Epidermal Ornithine Decarboxylase Induction Research Articles

Relation of the induction of epidermal ornithine decarboxylase and hyperplasia to the different skin tumor-promotion susceptibilities of protein kinase C alpha, -delta and -epsilon transgenic mice.

To define the in vivo role of individual PKC isoforms in mouse skin carcinogenesis, we previously characterized FVB/n transgenic mice that over-expressed epitope-tagged PKC delta (T7-PKC delta) or PKC epsilon (T7-PKC epsilon) isoforms under the regulation of the human K14 promoter. In continuation of our prior PKC isoform specificity studies, we now report the generation of FVB/n transgenic mice with K14-regulated, epitope-tagged PKC alpha (T7-PKC alpha). T7-PKC alpha transgenic mice (line 115) express 8-fold more PKC alpha protein than wild-type mice. Using high-resolution immunogold cytochemistry, we determined that transgenic over-expression of T7-PKC alpha did not alter the subcellular localization of PKC alpha but that the density of PKC alpha staining increased. PKC alpha localized primarily to the cytoskeleton (tonofilaments, tight junctions) and cell membranes, with modest but definite nuclear labeling also identified. Also, PKC alpha over-expression did not alter the immunoreactive protein levels of other PKC isoforms (delta, epsilon, eta, zeta, mu) in the epidermis. Skin tumor-promotion susceptibility was compared among all 3 lines of T7-PKC transgenic mice (alpha, delta and epsilon). While T7-PKC alpha had no effect on skin tumor promotion by TPA, T7-PKC delta reduced papilloma burden by 76% compared to wild-type controls. T7-PKC epsilon further reduced papilloma burden to 93% compared to wild-type controls but still resulted in the development of squamous-cell carcinoma. To find potential mechanisms of PKC-associated differences in tumor promotion, the induction of known downstream effectors of tumor promotion, ornithine decarboxylase (ODC) activity and epidermal hyperplasia, was determined. Despite long-term papilloma inhibition in both PKC delta and PKC epsilon transgenic mice, the induction of ODC by TPA was not attenuated in PKC delta and epsilon mouse lines. Both PKC transgenic and wild-type mice exhibited sustained hyperplasia after repeated TPA treatments. However, TPA-induced epidermal hyperplasia in T7-PKC epsilon mice was significantly increased (52%) compared with T7-PKC alpha, T7-PKC delta and wild-type mice. TPA-induced ODC activity and the resultant accumulation of polyamines may play different roles (e.g., induction of apoptosis vs. proliferation) in the pathways leading to the induction of cancer in PKC alpha, PKC delta and PKC epsilon transgenic mice.

Inhibitory effect of Hibiscus protocatechuic acid on tumor promotion in mouse skin

Hibiscus protocatechuic acid (PCA), a phenolic acid isolated from Hibiscus sabdariffa L., was evaluated for its ability to inhibit the 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion in skin tumors of female CD-1 mice. Topical application of PCA (5, 10 or 20 μmol) 5 min prior to TPA (15 nmol) treatment twice weekly for 20 weeks to mice which were initiated with benzo[ a]pyrene (B[ a]P) inhibited the incidence of tumors in mice to 81.3, 62.5 and 56.3%, respectively, while all mice in the TPA-treated group developed tumors. The average number of tumors in mice pretreated with PCA was 2–4 and that of mice treated only with TPA was 6.6. The protection effects of PCA were also presented by its significant suppression on the TPA-induced hyperplasia in the skin and edema of mouse ears by 65 and 73% at doses of 10 and 20 μmol, respectively. When it was applied to the dorsal surface of CD-1 mice before TPA application, PCA (5, 10 or 20 μmol) inhibited the induction of epidermal ornithine decarboxylase (ODC) activity by 5 nmol TPA and myeloperoxidase (MPO) activity by 6.5 nmol TPA. The same doses of PCA also reduced the formation of hydrogen peroxide in the mouse skin to an inhibition of 61, 84 and 89%, respectively, when compared with that of the TPA-treated group. These results indicate that PCA possesses potential as a cancer chemopreventive agent against tumor promotion.

