Mouse Skin Tumor Promotion Research Articles

Skin Cancer Chemopreventive Effects of a Flavonoid Antioxidant Silymarin Are Mediated via Impairment of Receptor Tyrosine Kinase Signaling and Perturbation in Cell Cycle Progression

Enhanced tyrosine kinase activity due to aberrant or overexpression of receptor and/or non-receptor tyrosine kinases has been implicated in a variety of human malignancies including cutaneous neoplasms. Epidermal growth factor receptor (EGFR)-mediated tyrosine phosphorylation may be a primary indicator of signal transduction regulating cell growth and proliferation. Recent studies have shown that skin tumor promoters such as phorbol ester and ultraviolet B radiation activate EGFR in mouse skin as well as in cell culture. Similarly, oxidative stress, which is implicated in skin tumor promotion, also activates EGFR-mediated cell signaling. Since this signaling pathway has been suggested to be involved in skin tumor promotion, its impairment by antioxidants may lead to an efficient way for skin cancer prevention and therapy. Recently, we showed that silymarin, a flavonoid antioxidant, affords exceptionally high to complete protection against several skin tumor promoters caused tumor promotion in mouse skin. Employing human epidermoid carcinoma cells A431 that contain overexpressed EGFR, in this study, we assessed whether the anti-skin tumor promoting effects of silymarin are due to its inhibitory effect on EGFR activation and down stream signaling pathway leading to perturbations in cell cycle progression. Treatment of cells with silymarin resulted in a significant inhibition of ligand-induced activation of EGFR with no change in its protein levels. Silymarin treatment also resulted in a significant decrease in tyrosine phosphorylation of Shc, an immediate downstream target of EGFR, but no change in the protein levels of Shc. The inhibition of EGFR activation by silymarin was associated with a highly significant to complete inhibition of EGFR intrinsic kinase activity. Cells treated with silymarin also showed a significant G2-M arrest in cell cycle progression, and a highly significant inhibition of DNA synthesis and cell growth in a dose-dependent manner. Taken together, these results suggest that skin cancer chemopreventive effects of silymarin are mediated via impairment of EGFR signaling which ultimately leads to perturbation in cell cycle progression resulting in the inhibition of proliferation and induction of growth arrest.

Inhibitory effects of [6]-gingerol, a major pungent principle of ginger, on phorbol ester-induced inflammation, epidermal ornithine decarboxylase activity and skin tumor promotion in ICR mice

A wide array of phytochemicals have been shown to possess potential cancer chemopreventive properties. Ginger contains pungent phenolic substances with pronounced antioxidative and antiinflammatory activities. In the present study, we have determined the antitumor promotional activity of [6]-gingerol, a major pungent principle of ginger, using a two-stage mouse skin carcinogenesis model. Topical application of [6]-gingerol onto shaven backs of female ICR mice prior to each topical dose of 12- O-tetradecanoylphorbol-13-acetate (TPA) significantly inhibited 7,12-dimethylbenz[ a]anthracene-induced skin papillomagenesis. The compound also suppressed TPA-induced epidermal ornithine decarboxylase activity and inflammation.

Conjugated linoleic acid reduces arachidonic acid content and PGE 2 synthesis in murine keratinocytes

Dietary conjugated linoleic acid (CLA) is associated with decreased 12- O-tetradecanoyl-phorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. In addition, CLA decreases TPA-induced prostaglandin E synthesis and ornithine decarboxylase activity in cultured keratinocytes compared with linoleic acid (LA) and arachidonic acid (AA). When LA or CLA was added to keratinocyte cell cultures, the amounts of each of these cellular fatty acids increased significantly in a dose-dependent manner. Furthermore, LA treatment was associated with increased cellular AA while the AA content of keratinocytes was reduced when cultures were treated with CLA. Moreover, CLA (16 μg/ml) was more potent than LA at decreasing the level of 14C-AA incorporated into cellular phosphatidylcholine. In order to determine the effect of CLA on arachidonate-derived PGE 2, the release of 14C-AA and 14C-PGE 2 synthesis was measured in cultures pre-treated with LA/ 14C-AA or CLA/ 14C-AA for 12 h. The amount of 14C-AA release induced by TPA in CLA/ 14C-AA pre-treated cultures was significantly lower than cultures pre-treated with LA/ 14C-AA. Furthermore, TPA-induced 14C-PGE 2 was significantly lower in cultures pre-treated with CLA/ 14C-AA compared with cultures pre-treated with LA/ 14C-AA. The effects of LA and CLA on AA composition of phospholipids and subsequent arachidonate-derived PGE 2 synthesis will provide insight into the anti-promoter mechanisms of CLA.

