Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and ornithine decarboxylase activity by quercetin: possible involvement of lipoxygenase inhibition

Quercetin (30 µmol/mouse) markedly suppressed the effectof 12-O-tetradecanoylphorbol-13-acetate (TPA, 20 nmol/mouse)on skin tumor formation in the CD-1 mice initiated by 7,12-dimethylbenz[a]anthracene(200 nmol/mouse). TPA (20 nmol/mouse)-induced epidermal ornithinedecarboxylase (ODC) activity was also inhibited by quercetin(10–30 µmol/mouse), but it failed to inhibit thestimulation, of epidermal DNA synthesis by TPA. In addition,quercetin potently inhibited lipoxygenase from 105 000 g supernatantof epidermal homogenate of mice. The 50% inhibition of lipoxygenasewas observed by quercetin at 1.3 µM. These results suggestthat the inhibition of lipoxygenase by quercetin is one of themajor actions of the above agent to inhibit tumor promotionand TPA-induced ODC activity.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity by lipoxygenase inhibitors: possible role of product(s) of lipoxygenase pathway

Induction of epidermal ornithine decarboxylase (ODC) by a topicalapplication of 12-O-tetradecanoylphorbol-13-acetate (TPA), atumor promoter, was inhibited by treatment of mouse skin withphenidone (3–90 µ mol/mouse), nordihydroguaiareticadd (30 µmol/mouse) or 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline(BW 755C, 30 µ/mouse), which are well-known lipoxygenaseinhibitors. Phenidone and BW 755C are also to be cyclooxygenaseinhibitors. Inhibition of TPA-induced ODC by indomethacin (1.12µmol/mouse), a selective cyclooxygenase inhibitor, wascounteracted by prostaglandin E₂(PGE₂) (140 nmol/mouse). Thiscounteracting effect of PGE₂ was reversed by the treatment ofmice with nordihydroguaiaretic acid (30 µmol/mouse) orphenidone (30 umol/mouse). ODC activity which was suppressedby nordihydroguaiaretic add or phenidone at a dose of 180 umol/mousewas not further inhibited by indomethadn (1.12 µmol/mouse).In addition, the counteracting action of PGE₂ (140 nmol/mouse)was not observed in mice treated with nordihydroguaiaretic acidor phenidone at a dose of 180 umol/mouse. Thus, the suppressiveeffect of nordihydroguaiaretic add or phenidone on the ODC inductionby TPA would be due to the inhibition of lipoxygenase. The abovefindings strongly suggest that not only cyclooxygenase product(i.e., PGE₂) but also lipoxygenase produces(s) are involvedin the mechanism of ODC induction in mouse epidermis, and alack of either cyclooxygenase product or lipoxygenase product(s)causes a failure of ODC induction by TPA.     

Comparison of the effect of sn-1,2-didecanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate on cutaneous morphology, inflammation and tumor promotion in CD-1 mice

Since sn-1,2-didecanoylglycerol mimics 12-O-tetradecanoylphorbol-13-acetate(FPA) by inducing ornithine decarboxylase activity and stimulatingDNA synthesis in mouse epidermis [Smart,R.C., Huang,M.-T. andConney,A.H. Carcinogenesis, 7, 1865(1986)], we have investigatedmorphological changes induced by TPA and sn-1,2-didecanoylglycerolin the epidermis and we have also examined sn-1,2-didecanoylglycerolas a possible complete tumor promoter. It wasdetermined thattopical application of 2.5 or 10 µmol of sn-1,2-didecanoylglycerolinduced epidermal ornithine decarboxylase activity to aboutthe same extent as the application of 1 or 2 nmol of TPA respectively.Therefore, these doses of TPA and sn-1,2-didecanoylglycerolwere used in most of our studies. Single or multiple application(2 x /week for 4 weeks) of 1, 2 or 5 nmol of TPA to the skinof CD-1 mice produced a dose-dependent increase in the numberof epidermal non-cornified cell layers, epidermal thickness,leukocyte infiltration and intracellular edema. In contrast,neither single nor multiple application (2 x /week for 4 weeks)of 2.5 or 10 µmol sn-1,2-didecanoylglycerol produced anyof these responses. However, when 5 µmol sn-1,2-didecanoyl-glycerol was applied topically twice a day (10 µmol/day)for 5 days there was a significant increase in the number ofepidermal non-cornified cell layers and epidermal thickness.The effects of TPA and sn-1,2-didecanoylglycerol were comparedusing the mouse ear inflammation model. Application of TPA causededema, but sn-1,2-didecanoylglycerol had little or no effect.sn-1,2-Didecanoylglycerol was then evaluated as a complete tumorpromoter utilizing the mouse skin two-stage model. CD-1 micewere initiated with 200 nmol 7,12-dimethythenz[a]anthraceneand then treated wIth 1 nmol TPA or 2.5 µmol sn-1,2-didecanoylglyceroltwice a week for 28 weeks. A28 weeks, 28% of the mice treatedwith TPA had developed tumors, while none of the mice treatedwith 2.5 µmol sn-1,2-didecanoylglycerol developed tumors.The data indicate that topical application of 2.5 µmolsn-1,2-didecanoylglycerol induced ornithine decarboxylase activityto the same extent as a tumor-promoting dose of 1 nmol TPA,but it did not cause morphologial changes in the epidermis whenapplied once or when applied twice a week for 4 weeks and didnot function as a complete tumor promoter when applied twicea week for 28 weeks. Since more frequent applications of sn-1,2-didecanoylglycerol(5 µmol twice a day     

Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol

We assessed the anti-mutagenic and anti-promotion propertiesof two flavones, apigenin and robinetin, and of indole-3-carbinol,because these compounds have been reported in vegetables, theconsumption of which has been associated with reduced ratesof cancer. However, the active components of these foods andtheir effects on carcinogenesis have not been established. Anti-mutagenicitywas determined in the Salmonella typhimurium assay by measuringthe effects of the test compounds on bacterial mutagenesis inducedby methyl-nitrosourea (MNU), methyl-n-nitro-N-nitrosoguanidine(MNNG), benzo[a]pyrene (BaP) or 2-aminoanthracene (2-AA). Inclusionof apigenin resulted in a 62% and a 43% inhibition of mutagenicitywith 13 nmol of 2-AA and 30 nmol BaP respectively. Robinetincaused an 87% inhibition of mutagenicity by 2-AA, but indole-3-carbinolhad little or no effect on the mutagenicity of any of the compounds.None of the three compounds inhibited mutagenesis by MNU orMNNG and none were mutagenic or toxic when tested in the absenceof mutagenic compounds at doses up to 20 µg/plate. Anti-promotionproperties were assessed by measuring the effects of apigenin,robinetin and indole-3-carbinol on induction of ornithine decarboxylaseactivity (ODC) in mouse epidermis by 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment of the skin half an hourbefore TPA with apigenin, robinetin, butylated hydroxyanisole,13-cis-retinoic acid (all at 50 µol) or di-fluoromethylornithine(1.6 µmol) inhibited ODC induction at 6 h after TPA by67–80%. Pretreatment with 50 µmol indole-3-carbinolcaused a 78% elevation in the TPA induction at this time. Doseresponse measurements were conducted with apigenin, indole-3-carbinoland robinetin. Inhibition by 30–90% of TPA-induced ODCwas observed at 6 h after TPA in mice pre-treated with 12.5–100µmol apigenin. Pretreatment with 37.5 or 50 µmolindole-3-carbinol or 0.5, 12.5 or 25 µmol robinetin resultedin elevated induction of epidermal ODC by TPA at 6 h after TPA.However, treatment with 50 or 100 µmol robinetin diminishedODC induction at 6 h after TPA. Treatment with 100 µmolapigenin or 50 or 100 µmol indole-3-carbinol in non-TPA-treatedmouse skin caused elevations in epidermal ODC. In comparingthe time course of ODC induction, indole-3-carbinol (50 µmol)pretreatment shifted the induction of epidermal ODC to earliertimes, in addition to elevating ODC induction by TPA. However,apigenin (50 µmol) pretreatment inhibited TPA-inducedODC activity at 4, 6 and 8 h, indicating no shift in ODC induction.In conclusion, indole-3-carbinol showed no potential for inhibitionof mutagenesis in the present study and presented potentialfor enhancement of promotion. In contrast, the potential ofapigenin and robinetin as inhibitors of the initiation and promotionphases of carcinogenesis merits further study.     

Inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced increase in ornithine decarboxylase mRNA in mouse epidermis

Application of 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA)to the skin of female CD-1 mice led to a rapid increase in theconcentration of epidermal ornithine decarboxylase (ODC) mRNAfrom an undetectable level in control mice to a high maximumlevel at 4–5 h after TPA administration. The concentrationof epidermal ODC mRNA then decreased rapidly during the next5 h. The time course for TPA-induced increases in epidermalODC enzyme activity paralleled very closely the time coursefor TPA-induced increases in ODC mRNA. Topical administrationof 1, 3 or 10µmol curcumin together with 5 nmol TPA inhibitedby 66, 81 and 91% respectively TPA-induced increases in epidermalODC enzyme activity measured 5 h later. In a parallel study,TPA-induced increases in the concentration of epidermal ODCmRNA was inhibited by 54, 85 and 82% respectively. Intraperitonealinjection of 10 or 30 µmol curcumin 1h before topicalapplication of 5 nmol TPA inhibited TPA-induced increases inepidermal ODC enzyme activity by 75 or 89% respectively. Ina parallel study, the induction of epidermal ODC mRNA was inhibitedby 53 and 65% respectively. The results indicate that curcumininhibits TPA-induced increases in epidermal ODC enzyme activityby inhibiting the synthesis and/or enhancing the breakdown ofODC mRNA.     

Possible dissociation of the phorbol ester-induced oxidant response and tumor promotion in the F1 offspring of SSIN x C57BL/6J mice

The F₁ progeny of a cross between 12-O-tetradecanoyl-phor-bol-13-acetate(TPA) tumor promotion-sensitive SSIN mice and TPA promotion-resistantC57BL/6J mice were found to be sensitive to TPA as a tumor promoter.The tumor response was substantial, with an average of 15 papillomasper mouse and a 100% incidence following initiation with 400nmol dimethylbenz[a]anthracene and promotion with 6.5 nmol (4µg) TPA. To determine whether tumor promotability of theF₁ mice correlates with other parameters believed to be associatedwith TPA responsiveness, oxidant generation, epidermal hyperplasiaand edema were compared in the parents and F₁ hybrids. The SSINproduced a strong hyperplastic response to TPA, the C57BL/6Ja negligible response and the F₁ hybrids a moderate response.In the SSIN, 6.5 nmol (4 µg) TPA caused an 18% increasein the water content of the skin (edema) while no change wasseen for either the C57BL/6J or F₁ hybrids. The oxidant responseof the F₁ hybrids to either TPA or phospholipase C was markedlyless than that observed for the SSIN and was similar to theresponse previously observed for the C57BL/6J mice. These findingssuggest that the oxidant response may not be an essential aspectof TPA tumor promotion. It may be related to the edema response,suggesting that at least this aspect of inflammation is notnecessary for promotion.     

Cytogenetic effects caused by phorbol ester tumor promoters in HeLa cells: mechanistic aspects

The effect of the convertogenic (‘first-stage’)tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) andthe nonconvertogenic (‘second-stage’) tumor promoterRPA (12-O-retinoylphorbol-13-acetate) on chromosomes was investigatedin HeLa cells which have previously been shown to exhibit aradiomimetic response to TPA. As in the case of mouse keratinocytes,only TPA had a significant clastogenic activity at non-cytotoxicconcentrations ranging from 10^–8 to 10^–6 M measuredafter 24 and 48 h. The values observed with RPA did not differsignificantly from values observed in the presence of the solvent(acetone, 0.2%). The response to TPA was saturable with respectto the dose of TPA. The chromosomal aberrations (mostly gapsand breaks) were predominantly of the chromatid type. Isochromatidaberrations were caused by a 24 or 48 h treatment with 10^–6M TPA. The aberrations appear as early as 6–8 h afterTPA application, i.e. as soon as the cells have recovered fromTPA-induced inhibition in G₂-phase. Even a 30 min exposure to10^–7 M TPA gives the same yield of aberrations as longertreatment, i.e. the response to TPA is ‘saturable’with respect to time. Both TPA and the non-clastogenic RPA causea temporal G₂-delay thus indicating that the G₂-inhibition isnot related to induction of chromosomal aberrations by TPA.The data are consistent with the hypothesis that TPA induceschromosomal aberrations via a receptor-mediated pathway.     