Short-Term Biomarkers of Tumor Promotion in Mouse Skin Treated with Petroleum Middle Distillates

Topical application of certain petroleum middle distillates (PMD) to mice produces skin tumors after long latency, and initiation/promotion protocols indicate that effect is associated with their tumor promoting activity. Since induction of sustained, potentiated epidermal hyperplasia is predictive of promoting activity, five compositionally distinct PMD [hydrodesulfurized kerosene (API 81-70); hydrodesulfurized PMD (API 81-10); odorless light petroleum hydrocarbons; severely hydrotreated light vacuum distillate (LVD); and lightly refined paraffinic oil (LRPO)] were assessed for their effects on epidermal hyperplasia PMD were administered (2 ×/week for 2 weeks) to skin of CD-1 mice Four quantitative biomarkers of epidermal hyperplasia were evaluated: epidermal thickness, number of nucleated epidermal cells per unit length of basement membrane, labeling (BrdUrd) index of epidermal cells, and induction of epidermal ornithine decarboxylase (ODC) activity. As positive controls, 12-O-tetradecanoylphorbol-13-acetate (TPA) and n-dodecane were utilized. PMD-induced skin irritation was evaluated visually and/or histopathologically. All five PMD produced dose-dependent, skin irritation and epidermal hyperplasia. On a weight basis the magnitude of the maximal PMD-induced effects was similar to that produced by n-dodecane, but >1000-fold less than that produced by TPA. Epidermal hyperplasia and subacute skin irritancy produced by the five PMD were similar. Of the four short-term markers of tumor promotion assessed, labeling index and epidermal ODC activity were predictive of the relative promoting activities of those PMD for which tumorigenicity bioassay data are available, i.e, API 81-07 > API 81-10 > LRPO. An apparent discrepancy to the predictability of epidermal ODC activity occurred with LRPO:toluene [1:1(v/v)]. This mixture is nontumorigenic, yet significantly induced epidermal ODC activity. This mixture, however, produced severe epidermal toxicity that precluded any meaningful analysis of short-term biomarkers in relationship to biological activity.

Inhibition of phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate-caused inflammatory responses in SENCAR mouse skin by black tea polyphenols.

Over the past 10 years many studies from several laboratories defined anticarcinogenic and anti-inflammatory effects of tea, a widely consumed beverage by the human population. Much of such work has been conducted with green tea or its polyphenolic constituents. Regarding black tea, studies have shown that its water extract affords protection against tumor promotion caused by chemical carcinogens or ultraviolet B radiation in murine skin carcinogenesis models. Several studies have shown that topical application of chemical tumor promoters to murine skin results in the induction of epidermal edema, hyperplasia and ornithine decarboxylase (ODC) and cyclo-oxygenase activities, and interleukin-1 alpha (IL-1alpha) and ODC mRNA expression. In this study, we assessed whether topical application of polyphenols isolated from black tea leaves (hereafter referred to as BTP) mainly consisting of theaflavine gallates and (-)-epigallocatechin-3-gallate, inhibits phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-caused induction of these markers of inflammatory responses in murine skin. Topical application of BTP (6 mg in 0.2 ml acetone/animal) 30 min prior to TPA application on to the mouse skin resulted in significant inhibition against TPA-caused induction of epidermal edema (40%, P < 0.01), hyperplasia (57%, P < 0.005), leukocytes infiltration (50%), and induction of epidermal ODC (57%) and pro-inflammatory cytokine IL-1alpha mRNA expression (69%). Pre-application of BTP to that of TPA also resulted in significant inhibition of TPA-caused induction of epidermal ODC (23-73%, P < 0.005-0.0001), and cyclo-oxygenase, in terms of prostaglandins metabolites formation (38-65%, P < 0.01-0.0005), enzyme activities. Our data indicate that the inhibition of TPA-caused changes in these markers of inflammatory responses in murine skin by BTP may be one of the possible mechanisms of chemopreventive effects associated with black tea against tumorigenesis. The results of this study suggest that black tea, specifically polyphenols present therein, may be useful against cutaneous inflammatory responses in human population.

