Potent inhibitory effects of suicide inhibitors of P450 isozymes on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene initiated skin tumors

A single dose of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) or 2-ethynylnaphthalene (EN) was applied to the skin of SENCAR mice 5 min before an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P) and the development of skin tumors then promoted with biweekly topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The application of EP strongly inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner. Application of 44 pmol of EP inhibited tumor initiation by 10 nmol of DMBA approximately 25%; application of 440 nmol of EP inhibited tumor initiation by 200 nmol of B[a]P approximately 51%. A high single dose of EP (4.4-44 mumol) nearly eliminated skin tumor initiation by either 10 nmol of DMBA or 200 nmol of B[a]P. Application of VP also inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner, but higher doses of VP than of EP were required to produce comparable inhibitions. Application of 44 nmol of VP inhibited tumor initiation by 10 nmol of DMBA approximately 30%; application of 4.4 mumol of VP inhibited tumor initiation by 200 nmol of B[a]P approximately 56%. Application of EN yielded contrasting results. EN inhibited the formation of skin tumors initiated by 10 nmol of DMBA, but the observed dose-dependence was minimal; tumors were decreased about 40% by 3.3 mumol of EN and only about 65% by 132 mumol of EN. A high single dose of EN (132 mumol) increased both the mean number of tumors per mouse and the percentage of mice that developed tumors after initiation by 200 nmol of B[a]P. Topical application of 4.4 mumol of EP, 22 mumol of VP or 33 mumol of EN to the skin of SENCAR mice 5 min before a single initiation dose of 2.5 mumol of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had a minimal inhibitory effect (14-28%) on the development of skin tumors produced by subsequent biweekly promotion with TPA. A single dose of 44 mumol of EP or 132 mumol of EN followed by biweekly applications of TPA did not produce skin tumors; however, a dose of 44 mumol of VP followed by promotion with TPA produced a low but significant number of skin tumors.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Comparison of the effects of 3-isobutyl-1-methylxanthine and adenosine cyclic 3':5' -monophosphate on the induction of skin tumors by the initiation-promotion protocol and by the complete carcinogenesis process

Topical application of either 5 µmol of 3-isobutyl-1-methylxanthine(IBMX) or 0.25 µmol of adenosine cyclic 3':5' -monophosphate(cyclic AMP) to the initiated skin of the mouse prior to eachpromotion with 8.5 nmol of 12-O-tetradecanoylphorbol-13-acetate(TPA) inhibited the formation of papillomas and carcinomas.Combined treatments including IBMX and cyclic AMP caused additivereduction of the incidence of skin papillomas and carcinomaspromoted by TPA. A good correlation was observed between thereduction of the tumor incidence by IBMX and cyclic AMP andtheir inhibition of TPA-stimulated polyamine, RNA, protein,and DNA synthesis. Since repeated treatments with these agentsbefore or after initiation with a single subcarcinogenic doseof 7,12-dimethylbenz[a]anthracene (DMBA) did not alter significantlythe development of skin tumors (Curtis et al.; Perchellet andBoutwell), the present results suggest that, in the two-stepinitiation-promotion protocol, both IBMX and cyclic AMP treatmentsmay decrease specifically the promoting stimulus of skin carcinogenesis.The same doses of IBMX and cyclic AMP inhibited the accumulationof polyamines and the increase in macromolecular synthesis observedin DMBA-treated skin, but their effect on DMBA-induced skincarcinogenesis was dependent upon the protocol used and thedose of carcinogen applied. IBMX and cyclic AMP treatments failedto inhibit the induction of skin tumors by weekly applicationsof 0.2 µmol of DMBA. In contrast to the inhibitory effectof cyclic AMP treatment, IBMX enhanced the carcinogenic responseto a single topical application of 3.6 µmol of DMBA. Theopposite effects of these agents on the carcinogenicity of DMBAcorrelated well with their different alteration of DMBA-inducedunscheduled DNA synthesis in vitro. Cyclic AMP (0.5 mM) enhanced,whereas IBMX (0.5 mM) inhibited, the DMBA-induced incorporationof labeled precursor into DNA of isolated epidermal cells duringincubation in the presence of hydroxyurea. Therefore, it issuggested that the different modulation of DMBA carcinogenesisby IBMX and cyclic AMP may result from concomitant effects onboth the initiating and promoting components of the carcinogen.     

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.     