Effects of lipoxygenase and cyclooxygenase inhibitors on the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate and isoproterenol in mouse tissues in vivo

I.p. administration of 12-O-tetradecanoylphorbol-13-acetate(TPA) (400 µg/kg) caused a remarkable increase in ornithinedecarboxylase (ODC) activity in CD-1 mouse liver (8.3-fold),spleen (17.8-fold), kidney (4-fold), lung (7.7-fold) and brain(2.7-fold). TPA induced an increase in ODC activity in liver,spleen and kidney in a dose-dependent manner (100–800µg/kg). The putrescine contents of these tissues werealso increased by TPA injection. BW755C, an inhibitor of cyclooxygenaseand lipoxygenase, prevented the TPA-induced increase in ODCactivity in liver, spleen and kidney in a dose-dependent manner.AA861-a selective lipoxygenase inhibitor, also showed the inhibitionof TPA-induced increase in ODC activity in these tissues. Significantinhibition was observed either by BW755C or AA861 at the doseof 30 mg/kg. On the other hand, indomethacin, a selective cyclooxygenaseinhibitor, enhanced the TPA-induced increase in ODC activityin these tissues dose-dependently. Significant enhancement wasobserved at 3 mg/kg for liver and spleen and 1 mg/kg for kidney.The subcutaneous administration of isoproterenol (1 mg/kg) causedan increase in ODC activity in both liver (11-fold) and spleen(3.4-fold). Both AA861 and BW755C failed to inhibit the isoproterenol-inducedincrease in ODC activity in these tissues. These results indicatethat product(s) of lipoxygenase pathway play an important rolein ODC induction caused by TPA in liver, spleen and kidney,while the lipoxygenase pathway does not play an essential rolein the isoproterenol-induced increase in ODC activity.     

Sensitivity to tumor promotion of SENCAR and C57BL/6J mice correlates with oxidative events and DNA damage

Significant differences in sensitivity to multistage carcinogenesishave been noted between mice that are sensitive (SENCAR) andresistant (C57BL/6J) to 12-O-tetradecanoyl-phorbol-13-acetate(TPA). However, the mechanism of this sensitivity has not yetbeen established. Recent studies from this laboratory have shownthat TPA significantly enhances formation of hydrogen peroxide(H₂O₂) and oxidized DNA bases in SENCAR mouse skin, as it increasesthe infiltration of polymorphonuclear leukocytes (PMNs), asquantitated by myeloperoxidase (MPO). In the studies reportedhere, we compared SENCAR and C57BL/6J mice with respect to TPA-mediatededema, hyperplasia, PMN infiltration, oxidant formation andoxidative DNA damage in mouse skin. Topical application of twoTPA doses (2x2–40 µg, 20 h apart) dose-dependentlyincreased PMN infiltration and oxidant formation in both mousestrains, which was consistent with TPA-induced morphologicalalterations (edema and hyperplasia). However, at low TPA doses(2–4 µg), the increases over controls in the SENCARmice were significantly greater (P < 0.01) than those inC57BL/6J mice. Comparison of the net values indicated that 4µg TPA enhanced PMN infiltration (MPO units/cm²) and oxidantformation (nmol H₂O₂/cm²) in SENCAR mice by 7.7- and 11-foldrespectively over those present in TPA-treated C57BL/6J mouseskin. At the same dose, TPA also significantly increased formationof thymidine glycol (dTG; 5.5-fold), 5-hydroxymethyl-2'-deoxy-uridine(HMdU; 4.9-fold) and 8-hydroxyl-2-deoxyguanosine (8-OHdG; 11.4-fold)in SENCAR mouse epidermis. Then, the levels of all three declined.In C57BL/6J mice, there were virtually no increases at 4 µgTPA, but their levels gradually increased with higher TPA dosesand reached maxima at 10 µg TPA for dTG (1.9-fold increase),at 20 µg TPA for 8-OHdG (6.0-fold), and at 30 µgTPA for HMdU (1.8-fold). We conclude that the TPA-mediated oxidativeevents and oxidative DNA modification by different doses ofTPA correlate with the promoting potencies of those doses inboth mouse strains. Therefore, they could be, at least in part,responsible for the strain-dependent sensitivity to tumor promotion.     