Evaluation of a topical iron chelator in animals and in human beings: Short-term photoprotection by 2-furildioxime

In previous work we found that iron is a factor in skin photodamage, apparently by way of its participation in oxygen radical production. Here we report topical photoprotection with the iron chelator 2-furildioxime (FDO). Our purpose was to determine the level of photoprotection provided by FDO in both animal and human testing. Mice, guinea pigs, and human beings were treated topically with 5% simple vehicle solutions of FDO versus vehicle. The skin was then exposed to doses of simulated solar UV radiation greater than the minimal erythema dose. Mouse skin was harvested for analysis of ornithine decarboxylase (ODC); guinea pig skin was graded for erythema; and human skin was graded for erythema and biopsy specimens taken for analysis of ODC and for histologic evaluation. In animal testing with simulated solar UV radiation, topical 5% FDO provided 90% protection against induction of ODC in the hairless mouse and sun protection factor 3.5 against erythema in the guinea pig. In a double-blind, paired-comparison, vehicle-controlled clinical test, 5% FDO applied topically before a single dose of simulated solar radiation at three times minimal erythema dose prevented UV-induced erythema, sunburn cell formation, epidermal thickening, infiltration of inflammatory cells, and induction of epidermal ODC. The high level of protection provided by FDO indicates that metal chelation is a significant approach to providing photoprotection.

PROTECTION AGAINST ULTRAVIOLET B RADIATION‐INDUCED EFFECTS IN THE SKIN OF SKH‐1 HAIRLESS MICE BY A POLYPHENOLIC FRACTION ISOLATED FROM GREEN TEA

In prior studies we and others have shown that oral feeding of a polyphenolic fraction isolated from green tea (GTP) or water extract of green tea affords protection against ultraviolet B (UVB) radiation-induced carcinogenesis in SKH-1 hairless mice (Wang et al., Carcinogenesis 12, 1527-1530, 1991). It is known that exposure of murine skin to UVB radiation results in cutaneous edema, depletion of the antioxidant-defense system and induction of ornithine decarboxylase (ODC) and cyclooxygenase activities. In this study we assessed the protective effect of GTP on these UVB radiation-caused changes in murine skin. Oral feeding of 0.2% GTP (wt/vol) as the sole source of drinking water for 30 days to SKH-1 hairless mice followed by irradiation with UVB (900 mJ/cm2) resulted in significant protection against UVB radiation-caused cutaneous edema (P < 0.0005) and depletion of the antioxidant-defense system in epidermis (P < 0.01-0.02). The oral feeding of GTP also resulted in significant protection against UVB radiation-caused induction of epidermal ODC (P < 0.005-0.01) and cyclooxygenase activities (P < 0.0001) in a time-dependent manner. Our data indicate that the inhibition of UVB radiation-caused changes in these markers of tumor promotion in murine skin by GTP may be one of the possible mechanisms of chemopreventive effects associated with green tea against UVB-induced tumorigenesis. The results of this study suggest that green tea, specifically polyphenols present therein, may be useful against inflammatory responses associated with the exposure of skin to solar radiation.

The induction of epidermal ornithine decarboxylase following UV-B irradiation is inhibited by estriol.

The induction of epidermal ornithine decarboxylase (ODC) can be partially blocked by corticosteroids, retinoic acid or active vitamin D3. The influence of the other members of this so-called "steroid hormone receptor superfamily", namely the sex-steroids and thyroid hormone, is unknown in epidermis, but they enhance ODC induction in certain other tissues. Here we investigated whether topical estriol leads to a spontaneous and/or enhanced epidermal ODC induction 8 h after UV-B irradiation of 6 postmenopausal women. Contrary to expectation, estriol did not stimulate induction but reduced induction by 44%. This observation raises the possibility that all members of the steroid hormone receptor superfamily may share a common AP-1 binding site.

Inhibition of chrysarobin skin tumor promotion in SENCAR mice by antioxidants.

