Palmitoylcarnitine reverses 12-O-tetradecanoylphorbol-13-acetate-induced refractory state for the TPA-caused ornithine decarboxylase induction in mouse epidermis

When a single topical application of 12-O-tetradecanoylphorbol-13-acetate(TPA) was performed 12 h before the second application, ornithinedecarboxylase (ODC) induction by the second application of TPAwas markedly suppressed (refractory state). However, at intervalsof 96 h between the first and the second application, the ODCactivity induced by the second application of TPA was higher(enhanced state) than the activity induced by the single application.When various antitumor promoting agents, i.e. p-bromophenacylbromide, nordihydroguaiaretic acid, quercetin, 1-tosylamide-2-phenylethylchloromethyl ketone, retinoic acid and palmitoylcarnitine, wereapplied concurrently with the first TPA application, the ODCinduction in the refractory state was restored only by palmitoylcarnitine,but not by other anti-tumor promoting agents. None of theseanti-tumor promoting agents affected the ODC induction in theenhanced state. Stearoylcarnitine also had the restorative effectbut was less effective than palmitoylcarnitine. Acetylcarnitineand palmitic acid were not effective. Pretreatment of mice withTPA 12 h or 96 h before the second TPA application resultedin the reduction or the increase in the V_max values of ODC bothfor ornithine and pyridoxal-5'-phosphate, respectively. Palmitoylcarnitinerestored these reduced V_max values to the control values. Twelvehours after TPA treatment, the epidermal protein kinase C activityof both cytosol and particulate fractions decreased moderately.At 96 h after TPA application, protein kinase C activities ofboth cytosol and particulate fractions were fully or at leastpartially restored to the control levels. Protein kinase C activitiesboth in the cytosol and the particulate fractions tended tobe restored by palmitoylcarnitine, but the effect was not alwaysreproducible. The TPA-induced refractory state and the enhancedstate for ODC induction appear to result from the changes inthe protein kinase C activities caused by TPA. However, it isnot known whether such changes in the protein kinase C activitiesare the major causes for the TPA-induced refractory and/or enhancedstate for ODC induction and whether or not the restorative effectof palmitoylcarnitine is due to its modulating action on proteinkinase C activity.     

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.     

Tumor promoter-induced refractory state against ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate in mouse epidermis

More than one application of the potent tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), to mouse skin at intervals of more than 48 h led to a larger induction of ornithine decarboxylase (EC 4.1.1.17; ODC) than did a single application. In contrast, at intervals of less than 24 h, the first application of TPA appeared to induce a refractory state; the second application of TPA did not induce ODC. The extent of the inhibitory effect caused by the first application of TPA was dependent on the dose. The abilities of a series of phorbol esters to induce the refractory state correlated with their promoting abilities. However, both mezerein and ethylphenylpropiolate, potent hyperplastic agents with little or no promoting properties, induced the refractory state. On the other hand, pretreatment with TPA caused a refractory effect on ODC induction by mezerein but potentiated ODC induction by ethylphenylpropiolate. The epidermal cells escaped from the refractory state by repeated application of TPA at intervals of 24 h as well as at intervals of twice a week; that is, there was a full induction of ODC activity following a second application within 24 h of a prior application. TPA did not elicit production of detectable ODC-antizyme activity in mouse epidermis. Mixing of a soluble extract from mouse epidermis in the refractory state with that from TPA-stimulated epidermis gave essentially additive ODC activity. Elimination of ODC induction by topical application of retinoic acid or injection of cycloheximide concurrent with the first application of TPA did not restore the ability of a second application of TPA to induce ODC. These results suggest that the refractory effect on ODC induction by TPA does not result from feedback regulation of ODC.     

