TPA-induced Ornithine Decarboxylase Activity Research Articles

Geraniol inhibits murine skin tumorigenesis by modulating COX‐2 expression, Ras‐ERK1/2 signaling pathway and apoptosis

Geraniol (GOH), a naturally occurring monoterpene, has been shown to have antiproliferative, cell cycle arrest and apoptosis-inducing effects, and represents a promising cancer chemopreventive agent. In the present study, we investigated the chemopreventive potential of GOH (50 and 100 mg kg(-1) body weight) against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated skin tumorigenesis in Swiss albino mice. The topical treatment of GOH, 30 min prior to TPA (2 µg per 200 µl of acetone) treatment significantly inhibited TPA-induced skin edema, hyperplasia, COX-2 induction and oxidative stress response. The GOH treatment also resulted in reduction of TPA-induced ornithine decarboxylase activity and [(3) H] thymidine incorporation by 53% (P < 0.001) and 41% (P < 0.001), respectively. We found that GOH treatment significantly inhibited the tumor incidence and number of tumors (P < 0.001) and extended the latency period from 4 weeks in DMBA/TPA treatment group to 10 weeks in GOH-pretreated mice. Furthermore, we observed that GOH treatment significantly suppressed the Ras/Raf/ERK1/2 signaling pathway in skin tumor. Consistently, GOH-treated skin tumors showed reduced expression of Bcl-2 and increased expression of Bax in these lesions. Thus, it was concluded that GOH inhibits DMBA/TPA-mediated skin tumorigenesis by attenuating the Ras proliferation pathway and inducing pro-apoptotic state via inhibition of oxidative stress response and inflammation.

Effects of psoralen from Psoralea corylifolia on quinone reductase, ornithine decarboxylase, and JB6 cells transformation promotion

The cancer chemopreventive effect of psoralen isolated from the seeds of Psoralea corylifolia was investigated in the induction of quinone reductase (QR) activity, intracellular detoxification enzyme, inhibition of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, a key regulatory enzyme for polyamine metabolism, and tumor promotion in mouse epidermal JB6 cells, sensitive to tumor promoters (clone 415a P+ cells), which are related to suppress multistage carcinogenesis including initiation and promotion. Psoralen was isolated and identified from the ethyl acetate-soluble fraction of the methanolic extract from the seeds. Psoralen was active in induction of QR activity, the concentration of psoralen required to induce 1.5 fold QR activity was 14.8 μg/mL. Also, this pure compound inhibited TPA-induced ODC activity by 50% (designated IC(50)) at the concentration 15.6 μg/mL and exhibited inhibition of TPA-induced tumor promotion in mouse epidermal JB6 cells with an IC(50) value of 17.1 μg/mL. Therefore, it is extrapolated that psoralen has the potential capable of inhibiting the initiation and/or promotion stage of carcinogenesis by induction of QR activity, inhibition of TPA-induced ODC activity and mouse epidermal JB6 cells tumor promotion.

Chemopreventive effects of various concentrations of α-santalol on skin cancer development in CD-1 mice

Previous studies from this laboratory have indicated that alpha-santalol (5%) provides chemopreventive effects in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin cancer in CD-1 and SENCAR mice. Skin cancer development is associated with increased ornithine decarboxylase (ODC) activity, DNA synthesis and rapid proliferation of epidermal cells. The purpose of this investigation was to determine the effects of various concentrations (1.25% and 2.5%) of alpha-santalol on DMBA-initiated and TPA-promoted skin cancer development, TPA-induced ODC activity, and DNA synthesis in CD-1 mice. alpha-Santalol treatment at both concentrations (1.25% and 2.5%) prevented the skin cancer development. alpha-Santalol treatment (1.25% and 2.5%) resulted in a significant decrease in the TPA-induced ODC activity and incorporation of [3H]thymidine in DNA in the epidermis of CD-1 mice. There was no significant difference in the effects of 1.25% and 2.5% alpha-santalol on tumour incidence, multiplicity, epidermal TPA-induced ODC activity, or DNA synthesis in CD-1 mice.