The present study was designed to further investigate the role of reactive oxygen species in the mechanism of action of anthrone tumor promoters. To accomplish this, the effects of several antioxidants on the induction of epidermal ornithine decarboxylase (ODC) activity, epidermal hyperplasia, skin edema, and skin tumor promotion by chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) were tested. Ascorbyl palmitate (AP), given 5 min prior to the promoter at 1 and 4 mumol doses, effectively inhibited the induction of ODC activity (28% and 59%, respectively) by 220 nmol of chrysarobin. Using a similar protocol, alpha-tocopherol acetate (alpha-TA) at 10 and 40 mumol doses also effectively inhibited the induction of ODC activity (36% and 70%, respectively) by 220 nmol of chrysarobin. In contrast, butylated hydroxyanisole (BHA) at doses up to 56 mumol per mouse was ineffective at inhibiting the induction of ODC by chrysarobin. AP at the 4 mumol dose significantly inhibited the induction of edema by chrysarobin by 24% and the induction of epidermal hyperplasia by 23%. alpha-TA at the 40 mumol dose also significantly inhibited chrysarobin-induced edema by 22% and epidermal hyperplasia by 17%. Skin tumor promotion in mice initiated with 25 nmol of 7,12-dimethylbenz[a]anthracene and promoted with once-weekly treatments of 220 nmol chrysarobin was markedly inhibited by treating mice with either AP or alpha-TA 5 min prior to promoter treatment. AP at 1 and 4 mumol doses significantly reduced the number of papillomas per mouse, by 48% and 44%, respectively. alpha-TA at 10 and 40 mumol doses also significantly reduced the number of papillomas per mouse, by 33% and 59%, respectively. In two separate tumor experiments, BHA at 2.8 and 5.6 mumol failed to inhibit chrysarobin tumor promotion. The current results provide further support for a role of reactive oxygen species in the tumor promoting activity of anthrones. In addition, the data indicate that the phenolic antioxidant BHA is an ineffective inhibitor of anthrone tumor promotion.

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

The effect of FK506, a potent immunosuppressive agent, on 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13- acetate (TPA)-promoted skin papilloma formation was examined in CD-1 mice. A topical application of FK506 to mouse skin 15 min before each TPA treatment resulted in a dose-related inhibition of tumor formation. FK506 (1 mumol) almost completely inhibited tumor formation. This inhibitory effect of FK506 was not due to any damage inflicted on the initiated cells but due to its anti-tumor-promoting action. A topical application of FK506 also inhibited epidermal ornithine decarboxylase induction and skin inflammation caused by TPA in a dose-related manner. Significant inhibition by FK506 of TPA-induced endogenous protein phosphorylation in intact epidermal cells was not detected. These results indicate that FK506 inhibits TPA-induced tumor promotion at a step distal to the endogenous protein phosphorylation by TPA.

The Induction of Ornithine Decarboxylase in Human Epidermis Is Independent of Lipoxygenase and Cyclo‐oxygenase Pathways

In vivo studies in rodents suggest that prostaglandins and/or leukotrienes are involved in the epidermal induction of ornithine decarboxylase (ODC). Recently, we have shown that, in human epidermis, prostaglandins are not involved in this process. Here we report the role of leukotrienes in epidermal ODC induction in human skin. Topical flufenamic acid (Dignodolin), vehicle, or nothing was applied under plastic occlusion to three sites on the backs of healthy volunteers. This was followed 1 h later by Sellotape stripping. After renewed application and occlusion for 8 h, biopsies were carried out for the estimation of ODC levels. There were no significant differences in the levels of ODC between the flufenamic acid treated and control sites. To confirm this finding, test sites were irradiated with 3 MED of UVB. This was immediately followed by the application of flufenamic acid, vehicle, or nothing to the three irradiated sites. After 8 h, biopsies were taken, and the levels of ODC were again similar in the flufenamic acid- and the vehicle-treated sites. The data indicate that, following Sellotape stripping or UVB irradiation, neither lipoxygenase not cyclooxygenase products contribute to the in vivo induction of ODC in human epidermis.

