Mouse Skin Tumorigenesis Research Articles

The role of trivalent dimethylated arsenic in dimethylarsinic acid-promoted skin and lung tumorigenesis in mice: Tumor-promoting action through the induction of oxidative stress

We investigated the relationship between lung- and skin-tumor promotion and oxidative stress caused by administration of dimethylarsinic acid (DMA(V)) in mice. The incidence of lung tumors induced by lung tumor initiator (4NQO) and DMA(V) were, as well as 8-oxo-2′-deoxyguanosine (8-oxodG), suppressed by cotreatment with (−)epigallocatechin gallate (EGCG). When mice were topically treated with trivalent dimethylated arsenic (DMA(III)), a further reductive metabolite of DMA(V), not only an increase in skin tumors but also an elevation of 8-oxodG in epidermis were observed. These results suggest that tumor promotion due to DMA(V) administration is mediated by DMA(III) through the induction of oxidative stress.

Haploinsufficiency for Odc Modifies Mouse Skin Tumor Susceptibility

Numerous studies have linked overexpression of ornithine decarboxylase (Odc) gene with enhanced susceptibility to mouse skin tumorigenesis. However, there is little experimental evidence suggesting that modest reductions in Odc expression might reduce tumor susceptibility. To address this issue, here we report the use of the Odc(+/-) haploinsufficiency model, in which one copy of the murine Odc gene has been inactivated by a homologous recombination. Compared with Odc(+/+) mice, Odc(+/-) mice exhibit reduced epidermal ODC enzyme activity and polyamine accumulation following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, following chronic TPA treatment, the characteristic hyperplastic response of the epidermis was diminished in Odc(+/-) mice. Finally, when subjected to a two-stage initiation-promotion protocol, substantially fewer skin papillomas developed in Odc(+/-) mice compared with wild-type littermates. These results support the concept that differences in tissue polyamine levels, resulting from either overexpression or reductions in ODC, are important modifiers of tumor susceptibility.

Effect of radiofrequency radiation exposure on mouse skin tumorigenesis initiated by 7,12-dimethybenz[α]anthracene

Purpose: Although radiofrequency (RF) radiation is not considered mutagenic, it has been suggested as a promoter of tumorigenesis. To study if RF radiation has a tumor promoting effect, we exposed mice with skin tumorigenesis initiated by 7,12-dimethybenz[α]anthracene (DMBA) to RF radiation.Materials and methods: Eighty male ICR mice were subjected to a single DMBA application (100 μg/100 μl acetone/mouse) on shaved dorsal skin at the age of 7 weeks. After one week, the mice were randomized into four equal groups of 20 mice each: i.e., sham-, 849 MHz-, 1,763 MHz-exposed, and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated groups. The RF exposure was conducted at a whole body average specific absorption rate (SAR) of 0.4 W/Kg, for 2 cycles of 45 min exposure with a 15 min interval each day, 5 days a week for 19 weeks. The TPA-treated group served as a positive control for skin tumorigenesis and were administered TPA (4 μg/100 μl acetone/mouse) twice weekly without RF exposure.Results: All mice were examined weekly at a macroscopic level. No skin tumors were observed in any groups except in the TPA-treated positive control group. TPA is known tumor promoter in DMBA-induced skin carcinogenesis and tumor incidence in the TPA treated group was 95%. At week 20 after DMBA initiation, skin tissues were analyzed immunohistochemically using anti-proliferating cell nuclear antigen (PCNA) antibody. No differences were observed by pathological examination or by PCNA staining between the sham- and the RF-exposed groups. The expression of cyclin D1 and c-fos were detected only in the tumorous skin tissues of the TPA-treated group.Conclusion: No evidence was found that RF radiation serves as a tumor promoter for skin tumors. Our data suggests that 849 MHz and 1,763 MHz RF radiations, similar to those emitted from mobile phones, do not have any promoting effect on skin tumor development in DMBA-initiated mice.