Sulforaphane prevents mouse skin tumorigenesis during the stage of promotion

Sulforaphane (SF), a natural product from broccoli, is known to enhance detoxification of carcinogens and block initiation of chemically-induced carcinogenesis in animal models. Cell culture and xenograft studies suggest additional roles for SF, inhibiting growth of tumors, arresting the cell cycle and enhancing apoptosis. As currently reported, topical SF (1, 5 or 10 μmol/mouse) significantly inhibited 7,12-dimethylbenz( a)anthracene/12- O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse skin tumorigenesis, using either an anti-promotion protocol (SF from 1 week after carcinogen until the end of the study) or a combined anti-initiation, anti-promotion protocol (SF 7 days prior to carcinogen until the end of the study). Surprisingly, no significant effect was observed in an anti-initiation protocol (SF from 7 days prior to 7 days after carcinogen). Separately, SF inhibited TPA-induced ornithine decarboxylase activity in mouse skin, an obligate step in TPA-induced promotion of carcinogenesis. These data link this molecular mechanism to SF-dependent inhibition of the promotion of tumorigenesis.

Biologic evaluation of curcumin and structural derivatives in cancer chemoprevention model systems

Curcumin is a natural product widely used as a spice in food. It has been shown to inhibit cyclooxygenase (COX)-1 and -2 and to suppress lipopolysaccharide-induced COX-2 and iNOS gene expression. In the present study, curcumin and 22 of its derivatives were evaluated for their chemopreventive potential. Based on COX-2 inhibition, curcumin (IC 50 = 15.9 μM), 1,7- bis(3-fluoro-4-hydroxyphenyl)-1,6-heptadiene-3,5-dione ( 19) (IC 50 = 23.7 μM) and 2,6- bis(3-fluoro-4-hydroxybenzylidene)cyclohexanone ( 23) (IC 50 = 5.5 μM) were found to be most potent. Tricyclic derivatives 2,6- bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone ( 10), 2,6- bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclohexanone ( 13) and 2,5- bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclopentanone ( 21) inhibited LPS-induced COX-2 and iNOS gene expression in murine macrophages with potency equal to curcumin. RT-PCR experiments demonstrated suppression of COX-2 and iNOS gene expression occurred at the transcriptional level. The most active compounds in the macrophage assays, 13 and 23, were also the most cytotoxic, however. Topical application of curcumin, 10, 13, 21, and 6, a methoxy derivative of curcumin, showed strong inhibition of 12- O-tetradecanoyl-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in mouse skin. These data suggest that structural elements responsible for COX-1 and COX-2 inhibition do not correlate well with those responsible for inhibiting COX-2 and iNOS gene expression, but elements capable of inhibiting COX-2 and iNOS gene expression also contribute to inhibition of TPA-induced ODC activity. The most potent compounds in these assays, 10, 13 and 21, as well as curcumin, were further evaluated for inhibition of 7,12-dimethylbenz( a)anthracene (DMBA)-induced preneoplastic lesion formation in a mouse mammary organ culture model, and dose-dependent responses were observed. Most potent effects were at concentrations between 1 and 5 μM for 10, 13 and 21, and at 10 μM for curcumin. These data demonstrate the substitution pattern on the aromatic moiety is especially crucial for activity.