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.

Effect of iron overload on the benzoyl peroxide-mediated tumor promotion in mouse skin

In this communication, we report that iron overload augments benzoyl peroxide (BPO)-mediated tumor promotion in 7,12-dimethylbenz[ a]anthracene (DMBA)-initiated mouse skin. Female albino Swiss mice were overloaded with iron and tumors were initiated by applying a single topical application of DMBA. A week after the initiation, promoting agent, BPO, was applied three times/week for 46 weeks. The appearance of the first tumor (papilloma) and the number of tumors/mouse were recorded. When compared to the control group, the iron-overloaded mice showed an increased incidence of tumors at various time intervals. In iron-overloaded animals, tumors appeared earlier and also the number of tumors/mouse was significantly higher. These data could be correlated with the iron levels of mouse skin in the two groups. Further, BPO-mediated induction in ornithine decarboxylase (ODC) activity and [ 3H]thymidine incorporation in cutaneous DNA were higher in the iron overload group. In addition, in iron-overloaded mice, cutaneous lipid peroxidation (LPO) and xanthine oxidase (XOD) activities were higher, whereas catalase activity was reduced. Similar to papilloma induction, a significant increase in carcinoma yield and incidence was observed in iron-overloaded animals. Based on this study, we propose that iron overload significantly increases the tumor promotion and progression potential of BPO. We suggest that oxidative stress generated by iron overload is responsible for the augmentation of BPO-mediated cutaneous tumorigenesis.

Potent suppressive activity of chlorophyll a and b from green tea (Camellia sinensis) against tumor promotion in mouse skin.

Potent antigenotoxic and anti-tumor promoting activities of chlorophyll a from green tea (camellia sinensis) have been shown using in vitro cell culture experiments (Okai Y. et al. (1996) Mutation Res., 370, 11-17). In the present study, the authors analyzed in vivo effects of chlorophyll a and b from green tea on tumor promotion in mouse skin in the following ways. 1. When chlorophyll a and b from green tea were applied before each treatment by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on BALB/c mouse skin initiated by 7, 12-dimethylbenz [a] an-thracene (DMBA), they caused significant suppression in a dose-dependent manner against BALB/c mouse skin tumorigenesis. 2. Chlorophyll a and b showed significant suppressive effects against TPA-induced inflammatory reaction such as edema formation in BALB/c mouse ear skin in a dose-dependent fashion. These results suggest that chlorophyll a and b possess potent suppressive activities against tumor promotion in mouse skin.

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.

Novel Cancer Chemopreventive Effects of a Flavonoid Antioxidant Silymarin: Inhibition of mRNA Expression of an Endogenous Tumor Promoter TNFα

In this study we describe exceptionally high protective effects of silymarin, a flavonoid antioxidant isolated from milk thistle, against 12-O-tetradecanoylphorbol 13-acetate (TPA)- and okadaic acid (OA)-caused tumor promotion in SENCAR mouse skin. Pre-application of silymarin to that of TPA in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin resulted in almost complete protection in terms of tumor incidence (85%) as well as multiplicity (94%). In OA-caused tumor promotion studies, application of silymarin prior to that of OA in DMBA-initiated mouse skin resulted in a complete protection against tumorigenicity. We next assessed the effect of silymarin on TPA- and OA-caused induction of mRNA expression of tumor necrosis factor α (TNFα) which is an endogenous tumor promoter and a central mediator of tumor promotionin vivoin the case of both TPA and OA tumor promotion. Topical application of silymarin on mouse skin prior to that of TPA or OA resulted in a highly significant to complete inhibition in a dose-dependent manner against both TPA- and OA-caused induction of TNFα mRNA expression in mouse epidermis. These results indicate that silymarin exerts novel chemopreventive effects against tumorigenicity by inhibiting endogenous tumor promoter TNFα. Additional studies are warranted in other tumor models to further evaluate the cancer chemopreventive effect of silymarin and to define the involvement of TNFα as a molecular target for such an effect.