Moderate enhancement of the promotion phase of skin tumorigenesis in hairless mice by topical pretreatment with a mitosis-inhibiting epidermal pentapeptide

The effect of the physiological epidermal proliferation inhibitorysubstance (EPP) pGlu-Glu-Asp-Ser-GlyOH on the promotion phasein two-stage carcinogenesis was investigated. EPP could be theactive component in what has been called epidermal chalone.Hairless mice were given an initial application of 100 nmol7, 12-dimethylbenz[a]anthracene in 200 µl acetone. Oneweek later promotion was started with topical applications of17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice a week.Ninety minutes before each TPA application the control groupreceived a topical application of 200 µl reagent gradeacetone and the two experimental groups were given either 0.005%or 0.01% EPP in 200 µl acetone. The mice were observedfor time of occurrence and time of regression of papillomas.The number of tumors produced by the group given the inhibitorysubstances before TPA increased, and so did the percentage oftumor-bearing animals. There was also a tendency towards a higherdegree of papilloma regression in the animals treated with EPPbefore TPA. We have previously shown that EPP enhances methylnitrosourea(MNU) carcinogenesis. Since we regard TPA as a skin-irritatingpromoter with weak carcinogenic potency, very different fromMNU, the fact that EPP has the same enhancing effect on promotionas it has on complete MNU carcinogenesis raises some very interestingquestions, and may indicate a similarity between the mechanismsin promotion and complete carcinogenesis. Some possible explanationsof the results are discussed, e.g. whether the transit zonelate G₁/S of the cell cycle is the most sensitive one for carcinogenicor tumorigenic effects.     

Effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate- and ultraviolet B light-induced expression of c-Jun and c-Fos in JB6 cells and in mouse epidermis

Expression of c-jun was observed in normally Proliferating JB6cells but not in confluent cells. Reduction of the serum concentrationfrom 5% to 2% in the cell culture medium caused JB6 cells toenter a quiescent non-proliferating state and down-regulatedthe expression of c-Jun. Treatment of quiescent JB6 cells with12-O-tetradecanoylphorbol-13-acetate (TPA) (10 ng/ml) for 24h markedly stimulated the formation of c-Jun and caused morphologicalchanges. Treatment of JB6 cells with TPA for 48 h resulted intransformed foci with mixed cell populations. Although somecells in these foci expressed high levels of c-Jun, many othercells did not. The increased expression of c-Jun and morphologicalchanges observed at 24 h after treatment of JB6 cells with TPA(10 ng/ml) was inhibited by curcumin (10 nmol/ml). TreatmentofJB6 cells with 2.5, 5 or 10 nmol curcumin/ml inhibited the formationof TPA-induced anchorage-independent colonies that grow in softagar by 31%, 43% and 77%, respectively. Although inhibitionof cell proliferation was not observed with 2.5 nmol curcumin/ml,higher concentrations did inhibit cell proliferation. Topicalapplication of 5 nmol TPA to the backs of CD-I mice once a dayfor 5 days caused epidermal hyperplasia and the levels of c-Junwere increased in the suprabasal layer of the epidermis andin the muscle layer of the dermis. This treatment also increasedc-fos protein (c-Fos) expression in the muscle layer, but therewas little or no increase in the expression of c-Fos in thebasal or suprabasal layer of the epidermis. Topical applicationof 10 µmol curcumin together with 5 nmol TPA once a dayfor 5 days strongly inhibited TPA-induced epidermal hyperplasiaand c-Jun and c-Fos expression. A single application of 180mJ/cm² of ultraviolet B light (UVB) to the backs of SKH-1 micecaused epidermal hyperplasia and expression of c-Fos and c-Junin the muscle layer of the dermis and of c-Fos in the suprabasallayer of the epidermis. Maximum effects were observed at 6 daysafter UVB exposure. Application of 10 µmol curcumin tomouse skin twice a day for 5 days immediately after UVB exposurehad only a small/variable inhibitory effect on UVB-induced increasesin the expression of c-Fos and c-Jun and on epidermal hyperplasia.These data suggest that induction of hyperplasia and c-Jun andc-Fos expression in mouse skin by TPA and UVB may involve differentpathways and that inhibition of TPA-induced skin tumorigenesisby curcumin may be associated with inhibition of TPA-inducedincreases in the expression of c-Jun and c-Fos.     

