Characterization of the tumor-promoting activity of m-chloroperoxybenzoic acid in SENCAR mouse skin and its inhibition by gallotannin, oligomeric proanthocyanidin, and their monomeric units

m-Chloroperoxybenzoic acid (CPBA),which induces ornithine decarboxylase activity as much as 12-O-tetradecanoylphorbol-13-acetate (TPA), was tested for its ability to induce DNA synthesis, hydroperoxide (HPx) production, and tumor promotion in mouse epidermis in vivo. After an early inhibition, CPBA stimulates DNA synthesis, a response which is maintained between 16 and 72 h and maximal after two treatments. CPBA at 0.6-5 mg stimulates DNA synthesis more than other organic peroxides, and nearly as much as TPA. The HPx-producing activity of the epidermis is maximally stimulated 48 h after two CPBA treatments at a 24-h interval. However, the HPx response to CPBA is much smaller than that to TPA. Aleppo gall tannic acid (AGTA) and loblolly pine bark condensed tannin (LPCT) inhibit both the DNA and HPx responses to CPBA. In contrast, their respective monomeric units, gallic acid (GA) and catechin (Cat) inhibit the DNA response to CPBA but fail to alter CPBA-stimulated HPx production. Although it is more potent than benzoyl peroxide, CPBA is a complete tumor promoter much weaker than TPA and even less effective than mezerein (MEZ). CPBA in stage 1 cannot enhance like TPA the tumor-promoting activity of MEZ in stage 2. And in contrast to that of MEZ, the very weak tumor-promoting activity of CPBA is not enhanced after stage 1 treatment with TPA. At equal mg doses, AGTA, GA, LPCT, and Cat pretreatments all remarkably inhibit complete skin tumor promotion by CPBA. In spite of their antioxidant activities, AGTA post-treatments have no or very little inhibitory effects on the development of skin tumors by CPBA during 2-stage or complete tumor promotion.     

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.     

Ability of tannins extracted from the leaves of various trees and shrubs to inhibit the biomarkers of tumor promotion in mouse skin in vivo

Eight heterogeneous tannin samples (HTSs) extracted from various tree/shrub leaves of African and Himalayan origin were tested topically for their ability to inhibit the biomarkers of tumor promotion in mouse skin in vivo. HTS2 (from Dichostachys cinerea) and HTS6 (from Cassia sieberiana) consistently inhibit tumor promoter-stimulated ornithine decarboxylase activity, DNA synthesis, hydroperoxide production, and edema formation almost as much as loblolly pine bark condensed tannin (LPB-CT), which is known to inhibit skin tumor promotion. The other HTSs tested have lesser or only partial inhibitory effects. The ability of HTSs to inhibit the biomarkers of tumor promotion may be related to their reducing power but there is no apparent correlation between their inhibitory effects and their proanthocyanidin contents expressed as absorbance units, protein precipitation activities, and relative degrees of polymerization. HTS6 is effective against a wide spectrum of tumor-promoting agents unrelated to 12-O-tetradecanoyl-phorbol-13-acetate. The antioxidant effects of HTS6 and LPB-CT are similar but do not resemble that of tannic acid. HTS6 and LPB-CT both fail to alter the covalent binding of a tumor-initiating dose of 7,12-dimethylbenz[a] anthracene to DNA but inhibit the stimulation of DNA synthesis caused by a carcinogenic dose of this compound. Some foliage tannins, therefore, have potent antioxidant and anti-inflammatory activities and may inhibit hyperplasia and tumor promotion but their efficacy may vary considerably depending on their origin, chemical composition, and biological properties.     

Modulation by adriamycin, daunomycin, verapamil, and trifluoperazine of the biochemical processes linked to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