Association of a murine chromosome 9 locus (Psl1) with susceptibility to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

It has been known for many years that there are dramatic differences in the susceptibility of mouse stocks and strains to two-stage skin carcinogenesis and that these differences are due the animals' responsiveness to tumor-promoting agents. In earlier studies using several inbred mouse strains, we found that susceptibility to skin tumor promotion by phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) is a multigenic trait. To extend this work, we conducted a genome scan of (C57BL/6 x DBA/2)F1 x C57BL/6 mice previously scored for sensitivity to skin tumor promotion by TPA. As a result of this scan, we now report an association of increased TPA promotion susceptibility with inheritance of the DBA/2 alleles of markers on the distal portion of mouse chromosome 9. Additional linkage analyses using (C57BL/6 x DBA/2)F2 and B x D recombinant inbred mice confirmed this association and suggested that a TPA promotion susceptibility locus maps near D9Mit51 (LODw = 4.1). We designated this locus promotion susceptibility locus 1 (Ps/1).

Conjugated linoleic acid modulation of phorbol ester-induced events in murine keratinocytes.

Recent work in our lab has shown that the chemoprotective fatty acid, conjugated linoleic acid (CLA), inhibits phorbol ester skin tumor promotion in mice. Because little is known about the deposition of CLA into tissues as well as its biological activity, this study compared the incorporation and biological activity of CLA to linoleic acid (LA; 18:2, c9,c12) and arachidonic acid (AA; 20:4 c5,c8,c11,c14) in cultured keratinocytes. When keratinocytes (HEL-30) were grown in media containing 14C-CLA for various periods, more than 50% of the 14C-CLA was incorporated into cellular lipids by 9 h. The distribution of CLA in phospholipid classes was similar to LA, Approximately 50% of 14C-LA and 14C-CLA were incorporated into phosphatidylcholine (PC), while the remainder was taken up by phosphatidylethanolamine (PE) and phosphatidylserine/phosphatidylinositol (PS/PI). In contrast, 14C-AA was more equitably distributed into PC, PE, or PS/PI (27, 30, or 38%, respectively). When keratinocytes were prelabeled with radiolabeled fatty acids, phorbol ester-induced release of 14C-CLA was 1.5 times higher than 14C-LA and 14C-AA. However, 14C-prostaglandin E (PGE) release in 14C-CLA prelabeled cultures was 6 and 13 times lower than cultures treated with 14C-LA and 14C-AA, respectively. Moreover, the ability of non-radiolabeled CLA to support ornithine decarboxylase activity, a hallmark event of tumor promotion, was significantly lower than in LA- and AA-treated cultures. These studies suggest that CLA inhibits skin tumor promotion, in part, through a PGE-dependent mechanism.

Auraptene, a citrus coumarin, inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in ICR mouse skin, possibly through suppression of superoxide generation in leukocytes.

Coumarin‐related compounds, auraptene and umbelliferone, have been isolated from the cold‐pressed oil of natsumikan (Citrus natsudaidai HAYATA), and tested as inhibitors of tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced Epstein‐Barr virus activation in Raji cells. The 50% inhibitory concentration (IC50) of auraptene (18μM)was almost equal to that of genistein. Umbelliferone, which lacks a geranyloxyl group present in auraptene, was less active (IC50=450 μM). In a two‐stage carcinogenesis experiment with 7, 12‐dimethylbenz[α]anthracene (topical application at 0.19 μmol) and TPA (topical application at 1.6 nmol) in ICR mouse skin, topical application of auraptene (at 160 nmol) significantly reduced tumor incidence and the numbers of tumors per mouse by 27% (P < 0.01) and 23% (P < 0.05), respectively. Auraptene at a concentration of 50 μM markedly suppressed superoxide (O2−) generation induced by 100 nM TPA in differentiated human promyelocytic HL‐60 cells. Having no O2− ‐scavenging potential, auraptene may inhibit the multicomponent NADPH oxidase system. Inhibition of intracellular hydroperoxide formation in differentiated HL‐60 cells by auraptene was also confirmed by flow‐cytometric analysis using 2′,7′‐dichlorofluorescein diacetate as a fluorescence probe. Quantitative analyses using high‐performance liquid chromatography showed the occurrence of auraptene not only in both the peels and sarcocarps of natsumikan, but also in those of hassaku orange (C. hassaku) and grapefruit (C. paradisi,) and even in their bottled fresh juice form. These results indicate that auraptene is a chemopreventer of skin tumorigenesis, and implies that suppression of leukocyte activation might be the mechanism through which it inhibits tumor promotion.