Inhibitory effects of topical application of low doses of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and oxidized DNA bases in mouse epidermis

The effects of topical applications of very low doses of curcumin (the major yellow pigment in turmeric and the Indian food curry) on 12-O- tetradecanoylphorbol-13-acetate (TPA)-induced oxidation of DNA bases in the epidermis and on tumor promotion in mouse skin were investigated. CD-1 mice were treated topically with 200 nmol of 7,12- dimethylbenz[a]anthracene followed one week later by 5 nmol of TPA alone or together with 1, 10, 100 or 3000 nmol of curcumin twice a week for 20 weeks. Curcumin-mediated effects on TPA-induced formation of the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HMdU) and tumor formation were determined. All dose levels of curcumin inhibited the mean values of TPA-induced HMdU formation in epidermal DNA (62-77% inhibition), but only the two highest doses of curcumin strongly inhibited TPA-induced tumor promotion (62-79% inhibition of tumors per mouse and tumor volume per mouse). In a second experiment, topical application of 20 or 100 nmol (but not 10 nmol) of curcumin together with 5 nmol TPA twice a week for 18 weeks markedly inhibited TPA- induced tumor promotion. Curcumin had a strong inhibitory effect on DNA and RNA synthesis (IC50 = 0.5-1 microM) in cultured HeLa cells, but there was little or no effect on protein synthesis.      

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

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

Inositol hexaphosphate reduces 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase independent of protein kinase C isoform expression in keratinocytes.

High-fiber diets have been shown to have beneficial effects on preventing tumorigenesis. Inositol hexaphosphate (InsP₆ or phytic acid) which is a fiber-associated component of cereals and legumes has been demonstrated to inhibit cell proliferation and enhance cell differentiation, indicating its potential for chemopreventive roles. In this study, we investigated the effect of InsP₆ on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, an essential event in tumor promotion in HEL-30 cells, a murine keratinocyte cell line and SENCAR mouse skin. ODC activity was significantly reduced by 0.5 mM InsP₆ in keratinocytes (P<0.01). Furthermore, when mouse skin was treated with 10 mM InsP₆, ODC induction was significantly inhibited (P<0.05). In addition, the expression of TPA-induced c-myc mRNA was significantly inhibited by the same InsP₆ treatments in HEL-30 cells and CD-1 mouse skin (P<0.01). No changes in protein kinase C (PKC) isoform expression and phorbol dibutyrate binding due to InsP₆ treatment were found in HEL-30 cells. These results indicate that InsP₆ reduces TPA-induced ODC activity independent of PKC isoform expression.     

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.     

Heterogeneity of ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin and in epidermal tumors

One of the earliest events after treatment of mouse skin with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is the induction of ornithine decarboxylase (ODC). Using an immunoperoxidase technique with a rabbit antiserum specific for ODC, the localization of cells containing high levels of ODC following TPA treatment was determined. CD-1 female mice treated with multiple topical applications of TPA and killed 4.5 h after the last TPA treatment exhibited a heterogeneous localization of ODC in this hyperplastic epidermis. The cells which exhibited intense immunostaining were found predominantly in the suprabasal cells lining the hair follicles. This specific ODC staining in cells surrounding hair follicles was inhibited by pretreatment of mice with either retinoic acid or cycloheximide 1 h before TPA treatment. The induction of ODC-specific staining after TPA treatment in hyperplastic mouse skin was transient, since no staining was observed 16 or 24 h after TPA treatment. In contrast, benign papillomas produced by two-stage tumorigenesis contained some cells demonstrating high levels of ODC a week after the last TPA application. These results indicate that both normal mouse epidermal cells as well as tumor tissue display cellular heterogeneity of ODC expression.     

Inhibitory effect of ailanthoidol on 12-O-tetradecanoyl-phorbol-13-acetate-induced tumor promotion in mouse skin

Many components derived from dietary or medicinal plants showing antioxidant and anti-inflammatory potential have been found to possess chemopreventive properties. In our previous study, we achieved the total synthesis of ailanthoidol (AT), a neolignan from Zanthoxylum ailanthoides or Salvia miltiorrhiza Bunge, which are used in Chinese traditional herbal medicine. In the present study, preliminarily, AT exhibited a radical quenching property by DPPH assay. Following this, we assessed the effect of AT on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammation in female CD-1 mouse skin which was closely linked to tumor promotion. The topical application of AT (0.5-2.5 mM; 200 microl) reduced the formation of hydrogen peroxide and inhibited the myeloperoxidase (MPO) activity in the mouse skin when compared with that of the TPA-treated alone group. In addition, AT presented a suppression effect on the TPA-induced hyperplasia and leukocyte infiltration in the epidermis and edema of mouse ears. Furthermore, it showed that AT inhibited the TPA-induced expression of COX-2 protein and ornithine decarboxylase (ODC) activity in epidermis. Finally, AT was evaluated for its ability to inhibit the TPA-induced promotion in skin tumors of female CD-1 mice. Topical application of AT 5 min prior to TPA (5 nmol) three times weekly for 12 weeks to mice which were initiated with benzo[a]pyrene (B[a]P) inhibited the incidence of skin tumors in mice and the average number of tumors per mice as compared to TPA-treated alone. These results indicate that AT possesses potential as a chemopreventive agent against tumor promotion.     

