Characterization of the tumor-promoting activity of thapsigargin in cf-1 mouse skin

The ornithine decarboxylase (ODC), hydroperoxide (HPx) and DNA responses to 12-O-tetradecanoylphorbol-13-acetate (TPA) and mezerein (MEZ) are similar in vivo. Thapsigargin (TG) respectively mimics about 15, 75 and 75% of the ODC, HPx and DNA responses to TPA and these differences persist after chronic treatments. The peak of ODC induction 5 h after 2 TG treatments at 12 to 48-h intervals is twice that produced 16 h after a single TG treatment or 5 h after 2 TG treatments at a 72-h interval. The ODC-inducing activity of TG is dose dependent from 1 to 15 nmol and its magnitude is maximal after 2 applications. The biochemical effects of TG and TPA are neither synergistic nor additive. But the ODC response to TG is greater when this compound is applied 48 h after TPA than after another TG treatment. In initiated skins promoted 2x/week with 5 nmol of TPA, MEZ, or TG, the Ist papillomas (PAs) appear at 7, 12 and 17 weeks, the tumor incidences are 100, 40 and 24%, and there are 17, 1 and 0.6 PAs/mouse, respectively. The tumor-promoting activity of TG is increased at a higher dose (15 nmol), slightly accelerated at a higher frequency (1x/2 days) and decreased at a lower frequency (1x/week). TPA applied 1x/3 weeks is insufficient to promote tumors but slightly enhances the tumor-promoting activity of TG. MEZ applied 1x/4 weeks in stage 2 also accelerates the tumor-promoting activity of TG. TG is effective as a stage 1 promoter but its promoting activity is not enhanced by stage 1 treatments with TPA. In contrast to TPA or even MEZ, TG may be a very weak tumor promoter because it is a very weak ODC inducer and mobilizes enough intracellular free Ca2+ to impair tumor cell proliferation.     

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 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.     

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.     

Antitumorigenic and antipromoting activities of ellagic acid, ellagitannins and oligomeric anthocyanin and procyanidin

We previously showed that ellagic acid (EA) was inhibiting lung tumorigenesis induced by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. In the present study, we observed that the inhibition of lung tumorigenesis was independent of the solvent used to purified EA. Pomegranate peels extract containing punicalagin (alpha and beta anomers) (10 g/kg diet) and oligomeric anthocyanins (6 g/kg diet) did not inhibit lung tumorigenesis. Raspberry extract (2x15 mg) containing sanguiin H6 and lambertianin D as well as oligomeric procyanidins (2x15 mg) inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity by about 30%. The same treatments inhibit TPA-stimulated hydroperoxide (HPx) production by about 30 and 70%. Raspberry ellagitannins and oligomeric procyanidins respectively inhibit TPA stimulated DNA synthesis by 42 and 26%. Our results su est that hydrolyzable and condensed tannins from various sources, which can inhibit the ODC, HPx, and DNA responses to TPA, might also inhibit the tumor-promoting activity of this agent. The results of this study show that EA and ellagitannins have different antitumorigenic and antipromoting activities.     

Effects of retinoic acid on epiderma DNA synthesis induced by 12-O-tetradecanoylphorbol-13-acetate, mezerein or ethylphenylpropiolate in hairless mice

We examined the effects of retinoic acid (RA) on epidermal DNAsynthesis, induced by 12-O-tetradecanoylphorbol-13- acetate(TPA), a strong tumor promoter; mezerein (MEZ), a strong secondstage promoter or ethylphenylpropiolate (EPP), a weak tumorpromoter. RA reduced the initial wave of epideniial DNA synthesisin a dose-dependent manner after a single application of TPA,MEZ or EPP. Doses of RA that maximally depressed epidermal DNAsynthesis after single applications had an unexpected stimulatoryeffect when given as five applications over a period of 2 weeks.This might be due to synergistic actions of RA, since RA perse was mitogenic after repeated applications. However, the non-stimulatory17 nmol dose of RA potentiated DNA synthesis in MEZ-treatedepidermis to the same degree as the stlmulatory 170 nmol dosedid in TPA-treated epidermis. We therefore suggested that thepotentiatlon of DNA synthesis seen in the long-term study couldbe mainly due to compensatory growth as a response to initialInhibition. Some observations distinguished the actions of RAin TPA- or MEZ- treated epidermis on the one hand from thosein EPP-treated epidermis on the other: the dose of RA neededfor inhibition was much larger in EPP-treated epidermis; thecombination of RA and EPP was toxic, as observed in the long-termstudy; further reduction of the specific activity of DNA/labelingindex (SA/LI) ratio was only demonstrated in TPA- or MEZ-treatedepidermis. Compared with controls the epidermal SA/LI ratiowas depressed after TPA, MEZ or EPP, indicating that the increasednumber of basal cells with DNA synthesis (LI) displayed a depressedrate of DNA synthesis.     