Staurosporine, a potent protein kinase C inhibitor, augments phorbol ester-caused ornithine decarboxylase induction in mouse epidermis.

A single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin caused an induction of epidermal ornithine decarboxylase (ODC) activity. When mice were topically pretreated with staurosporine, a most potent protein kinase C inhibitor, 6-84 h prior to TPA treatment, TPA-caused ODC induction was markedly enhanced. The enhancement of TPA-caused ODC induction by staurosporine was most pronounced when the time interval between staurosporine and TPA treatment was 36 h. Staurosporine elicited this enhancing effect in a dose-related manner. Staurosporine by itself also induced epidermal ODC activity. But the activity induced was very slight and would not directly contribute to the enhancing effect of this compound. Although staurosporine markedly augmented TPA-caused ODC induction, staurosporine-caused ODC induction was not augmented by this compound. Other protein kinase C inhibitors, such as 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, sphingosine and palmitoylcarnitine did not mimic the enhancing effect of staurosporine. These results indicate that the enhancement of ODC induction by staurosporine is specific for the induction caused by TPA and that this enhancing effect is not related to the protein kinase C inhibitory action of staurosporine. TPA-caused epidermal ODC induction was inhibited by indomethacin, and this inhibition was reversed by prostaglandin E2 (PGE2). Staurosporine-caused ODC induction was also inhibited by indomethacin but the inhibition was not reversed by PGE2, indicating that the mechanism of staurosporine-caused ODC induction is different from that of TPA.

(-)-Epigallocatechin-3-gallate in Camellia sinensis leaves from Himalayan region of Sikkim: inhibitory effects against biochemical events and tumor initiation in Sencar mouse skin.

Recently, we and others showed that the components of green tea may be useful cancer chemopreventive agents. It has been suggested that (-)-epigallocatechin-3-gallate (EGCG), the major constituent in green tea, may possess antitumor-promoting and/or anticarcinogenic effects in rodent tumor bioassay systems. During the chemical analysis of various green tea products, we found a traditionally preserved preparation of green tea used by tribes in the Himalayan region of Sikkim, India that was rich in EGCG. EGCG was isolated from this tea product, and its inhibitory effects were evaluated against the binding of topically applied 3H-labeled polycyclic aromatic hydrocarbons (PAHs) to epidermal DNA and 12-O-tetradecanoylphorbol-13-acetate (TPA) caused induction of epidermal ornithine decarboxylase (ODC) activity in Sencar mice, the short-term markers of tumor initiation and tumor promotion, respectively. Preapplication of EGCG resulted in significant inhibition (p less than 0.05) in the binding of [3H]PAH to epidermal DNA. Similarly, the topical application of EGCG resulted in significant inhibition (p less than 0.005) in TPA-caused induction of epidermal ODC activity. In further studies, we assessed the anti-skin tumor-initiating effect of EGCG in Sencar mice in an initiation-promotion protocol. The application of EGCG before challenge with 7,12-dimethylbenz[a]anthracene as tumor initiator resulted in significant reduction both in percentage of mice with tumors and number of tumors per mouse compared with a non-EGCG-pretreated group of animals. The results of the present study suggest that the green tea preparation from Sikkim may be a good source for the isolation of EGCG and that this compound may have significant potential as a cancer chemopreventive agent.

Induction of ornithine decarboxylase in specific subpopulations of murine epidermal cells following multiple exposures to 12-O-tetradecanoylphorbol-13-acetate, mezerein and ethyl phenylpropriolate