Comparison of the inhibitory effects of retinoids on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activities in CD-1 and Sencar mice

Linear regression analysis of the dose levels of a series of retinoids required ornithine decarboxylase (ODC) induction by 50% indicated that there was no significant difference in inhibitory activity between CD-1 and Sencar mice (r = 0.9699; P less than 0.001). The ID50 values (nmol) found were as follows: retinoic acid (0.20, CD-1; 0.29, Sencar); 13-cis-retinoic acid (1.4, CD-1; 1.9, Sencar); 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1E-propenyl]benzoic acid (0.04, CD-1; 0.03; Sencar); 2-[1-(4-carboxyphenyl)-1E-propeny-2-yl]-4,5,6,7-tetrahydro-4, 4-dimethylbenzothiophene (0.08, CD-1; 0.14, Sencar); 2-[1-(4-carboxyphenyl)-1E-propen-2-yl]3, 4-dihydro-4,4-dimethyl-2H-1-benzothiopyran (0.56, CD-1; 0.70, Sencar); 6-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-2-naphthalenecarboxylic acid (1.0, CD-1; 0.90, Sencar).     

Effect of retinoic acid pretreatment on 12-O-tetradecanoylphorbol-13-acetate-induced cell population kinetics and polyamine biosynthesis in hairless mouse epidermis

A single topical application of 17 nmol 12-O-tetradecanoyl-phorbol-13-acetate(TPA) to the skin of hairless mice induces characteristic transientalterations in the epidermal cell turnover and maturation (0–96h), associated in time with characteristic changes in the activitiesof L-ornithine carboxylyase (E.C. 4.1.1.17 [EC] ) (ODC) and S-adenosyl-L-methioninecarboxy-lyase (E.C. 4.1.1.50 [EC] ) (SAM-D) and in the accumulationof polyamines. The effects on these responses of local pretreatmentof the skin with retinoic acid 1 h prior to TPA were investigatedat selected time points. Retinoic acid inhibited the TPA-inducedODC activity and the ensuing accumulation of putrescine, butdid not alter the TPA-induced SAM-D activity or the molar ratioof spermidine/spermine. This pretreatment also decreased thenumber of dividing basal cells in the first TPA-induced synchronizedwave of proliferating cells. However, during the subsequentperiod of proliferation, the number of dividing cells in theretinoic acid pretreated group was comparatively increased.Hence, at four dose levels of retinoic acid (0.17, 1.70, 17.0and 170 nmol), which all inhibited the TPA-induced ODC effectively,there was no change in the total number of basal cells thatdivided during 16–48 h after TPA-application. The theoryis put forward that retinoic acid might exert its antitumorigeniceffect during tumor promotion with TPA by interfering with therate and/or quality of epidermal cell maturation, rather thanby inhibiting cell proliferation.     

Induction of mouse brain ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate is independent of TPA receptor concentration

Ornithine decarboxylase (ODC, E.C. 4.1.1.17) activity was measured in a 35,000 X g brain supernatant fraction, prepared 5 h after intracisternal injection of 12-O-tetradecanoylphorbol-13-acetate (TPA) into developing mouse brain. TPA-dependent induction of ODC activity was maximal on days 5 and 9 postnatally while on day 7, the developmental (endogenous) level of ODC in brain was high and, concurrently, the ability of TPA to induce ODC was reduced. Both TPA-dependent and developmental increases in mouse brain ODC activity were significantly reduced by intracisternal injection of retinoic acid (RA). The efficacy of TPA in elevating ODC activity at postnatal ages 1-220 days-old was independent of both soluble-and particulate-associated TPA receptor concentration. These observations suggest that although TPA receptor activation may be an obligatory event in ODC induction, TPA receptor activation and its concentration per se, are not sufficient determinants for ODC induction and tumorigenesis. Furthermore, the endogenous mechanism of ODC induction is distinct from that of the TPA-dependent increase in ODC enzyme activity.     