The antitumor antibiotics Adriamycin (ADR) and daunomycin (DAU) were tested for their ability to alter some of the molecular events linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). When applied topically to mouse skin, DAU is a more effective inhibitor of the basal level of epidermal DNA synthesis than ADR. However, these drugs alone are unable to inhibit the sequential induction of RNA, protein, and DNA synthesis caused by TPA in mouse epidermis in vivo. Moreover, ADR enhances substantially the induction of epidermal ornithine decarboxylase (ODC) activity by TPA. In vitro, the incorporation of [3H]DAU into isolated epidermal cells resembles more that of the HL-60 cells resistant to vincristine than that of the parental cell line. TPA does not alter the incorporation of [3H]DAU into epidermal cells. The Ca2+ antagonists verapamil (VRP) and trifluoperazine (TFP) enhance significantly the amount of [3H]DAU associated with the epidermal cells after 1 h. When applied shortly before TPA in vivo, VRP and TFP inhibit TPA-induced ODC activity at 5 h and TPA-induced DNA synthesis at 17 h. Moreover, the combinations of Ca2+ antagonists and anthracycline antibiotics administered before TPA inhibit synergistically these ODC and DNA responses to the tumor promoter. When they are applied at various times after TPA treatment, the same combinations of ADR or DAU and VRP or TFP fail to alter TPA-induced RNA and protein synthesis but still exert synergistic inhibitory effects on the peak of DNA synthesis observed 17 h after TPA. However, the chronic administration of ADR and DAU alone or in combination with VRP prior to the peak of TPA-induced DNA synthesis 16 h after each promotion treatment with TPA fails to alter the promotion of skin papillomas in the two-stage protocol of mouse skin carcinogenesis. In contrast, when administered alone or in combination with DAU prior to each TPA treatment, VRP inhibits skin tumor promotion and reveals the antitumor-promoting activity of DAU. These results point to the modulatory role of Ca2+ in the action of ADR and TPA and demonstrate the refractory nature of mouse epidermis to cancer chemotherapy by anthracycline antibiotics. However, ADR and DAU may be effective against skin tumor promotion if they are applied in combination with Ca2+ antagonists and at a time when they can inhibit the inductions of both ODC activity and DNA synthesis by TPA.     

Inhibition of multistage tumor promotion in mouse skin by diethyldithiocarbamate

Diethyldithiocarbamate (DDTC) injected i.p. inhibits remarkably and in a dose-dependent manner 12-O-tetradecanoylphorbol-13-acetate (TPA)-decreased glutathione (GSH) peroxidase and TPA-induced ornithine decarboxylase (ODC) activities in mouse epidermis in vivo. DDTC is more potent in inhibiting these effects of TPA than 16 other antioxidants, free radical scavengers, thiol-containing compounds, and reduced glutathione (GSH) level-raising agents, even though some of these treatments are applied directly to the TPA-treated skin. DDTC also inhibits the effects of several structurally different tumor promoters and the greater GSH peroxidase and ODC responses produced by repeated TPA treatments. The inhibitory effects of DDTC on TPA-decreased GSH peroxidase and TPA-induced ODC activities are additive with those of Na2SeO3 and D-alpha-tocopherol (vitamin E). Interestingly, DDTC is a more effective inhibitor when it is administered after TPA, suggesting that DDTC may supplement, facilitate, and/or enhance the activity of the natural GSH-dependent detoxifying system protecting the epidermis against the oxidative challenge presumably linked to the tumor-promoting activity of TPA. When tested in the initiation-promotion protocols, DDTC inhibits to the same degree complete tumor promotion by TPA and stage 2 tumor promotion by mezerein, in relation with its identical inhibition of the GSH peroxidase and ODC responses to both TPA and mezerein. Moreover, the inhibition of the first stage tumor-promoting activity of TPA by DDTC may be attributed to its ability to inhibit TPA-induced DNA synthesis, a postulated component of the conversion phase of skin carcinogenesis when TPA is used as a stage 1 tumor promoter.     

Camptothecin post-treatments inhibit the biochemical events linked to the tumor-promoting component of carcinogenesis in mouse epidermis in vivo

20(S)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S-phase cells, was tested topically for its ability to inhibit the biochemical markers of skin tumor promotion. CPT has no or very little inhibitory effect on the covalent binding of an initiating dose of 7,12-dimethylbenz-[a]anthracene (DMBA) to DNA at 24 hr, but CPT post-treatments remarkably inhibit stimulations of DNA synthesis caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 16 hr and a carcinogenic dose of DMBA at 7 days. CPT is a much more potent inhibitor if it is applied 10–14 hr after TPA or 4–6 days after DMBA, when DNA synthesis starts being stimulated after the periods of early inhibition caused by TPA and DMBA. When applied 12 hr after the tumor promoter, the ability of 3–3,000 nmol of CPT to inhibit TPA-stimulated DNA synthesis at 16 hr is dose-dependent. A single dose of 500 nmol of CPT inhibits the entire time course for the stimulation of DNA synthesis observed 16–64 hr after TPA. CPT also reduces the various DNA responses to chronic TPA treatments and structurally different non-TPA-type tumor promoters. CPT may indirectly decrease the ornithine decarboxylase-inducing activity of multiple TPA treatments because it can inhibit the stimulation of RNA synthesis by this compound. However, CPT fails to alter TPA-stimulated hydroperoxide production in relation to its inability to inhibit TPA-stimulated protein synthesis. On an equal dose basis, topotecan and 10-hydroxycamptothecin are more and less effective than CPT, respectively, whereas 10,11-methylenedioxycamptothecin is much more potent than its parent compound at inhibiting the DNA response to TPA. A single dose of 400 nmol of CPT has no effect on tumor initiation when applied 4 hr before or 1 hr after a single subcarcinogenic dose of DMBA. In contrast, 400 nmol of CPT chronically applied 1 hr before or 24 hr after each treatment with TPA remarkably inhibits the complete tumor-promoting activity of this agent. CPT post-treatments also inhibit the respective activities of TPA and mezerein in the 1st and 2nd stages of skin tumor promotion. © 1996 Wiley-Liss, Inc.     