In situ reduction of oxidative damage, increased cell turnover, and delay of mitochondrial injury by overexpression of manganese superoxide dismutase in a multistage skin carcinogenesis model.

To study early subcellular pathologic changes of tumorigenesis in mouse skin and possible modulation by overexpression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD), skin keratinocytes from nontransgenic (Ntg) and transgenic (TgH) mice overexpressing MnSOD topically treated with one dose of 7,12-dimethylbenz(a)anthracene (DMBA) and a subsequent dose of 12-O-tetradecanoylphorbol 13-acetate (TPA) were analyzed in situ for levels of MnSOD and the oxidative damage product 4-hydroxy-2-nonenal (4HNE)-modified proteins using specific antibodies and immunogold electron microscopy. At all selected time points analyzed after TPA treatment, there was more MnSOD immunoreactive protein in mitochondria of keratinocytes of TgH mice than Ntg mice. Compared with untreated groups, there was a large increase in 4HNE-modified proteins at 6-24 h after TPA treatment, and this increase was larger in Ntg than TgH mice. Indices of mitosis and apoptosis of keratinocytes were greater in DMBA/TPA-treated TgH than Ntg mouse skin. Mitochondrial injury detected by transmission electron microscopy was delayed in keratinocytes of TgH compared with Ntg mice. The present study demonstrated that overexpression of MnSOD not only protected cells from oxidative damage, but also affected cell turnover kinetics. Thus, previously identified reduction in papilloma formation observed in TgH mice is correlated with mitochondrial events.

Lupeol modulates NF-kappaB and PI3K/Akt pathways and inhibits skin cancer in CD-1 mice.

Chemoprevention has become an effective cancer control modality; however, the search for novel agent(s) for the armamentarium of cancer chemoprevention continues. We argue that agents capable for inhibition of promotion stage of tumorigenesis with the ability to intervene at several critical pathways in the tumorigenesis process will have greater advantage over other single-target agents. Lupeol, a triterpene, is the principal constituent of common fruit plants such as olive, mango, fig and medicinal herbs that have been used to treat skin aliments. Lupeol has been reported to possess a wide range of medicinal properties that include strong antioxidant, antimutagenic, anti-inflammatory and antiarthritic effects. In the present study, we show that Lupeol possesses antitumor-promoting effects in a mouse skin tumorigenesis model. We first determined the effect of topical application of Lupeol to CD-1 mouse against 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of Lupeol (1-2 mg/mouse) 30 min prior to TPA (3.2 nmol/mouse) application onto the skin of CD-1 mice afforded significant inhibition, in a time- and dose-dependent manner, against TPA-mediated increase in (i) skin edema and hyperplasia, (ii) epidermal ornithine decarboxylase (ODC) activity, and (iii) protein expression of ODC, cyclo-oxygenase-2 and nitric oxide synthase. As of the role of nuclear factor kappa B (NF-kappaB) and phosphatidyl inositol 3-kinase (PI3K)/Akt signaling in tumor promotion, we next determined the effect of topical application of Lupeol to mouse skin against these signaling pathways. We found that Lupeol treatment to mouse skin resulted in the inhibition of TPA-induced (i) activation of PI3K, (ii) phosphorylation of Akt at Thr(308), (iii) activation of NF-kappaB and IKKalpha, and (iv) degradation and phosphorylation of IkappaBalpha. The animals pretreated with Lupeol showed significantly reduced tumor incidence, lower tumor body burden and a significant delay in the latency period for tumor appearance. At the termination of the experiment at 28 weeks, 100% of the animals in TPA-treated group exhibited seven to eight tumors/mouse, whereas only 53% of the mice receiving Lupeol prior to TPA treatment exhibited one to three tumors/mouse. These results for the first time provide evidence that Lupeol possesses antiskin tumor-promoting effects in CD-1 mouse and inhibits conventional as well as novel biomarkers of tumor promotion. We suggest that Lupeol is an attractive antitumor-promoting agent that must be evaluated in tumor models other than skin carcinogenesis.

