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.     

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.     

Evaluation of pentacyclic triterpenes found in Perilla frutescens for inhibition of skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

A series of pentacyclic tritperpenes found in Perilla frutescens (P. frutescens), including ursolic acid (UA), oleanolic acid (OA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) were evaluated for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and compared to UA as the prototype compound. All compounds were given topically 30 min prior to each TPA application and significantly inhibited skin tumor promotion. 3-epiCA and MA were significantly more effective than UA at inhibiting tumor development. All of these compounds significantly inhibited epidermal proliferation induced by TPA, however, CA, 3-epiCA and MA were more effective than UA. All compounds also reduced skin inflammation (assessed by infiltration of mast cells and T-cells) and inflammatory gene expression induced by TPA, however, 3-epiCA and MA were again more effective than UA. The greater ability of 3-epiCA and MA to inhibit skin tumor promotion was associated with greater reduction of Cox-2 and Twist1 proteins and inhibition of activation (i.e., phosphorylation) of IGF-1R, STAT3 and Src. Further study of these compounds, especially 3-epiCA and MA, for chemopreventive activity in other cancer model systems is warranted.     

Inhibitory effect of topical application of polymerized ferulic acid, a synthetic lignin, on tumor promotion in mouse skin two-stage tumorigenesis

A dehydrogenation polymer of ferulic acid (DHP-FA), a syntheticlignin, was evaluated for its ability to inhibit tumor promotionby 12-O-tetradecanoylphorbol-13-acetate (TPA) in the 7,12-dimethylbenz[a]anthracene-treatedskin of female ICR mice. Topical application of DHP-FA inhibitsTPA-induced tumor promotion, whereas a monomeric ferulic aciddoes not show the inhibitory effect.     

Inhibition of both stage I and stage II mouse skin tumour promotion by retinoic acid and the dependence of inhibition of tumor promotion on the duration of retinoic acid treatment

Retinoic acid is a potent inhibitor of mouse skin tumor promotion by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have further evaluated the effect of retinoic acid on the stages of tumor promotion and also analyzed the effect of duration of retinoic acid treatment on mouse skin tumor promotion by TPA. In a number of independent experiments, either with female CD-1 or SENCAR mice, we failed to observe a specificity of inhibition by retinoic acid of either Stage I or Stage II tumor promotion. In a typical experiment with SENCAR mice, application of 34 nmol of retinoic acid concurrently with each application of either TPA (3.2 nmol) or mezerein (3.2 nmol) to initiated (with 10 nmol 7,12-dimethylbenz(a)anthracene) skin equally inhibited promotion of skin papilloma formation. Furthermore, sustained inhibition of tumor promotion by retinoic acid required a continuous application of retinoic acid in conjunction with each promotional treatment with TPA; if retinoic acid treatment was discontinued, TPA treatment elicited tumor formation. These results indicate: (a) retinoic acid inhibits both Stage I and Stage II of tumor promotion; and (b) inhibition of tumor promotion exhibits retinoic acid dependency.     

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.     

Stimulation of arachidonic acid release and prostaglandin synthesis by bryostatin 1

The mechanism of tumor promotion may involve stimulation ofprostagladin production. Previous studies with the tumor promotor12-O-tetradecanoylphorbol-13-acetate (TPA) have identified twoeffects of TPA on prostaglandin production. TPA stimulates botharachidonic acid release and de novo synthesis of prostaglandinH synthase. Activation of protein kinase C by TPA appears tobe part of the mechanism to cause archidonic acid release. However,it is unclear if induction of prostaglandin H synthase alsoinvolves activation of protein kinase C. Bryostatin 1 is knownto activate protein kinase C and to mimic some of the effectsof TPA. We compared bryostatin 1 with TPA for the ability tocause arhidonic acid release and induced synthesis of prostaglandinH synthase. Bryostatin 1 induced arachidonic acid release andcaused some prostaglandin production but only marginally inducedsynthesis of prostaglandin H synthase. Furthermore, we foundthat bryostatin 1 could inhibit the effect of TPA both in stimulationof arachidonic acid release and in the induction of prostaglandinH synthase.     

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.     

Hydrolyzable tannins: potent inhibitors of hydroperoxide production and tumor promotion in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate in vivo.