Single applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein or ethyl phenylpropriolate (EPP) to mouse skin at appropriate doses cause similar degrees of hyperplasia and comparable levels of induction of epidermal ornithine decarboxylase (ODC) activity. Multiple (n = 5) treatments with these agents, in contrast, resulted in large differences in induced ODC activity (TPA much greater than mezerein greater than EPP) with no differences in the degree of hyperplasia or [3H]thymidine pulse-labeling among the multiple treatment groups. To attempt to explain the cellular basis for the greater ODC-inducing ability of TPA relative to mezerein and EPP in chronic exposure protocols, immunocytochemical and flow cytometric analyses were performed. Immunocytochemistry using an ODC-specific polyclonal antibody revealed substantially different pattern of ODC-positive cells in chronically exposed epidermis than observed with single exposures. TPA treatment resulted in very pronounced immunostaining of the perifollicular cells, with little evidence of specific staining in the interfollicular epidermis mezerein treatment yielded staining in both interfollicular and some perifollicular areas, while EPP treatment produced the least amount of specifically stained cells, all of which were in the interfollicular epidermis. Flow cytometric analysis of keratinocytes isolated from chronically treated skin identified three distinct subpopulations that bound varying amounts of ODC antibody. Chronic treatment of CD-1 murine epidermis with TPA appeared to cause the expansion of an intermediate sized cell subpopulation that was not apparent with EPP or mezerein. Our results suggest that chronic treatment of murine epidermis with the potent complete tumor promoter TPA leads to the selective expansion of a keratinocyte subpopulation that is hyperinducible for ODC and may be identical to the cells in the perifollicular region previously identified. These observations also suggest that the weaker tumor promoters mezerein and EPP are less capable of causing expansion of this specific subpopulation, which may be an important target cell population for neoplastic transformation in mouse epidermis.

Induction of epidermal ornithine decarboxylase activity in mouse skin exposed to biogenic silica fibers.

The present study demonstrates that biogenic silica fibers (BSF), previously shown to promote skin tumors in mice and more recently to promote the induction of mesotheliomas when injected into the pleural cavity of rats, rapidly induces epidermal ornithine decarboxylase (ODC) activity in SENCAR mice following topical application. The time course for induction of epidermal ODC by BSF was very similar to that observed following topical treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). Maximal ODC activity was observed 4-6 h following treatment with BSF. Cycloheximide (70 mg/kg i.p.) partially inhibited (61%) the induction of ODC by BSF at 5 h. In addition, retinoic acid (RA, 5 micrograms per mouse given 30 min before BSF) effectively inhibited BSF-induced ODC by 68%, while indomethacin (100 micrograms per mouse 2 h before BSF) had little or no effect. Copper(II) bis(diisopropylsalicylate) (2 mumol 30 min before BSF), an effective inhibitor of TPA-induced ODC activity and tumor promotion, also had little or no effect on BSF-induced ODC. The work described in this paper suggests that BSF induces epidermal ODC by a very specific mechanism that exhibits both similarities and differences with that of the phorbol ester, TPA. Nevertheless, this response strongly supports the conclusion that BSF is an effective tumor promoter in mouse skin and that ODC induction is an integral part of the mechanism of action of this environmental promoter.

O-321 - Anti-tumor-promoting action of FK506, a potent immunosuppressive agent

The effect of FK506, a potent immunosuppressive agent, on 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13- acetate (TPA)-promoted skin papilloma formation was examined in CD-1 mice. A topical application of FK506 to mouse skin 15 min before each TPA treatment resulted in a dose-related inhibition of tumor formation. FK506 (1 mumol) almost completely inhibited tumor formation. This inhibitory effect of FK506 was not due to any damage inflicted on the initiated cells but due to its anti-tumor-promoting action. A topical application of FK506 also inhibited epidermal ornithine decarboxylase induction and skin inflammation caused by TPA in a dose-related manner. Significant inhibition by FK506 of TPA-induced endogenous protein phosphorylation in intact epidermal cells was not detected. These results indicate that FK506 inhibits TPA-induced tumor promotion at a step distal to the endogenous protein phosphorylation by TPA.