Celecoxib inhibits phorbol ester-induced expression of COX-2 and activation of AP-1 and p38 MAP kinase in mouse skin.

Celecoxib, the first US FDA-approved selective cyclooxygenase-2 (COX-2) inhibitor initially developed for the treatment of adult rheumatoid arthritis and osteoarthritis, was reported to reduce the polyp burden in patients with familial adenomatous polyposis. This specific COX-2 inhibitor also protects against experimentally induced carcinogenesis, but molecular mechanisms underlying its chemopreventive activities remain largely unresolved. In the present work, we found that celecoxib inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of COX-2 in female ICR mouse skin when applied topically 30 min prior to TPA as determined by both immunoblot and immunohistochemical analyses. In another study, celecoxib attenuated the DNA binding activity of activator protein 1 (AP-1) through suppression of c-Jun and c-Fos expression in TPA-treated mouse skin. In addition, celecoxib inhibited both the catalytic activity and phosphorylation of p38 mitogen-activated protein (MAP) kinase. In the same animal model, TPA treatment resulted in rapid activation via phosphorylation of extracellular signal-regulated protein kinase (ERK)1/2 and p38 MAP kinase, which are upstream of AP-1 in mouse skin. In order to clarify the roles of p38 and ERK in TPA-induced AP-1 activation, we utilized the pharmacologic inhibitors of these enzymes. The p38 inhibitor SB203580 blocked TPA-mediated AP-1 activation, while the MEK1/2 inhibitor U0126 was not inhibitory despite suppression of c-Fos expression in mouse skin. Furthermore, SB203580 markedly inhibited COX-2 expression induced by TPA. Taken together, these findings suggest that celecoxib down-regulates COX-2 by blocking activation of p38 MAP kinase and AP-1, which may represent molecular mechanisms underlying antitumor promoting effects of this drug on mouse skin tumorigenesis.

Dietary Lutein/Zeaxanthin Decreases Ultraviolet B-Induced Epidermal Hyperproliferation and Acute Inflammation in Hairless Mice

Lutein and zeaxanthin are carotenoids found in green leafy vegetables with interesting antioxidant properties. They are present in high concentrations in the fovea centralis of the human retina and their role in the prevention of age-related macula degeneration has been reported. We have investigated the effect of orally administered lutein and zeaxanthin in the cutaneous response to ultraviolet B irradiation. Female hairless SKh-1 mice receiving 0.4% and 0.04% lutein plus zeaxanthin-enriched diet for 2 wk were exposed to single doses of ultraviolet B radiation. Skin biopsies were taken at 24 and 48 h after irradiation and analyzed for the presence of apoptotic cells, proliferating cells, and expression of proliferating cell nuclear antigen. Our results show a clear ultraviolet-induced dose-dependent inflammatory response. Orally administered 0.4% lutein and zeaxanthin decreased significantly the edematous cutaneous response (p<0.01) as determined by the reduction of the UVB-induced increase of ear bifold thickening. Additionally, dietary carotenoids were efficient in reducing the ultraviolet B-induced increases in the percentage of proliferating cell nuclear antigen (p<0.05), bromodeoxyuridine (p<0.05), and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells (p<0.01). These data demonstrate that oral supplementation of lutein and zeaxanthin diminishes the effects of ultraviolet B irradiation by reducing acute inflammatory responses and ultraviolet-induced hyperproliferative rebound.

Curcumin inhibits phorbol ester-induced expression of cyclooxygenase-2 in mouse skin through suppression of extracellular signal-regulated kinase activity and NF-kappaB activation.