The anti-oxidant and the anti-tumor-promotion activities of several hydrolyzable tannins (HTs), including a commercial tannic-acid (TA) mixture, were examined in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo. A single application of TPA gradually increases the hydroperoxide (HPx)-producing activity of the epidermis, which is maximally stimulated at 3 days and returns to control levels at 9 days. Pre-treatments with TA and ellagic acid (EA) strongly inhibit, in a dose-dependent manner, this HPx response to TPA. Total inhibition by TA lasts for about 16 hr, beyond which it is substantially reduced but not completely lost. TA can also reduce the level of epidermal HPx when it is applied 36 hr after the tumor promoter. EA is an antioxidant 10 times more potent than TA and n-propyl gallate (PG), which are equally effective against TPA-induced HPx production. Gallic acid is the least effective of the HTs in inhibiting HPx formation. TA also inhibits the production of HPx induced by several structurally different tumor promoters and the greater HPx responses produced by repeated TPA treatments. When applied 20 min before each promotion treatment, twice a week for 45 weeks, several HTs inhibit the incidence and yield of papillomas and carcinomas promoted by TPA in initiated skin. Overall, TA is more effective than EA and PG in inhibiting skin-tumor promotion by TPA, suggesting that the anti-oxidant effects of HTs are essential but not sufficient for their anti-tumor-promotion activity.     

Antitumor-promoting activity of garlic extracts

Garlic extract was proved to inhibit one of the earliest phenomena caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, in vitro; i.e., the enhancement of phospholipid metabolism. And also the first stage of tumor promotion in two-stage mouse skin carcinogenesis in vivo was suppressed by the treatment with garlic extract. Thus, garlic extract seems to be effective to inhibit initial events caused by TPA type tumor promoters in vitro and in vivo.     

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.     

Influence of the duration of topical 13-cis-retinoic acid treatment on inhibition of mouse skin tumor promotion

The effect of the time and duration of retinoid treatment on the inhibition of Stage II tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied in CD-1 mice. All mice were initiated with 400 nmol of benzo(a)pyrene and received Stage I tumor promotion (3.2 nmol of TPA twice weekly for 2 wk). Animals were then randomized into groups which received 13-cis-retinoic acid during early, middle, or late Stage II promotion. 13-cis-Retinoic acid pretreatments starting on Day 1, Wk 8, or Wk 23 of Stage II promotion resulted in 47, 28, or 19% inhibition, respectively, of TPA-induced tumor formation. One-half of the mice receiving 13-cis-retinoic acid at Day 1 or Wk 8 were removed from the retinoid treatments at Wk 23, the time of cessation of TPA promotion. The inhibition of tumor formation remained constant during the 15-wk observation period after cessation of retinoid treatment, suggesting that retinoid inhibition of mouse skin tumor promotion is stable in the absence of further promotion and preceded the step of irreversible conversion of promoter dependence to promoter independence.     

Inhibitory effects of ursolic and oleanolic acid on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

Ursolic acid (UA) and oleanolic acid (OA), which had been isolated from Glechoma hederacea as inhibitors of Epstein-Barr virus (EBV) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), were tested against inhibitory effect on tumor promotion by TPA in vivo. They inhibited effectively the tumor promotion in mouse skin and the activities were comparable to that of a known inhibitor of tumor promotion, retinoic acid (RA). Interestingly, UA was more effective on a single application before initial TPA-treatment than on a continuous application before each TPA-treatment, while OA and RA were ineffective in the same treatment. These data suggest that the role of UA for inhibitory action on tumor promotion differs slightly from those of RA and OA.     

Inhibition of protein kinase C by the 12-O-tetradecanoylphorbol-13-acetate antagonist glycyrrhetic acid

Glycyrrhetic acid is an anti-inflammatory agent isolated from licorice root that inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion in mouse skin. Although it has been established that glycyrrhetic acid inhibits a number of events induced by the phorbol ester tumor promoter TPA in cultured cells, its mechanisms of action has remained obscure. In this report, we demonstrate that glycyrrhetic acid inhibits the Ca2+-and phospholipid-dependent phosphotransferase activity of protein kinase C (PKC), the phorbol ester tumor promoter receptor. Therefore, inhibition of PKC may play a role in the anti-promoting activity of glycyrrhetic acid.     