Inhibition of chrysarobin skin tumor promotion in SENCAR mice by antioxidants

The present study was designed to further investigate the roleof reactive oxygen species in the mechanism of action of anthronetumor promoters. To accomplish this, the effects of severalantioxidants on the induction of epidermal ornithine decarboxylase(ODC) activity, epidermal hyperplasia, skin edema, and skintumor promotion by chrysarobin [1, 8-di-hydroxy-3-methyl-9-anthrone)were tested. Ascorbyl palm-itate (AP), given 5 min prior tothe promoter at 1 and 4 µmol doses, effectively inhibitedthe induction of ODC activity (28% and 59%, respectively) by220 nmol of chrysarobin. Using a similar protocol,     

Generation of the arachidonic acid metabolite 8-HETE by extracts of mouse skin treated with phorbol ester in vivo; identification by 1H-n.m.r. and GC-MS spectroscopy

After a single in vivo application of 20 nmol of the ‘complete’tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) orof the ‘incomplete’ promoter 12-O-retinoylphorbol-13-acetate(RPA) to the back skin of mice, a major arachidonic acid metabolite(AAMX) was found in cell-free epidermal preparations after additionof arachidonic acid (AA). Studies with cyclooxygenase and lipoxygenaseinhibitors indicated that this metabolite is a lipoxygenaseproduct. The metabolite has been purified by sequential useof t.l.c. and h.p.l.c. and identified as 8-hydroxy-5Z, 9E, 11Z,14Z-eicosatetraenoic acid (8-HETE) by means of GC-MS and ¹H-n.m.r.spectroscopy. To assist in the characterization of AAMX, a referencemixture of 8-HETE and 9-HETE was used for which a complete structuralanalysis could be performed using one- and twodimensional ¹H-n.m.r.at 500 MHz. The production of 8-HETE has been shown to startwith a lag phase of 3 h after TPA treatment and to reach a maximumafter 24–48 h. Nonpromoting phorbol esters are 10-fold,the Ca²⁺-ionophore A 23187 100-fold, less efficient in inducingin vivo the lipoxygenase responsible for 8-HETE production.3-Day-old neonatal mice cannot be induced to generate 8-HETEin response to TPA, whereas 8-day-old mice show an extremelystrong response. Neither primary basal keratinocytes, isolatedfrom TPA-treated mouse epidermis nor TPA-treated epidermal celllines generate 8-HETE, indicating that the metabolite may notoriginate from these epithelial cells.     

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.     

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.     

Tumour-promoting and hyperplastic effects of phorbol and daphnane esters in CD-1 mouse skin and a synergistic effect of calcium ionophore with the non-promoting activator of protein kinase C, sapintoxin A

Using an 18 week two-stage protocol we have compared the tumour-promotingproperties of a range of phorbol and daphnane esters on femaleCD-1 mice. The induction of epidermal hyperplasia in this mousestrain by these compounds has also been assessed by comparisonwith the standard phorbol ester, 12-O-tetradecanoylphorbol-13-O-acetate(TPA). Two compounds, sapintoxin D (SAP D) and thymeleatoxinA (TA) (a daphnane structurally related to the second-stagepromoter mezerein) were shown to be second-stage promoters using5 nmol TPA as a first-stage promoter and 0.2 µmol 7, 12-dimethylbenz(a)anthracene (DMBA) as initiator. Both compounds at a dose of17 nmol were hyperplasiogenic. Two further derivatives, sapintoxinC (SAP C) and 4-sapinine (-SAP) were inactive as promoters andhyperplastic agents. 4-sapinine, which prevents in vitro stimulationof protein kinase C (PKC), by 12-O-tetra decanoylphorbol-13-O-acetate(TPA) failed to inhibit significantly TPA-induced promotionand hyperplasia at a dose of 20 nmol and 100 nmol respectively.Sapintoxin A (SAP A), a potent activator of PKC, was neithera complete nor second-stage promoter at doses of up to 20 nmol.A series of in vivo and in vitro experiments which were carriedout to determine the metabolic fate of this compound under experimentalconditions showed that SAP A was not metabolized to any significantextent up to 48 h. When SAP A was co-administered with sub-hyperplasticdoses of the calcium ionophore A23187 (5 µg and 10 µg)tumours appeared in a dose-dependent manner. This combinationwas also hyperplasiogenic in mouse skin. SAP A may be a usefulprobe for studying the involvement of PKC isozymes in tumourpromotion and cell proliferation.     