Recently, there have been considerable efforts to search for naturally occurring substances for the intervention of carcinogenesis. Many components derived from dietary or medicinal plants have been found to possess substantial chemopreventive properties. Curcumin, a yellow coloring ingredient of turmeric (Curcuma longa L., Zingiberaceae), has been shown to inhibit experimental carcinogenesis and mutagenesis, but molecular mechanisms underlying its chemopreventive activities remain unclear. In the present work, we assessed the effects of curcumin on 12-O- tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2) in female ICR mouse skin. Topical application of the dorsal skin of female ICR mice with 10 nmol TPA led to maximal induction of cox-2 mRNA and protein expression at approximately 1 and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, curcumin inhibited the expression of COX-2 protein in a dose-related manner. Immunohistochemical analysis of TPA-treated mouse skin revealed enhanced expression of COX-2 localized primarily in epidermal layer, which was markedly suppressed by curcumin pre-treatment. Curcumin treatment attenuated TPA- stimulated NF-kappaB activation in mouse skin, which was associated with its blockade of degradation of the inhibitory protein IkappaBalpha and also of subsequent translocation of the p65 subunit to nucleus. TPA treatment resulted in rapid activation via phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein (MAP) kinases, which are upstream of NF-kappaB. The MEK1/2 inhibitor U0126 strongly inhibited NF-kappaB activation, while p38 inhibitor SB203580 failed to block TPA-induced NF-kappaB activation in mouse skin. Furthermore, U0126 blocked the IkappaBalpha phosphorylation by TPA, thereby blocking the nuclear translocation of NF-kappaB. Curcumin inhibited the catalytic activity of ERK1/2 in mouse skin. Taken together, suppression of COX-2 expression by inhibiting ERK activity and NF-kappaB activation may represent molecular mechanisms underlying previously reported antitumor promoting effects of this phytochemical in mouse skin tumorigenesis.

Silk protein, sericin, suppresses DMBA-TPA-induced mouse skin tumorigenesis by reducing oxidative stress, inflammatory responses and endogenous tumor promoter TNF-alpha.

This study was conducted to assess protective effect of an antioxidant protein, sericin, on tumor promotion in the 7,12-dimethylbenz (alpha) anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol 13-acetate (TPA)-promoted mouse skin tumorigenesis model. In experiment 1, sericin was applied topically to DMBA-initiated female ICR mouse skin at the doses of 2.5 and 5 mg twice per week for 16 weeks, 30 min prior to each promotion treatment with TPA. The protective effect of sericin was evident in terms of significant reduction in tumor incidence and tumor multiplicity at the doses of 2.5 and 5 mg per application, compared to the control group without receiving sericin. The expression of tumor necrosis factor (TNF)-alpha protein and the level of 4-hydroxynonenal (4-HNE) in normal epidermis were significantly reduced in both sericin treatment groups. In experiment 2, sericin at the dose of 5 mg was applied topically to the dorsal mouse skin 30 min before application of a TPA, and the same doses of TPA and sericin were applied twice at an interval of 24 h. Sericin treatment inhibited double TPA treatment-induced morphological changes reflecting inflammatory response, including leukocyte infiltration, hyperplasia and cell proliferation. Furthermore, sericin treatment significantly suppressed the elevation in 4-HNE level and elevated expressions of c-fos, c-myc and cyclooxygenase-2 (COX-2) in normal epidermis induced by double application of TPA. The results suggest that sericin possesses protective effect against tumor promotion in mouse skin by suppressing oxidative stress, inflammatory responses and TNF-alpha.