S/RV Cri-ba, a hairless mouse strain sensitive to skin tumorigenesis by suboptimal doses of 7,12-dimethylbenz[a]anthracene, initiation-promotion and two stage promotion protocols

Susceptibility of hairless inbred S/RV Cri-ba or Bare mice to skin tumor development with suboptimal doses of 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-TPA two stage protocol and two stage promotion using 12-O-tetradecanoylphorbol-13-acetate (TPA) as sub-stage 1 promoter and mezerein (MEZ) or phorbol retinoate acetate (PRA) as substage 2 promoter was determined. A single application of 40 or 20 nmol DMBA induced 4-5 papillomas per mouse 40 weeks after initiation while no tumors appeared after similar treatment with 10 or 4 nmol DMBA. Dose response studies for DMBA initiation revealed that 10 nmol DMBA dose saturated the sites for initiation in the resting epidermis. In two stage promotion experiments, MEZ was found to be a potent stage 2 promoter, while PRA acted as a weak complete promoter.     

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.     

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.     

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.     

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 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.     

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.     

Elevated expression and point mutation of the Ha-ras proto-oncogene in mouse skin tumors promoted by benzoyl peroxide and other promoting agents

The two-stage skin carcinogenesis model of initiation and promotion in SENCAR mice has been used to examine the effects of various tumor-promoting agents on the expression of the Ha-ras oncogene in early stages of tumorigenesis in vivo. Papillomas were induced in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated SENCAR mouse epidermis by (i) complete promotion with benzoyl peroxide; (ii) complete promotion with 12-O-tetradecanoyl phorbol-13-acetate (TPA); and (iii) two-stage promotion with TPA for 2 weeks followed by mezerein for 9 weeks. Results of Northern blot hybridization analyses show that early papillomas contain significantly elevated levels of Ha-ras polyadenylated [poly(A)+] RNA, irrespective of the type of tumor promotion regimen used. This pattern holds for promoters of the phorbol ester class as well as for the free radical generating agent benzoyl peroxide. Furthermore, digestion of tumor DNA with diagnostic restriction endonucleases demonstrated that 9-week-old papillomas induced by DMBA contained a point mutation in the 61st codon of one allele of the Ha-ras gene. The results represent the earliest stage in the development of a papilloma at which a Ha-ras point mutation has been reported.     

Antitumor-promoting activities of hydrolyzable tannins in mouse skin.

Ellagic acid and gallic acid and its derivatives, applied topically to female CF-1 mice 20 min before each 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment inhibit the inductions of epidermal ornithine decarboxylase activity, hydroperoxide production and DNA synthesis caused by this potent tumor promoter in relation with their abilities to inhibit the promotion of skin papillomas and carcinomas in the two-step initiation-promotion protocol. Because of its potency against TPA promotion, tannic acid, which is already known to inhibit tumor initiation, may inhibit the multistage process of carcinogenesis.     

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.     

Stimulatory effect of thapsigargin, a non-TPA-type tumor promoter, on arachidonic acid metabolism in the murine keratinocyte line HEL30 and on epidermal cell proliferation in vivo as compared to the effects of phorbol ester TPA

The effect of thapsigargin (Tg), a non-12-O-tetradecanoylphorbol-13-acetate (TPA) type skin tumor promoter, on arachidonic acid and prostaglandin E2 (PGE2) formation in HEL30 keratinocytes and on epidermal DNA synthesis in vitro and in vivo (mouse skin) was investigated and compared with that of the phorbol ester TPA. On a molar basis Tg was 30-fold more potent in inducing the arachidonic acid/PGE2 release than TPA. Applied together, the two agents showed a strong synergistic action. The response critically depended on the presence of Ca2+ in the extracellular medium. While the TPA-induced release was mediated by protein kinase C (PKC) the Tg-induced release was not. In contrast to TPA (1 microM), which is a stimulator of HEL30 DNA synthesis, Tg (0.1-1 microM) inhibited DNA labeling in vitro due to a pronounced cytotoxic effect. TPA did not exhibit such an effect. In vivo both agents were practically equipotent in inducing epidermal DNA synthesis and hyperplasia with TPA having an approximately 10-fold higher irritating potential than Tg. It is concluded that the hyperplasiogenic and tumor-promoting effect of Tg in vivo is due to cytotoxicity causing cell death and regenerative hyperproliferation. Thus, Tg-induced skin tumor promotion seems to resemble tumor promotion by mechanical skin wounding, whereas TPA evokes a more specific, i.e. PKC-mediated response. Since despite these mechanistic differences both agents induce an immediate release of arachidonic acid/PGE2 in keratinocytes, this response may be considered to provide an in vitro parameter for irritancy and tumor promotion.     