Synergistic interaction between the non-phorbol ester-type promoter mixer and 12-0-tetradecanoylphorbol-13-acetate in mouse skin tumor promotion

Mirex, an organochlorine pesticide and non-genotoxic rodenthepatocarcinogen, is also a potent non-phorbol ester-type promoterof mouse skin tumors. Mirex, unlike most other skin tumor promoters,is not a significant epidermalhyper plasiogen even at a maximallypromoting dose (200 nmol). Experiments described here examinedwhether tumor promotion by mirex and 12-O-tetradecanoylphorbol-13-acetate(TPA) are mediated through different mechanisms as indicatedby their additivity when co-applied to 7,12-dimethylbenz[a]anthracene(DMBA, 200 nmol)-initiated female CD-1 mouse skin. Instead ofthe additive response of 14 plus 5 tumors/mouse predicted frommice promoted for 20 weeks (2x/week) with either mirex (200nmol) or TPA (2 nmol) respectively, their co-application yielded35 tumors/mouse. This synergy with TPA was specific to mirexsince a structurally related compound chlordecone (Kepone) wasinactive. Mirex plus TPA-promoted papillomas contained a c-Ha-rasA¹⁸² - T mutation as frequently (13/14) as those promoted bymirex or TPA alone, suggesting that these DMBA initlated/co-promotedpapillomas were not atypical in this genotypic marker. Promotionalsynergy with mirex was only observed with a submaximal promotingdose of 2 nmol TPA; 5 or 8 nmol TPA plus mirex gave additiveor less tumor multiplicitles. This synergistic multiplicitywith mirex plus 2 nmol TPA (35 tumors/mouse) approximated thesum of individual responses to 200 nmol mirex (14 tumors/mouse)and the maximally promoting dose of TPA (12 mnol), 24 tumors/mouse,suggesting that mirex potentiated the promotional activity ofTPA, as well as promoted through a mirex-specific mechanism.Epidermal DNA synthesis induced by 2 nmol TPA was potentiatedby mirex, further supporting a role for mirex in potentiationof epidermal TPA activity. Collectively, these studies suggestthat mirex affects two possibly related responses: (i) promotionthrough a distinct mirex-specific mechanism, and (ii) potentiationof a mechanism mediating the promotional activity of TPA.     

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.     

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.     

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

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

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.     

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.     

Inhibition of 7-bromomethylbenz[a]anthracene-promoted mouse skin tumor formation by retinoic acid and dexamethasone

Retinoic acid, a potent inhibitor of mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate, fails to inhibit tumor formation by the complete carcinogen, 7, 12-dimethylbenz[a]anthracene (DMBA). To obtain further clues about the nature of the mechanism of the carcinogenic process as well as the mechanism of the effect of retinoic acid on tumor promotion, the effect of retinoic acid and two other modifiers (dexamethasone and 7,8-benzoflavone) of tumor formation on tumor promotion by 7-bromomethylbenz[a]anthracene (BrMBA) was determined. BrMBA, a structural analogue of DMBA, is a weak mouse skin tumor-initiating agent but is a good skin tumor promoter. Application of 10, 100, and 200 nmol of BrMBA twice weekly to DMBA-initiated skin resulted in 0, 1.6, and 2.5 papillomas per mouse, and 0, 44, and 60% of mice had papillomas at the 25th week of promotion treatment, respectively. Application of 17 nmol of retinoic acid or 76 nmol of dexamethasone 30 min prior to each twice weekly application of 100 nmol of BrMBA to DMBA-initiated skin inhibited the formation of skin papillomas by 73 and 100%, respectively. 7,8-Benzoflavone, at a 367-nmol dose, did not inhibit tumor promotion by BrMBA. Application of 200 nmol of BrMBA to mouse skin induced epidermal ornithine decarboxylase activity; a peak activity was observed between 8 and 18 hr following BrMBA treatment. Application of 17 nmol of retinoic acid or 76 nmol of dexamethasone inhibited the induction of ornithine decarboxylase activity by BrMBA. 7,8-Benzoflavone did not inhibit the induction of ornithine decarboxylase activity by BrMBA. Retinoic acid and dexamethasone, which inhibit tumor promotion by 12-O-tetradecanoylphorbol-13-acetate, also inhibited tumor promotion by BrMBA, but the nature of the mechanism of tumor promotion by BrMBA is unclear; BrMBA did not inhibit specific binding of 12-O-[3H]tetradecanoylphorbol-13-acetate to the cellular membrane fraction of mouse epidermis.     