Effects of a biomimetic superoxide dismutase on complete and multistage carcinogenesis in mouse skin

The effects of a selective detoxifier of the proximate oxygenradical, superoxide anion, on the induction of tumors in theskin of CD-1 mice by either the initiation-promotion regimenor the complete carcinogenesis process were investigated. Theprinciple agent of interest, copper (II) (3,5-diisopropylsalicylate)₂(CuDIPS), is a low molecular weight, lipophilic copper coordinationcomplex that catalytically disproportionates superoxide anionat a rate comparable to native CuZn superoxide dismutase (SOD).The protocols used to elicit tumors were: (i) a single applicationof 0.2 µmol of 7,12-dimethylbenz[a]anthracene (DMBA) followedby twice-weekly applications of 12-O-tetradecanoylphorbol-13-acetate(TPA) in an initiation-promotion study, and (ii) either a singleapplication of 3.6 µmol DMBA followed by no further treatmentor weekly applications of 0.2 µmol DMBA in complete carcinogenesisprotocols. Application of 2 µmol CuDIPS 15 min prior tothe initiating dose of DMBA was without significant effect ontumor yield or incidence, whereas application prior to eachdose of TPA substantially reduced tumor incidence and yield.This anti-promoting property of CuDIPS can be attributed toits SOD-mimetic activity in as much as the corresponding zinccoordination complex lacking in SOD activity, zinc (II) (3,5-diisopropylsalicylate)₂,was non-inhibitory. Significant reductions in tumor yield werealso observed when CuDIPS was applied prior to DMBA in eitherof the complete carcinogenesis protocols. Additionally, covalentbinding of [³H]DMBA to epidermal DNA was markedly reduced byCuDIPS pre-treatment, suggesting that the anti-carcinogenicproperties may reflect a perturbation in superoxide anion-dependentmetabolic activation of DMBA. The induction by DMBA of ornithinedecarboxylase activity, a biochemical marker of tumor promoteractivity, was not affected by CuDIPS; however, induction ofornithine decarboxylase by TPA was potently blocked. Collectively,these effects of a biomimetic SOD further implicate reactiveoxygen species at multiple stages in chemical carcinogenesis.     

Inhibitory effect of silymarin, an anti-hepatotoxic flavonoid, on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and mRNA in SENCAR mice

In recent years, considerable emphasis has been placed on identifyingnew cancer chemopreventive agents which could be useful forhuman populations. Silymarin, an anti-oxidant flavonoid isolatedfrom artichoke, has been shown to possess siginificant activityagainst hepatotoxicity and other pharmacological and physiologicaldisorders. Since many antioxidants inhibit tumor promotion,and because ornithine decarboxylase (ODC) is a well known biochemicalmarker of tumor promotion, we assessed the effect of skin applicationof silymarin on 12-O-tetradecanoylphorbol-13-acetate (TPA) inducedepidermal ODC activity and ODC mRNA levels in SENCAR mice. Applicationof silymarin at doses of 0.5–18 mg (1–37 µmol)/mouseprior to that of TPA (2.5 µg) treatment resulted in significantinhibition of TPA-induced epidermal ODC activity in a dose-and time-dependent manner. Northern blot analysis revealed thattopical application of silymarin at the dose of 2 mg/mouse resultedin almost complete inhibition of TPA-induced epidermal ODC mRNA.In other studies, silymarin also showed significant inhibitionof epidermal ODC activity induced by several other tumor promoters,including free radical-generating compounds. Our data suggestthat silymarin could be a useful anti-tumor promoting agentcapable of ameliorating the tumor promoting effects of a widerange of tumor promoters.     

Anti-tumor-promoting action of FK506, a potent immunosuppressive agent

The effect of FK506, a potent immunosuppressive agent, on 7,12-dimethylbenz[a]anthracene-initiatedand 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skinpapilloma formation was examined in CD-1 mice. A topical applicationof FK506 to mouse skin 15 min before each TPA treatment resultedin a dose-related inhibition of tumor formation. FK506 (1 µmol)almost completely inhibited tumor formation. This inhibitoryeffect of FK506 was not due to any damage inflicted on the initiatedcells but due to its antitumor-promoting action. A topical applicationof FK506 also inhibited epidermal ornithine decarboxylase inductionand skin inflammation caused by TPA in a dose-related manner.Significant inhibition by FK506 of TPA-induced endogenous proteinphosphorylation in intact epidermal cells was not detected.These results indicate that FK506 inhibits TPA-induced tumorpromotion at a step distal to the endogenous protein phosphorylationby TPA.