In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols

Several herbal teas contain bioactive compounds that have been associated with a lower risk of chronic diseases including cancer. The aim of this study was to evaluate the chemopreventive activity of tea aqueous extracts and selected constituent pure polyphenols using a battery of in vitro marker systems relevant for the prevention of cancer. The effects of (−) epigallocatechin gallate (EGCG), quercetin (Q), gallic acid (GA), green tea (GT, Camellia sinensis), ardisia tea (AT, Ardisia compressa) and mate tea (MT, Ilex paraguariensis) extracts were tested. Cytotoxicity, TPA-induced ornithine decarboxylase (ODC) and quinone reductase (QR) activities were evaluated in vitro using HepG2 cells. The topoisomerase inhibitory activity was also tested, using the Saccharomyces cerevisiae yeast system. Results suggest that MT, AT and GT are cytotoxic to the HepG2 cells, with MT demonstrating dominant cytotoxicity. EGCG showed greater cytotoxicity than Q and GA against HepG2 cells. The greatest inhibition (82%) of TPA-induced ODC activity was shown by Q, with 25 μM (IC 50=11.90 μM). Topoisomerase II, but not topoisomerase I, was the cellular target of MT, AT, EGCG, Q and GA, which acted mainly as true catalytic inhibitors. The cytotoxic activity and the inhibition of topoisomerase II may contribute to the overall chemopreventive activity of AT and MT extracts. Ardisia and mate teas may thus share a public health potential as chemopreventive agents.

Salix caprea inhibits skin carcinogenesis in murine skin: inhibition of oxidative stress, ornithine decarboxylase activity and DNA synthesis

Chemoprevention of free radical-mediated diseases including cancer by natural products is an emerging discipline due to its wider applicability and acceptance. The present study deals with the chemopreventive effect of Salix caprea against phorbol ester-induced oxidative stress and tumor promotion in murine skin. In the present investigation, it was observed that a single application of 12- O-tetradecanoyl-13-phorbol acetate (TPA) (20 nmol/0.2 ml acetone/animal) caused a significant ( P<0.05) depletion of cutaneous antioxidants viz., glutathione, glutathione reductase, glutathione peroxidase, catalase and phase II drug metabolizing enzymes viz., glutathione- S-transferase, quinone reductase. An increase in the hydrogen peroxide generation and protein oxidation (measured in terms of protein carbonyl content) was also observed with a single application of TPA. However, the pretreatment of animals with different doses of Salix caprea (0.5, 1.0 and 1.5 mg/kg/0.2 ml acetone) caused a significant recovery in the TPA-mediated depletion in antioxidant levels. The pretreatment of animals with Salix caprea was observed to inhibit the TPA-mediated depletion in phase II enzymes. It was also observed that Salix caprea reversed the TPA-mediated depletion in the activity of phase II enzymes that is an important characteristic of cancer chemopreventive agents. Phorbol esters are known to induce the tumor promotion by increasing rate of DNA synthesis, ornithine decarboxylase activity (ODC), and xanthine oxidase activity. In the present investigation, it was observed that the pretreatment of animals with Salix caprea caused a significant ( P<0.05) depletion in the TPA-induced DNA synthesis, ODC and xanthine oxidase activity in mice skin. Salix caprea significantly reduced the tumor promotion in mice skin when tested in two-stage chemical carcinogenesis model. It was observed to inhibit significantly ( P<0.05) the 7,12-dimethyl benz[ a] anthracene (DMBA)-initiated phorbol ester promoted skin carcinogenesis. It was concluded from the results that Salix caprea is an effective antioxidant and chemopreventive agent against phorbol ester-induced tumor promotion.

Chemopreventive effects of pomegranate seed oil on skin tumor development in CD1 mice.

Pomegranate seed oil was investigated for possible skin cancer chemopreventive efficacy in mice. In the main experiment, two groups consisting each of 30, 4-5-week-old, female CD(1) mice were used. Both groups had skin cancer initiated with an initial topical exposure of 7,12-dimethylbenzanthracene and with biweekly promotion using 12-O-tetradecanoylphorbol 13-acetate (TPA). The experimental group was pretreated with 5% pomegranate seed oil prior to each TPA application. Tumor incidence, the number of mice containing at least one tumor, was 100% and 93%, and multiplicity, the average number of tumors per mouse, was 20.8 and 16.3 per mouse after 20 weeks of promotion in the control and pomegranate seed oil-treated groups, respectively (P <.05). In a second experiment, two groups each consisting of three CD(1) mice were used to assess the effect of pomegranate seed oil on TPA-stimulated ornithine decarboxylase (ODC) activity, an important event in skin cancer promotion. Each group received a single topical application of TPA, with the experimental group receiving a topical treatment 1 h prior with 5% pomegranate seed oil. The mice were killed 5 h later, and ODC activity was assessed by radiometric method. The experimental group showed a 17% reduction in ODC activity. Pomegranate seed oil (5%) significantly decreased (P <.05) tumor incidence, multiplicity, and TPA-induced ODC activity. Overall, the results highlight the potential of pomegranate seed oil as a safe and effective chemopreventive agent against skin cancer.