Ability of the Ca2+ ionophores A23187 and ionomycin to mimic some of the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on hydroperoxide production, ornithine decarboxylase activity, and DNA synthesis in mouse epidermis in vivo

When applied topically to the skin twice at a 48-h interval or thrice at 24-h intervals, 17 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 mumol of A23187 or ionomycin induce the same 3-fold increases of hydroperoxide (HPx) production in mouse epidermis. In contrast, these doses of Ca2+ ionophores applied once or twice at a 48-h interval produce only 3-8% of the 16- or 34-fold inductions of epidermal ornithine decarboxylase (ODC) activities caused by similar TPA treatments. However, these various Ca2+ ionophore treatments mimic entirely the stimulatory effects of TPA on epidermal DNA synthesis at 16 h and produce from 30 to 70% of the DNA responses to TPA at 32 h. Interestingly, the Ca2+ ionophore and TPA treatments applied thrice at 24-h intervals still produce above maximal or submaximal DNA responses, in spite of their very weak ODC-inducing activities or refractoriness against ODC induction. Treatment with alpha-difluoromethylornithine plus methylglyoxal bis-(guanylhydrazone) (1.25 mumol each), which inhibits the activity of the polyamine-synthesizing enzymes, does not block the HPx and DNA responses to TPA. Conversely, 1.6-25-nmol doses of fluocinolone acetonide inhibit both TPA-induced HPx production and DNA synthesis, without affecting ODC induction. The results suggest that the magnitudes of Ca2+ ionophore- and TPA-induced DNA synthesis may be linked to HPx production rather than ODC induction. Each of these three responses appears to be essential but not sufficient for tumor promotion. A23187 may be a poor or incomplete skin tumor promoter because it lacks sufficient ODC-inducing activity and cannot fully maintain the prolonged stimulation of DNA synthesis required for hyperproliferation.     

Inhibition of mouse skin tumor promotion and of promoter-stimulated epidermal polyamine biosynthesis by alpha-difluoromethylornithine

Application of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin leads to a manifold induction of ornithine decarboxylase (ODC) activity within 5 hr and an increased accumulation of putrescine. The relevance of these TPA-induced changes to the mechanism of tumor promotion was investigated using alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO applied to mouse skin (0.3 mg in 0.2 ml of solvent) or administered in the drinking water (1%) in conjunction with skin tumor promotion by TPA inhibited the formation of mouse skin papillomas by 50 and 90%, respectively. TPA-induced ODC activity and the accumulation of putrescine were almost completely inhibited. DFMO given in the drinking water decreased spermidine levels, but DFMO treatment by any route did not alter the spermine levels of mouse epidermis. DFMO decreased TPA-induced hyperplasia by 25 to 40%, and the TPA-caused increases in DNA synthesis and mitotic index were inhibited by 60 and 50%, respectively. Therefore, in mouse epidermis, enhanced cell proliferation can be dissociated from ODC induction and the accumulation of putrescine. At the tested dose levels and routes of administration, DFMO did not inhibit the inflammatory response to TPA in several tissues. These results provide evidence for an essential role of ODC induction and the accumulation of putrescine in tumor promotion by TPA and add strength to the proposal that DFMO may be a promising drug for the prevention and treatment of cancer in human beings.     