Palm oil alleviates 12- O-tetradecanoyl-phorbol-13-acetate-induced tumor promotion response in murine skin

Palm oil is a rich source of vitamin E, carotenoids, tocotrienols and tocopherols which are natural antioxidants and act as scavengers of oxygen free radicals. 12- O-Tetradecanoyl-phorbol-13-acetate (TPA) is a known oxidant that promotes tumorigenesis in mouse skin through the elaboration of oxidative stress. In this study we therefore assessed the anti-tumor promoting potential of palm oil against TPA-mediated skin tumorigenesis in 7,12-dimethylbenz[ a]anthracene-initiated Swiss albino mice. Topical application of palm oil 1 h prior to application of TPA resulted in a significant protection against skin tumor promotion. The animals pre-treated with palm oil showed a decrease in both tumor incidence and tumor yield as compared to the TPA (alone)-treated group. Palm oil application also reduced the development of malignant tumors. Since TPA-induced epidermal ornithine decarboxylase (ODC) activity and [ 3H]thymidine incorporation are conventionally used markers of skin tumor promotion, we also assessed the effect of pre-application of palm oil on these parameters, and it was observed that the application of palm oil prior to the application of TPA alleviated both these TPA-induced markers of tumor promotion. The effect of pre-application of palm oil on TPA-mediated depletion in the non-enzymatic and enzymatic molecules was also assessed and it was observed that palm oil application prior to TPA application resulted in the recovery of TPA-mediated depletion in the levels of these molecules viz. glutathione, glutathione peroxidase, glutathione reductase, glutathione- S-transferase and catalase. Similarly, palm oil also exhibited a protective effect against Fe 2+-ascorbate-induced lipid peroxidation in the epidermal microsomes. The results of the present study thus suggest that palm oil possesses anti-skin tumor promoting effects, and that the mechanism of such effects may involve the inhibition of tumor promoter-induced epidermal ODC activity, [ 3H]thymidine incorporation and cutaneous oxidative stress.

Expression, localization, and activity of glycogen synthase kinase 3beta during mouse skin tumorigenesis.

Glycogen synthase kinase 3 (GSK-3) is a protein kinase that plays essential roles in the control of several developmental, metabolic, and apoptotic processes. Owing to its negative actions on several oncogenic insults, it has been considered a putative functional tumor suppressor. We studied the expression, activity, and localization of GSK-3beta during the process of chemically induced two-stage mouse skin carcinogenesis and also in the tumors generated upon subcutaneous injection of Akt-transformed keratinocytes. We found that GSK-3 activity was downregulated at the later stages of promotion by tyrosine 216 dephosphorylation and serine 9 phosphorylation. The data obtained with Akt-transformed keratinocytes clearly suggested the involvement of Akt in serine 9 phosphorylation of GSK-3beta. Finally, besides functional inactivation, significant basal activity of GSK-3beta was detected in all cases, indicating that this enzyme provides essential functions to malignant keratinocytes.

Additive roles of XPA and MSH2 genes in UVB-induced skin tumorigenesis in mice

We have made xeroderma pigmentosum group A gene ( XPA)-knockout mice ( XPA −/− mice). The XPA −/− mice had no detectable activity for nucleotide excision repair (NER) and showed a high incidence of UVB-induced skin tumorigenesis. We have also found that cell lines derived from skin cancers in UVB-irradiated XPA −/− mice become tolerant to UV-irradiation and showed abnormal UV-induced cell cycle checkpoints and decreased mismatch repair (MMR) activity. These results suggested that the MMR-downregulation may help cells escape killing by UV-irradiation and thus MMR-deficient clones are selected for during the tumorigenic transformation of XPA −/− cells. In this report, we examined whether the incidence of UVB-induced skin tumorigenesis is enhanced in XPA −/− MSH2 −/−, XPA −/− and MSH2 −/− mice when compared with that in wild-type mice. Our results indicate that the MSH2-deficiency caused a high incidence of spontaneous and UVB-induced skin tumorigenesis and the XPA and MSH2 genes have additive roles in the UV-induced skin tumorigenesis.