Effects of N-acyl dehydroalanines on phorbol ester-elicited tumor development and other events in mouse skin

The free radical scavengers N-acyl dehydroalanines (AD compounds) were examined for their effect on several 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited events in mouse skin. The induction of oxidant production by TPA in isolated mouse epidermal cells was reduced by ∼ 70% by 1 mM paramethoxyphenyl-acetyl dehydroalanine (AD5) and 80% by 1 mM parasulfoxyphenyl-acetyl dehydroalanine (AD19). These AD compounds also completely suppressed the TPA-dependent stimulation of prostaglandin E 2 synthesis in primary cultures of epidermal cells. Single and multiple topical applications on the dorsal skin of SENCAR mice of either AD5 or AD 19 inhibited TPA-induced epidermal hyperplasia but failed to inhibit epidermal ornithine decarboxylase induction. When used with TPA on initiated mice, AD19 did not inhibit papilloma formation; however, after 40 weeks of promotion, the carcinoma incidence was reduced by 50% in the AD19 group. These results suggest that reactive oxygens may be more important to the conversion of benign to malignant tumors than in the initial development of the benign tumors.

The induction of epidermal ornithine decarboxylase following tape stripping is inhibited by a topical vitamin D3 analogue (MC903)

The efficacy of MC903, a vitamin D3 analogue, in reducing hyperproliferation as determined by levels of ornithine decarboxylase (ODC) was investigated in a double-blind study of 15 patients with chronic plaque psoriasis. The lesions of psoriasis were treated for 8 weeks with MC903 in one of two different cream bases or with a placebo cream. Biopsies were taken before and after treatment. In addition an uninvolved area of skin was treated during the last 3 weeks and this as well as control areas were then sellotape stripped and biopsied after 8 h. Clinical improvement was seen in eight out of 11 patients treated with MC903 but there was no reduction in the level of ODC in psoriatic lesions after 8 weeks of treatment. The levels of ODC in the tape-stripped uninvolved skin after 3 weeks of treatment with MC903 averaged 22.5 +/- 4.2 pmol/min/mg protein as compared to 58.6 +/- 12.6 pmol/min/mg protein (P = 0.004). The trauma-induced induction of ODC activity was markedly inhibited by the application of MC903.

The potent anti-tumor-promoting agent isoliquiritigenin

A topical application of a chalcone derivative, 4,2',4'-trihydroxychalcone (isoliquiritigenin) inhibited epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. inflammation, caused by a topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in CD-1 mice. In addition, isoliquiritigenin potently inhibited 7,12-dimethylbenz[alpha]anthracene (DMBA)-initiated and TPA-promoted skin papilloma formation. This inhibitory effect of isoliquiritigenin was not due to any damage inflicted on the initiated cells but due to its anti-tumor-promoting action. Isoliquiritigenin also inhibited epidermal ODC induction and skin tumor promotion caused by 7-bromomethylbenz[alpha]anthracene (BrMBA), a non-TPA type of tumor-promoting agent, in DMBA-initiated mice. Isoliquiritigenin inhibits neither 12-lipoxygenase nor cyclooxygenase in epidermal subcellular fractions. This compound, however, inhibited TPA-stimulated prostaglandin E2 (PGE2) production in intact epidermal cells. ODC induction caused by TPA was inhibited by a topical application of cyclooxygenase inhibitor, indomethacin. Inhibition of ODC induction by indomethacin was counteracted by a topical application of PGE2, while inhibition caused by isoliquiritigenin was not overcome by PGE2. The results suggest that a mechanism other than the inhibition of PGE2 production is involved in the anti-tumor-promoting action of isoliquiritigenin. Isoliquiritigenin failed to inhibit phospholipase A2 activity of platelet sonicates, but inhibited platelet 12-lipoxygenase and 5-lipoxygenase in polymorphonuclear leukocytes. Therefore, it might be possible that isoliquiritigenin exerts its anti-tumor-promoting action through the lipoxygenase inhibition by acting on cells other than the target epidermal cells. Our present results, in combination with our previous data, demonstrate that some chalcone derivatives and flavonoids which show a potent lipoxygenase inhibitory action act on a common step in the skin tumor promotion caused by two different types of tumor-promoting agents, i.e. TPA and BrMBA, and suggest that these compounds show promise as drugs to prevent tumor promotion.