Effects of dose and duration of treatment with the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate on mouse skin carcinogenesis

The effects of dose and duration of treatment with the potenttumor-promoting agent 12-O-tetra-decanoylphorbol-13-acetate(TPA) on the formation of skin tumors in Charles River CD-1mice was studied. Mice were initiated with a single applicationof 0.2 µmol of 7, 12-dimethylbenz[a]anthracene (DMBA)in 0.2 ml acetone. Beginning two weeks after initiation, micewere treated twice weekly with various doses (0.01 – 20nmol) of TPA in 0.2ml acetone. Application of either 0.01 or0.1 nmol of TPA did not elcity tumors during the 50 weeks durationof treatment. A dose-dependent increase in the number of papillomaswas observed through the range of 1 to 10 nmol of TPA. Twiceweekly applications of 20nmol of TPA did not further enhancethe papilloma incidence. A good correlation was observed betweenthe induction of ornithine decarboxylase (ODC) activity andthe formation of skin tumors by various doses of TPA. To determine the effect of promotion duration on the incidenceof papillomas and carcinomas, mice were treated with 10 nmolof TPA for various durations (6,12,18,24,30, or 36 weeks) beginning2 weeks after initiation with 0.2 µmol of DMBA. Mice promotedfor only 6 weeks developed papilllomas and carcinomas afterpromotion had been discontinued. There was an intermediate incidenceof tumors in the group treated for 12 weeks. Promotion for 18,24, 30, or 36 weeks elicited virtually identical yields of papillomas.The incidence of carcinomas was proportional to promotion durationtimes of 6, 12, and 18 weeks, but carcinoma incidence was lessthan maximal in mice promoted for 24 weeks or longer. The results indicate that a) the incidence of papillomas servesas a rapid (18 weeks) index for subsequent appearance of carcinomas,b) twice weekly applications of 10 nmol of TPA for 18 weeksfollowing initiation of female CD-1 mice with 0.2 µmnolof DMBA is an appropriate protocol for maximum tumor yield ininitiation-promotion experiments, and c) ODC induction may bean important component of the mechanism of skin tumor promotionby TPA.     

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.     

Three-stage tumorigenesis in mouse skin: DNA synthesis as a prerequisite for the conversion stage induced by TPA prior to initiation

Recent evidence shows that stage I of tumor promotion in NMRI-mouseskin induced by a single dose of 12-O-tetradecanoylphorbol-13-acetate(TPA) can be effected not only after initiation by 7,12-dimethylbenz(a)anthracene(DMBA) but also several weeks prior to initiation. In view ofthis partial inversion of the initiation-promotion sequencewe proposed to replace the term ‘stage I of promotion’by the term ‘conversion’. The convertogenic effectivityof a single dose of TPA applied after DMBA can be suppressedin a rather characteristic and non-toxic fashion by hydroxyurea(HU). In the present study we asked whether HU might also interferewith the converting effectivity of a single dose of TPA givenprior to DMBA. NMRI mice received a single dose of TPA 3 weeksprior to initiation by DMBA which was followed by twice weeklyapplication of skin irritant 12-O-retinoylphor-bol-13-acetate(RPA) in order to effect stage II of promotion. A single doseof HU given i.p. at different times after TPA was found to interferewith tumor formation exhibiting an almost complete inhibitionif administered 18 h after TPA. This inhibition did not preventa subsequent promotion by repetitive TPA treatment. The dataindicate that conversion can be inhibited by HU in the samecharacteristic fashion regardless of whether TPA was administeredafter or prior to initiation. The data also support the autonomouscharacter of the conversion process for which epidermal DNAsynthesis appears to be obligatory.     

Inhibition of mouse skin tumor promotion by adriamycin and daunomycin in combination with verapamil or palmitoylcarnitine

The anti-cancer drugs Adriamycin (ADR) and Daunomycin (DAU) alone were unable to inhibit the promotion of skin papillomas by repeated applications of 8.5 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mice. Pretreatments with 50 micrograms of ADR also failed to alter the tumor-promoting activities of smaller doses of TPA. Therefore, the effects of the anthracycline antibiotics on skin tumor promotion were evaluated in combination with the Ca2+ antagonist verapamil (VRP) and the protein kinase C (PKC) inhibitor palmitoylcarnitine (PC), compounds known to circumvent drug resistance. When applied simultaneously with each promotion treatment with 8.5 nmol of TPA, 2.5 mg of VRP inhibited the number of papillomas/mouse by 26%. But the combination of VRP + 50 micrograms of ADR or DAU inhibited the yields of papillomas by 50 or 47%, respectively, suggesting that VRP was required to reveal the antitumor-promoting activities of otherwise ineffective drugs. Similarly, the promotion of skin tumors by TPA was inhibited synergistically by the combinations of 2 mumol of PC + 50 micrograms of ADR or DAU. For instance, ADR and DAU had no effects alone but inhibited the incidence of skin papillomas by 78 and 86%, respectively, in the presence of PC, a compound which alone inhibited the tumor incidence by only 44%. The results indicate that ADR and DAU are effective against the promoting component of skin carcinogenesis only if they are applied in combination with Ca2+ antagonists or PKC inhibitors at a time when they can inhibit the early biochemical effects induced by TPA.     