Inhibition of ultraviolet light-induced oxidative events in the skin and internal organs of hairless mice by isoflavone genistein

We have previously demonstrated that soybean isoflavone genistein inhibits ultraviolet-B (UVB)-induced skin tumorigenesis in hairless mice. In the present study, we further investigated the possible mechanism(s) of action whereby genistein inhibits photocarcinogenesis with focuses on UVB-induced oxidative events, including hydrogen peroxide (H 2O 2) production, lipid peroxidation (as represented by malondialdehyde, MDA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in vivo. We demonstrated that subacute exposure to UVB substantially increased the level of H 2O 2, lipid peroxides, and 8-OHdG in skin of hairless mice. In addition, chronic exposure to low-dose UVB (0.9–1.2 kJ/m 2 for 20 weeks) substantially increased the levels of 8-OHdG not only in the epidermis, but also in the internal organs such as liver, brain, and spleen of mice with exception of kidney. However, genistein did not affect the level of UVB-induced pyrimidine dimmers in the same UVB exposed mouse skin, indicating selective inhibition of oxidative DNA damage by genistein. Induction of H 2O 2 was independent of UVB fluences whereas the levels of MDA and 8-OHdG were induced in an UVB fluence-dependent manner. The results suggest that H 2O 2 be generated as an acute cutaneous response to UVB irradiation, while MDA and 8-OHdG are accumulated with increasing UVB exposure and more closely related to chronic effects of UVB radiation. Pre-treatment of animals with 10 μmol of genistein 1 h prior to UVB exposure significantly inhibited UVB-induced H 2O 2 and MDA in skin and 8-OHdG in epidermis as well as internal organs. Suppression of 8-OHdG formation by genistein has been corroborated in purified DNA irradiated with UVA and B. In summary, our results suggest that UVB irradiation elicit a series of oxidative events, which can be substantially inhibited by isoflavonoid genistein through either direct quenching of reactive oxygen species or indirect antiinflammatory effects. Thus, the antioxidative properties of genistein may explain for the mechanisms of anti-photocarcinogenic action of genistein.

Functional roles of Akt signaling in mouse skin tumorigenesis

The mouse skin carcinogenesis protocol is a unique model for understanding the molecular events leading to oncogenic transformation. Mutations in the Ha-ras gene, and the presence of functional cyclin D1 and the EGF receptor, have proven to be important in this system. However, the signal transduction pathways connecting these elements during mouse skin carcinogenesis are poorly understood. This paper studies the relevance of the Akt and ERK pathways in the different stages of chemically induced mouse skin tumors. Akt activity increases throughout the entire process, and its early activation is detected prior to increased cyclin D1 expression. ERK activity rises only during the later stages of malignant conversion. The observed early increase in Akt activity appears to be due to raised PI-3K activity. Other factors acting on Akt such as ILK activation and decreased PTEN phosphatase activity appear to be involved at the conversion stage. To further confirm the involvement of Akt in this process, PB keratinocytes were transfected with Akt and subsequently injected into nude mice. The expression of Akt accelerates tumorigenesis and contributes to increased malignancy of these keratinocytes as demonstrated by the rate of appearance, the growth and the histological characteristics of the tumors. Collectively, these data provide evidence that Akt activation is one of the key elements during the different steps of mouse skin tumorigenesis.

Use of intercross outbred mice and single nucleotide polymorphisms to map skin cancer modifier loci

Car-R and Car-S outbred mouse lines, phenotypically selected for resistance and susceptibility to skin carcinogenesis respectively, show significant linkage disequilibrium (LD) at genetic markers mapping on chromosomal regions where skin cancer modifier loci (Skts3, Skts1, and Psl1 on Chrs 5, 7, and 9 respectively) have been mapped in standard crosses. Analysis of these regions for genetic linkage with skin cancer phenotypes in 245 (Car-R x Car-S)F2 intercross mice, by using single nucleotide polymorphisms (SNPs), revealed significant linkage at a possible allelic form of the Skts1 locus, whose mapping region was shortened to a <5.5-cM interval near the Tyr locus. The Car-derived Skts1 locus was linked with papilloma multiplicity and latency by a recessive inheritance of the susceptibility allele. Putative loci on Chr 5 (Skts3) and 9 (Psl1) showed no significant linkage. These results point to the important role of the Stks1 locus in mouse skin tumorigenesis in independent crosses. The shortened Skts1 mapping region should facilitate the identification of candidate genes.