Induction of mouse epidermal ornithine decarboxylase activity and skin tumors by 7,12-dimethylben

Application of a single large dose (3.6 micromol) or smaller weekly repeated doses (0.2 micromol) of 7,12-dimethylbenz[a]anthracene (DMBA) to the skin of CD-1 mice led to a 20 to 50-fold increase in epidermal ornithine decarboxylase (ODC) (EC 4.1.1.17) activity as well as tumor formation. Retinoic acid (0.17-68 nmol), a potent inhibitor of both the induction of ODC activity and tumor formation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), failed to inhibit both the induction of ODC activity and tumor formation by DMBA. In contrast, 7,8-benzoflavone (367 nmol), which did not inhibit the induction of ODC activity by TPA, effectively inhibited the induction of ODC activity as well as the formation of skin tumors caused by DMBA. These results indicate that (a) the mechanism of the induction of ODC activity and tumor formation by a complete carcinogen appears to be different from that of the tumor promoter TPA, (b) DMBA-induced ODC activity may be an important component of the mechanism of DMBA carcinogenesis, and (c) the protective effect of retinoic acid on skin carcinogenesis is not universal; it inhibits skin tumor formation by some agents and not by others.     

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.     

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.     

Characterization of the hydroperoxide response observed in mouse skin treated with tumor promoters in vivo

The production of hydroperoxides is rapidly increased and remains at 200-280% of the control 1-24 h after the second daily application of 17 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin in vivo. The levels of hydroperoxides are increased 1.63-, 2.64-, 4.07-, and 4.31-fold 18 h after one, two, three, or four applications of TPA at 24-h intervals, respectively. The hydroperoxide response to TPA observed in whole skin reflects almost entirely the increased hydroperoxide-producing activity of the epidermis. Such hydroperoxide responses are triggered to various degrees by the anthrone derivatives and the phorbol esters and diterpene with complete and/or stage 2 tumor-promoting activities but not by the agents with only inflammatory, hyperplastic or stage 1 tumor-promoting activities. However, the Ca2+ ionophores A23187 and ionomycin are potent inducers of hydroperoxide formation. Several discrepancies are observed between the hydroperoxide response to TPA and the known effects of the tumor promoter on ornithine decarboxylase (ODC) induction. In contrast to the refractory state against ODC induction caused by TPA treatments repeated at intervals of less than 48 h, the time interval required for recovery of the hydroperoxide response to TPA in TPA-pretreated skins is only 5 h. The stimulatory effects of A23187, ionomycin and various diacylglycerols (DAGs) on hydroperoxide production do not correlate with their ODC-inducing activities. The increasing susceptibilities of C57BL/6, CF-1, and SEN-CAR mice to skin tumor promotion correlate with their hydroperoxide responses but not with their ODC responses to TPA. alpha-Difluoromethylornithine (DFMO) and other inhibitors of TPA-induced ODC activity fail to alter hydroperoxide production whereas the compounds that inhibit the hydroperoxide response to TPA, such as fluocinolone acetonide, have no or only minimal inhibitory activity against ODC induction. This would suggest that the hydroperoxide response to TPA does not require ODC induction and may not be essential for ODC induction. The hydroperoxide response to TPA is mimicked, but to a lesser degree, by the activator of protein kinase C, 1,2-dioctanoyl-sn-glycerol, and inhibited by verapamil, trifluoperazine, and palmitoylcarnitine. Populations of TPA-treated keratinocytes, therefore, may be responsible not only for ODC activation but also for hydroperoxide production. However, these two responses, which involve, at least in part, Ca2+ mobilization and protein kinase C activation and play important roles in the mechanism of skin tumor promotion, do not appear to be correlated.     

Inhibitory effect of munetone, an isoflavonoid, on 12-O-tetradecanoylphorbol 13-acetate-induced ornithine decarboxylase activity

Starting with an extract derived from the bark of Mundulea sericea Willd. (Leguminosae) that was active in the process of inhibiting 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase activity (ODC) in cultured mouse epidermal ME 308 cells, the isoflavonoid munetone was isolated and identified as an active principle (IC50 = 46 ng/ml). Topical application of munetone (0.04-5 micromol) to the skin of CD-1 mice 2 h prior to treatment with TPA (10 nmol) resulted in dose-dependent inhibition of epidermal ODC activity. In addition, munetone inhibited TPA-independent c-Myc-induced ODC activity with cultured BALB/c c-MycER cells, as well as 7,12-dimethylbenz[a]anthracene (DMBA)-induced preneoplastic lesion formation in a mouse mammary gland organ culture (MMOC) system. These data suggest the potential of munetone to serve as a cancer chemopreventive agent by virtue of blocking the process of tumor promotion.     