Events associated with mouse skin tumor promotion with respect to arachidonic acid metabolism: a comparison between SENCAR and NMRI mice

NMRI and SENCAR, two stocks of mice commonly used in multistage skin carcinogenesis studies, were compared with respect to the effects of inhibitors of arachidonic acid metabolism for the following 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited events: tumor promotion, DNA synthesis in vivo and in vitro, ornithine decarboxylase induction, and prostaglandin (PG) E2 synthesis. Previous work had shown that the cyclooxygenase inhibitor indomethacin enhanced TPA promotion in SENCAR mice. We report here that over the same dose range (50 to 200 micrograms) indomethacin caused a dose-dependent inhibition of promotion in NMRI mice. Significant reversal of this inhibition was achieved with concomitant application of 10 micrograms PGF2 alpha but not PGE2. DNA synthesis studies showed that low doses of indomethacin and flurbiprofen increased TPA-stimulated DNA synthesis in primary cultures from SENCAR mice; indomethacin suppressed this response in NMRI cultures. In vivo DNA synthesis studies showed the same pattern: indomethacin enhanced TPA-stimulated DNA synthesis in SENCAR mice but inhibited in NMRI mice. Other classes of inhibitors of arachidonate metabolism (i.e., the cyclooxygenase-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and phenidone and the phospholipase A2 inhibitor dibromoacetophenone) had inhibitory activity in vitro and in vivo in both stocks of mice. Indomethacin was found to inhibit TPA-induced ornithine decarboxylase activity to the same extent in both mice. Indomethacin was also very effective in inhibiting TPA-induced PGE2 synthesis in both stocks of mice. 5,8,11,14-Eicosatetraynoic acid and phenidone were likewise suppressive in both stocks of mice. It is concluded that the NMRI and SENCAR mice respond similarly to TPA with respect to promotion, DNA synthesis, ornithine decarboxylase induction, and PG synthesis. The difference appears to be in the degree of involvement of the lipoxygenase pathway.     

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.     

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.     

Correlation between sensitivity to tumor promotion and sustained epidermal hyperplasia of mice and rats treated with 12-O-tetra-decanoylphorbol-13-acetate

The role of sustained hyperplasia in tumor promotion has beenreexamined in light of our previous report that the Syrian hamster,which is insensitive to two-stage epidermal carcinogenesis initiatedby DMBA and promoted by 12-O-tetradecanoylphorbol-13-acetate(TPA), does not respond with epidermal hyperplasia after multipletreatments with the promoter. Epidermal hyperplasia of severalother species and strains of animals, which vary in their responsivenessto TPA-mediated tumor promotion, was examined after single ormultiple treatments with the tumor promoter TPA. It was foundthat CD-1 mice, a strain which is responsive to TPA-inducedtumor promotion, reacted with a slight degree of hyperplasiaafter a single exposure to the chemical and this response waspotentiated after several treatments. C57B1 mice, a strain marginallyresponsive to TPA promotion, displayed only slight hyperplasiaafter single and multiple treatments. Ba1b/c mice, which areintermediately responsive to TPA-mediated promotion, displayedconsiderable epidermal hyperplasia after a single dose of TPAwhich was maintained after multiple treatments but at a reducedlevel. DBA mice, only marginally responsive to TPA promotion,showed considerable hyperplasia after a single dose of TPA whichwas maintained after multiple treatments but at a reduced level.DBA mice, only marginally responsive to TPA promotion, showedconsiderable hyperplaisa which was potentiated by multiple treatments.F344 rats, which are relatively insensitive to TPA promotion,showed a moderate hyperplastic response after a single dosewhich was reduced slightly after multiple treatments. A strikingdifference was observed in the response of the different animalspecies to single versus multiple exposures to the promoter.When the effects of single versus multiple doses of TPA werecompared, every possible combination of responses was observed,including potentiation, adaptation, and no change. Thus, theresults of a single exposure could not be used to predict theresponse with multiple treatments. While no correlation wasobserved between sensitivity to tumor promotion and hyperplasiainduced by a single exposure of TPA, a good correspondence wasfound between sensitivity to tumor promotion and TPA inducedsustained hyperplasia after 4 weeks of treatment. The role ofsustained hyperplasia in tumor promotion and the necessity tostudy persistent effects induced by TPA are discussed.