Capsaicin suppresses phorbol ester-induced activation of NF-κB/Rel and AP-1 transcription factors in mouse epidermis

Capsaicin, the principal pungent ingredient of hot chili peppers, has anti-inflammatory and analgesic properties and is currently used as a topical cream for the management of various neuropathic conditions. In the present study, topical application of capsaicin onto dorsal skin of female ICR mice strongly suppressed phorbol ester-stimulated activation of NF-κB via blockade of IκB-α degradation with subsequent inhibition of nuclear translocation of the functionally active NF-κB subunit, p65. Likewise, phorbol ester-induced activation of activator protein-1 (AP-1) was abolished by capsaicin pretreatment. Since altered transactivation of NF-κB and AP-1 has been implicated for neoplastic transformation and progression, the suppression of these transcription factors by capsaicin may account for its previously reported chemopreventive effects on mouse skin tumorigenesis as well as inflammation.

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.

Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes.

Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In this report we have used the mouse keratinocyte 308 cell line, which contains a wild-type p53 gene, to study the effect of apigenin treatment on p53 protein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 microM apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 protein was induced 27-fold after 4 h of apigenin treatment and levels remained elevated through 10 h of exposure. After 24 h of exposure to 70 microM apigenin, p53 protein levels returned to control levels. p21/waf1 protein levels increased approximately 1. 5-2-fold after 4 h and remained elevated at 24 h. To investigate the mechanism of p53 protein accumulation, we compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells were incubated for 4 h in the presence of apigenin, then cycloheximide was added to inhibit further protein synthesis and p53 protein levels were measured by western blot. The half-life of p53 protein was found to be increased an average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t(1/2) = 131 min versus 16 min in apigenin- versus vehicle-treated cells, respectively). The mechanism of p53 protein stabilization is currently being investigated. To determine whether p53 was transcriptionally active, we also performed gel mobility shift assays and transient transfection studies using a luciferase plasmid under the control of the p21/waf1 promoter. Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exert anti-tumorigenic activity by stimulating the p53-p21/waf1 response pathway.

The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice

The effect of dimethylarsinic acid (DMA) on skin tumorigenesis by UVB irradiation was examined. Hairless mice (Hos: HR-1) irradiated with UVB at a dose of 2 kJ/m 2 twice weekly, were fed with drinking water containing 1000 ppm DMA, a main metabolite of inorganic arsenics, produced more skin tumors than DMA-untreated mice. Histopathological examination revealed that the mouse malignant tumors with severe atypism appeared only in the treatment group of UVB plus 1000 ppm DMA. These positive results point out the importance of dimethylated metabolites of inorganic arsenic in the process of skin carcinogenesis.

The Phototumorigenic Fluoroquinolone Lomefloxacin Photosensitizes Pyrimidine Dimer Formation in Human Keratinocytes In Vitro

The fluoroquinolone antibiotic lomefloxacin is phototoxic, photogenotoxic, photomutagenic and photosensitizes tumorigenesis in mouse skin. We have used T4 endonuclease V to demonstrate that lomefloxacin photosensitizes pyrimidine dimer formation in a human keratinocyte line (HaCaT). A possible mechanism for this effect would be triplet-triplet energy transfer. However, there is indirect evidence that the lomefloxacin triplet yield is very low, making this reaction less likely. The finding that lomefloxacin photosensitizes production of highly mutagenic pyrimidine dimers correlates with its ability to initiate skin tumor formation in mice. Until the potential of other fluoroquinolones to photosensitize dimer formation is explored it may be unadvisable to prescribe these antibiotics to patients with defective DNA repair capacity (e.g. xeroderma pigmentosum).