Inhibition of 12-O-Tetradecanoylphorbol-13-Acetate-Induced Inflammatory Skin Edema and Ornithine Decarboxylase Activity by Theaflavin-3,3?-Digallate in Mouse

Among black tea polyphenols, theaflavins were generally considered to be the most effective in cancer chemoprevention. In this study, we examined the inhibitory effects of black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate and theaflavin-3′-gallate, theaflavin-3,3′-digallate (TF-3), and the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema and ornithine decarboxylase (ODC) activity. Topical application of these polyphenols onto the mouse resulted in inhibition of TPA-induced ear edema and skin epidermal ODC activity. The inhibitory order was as follows: TF-3 > TF-2 ≃ EGCG > TF-1. Western and Northern blots indicated that TF-3 significantly reduced the protein and mRNA levels of ODC in TPA-treated mouse skin and NIH 3T3 cells, whereas EGCG showed less activity. EGCG and TF-3 were able to inhibit the ODC enzyme activity in vitro. Furthermore, TF-3 also significantly reduced the basal promoter activity of the ODC gene in NIH 3T3 cells that were transiently transfected with ODC reporter plasmid. These results suggested that TF-3 was a potential inhibitor of ODC activity and TPA-induced edema and might be effective in cancer chemoprevention.

Inhibition of mouse skin tumor promotion by anti-inflammatory diarylheptanoids derived from Alpinia oxyphylla Miquel (Zingiberaceae).

Alpinia oxphylla Miquel, which belongs to the ginger family (Zingiberaceae), has been used in Oriental herbal medicine. Our recent studies have revealed that the methanolic extract of A. oxyphylla suppresses mouse skin tumor promotion and induces apoptosis in cultured human promyelocytic leukemia cells. In the present work, we have assessed effects of yakuchinone A and yakuchinone B, phenolic diarylheptanoids derived from A. oxyphylla, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation and epidermal ornithine decarboxylase (ODC) activity as well as on skin tumor promotion in female ICR mice. Thus, topical application of 2 or 6 micromol of the diarylheptanoids prior to each topical dose of TPA significantly ameliorated 7,12-dimethylbenz[a]anthracene-initiated mouse skin tumor formation. In parallel with suppression of tumor promotion, topically applied yakuchinone A and B markedly inhibited TPA-induced epidermal ODC activity and ODC mRNA expression. In another experiment, yakuchinone A and B reduced production of tumor necrosis factor-alpha in TPA-stimulated mouse skin. Furthermore, both compounds inhibited the TPA-induced expression of cyclooxygenase-2 at both transcriptional and translational levels. These findings indicate that pungent diarylheptanoids from A. oxyphylla Miquel have an antitumor promotional activity that might be related to their anti-inflammatory properties.

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.

Inhibition of 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhibits 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, as well as 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, TPA-promoted mouse skin tumorigenesis. The present studies were designed to further characterize the effect of time and dose of application of GPF on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 min prior to treatment with TPA resulted in a significant decrease in epidermal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet, ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were significantly increased by 1.8 and 1.9-fold, respectively, compared with controls. A similar response was observed with the ODC inhibitor 2-difluoromethylornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibited ODC activity by 62 and 68%, respectively, compared with controls ( P<0.05). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar to that observed when GPF application preceded TPA. When applied to mouse skin prior to TPA, GPF was associated with a decrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preceded the decrease in ODC activity. In a separate in vitro study, kinetic analyses indicated that GPF is a competitive inhibitor of ODC activity. Collectively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magnitude of inhibition is independent of epidermal ODC protein content. In addition, GPF is a competitive inhibitor of ODC activity in vitro. The decrease in TPA-induced ODC activity due to GPF treatment is preceded by an inhibition of TPA-induced PKC activity. Thus, the polyphenolic fraction from grapes warrants further examination as a skin cancer chemopreventive agent that interferes with cellular events associated with TPA promotion.

