TA are applied topically to the skin 20 min before the tumor promoter. Gallic acid and several of its derivatives inhibit the ODC response to TPA to a lesser degree than TA. Ellagic acid is the least effective inhibitor tested. TA also inhibits the ODC-inducing activities of several structurally different tumor promoters and the greater ODC responses produced by repeated TPA treatments. The ability of TA to inhibit by 85% the ODC marker of skin tumor promotion suggests that TA and other polyphenols may be effective not only against tumor initiation and complete carcinogenesis but also against the promotion phase of tumorigenesis.     

The potent anti-tumor-promoting agent isoliquiritigenin

A topical application of a chalcone derivative, 4,2',4'-trihydroxychalcone (isoliquiritigenin) inhibited epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. inflammation, caused by a topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in CD-1 mice. In addition, isoliquiritigenin potently inhibited 7,12-dimethylbenz[alpha]anthracene (DMBA)-initiated and TPA-promoted skin papilloma formation. This inhibitory effect of isoliquiritigenin was not due to any damage inflicted on the initiated cells but due to its anti-tumor-promoting action. Isoliquiritigenin also inhibited epidermal ODC induction and skin tumor promotion caused by 7-bromomethylbenz[alpha]anthracene (BrMBA), a non-TPA type of tumor-promoting agent, in DMBA-initiated mice. Isoliquiritigenin inhibits neither 12-lipoxygenase nor cyclooxygenase in epidermal subcellular fractions. This compound, however, inhibited TPA-stimulated prostaglandin E2 (PGE2) production in intact epidermal cells. ODC induction caused by TPA was inhibited by a topical application of cyclooxygenase inhibitor, indomethacin. Inhibition of ODC induction by indomethacin was counteracted by a topical application of PGE2, while inhibition caused by isoliquiritigenin was not overcome by PGE2. The results suggest that a mechanism other than the inhibition of PGE2 production is involved in the anti-tumor-promoting action of isoliquiritigenin. Isoliquiritigenin failed to inhibit phospholipase A2 activity of platelet sonicates, but inhibited platelet 12-lipoxygenase and 5-lipoxygenase in polymorphonuclear leukocytes. Therefore, it might be possible that isoliquiritigenin exerts its anti-tumor-promoting action through the lipoxygenase inhibition by acting on cells other than the target epidermal cells. Our present results, in combination with our previous data, demonstrate that some chalcone derivatives and flavonoids which show a potent lipoxygenase inhibitory action act on a common step in the skin tumor promotion caused by two different types of tumor-promoting agents, i.e. TPA and BrMBA, and suggest that these compounds show promise as drugs to prevent tumor promotion.     

Tumor promoter-induced refractory state against ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate in mouse epidermis

More than one application of the potent tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), to mouse skin at intervals of more than 48 h led to a larger induction of ornithine decarboxylase (EC 4.1.1.17; ODC) than did a single application. In contrast, at intervals of less than 24 h, the first application of TPA appeared to induce a refractory state; the second application of TPA did not induce ODC. The extent of the inhibitory effect caused by the first application of TPA was dependent on the dose. The abilities of a series of phorbol esters to induce the refractory state correlated with their promoting abilities. However, both mezerein and ethylphenylpropiolate, potent hyperplastic agents with little or no promoting properties, induced the refractory state. On the other hand, pretreatment with TPA caused a refractory effect on ODC induction by mezerein but potentiated ODC induction by ethylphenylpropiolate. The epidermal cells escaped from the refractory state by repeated application of TPA at intervals of 24 h as well as at intervals of twice a week; that is, there was a full induction of ODC activity following a second application within 24 h of a prior application. TPA did not elicit production of detectable ODC-antizyme activity in mouse epidermis. Mixing of a soluble extract from mouse epidermis in the refractory state with that from TPA-stimulated epidermis gave essentially additive ODC activity. Elimination of ODC induction by topical application of retinoic acid or injection of cycloheximide concurrent with the first application of TPA did not restore the ability of a second application of TPA to induce ODC. These results suggest that the refractory effect on ODC induction by TPA does not result from feedback regulation of ODC.