Anti-tumor promoting action of phthalic acid mono-n-butyl ester cupric salt, a biomimetic superoxide dismutase

Skin tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by a concurrent and topical application of phthalic acid mono-n-butyl ester cupric salt (PAMBCu) in CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene. PAMBCu inhibited TPA-caused epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. skin inflammation. However, neither PAMBCu nor superoxide dismutase (SOD) inhibited TPA-caused ODC induction in primary cultured mouse epidermal cells. 7-Bromomethylbenz[a]anthracene (BrMBA) is known to be a non-TPA type of tumor promoting agent. Epidermal ODC induction and inflammation caused by BrMBA were not inhibited by a concurrent application of PAMBCu. When mice were topically treated twice with PAMBCu, i.e. concurrently with and 7 h after BrMBA treatment, BrMBA-caused ODC induction was markedly suppressed. The same dose regimen of PAMBCu, however, failed to inhibit tumor promotion and inflammation caused by BrMBA. PAMBCu showed SOD-mimetic activity in superoxide generating systems, i.e. xanthine-xanthine oxidase reaction and TPA-stimulated polymorphonuclear leukocytes (PMN). Mono-n-butyl phthalate, which lacks SOD-mimetic activity, failed to inhibit TPA-caused ODC induction and skin inflammation. Therefore, inhibition by PAMBCu of TPA-caused tumor promotion, epidermal ODC induction and inflammation may be attributable to its SOD-mimetic activity. The results also support the contention that a superoxide anion of non-epidermal cell origin, such as PMN and macrophages, plays a role (probably some enhancing role) in in vivo ODC induction and tumor promotion caused by TPA. Failure of PAMBCu to inhibit BrMBA-caused tumor promotion suggests that superoxide anion generation is not involved in the tumor promoting action of this agent and that the anti-tumor promoting action of PAMBCu is dependent on the nature of the tumor promoting agents.

Epidermal ornithine decarboxylase induction and mouse skin tumor promotion by quinones

The generation of reactive oxygen species and the subsequent development of a pro-oxidant state, such as occurs during the redox cycling of quinones, has been suggested to play a role in the tumor promotion. Moreover, we have recently shown that the relative tumor promoting activity of a series of structurally related anthrones correlated with their ability to undergo base-catalyzed oxidation. We therefore analyzed dose-response relationships for skin tumor promoting activity and the ability to induce epidermal ornithine decarboxylase (ODC) with a series of structurally related quinones. Single topical applications of 1,4-naphthoquinone and its 5-hydroxy analog (juglone) produced dose-dependent increases in epidermal ODC activity in the dose range 880-3520 nmol/mouse. These two quinones also promoted papilloma formation in female SENCAR mice initiated with 25 nmol 7,12-dimethylbenz[a]anthracene at doses capable of inducing epidermal ODC. The tumor promoting response with juglone (1760 nmol) was dependent upon the frequency of application, with the highest tumor response obtained with a three times per week application regimen. In contrast, neither 1,8-dihydroxy-9,10-anthraquinone nor 1,4-benzoquinone, at doses up to 1760 nmol/mouse, had any effect on epidermal ODC, nor did they possess tumor promoting activity after 31 weeks of promotion. Interestingly, 3-methyl-1,4-naphthoquinone (meadione), a relatively good redox cycling quinone, at a dose of 3520 nmol had only very weak ODC inducing activity and after 31 weeks of promotion (1760 nmol) did not produce a significant papilloma response in SENCAR mice. Thus, there was a good correlation between the ability of structurally related quinones to induce epidermal ODC and their ability to behave as tumor promoters. In contrast, a relationship between quinone redox cycling and tumor promotion was not readily apparent. Finally, under optimal promoting conditions, juglone was very effective at supporting the conversion of papillomas to carcinomas (carcinoma/papilloma ratio of 0.35). In addition, histological examination of all tumors produced during promotion with juglone revealed the presence of both kerathoacanthomas and sebaceous squamous cell carcinomas. These latter tumors, not found in the anthrone group, may be indicative of a potentially unique site and/or mechanism of action for this class of compounds.