Tumor promoter-induced ornithine decarboxylase gene expression occurs independently of AP-1 activation

Activator protein 1 (AP-1) transactivation and ornithine decarboxylase (ODC) activity have been established as essential downstream effectors of mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Previous studies have shown that inhibition of either AP-1 transactivation or ODC activity suppressed tumor promoter-induced transformation. By utilizing the JB6 mouse epidermal cell system, the present study determined whether TPA-induced ODC gene expression and activity is independent of AP-1 transactivation. In three independent JB6 (P+) clones, stably expressing dominant negative c-jun, TPA-induced ODC gene expression and activity were similar compared to JB6 P+ cells expressing vector-control alone, while AP-1-dependent transcription was inhibited. Transformation-insensitive JB6 (P-) cells, which lack TPA-inducible c-jun expression, also exhibited similar induction of ODC activity by TPA. alpha-Difluoromethylornithine, an irreversible inhibitor of ODC, attenuated, at an equivalent IC50, both TPA-induced ODC activity and anchorage-independent growth of JB6 P+ cells, despite no inhibition of AP-1 transactivation. Taken together, the results presented indicate that TPA-induced ODC gene expression and activity are independent of AP-1 transactivation. Because inhibition of either AP-1 or ODC precludes TPA-induced transformation, and because ODC is independent of AP-1, we propose that there are at least two pathways to transformation. Each pathway is required but not sufficient for transformation.

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 (InsP 6 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 InsP 6 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 InsP 6 in keratinocytes ( P<0.01). Furthermore, when mouse skin was treated with 10 mM InsP 6, ODC induction was significantly inhibited ( P<0.05). In addition, the expression of TPA-induced c-myc mRNA was significantly inhibited by the same InsP 6 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 InsP 6 treatment were found in HEL-30 cells. These results indicate that InsP 6 reduces TPA-induced ODC activity independent of PKC isoform expression.

Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds

The anti-tumor promoting activity of a polyphenolic fraction from grape seeds (GSP) was examined in CD-1 mouse skin epidermis. Specifically, the ability of this fraction to inhibit 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and two markers of promotion in mouse skin, ornithine decarboxylase (ODC) and myeloperoxidase (MPO) activities, was evaluated. Pretreatment of mouse skin with 5, 10, 20 and 30 mg of GSP resulted in a dose-dependent reduction in TPA-induced epidermal ODC activity of 27, 37, 48 and 70%, respectively, compared to controls. In addition, pretreatment of mouse skin with 1, 5, 10 and 20 mg of GSP resulted in a significant 43, 39, 54 and 73% inhibition of MPO activity, respectively, compared to controls. In 7,12-dimethylbenz[a]anthracene (DMBA)-initiated CD-1 mice, biweekly treatment of mouse skin with 5, 10, and 20 mg of GSP 20 min prior to TPA application resulted in a 30, 40, and 60% inhibition of final skin tumor incidence, respectively, compared to controls. In addition, the final number of tumors per mouse in the 5, 10 and 20 mg GSP-treated animals was decreased 63, 51, and 94%, respectively, compared to controls. These studies indicate that GSP possesses anti-tumor promoting activity when applied to CD-1 mouse skin prior to treatment with TPA. The mechanism of this tumor inhibition is due, in part, to a GSP-associated inhibition of TPA-induced epidermal ODC and MPO activities. Thus, GSP warrants further evaluation as a skin cancer chemopreventative agent.

NADH: ubiquinone oxidoreductase inhibitors block induction of ornithine decarboxylase activity in MCF-7 human breast cancer cells.