sn-l, 2-Diacylglycerols mimic the effects of 12-0-tetradecanoylphorbol-13-acetate in vivo by inducing biochemical changes associated with tumor promotion in mouse epidermis

12-0-Tetradecanoylphorbol-13 acetate (TPA) or various acyl-glycerolswere applied topically to CD-I mice, and biochemical changesassociated with tumor promotion in the epidermis were examined.The topical application of 5 _µmol of sn-l, 2-didecanoylglycerolcaused a 40-fold increase in orni-thine decarboxylase activitywhich was similar to that found after the topical applicationof 2 nmol of TPA. The time course for the induction of ornithinedecarboxylase activity by TPA and the time course for its inductionby sn-l, 2-di-decanoylglycerol were similar; both compoundsproduced rapid increases in ornithine decarboxylase activitywith peak induction occurring 4–6 h after applicationof the inducing chemical. sn-l, 2-Dioctanoylglycerol and sn-l-oleoyl-2-acetyl-glycerolalso increased ornithine decarboxylase activity in mouse epidermis,but sn-l, 2-dioleoylglycerol, 1, 3-didecanoyl-glycerol and rac-1-monodecanoylglycerolwere inactive at the dose tested. trans-Retinoic acid, a potentinhibitor of tumor promotion, markedly inhibited the epidermalinduction of ornithine decarboxylase activity that resultedfrom the topical administration of sn-l, 2-didecanoylglycerolor TPA. The effects of TPA and the acylglycerols on epidermalDNA synthesis in vivo were determined by measuring the incorporationof [³H]thymidine into epidermal DNA. The application of sn-l,2-didecanoylglycerol or TPA to mouse skin stimulated epidermalDNA synthesis. The maximum increase occurred 18 h after administrationof the inducing chemical, and the increase in DNA synthesiswas proportional to the dose of sn-l, 2-didecanoylglycerol.Although sn-l, 2-didecanoyl-glycerol, sn-l, 2-dioctanoylglyceroland sn-l, 2-dk)leoylglycerol stimulated epidermal DNA synthesis,sn-l-oleoyl-2-acetylglycerol, 1, 3-didecanoylglycerol and rac-1-monodecanoylglycerolhad little or no effect. The increase in epidermal DNA synthesisinduced by sn-l, 2-di-decanoylglycerol or TPA was inhibitedby the simultaneous application of fluocinolone acetonide, apotent inhibitor of tumor promotion. The results indicate thatseveral sn-l, 2-diacylglycerols mimic TPA in vivo with respectto their effects on certain biochemical parameters associatedwith tumor promotion in mouse skin.     

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.     

Enhancement by adriamycin of the effects of 12-O-tetradecanoylphorbol-13-acetate on mouse epidermal glutathione peroxidase activity, ornithine decarboxylase induction and skin tumor promotion

Adriamycin (ADR) failed to inhibit and paradoxically enhanced the biological action of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo and in vitro. In the presence of ADR, the tumor promoter caused a greater sequential rapid increase and prolonged decrease in glutathione (GSH) peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity accompanied by a greater decrease in the ratio of reduced (GSH)/oxidized (GSSG) glutathione in isolated epidermal cells. The ability of ADR to deplete the intracellular level of GSH correlated with its ability to increase basal and TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxylase, EC 4.1.1.17) activities. In vivo, topical ADR treatments also enhanced TPA-induced ODC activity as well as the tumor-promoting ability of TPA in the two-stage system of mouse skin carcinogenesis. Since lipid peroxidation has been associated with ADR toxicity, these data suggest that the enhancement of the tumor-promoting ability of TPA by ADR may be the result of an increased oxidative challenge that overwhelms the GSH-dependent antioxidant protective system of the epidermal cells.