Inhibition of the effects of 12-O-tetradecanoylphorbol-13-acetate on mouse epidermal glutathione peroxidase and ornithine decarboxylase activities by glutathione level-raising agents and selenium-containing compounds

The present study was undertaken to determine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter known to inhibit superoxide dismutase (SOD) (superoxide: superoxide oxidoreductase, EC 1.15.1.1) and catalase (CAT) (H2O2: H2O2 oxidoreductase, EC 1.11.1.6) activities, on mouse epidermal glutathione (GSH) peroxidase (glutathione: H2O2 oxidoreductase, EC 1.11.1.9) activity in vivo and in vitro. TPA led to a rapid and transient increase in GSH peroxidase specific activity within 30 min followed by a decrease from 1 to 12 h. Incubation of isolated epidermal cells with GSH level-raising agents and/or selenium-containing compounds increased remarkably basal GSH peroxidase activity, and thus, abolished totally the prolonged inhibitory effects of TPA on this enzyme. The inhibitory effects of 0.2 mM cysteine (Cys) or 0.5 mM GSH and 2.5 microM Na2 SeO3 or 50 microM selenocystamine on TPA-decreased GSH peroxidase activity were additive, in relation with their additive inhibitory effects on TPA-induced ornithine decarboxylase (ODC) (L-ornithine carboxylase, EC 4.1.1.17) activity. These data support the hypothesis that the stimulators of the GSH-dependent antioxidant protective system of the epidermal cells may inhibit the oxidative challenge linked to skin tumor promotion by TPA.     

Inhibition of phorbol ester--induced human epidermal ornithine decarboxylase activity by oral compounds: a possible role in human chemoprevention studies

Extensive animal data have suggested that, in some systems, the induction of ornithine decarboxylase (ODC) is an essential, although not sufficient, aspect of tumor promotion and that compounds that inhibit ODC can inhibit tumor formation. Using fasting human volunteers, we report that human epidermal and dermal ODC are consistently induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in a manner similar to that seen in mouse skin. There is a marked intersubject variation in TPA-induced epidermal ODC activity levels. Orally administered compounds significantly inhibited TPA-caused human epidermal ODC induction. These data may be useful in the further development of drugs, doses, and dose schedules for use in human cancer chemoprevention studies.     

Protein kinase Cdelta-mediated signal to ornithine decarboxylase induction is independent of skin tumor suppression

Protein Kinase Cdelta (PKCdelta), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately eightfold) PKCdelta protein in basal epidermal cells are resistant to skin tumor formation by the 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. However, despite being resistant to skin tumor promotion by TPA, PKCdelta transgenic mice elicited a 3-4-fold increase in TPA-induced epidermal ODC activity and putrescine levels than their wild-type littermates. PKCdelta was observed to be the key component of the TPA signal transduction pathways to the induction of mouse epidermal ODC activity. To determine if TPA-induced ODC activity and associated putrescine levels in PKCdelta transgenic mice contributed to PKCdelta-mediated suppression of skin tumor promotion by TPA, the irreversible inhibitor of ODC, alpha-difluoromethylornithine (DFMO), was used. PKCdelta transgenic mice and their wild-type littermates were initiated with 100 nmol DMBA and then promoted twice weekly with 5 nmol TPA. The experimental group was given 0.5% DFMO in their drinking water, while the control group was given tap water. After 25 weeks, the number of papillomas (>2 mm) per mouse was counted. The DFMO treatment did not affect the skin tumor multiplicity of PKCdelta transgenic mice. These results indicate that PKCdelta-induced ODC activity is not involved in PKCdelta-mediated tumor suppression. Thus, the signaling pathways via PKCdelta to epidermal ODC induction and skin tumor suppression appear to be independent.     

Enhanced induction of epidermal ornithine decarboxylase activity in C57BL/6 compared to DBA/2 mice by protein kinase C-activating skin tumor promoters: relevance to genetically mediated differences in promotion susceptibility.