Rotenone is the classical inhibitor of NADH: ubiquinone oxidoreductase and its analogue deguelin is a potent inhibitor of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase mRNA steady state level and enzyme activity in mouse 308 cells (Gerhäuser et al. 1995). In MCF-7 human breast cancer cells, rotenone, deguelin and two structurally-unrelated miticides (pyridaben and fenazaquin) inhibit not only NADH: ubiquinone oxidoreductase but also induced ornithine decarboxylase activity with IC50 values of < 1 to 70 nM. Rotenone inhibits ornithine decarboxylase activity equally well as induced by TPA, insulin-like growth factor I and 17 beta-oestradiol. Pyridaben is the most potent of the four inhibitors not only for NADH: ubiquinone oxidoreductase activity (bovine heart enzyme) and TPA-induced ornithine decarboxylase activity and mRNA steady state level but also for TPA-induced reactive oxygen species. It is therefore proposed that NADH: ubiquinone oxidoreductase inhibitors block multiple and possibly reactive oxygen species-modulated pathways which regulate ornithine decarboxylase activity.

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

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

An ornithine decarboxylase activity assay to screen for potential chemopreventive agents

A unique assay method to detect ornithine decarboxylase (ODC) activity in normal tracheal epithelial cells induced by a classic tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) has been developed. Using this new assay, the inhibition of induced ODC activity is measured, thereby providing a screening system to identify potential chemopreventive or therapeutic agents. The assay is designed to be specific as it detects a radioactively labeled reaction product, putrescine from the decarboxylation of ornithine by ODC. As TPA is a classical tumor promoter, inhibition TPA-induced ODC activity can also be used to select antitumor promoter agents. Since the assay requires only 2 days to complete, it can be used to screen a large number of compounds for chemopreventive activity in a relatively short period of time.

Tacalcitol (1,24(OH)2D3, TV-02) inhibits phorbol ester-induced epidermal proliferation and cutaneous inflammation, and induces epidermal differentiation in mice.

In this study, we examined the cutaneous effects of tacalcitol [1,24(R)(OH)2D3] on epidermal proliferation, differentiation, and skin inflammation in vivo using hairless mice. Tacalcitol was shown to inhibit epidermal proliferation using TPA-induced ornithine decarboxylase activity and DNA synthesis as indices, and the induction of epidermal differentiation using type I transglutaminase activity as an index. Tacalcitol also displayed an antiinflammatory effect on TPA-induced inflammatory changes histopathologically. These results confirm the clinical efficacy of tacalcitol in psoriasis, and suggest that it may be efficacious in the treatment of other inflammatory skin diseases.

ANTITUMOR-PROMOTING ACTIVITY OF OLIGOMERIC PROANTHOCYANIDINS IN MOUSE EPIDERMIS IN-VIVO

The flavanoid catechin and heterogenous samples of oligomeric proanthocyanidins extracted from various sources were compared for their ability to inhibit the biochemical and biological effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo. Topical applications of catechin fail to alter the hydroperoxide response to TPA but inhibit the induction of ornithine decarboxylase (ODC) activity and, to a lesser degree, the stimulation of RNA, protein, and DNA synthesis caused by this tumor promoter. Under similar conditions, condensed tannins (CTs) from guamuchil, loblolly pine, and southern red oak barks inhibit to various degrees all these biochemical markers of TPA promotion. The most effective antioxidant, loblolly pine bark CT, also inhibits TPA-induced ODC activity and macromolecule synthesis to a much greater degree than catechin or the other CTs tested. Pecan nut pith CT, however, has no inhibitory activity in this system. Pretreatments with 4 and 12 mg of loblolly pine bark CT remarkably inhibit the incidence and yield of skin tumors promoted by TPA in initiated mice, whereas similar doses of catechin are ineffective. Loblolly pine bark CT inhibits the 2nd rather than the 1st stage of tumor promotion. In contrast to their monomer units, therefore, some naturally occurring polyflavanoids have antioxidant activities and may be valuable against tumor propagation but their efficacy may vary considerably depending on their origin and structure.