Induction of metallothionein mRNA by tumor promoters in mouse skin and its constitutive expression in papillomas

A single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to induce mRNA of a metallothionein (MT) gene or genes in the skin of Sencar mice, and papillomas produced by repeated applications of TPA were shown to have elevated levels of MT mRNA. Induction of MT mRNA was maximal 4-8 h after application of TPA and returned to the control level 24 h later. A dose-dependent increase of MT mRNA was observed with doses of TPA of 1-5 micrograms. Of the other promoters tested, phorbol-12, 13-didecanoate, mezerein, and the ionophore A23187 also induced MT mRNA, but 4-O-methyl-TPA and benzoyl peroxide did not. Phorbol and 4 alpha-phorbol-12,13-didecanoate, which are not promoters, also did not induce MT mRNA. Retinoic acid and 1 alpha, 25-dihydroxyvitamin D3, inhibitors of tumor promotion, did not induce MT mRNA themselves or inhibit the induction of MT mRNA by TPA. In C57BL/6 promotion-resistant mice, TPA caused only slight induction of MT mRNA. These data suggest a correlation between induction of MT mRNA and epidermal hyperplasia. The constitutive elevation of MT mRNA levels in papillomas may be due to the loss, during the process of tumor promotion, of some mechanism regulating MT gene expression.     

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.     

Nicotinamide and nicotinamide analogues as antitumor promoters in mouse skin

Phorbol ester-induced promotion of initiated NMRI mouse skin keratinocytes to papillomas could be largely prevented when nicotinamide-like inhibitors of poly(ADP-ribose)polymerase (nicotinamide, benzamide, 3-aminobenzamide) were applied simultaneously with 12-O-tetradecanoylphorbol-13-acetate (TPA). A similar suppression of tumor promotion by nicotinamide analogues was demonstrated in clone 41 JB6 epidermal cells which are promotable by TPA to anchorage-independent growth. The antipromotion effect of nicotinamide analogues, however, does not appear to come about by an inhibition of poly(ADP-ribose)polymerase. Acid analogues of nicotinamide, such as benzoic acid or 3-aminobenzoic acid which do not inhibit the polymerase, showed antipromotion activity similar to that of their corresponding amides. It could also be ruled out that these antipromoters mediate their effect on keratinocytes by a cytostatic action, by scavenging the promoter TPA in a chemical reaction, or by inhibiting protein kinase C. In initiated mouse skin, nicotinamide analogues strongly suppressed TPA-induced accumulation of inflammatory cells and vascular permeability, while epidermal hyperplasia was not significantly affected.     

Antitumor-promoting activity of oligomeric proanthocyanidins in mouse epidermis in-vivo

The flavanoid catechin and heterogenous samples of oligomeric proanthocyanidins extracted from various sources were compared for their ability to inhibit the biochemical and biological effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo. Topical applications of catechin fail to alter the hydroperoxide response to TPA but inhibit the induction of ornithine decarboxylase (ODC) activity and, to a lesser degree, the stimulation of RNA, protein, and DNA synthesis caused by this tumor promoter. Under similar conditions, condensed tannins (CTs) from guamuchil, loblolly pine, and southern red oak barks inhibit to various degrees all these biochemical markers of TPA promotion. The most effective antioxidant, loblolly pine bark CT, also inhibits TPA-induced ODC activity and macromolecule synthesis to a much greater degree than catechin or the other CTs tested. Pecan nut pith CT, however, has no inhibitory activity in this system. Pretreatments with 4 and 12 mg of loblolly pine bark CT remarkably inhibit the incidence and yield of skin tumors promoted by TPA in initiated mice, whereas similar doses of catechin are ineffective. Loblolly pine bark CT inhibits the 2nd rather than the 1st stage of tumor promotion. In contrast to their monomer units, therefore, some naturally occurring polyflavanoids have antioxidant activities and may be valuable against tumor propagation but their efficacy may vary considerably depending on their origin and structure.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced induction in Epstein-Barr virus early antigen in Raji cells

Retinol, 5 flavonoids, 3 steroids and 7 sweetening agents were studied for their effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced early antigen (EA) of Epstein-Barr virus (EBV) in Raji cells. Concomitant treatment of Raji cells with TPA and retinol showed inhibition of EA induction. Among flavonoids, quercetin resulted in effective inhibition of EA induction by TPA and α-naphthoflavone showed the weakly inhibitory effect. None of the other flavonoids such as rutin, catechin and β-naphthoflavone affected the induction of EBV-EA by TPA. β-Estradiol obviously inhibited EBV-EA induction by TPA, but hydrocortisone did not show any inhibitory effect on it. Glycyrrhetinic acid, steviol, phyllodulcin and perrillartine also showed the remarkable inhibition of EBV-EA induction. On the other hand, glycyrrhizin and stevioside, glycosides of glycyrrhetinic acid and steviol, did not inhibit the induction of EBV-EA by TPA. Some of the inhibitors reported here may be effective on the inhibition of the in vivo tumor promotion by TPA.