12-O-tetradecanoylphorbol-13-acetate-induced rapid loss of persistent 7,12-dimethylbenz[a]anthracene-DNA adducts in mouse epidermis and dermis

Twice-weekly application to mouse skin of 10 nmol of the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), beginning at 3 weeks after topical application of 1.2 mumol of 7,12-dimethylbenz[a]anthracene (DMBA), was found to cause a rapid loss of persistent DMBA-DNA adducts from both epidermal and dermal DNA. This effect is thought to reflect TPA-induced proliferation of quiescent initiated skin cells containing persistent adducts and may be causally related to the irreversibility of the initial phase of tumor promotion, which is known to require cell proliferation.     

Identification of epidermal cell subpopulations with increased ornithine decarboxylase activity following treatment of murine epidermis with 12-O-tetradecanoylphorbol-13-acetate

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.     

Inhibition of the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate in mouse skin by sphingosine sulfate

We investigated the effect of sphingosine sulfate on the inductionof ODC (ornithine decarboxylase) activity by TPA (12-O-tetradecanoylphorbol-13-acetate)in mouse skin. When applied topically to the shaved skin ofSENCAR mice at dosages of 10–40 µunol per animal,30 min before the superficial application of 8.5 nmol of TPA,sphingosine sulfate dramatically inhibited the induction ofODC activity by the tumor promoter. Significant inhibition ofTPA-induced ODC activity was observed at 4, 6 and 8 h afterTPA treatment in separate studies. The results indicate thatsphingosine sulfate is an effective inhibitor of ODC inductionby TPA in mouse skin.     

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.     

Effect of butyric acid on 12-O-tetradecanoylphorbol-13-acetate-(TPA) induced mouse skin ornithine decarboxylase (ODC)

Butyric acid was topically applied on the interscapular region of Swiss albino mice before and after application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in various doses and at different time intervals. Skin taken from the painted area, 4 h after TPA application, was subjected to ornithine decarboxylase (ODC) enzyme estimation. It was found that butyric acid inhibited the TPA-induced mouse skin ODC activity. The effect was dependent on the dose and duration of the butyric acid application.     

Inhibitory effects of glutathione level-raising agents and D-{alpha}-tocopherol on ornithine decarboxylase induction and mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

The constituent amino acids of reduced glutathione (GSH), GSHitself, and D--tocopherol inhibited 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced ornithine decarboxylase (ODC, L-omithine carboxy-lyase,EC 4.1.1.17 [EC] ) activity in mouse epidermis in vivo and in vitro.The inhibitory effects of cysteine (Cys), GSH and D--tocopherolon ODC induction were proportional to their abilities to decreasethe incidence of skin tumors in the initiation-promotion protocol.Moreover, the ability of the constituent amino acids of GSHand GSH to inhibit TPA-induced ODC activity correlated wellwith their ability to increase the ratio of GSW/oxidized glutathione(GSSG) in isolated epidermal cells. In vitro, various treatmentswith 1 mM GSH, 1 mM glutamic acid (Glu), 1 mM glycine (Gly),0.4 mM Cys and/or 0.2 mM cystine (CysCys) inhibited dramaticallythe sharp decline in the intracellular ratio of GSH/GSSG causedby 0.1 µM TPA. Since the inhibitory effects of Cys onboth the decrease in the ratio of GSH/GSSG and the inductionof ODC activity by TPA were greatly reduced by the inhibitorsof -glutamyl transpeptidase and -glutamylcysteine synthetase,it is suggested that some of the inhibitory effects of Glu,Cys and Gly on tumor promotion could result from their interferencewith the metabolism of the tripeptide GSH, a natural antioxidantwhich inhibits chemical carcinogenesis. The free radical scavengerD--tocopherol, which did not alter directly the intracellularratio of GSH/GSSG, also prevented completely the decrease inthe ratio of GSH/GSSG caused by TPA. These results, therefore,suggest that GSH level-raising agents and other antioxidantsmight inhibit by diverse means the effects of TPA on GSH metabolismand skin tumor promotion.