Previous work from our laboratory demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) or a synthetic diacylglycerol induced significantly higher epidermal ornithine decarboxylase (ODC) activity in C57BL/6 than in DBA/2 mice. To understand further the genetic basis for this strain difference, two tumor promoters were evaluated for their effects on epidermal ODC activity: teleocidin, which activates protein kinase C (PKC); and 1,8-dihydroxyl-3-methyl-9-anthrone (chrysarobin), which does not. In addition, the ODC induction response in B6D2F1 offspring and BXD recombinant inbred (RI) strains was examined following multiple treatments with TPA. A single topical application of teleocidin to mouse dorsal skin led to the hyperinduction of epidermal ODC activity in C57BL/6 mice. In contrast, while chrysarobin induced epidermal ODC activity, no significant differences in the magnitude of this response were observed in SENCAR, DBA/2 or C57BL/6 mice. Consistent with our previous findings, the magnitude of ODC induction by teleocidin in these three mouse lines (C57BL/6 greater than SENCAR greater than DBA/2) did not correlate with their susceptibility to tumor promotion by TPA (SENCAR greater than DBA/2 greater than C57BL/6). ODC activity induced by multiple application of TPA in B6DF1 mice, whose susceptibility to phorbol ester tumor promotion is inherited as an incomplete dominant trait, was comparable to that induced in C57BL/6 mice at all the doses examined. Cluster analysis of TPA-induced ODC activity in BXD RI strains allowed us tentatively to group them into four or five phenotypes and to estimate a minimum of two genetic loci controlling TPA-induced ODC activity. Furthermore, in BXD RI strains, there was no apparent relationship between the magnitude of ODC induction and responsiveness to tumor promotion or sustained hyperplasia. Collectively, these results suggest that hyperinducibility of ODC in response to PKC-activating tumor promoters is inherited as an autosomal dominant trait, and that genetic determinants for ODC induction, at least in C57BL/6 and DBA/2 mice, appear completely independent of those controlling tumor promotion susceptibility.     

Mouse skin ornithine decarboxylase induction and tumor promotion by cyclohexane

Cyclohexane, a frequently used solvent in industry, was assessed for its tumorigenic potential on mouse skin following multistage initiation-promotion protocols. The activity of ornithine decarboxylase (ODC), a marker of tumor promotion was found to be induced by the topical application of cyclohexane. This ODC induction was dependent on the dose of cyclohexane used and the duration of application. Effect of protein synthesis inhibitors and the modifiers of tumor promotion on the cyclohexane induced ODC activity was also studied. ODC induction was inhibited by cycloheximide and also, up to some extent, by actinomycin D. Inhibitors of stage II tumor promotion showed more effect on the ODC induction by cyclohexane as compared to the inhibitors of stage I tumor promotion. In chronic animal bioassay experiments topical application of cyclohexane to DMBA initiated mouse skin resulted in just 10% of tumor bearing animals while prior application of TPA for two weeks resulted in 45% of tumor bearing animals. Collectively, the present study demonstrates that cyclohexane is more effective as a stage II tumor promoter over mouse skin and possibly affects the biochemical events at the molecular level.     

Differential inhibition by staurosporine, a potent protein kinase C inhibitor, of 12-O-tetradecanoylphorbol-13-acetate-caused skin tumor promotion, epidermal ornithine decarboxylase induction, hyperplasia and inflammation

The effect of staurosporine on 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin papilloma formation was examined in CD-1 mice. A topical application of staurosporine 15 min prior to each TPA treatment resulted in a dose-related inhibition of tumor formation. Staurosporine by itself had no tumor producing activity in DMBA-initiated mice. Staurosporine failed to prevent TPA-induced edema formation, whereas quercetin markedly suppressed it. Staurosporine by itself did not induce a significant edema. Histological studies revealed that staurosporine failed to inhibit TPA-induced inflammation but rather augmented TPA-induced polymorphonuclear leukocyte (PMN) infiltration. Staurosporine by itself induced a slight PMN infiltration 1 h after the drug application, but the effect was only transient. Although staurosporine failed to inhibit the TPA-induced epidermal hyperplasia and DNA synthesis significantly, nuclear atypism of the superficial layer of the epidermis appeared to be less remarkable in staurosporine-pretreated mice. TPA-caused epidermal ornithine decarboxylase (ODC) induction was not inhibited by staurosporine but rather augmented by this agent. TPA enhanced the phosphorylation of 34 kd protein in intact epidermal cells in a concentration-dependent manner. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) suppressed the TPA-stimulated phosphorylation of 34 kd protein, but palmitoylcarnitine failed to suppress it. In addition, TPA-stimulated superoxide generation of rabbit peritoneal PMN was potently inhibited by staurosporine. It is possible that TPA induces inflammation, ODC activity, epidermal hyperplasia and tumor promotion through the activation of different type(s) of protein kinase C and staurosporine inhibits only certain type(s) of protein kinase C. Another possible explanation is that the protein kinase C inhibition by staurosporine depends on the nature of the substrate proteins or the intracellular localization of the enzyme.