Analysis of the Ability of 12-O-Tetradecanoylphorbol-13-Acetate to Induce Epidermal Hyperplasia, Transforming Growth Factor-α, and Skin Tumor Promotion in wa-1 Mice

Wa-1 mutant mice possess a defect in the production of transforming growth factor-alpha (TGF-alpha) that leads to a phenotype characterized by wavy hair and curly whiskers. In light of recent evidence indicating the importance of TGF-alpha in epithelial tumorigenesis, this study characterizes the responsiveness of wa-1 mice to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). The responsiveness of wa-1 mice to TPA was compared with that of SENCAR and C57BL/6 mice, representing mouse lines highly sensitive and resistant to skin tumor promotion, respectively. Wa-1 mice were found to be very resistant to skin tumor promotion by TPA after initiation with 10 nmol DMBA, similar to C57BL/6 mice. TPA failed to induce a dramatic increase in TGF-alpha mRNA and protein in the skin of wa-1 mice, whereas TGF-alpha mRNA and protein were dramatically induced in the skin (both epidermis and dermis) of SENCAR and C57BL/6 mice. TPA treatment dramatically increased mRNA levels of two other EGF receptor ligands, amphiregulin and heparin binding-EGF, however, in the skin of all three mouse lines. Comparison of histologic changes in skin revealed that wa-1 mice exhibited only modest sustained epidermal hyperplasia after multiple treatments with TPA, similar in magnitude to that of C57BL/6 mice and significantly lower than that of SENCAR mice. The current data indicate that wa-1 mice are relatively resistant to TPA promotion. Possible mechanisms for this resistance are discussed.

Activation of erbB2 and c-src in phorbol ester-treated mouse epidermis: possible role in mouse skin tumor promotion.

In recent work we showed that the EGF receptor (EGFr) was activated in tumor promoter treated mouse epidermis (Cell Growth & Differentiation, 6: 1447-1455, 1995). In the present study, we have investigated the possible role of other erbB family members in the process of tumor promotion. Both erbB2 and erbB3, but not erbB4, were expressed in cultured mouse keratinocytes and in mouse epidermis in vivo. In cultured mouse keratinocytes, EGF stimulated rapid tyrosine phosphorylation of erbB2 followed by a time-dependent degradation of erbB2 protein. Furthermore, an increase in erbB2:EGFr heterodimer formation was also induced by EGF. In contrast to the results with erbB2, EGF did not induce tyrosine phosphorylation, the degradation of erbB3, or erbB3:EGFr heterodimer formation in cultured keratinocytes. Further analyses revealed that c-src kinase activity was dramatically elevated in cultured mouse keratinocytes exposed to EGF. In mouse epidermis following multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), the phosphotyrosine content of erbB2 was significantly elevated in a dose-dependent manner. Concomittantly, erbB2:EGFr heterodimer formation and c-src kinase activity were also elevated in TPA-treated epidermis. Structure-activity relationships with several phorbol ester analogs showed that the elevated phosphorylation of erbB2 in mouse epidermis followed closely with tumor promoting ability. Activation of erbB2 and c-src kinase were also observed in the epidermis of TGF alpha transgenic mice where expression of human TGF alpha was targeted to basal keratinocytes with the human K14 promoter. Collectively, the current data suggest that the activation of erbB2 in phorbol ester treated skin can be explained solely by a mechanism involving elevation of EGFr ligands and activation of the EGFr. In addition, activation of c-src may be an important downstream effector in mouse keratinocytes both in vivo and in vitro, following activation of the EGFr, erbB2, or both.

Altered glucocorticoid receptor expression and function during mouse skin carcinogenesis

Glucocorticoids are the most potent inhibitors of tumor promotion in mouse skin, when applied with a promoting agent at the early stages of promotion. However, established skin papillomas become resistant to growth inhibition by glucocorticoids. Glucocorticoid control of cellular functions is mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Here we present data on GR expression and function in mouse papillomas and squamous cell carcinomas. Tumors were produced in SENCAR mice by a 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate two-stage protocol. In early papillomas (after 15-20 wk of promotion), northern blotting revealed a decrease in the GR mRNA level that was confirmed by a binding assay. However, in late papillomas (after 30-40 wk of promotion), and especially in squamous cell carcinomas, the level of GR in both assays was similar to or higher than the GR level in normal epidermis. To test the functional capability of GR in tumors, we compared the effect of the synthetic glucocorticoid fluocinolone acetonide (FA) on keratinocyte proliferation and on expression of glucocorticoid-responsive genes in normal epidermis, hyperplastic skin surrounding tumors, and mouse skin papillomas. FA strongly inhibited DNA synthesis in keratinocytes in normal skin and tumor-surrounding skin but had no effect on DNA synthesis in papillomas. In addition, FA strongly induced metallothionein 1 expression and inhibited connexin 26 expression in skin but did not affect expression of these genes in tumors. These data suggest that alteration of both the expression and function of GR may be an important mechanism of tumor promotion in skin.

Protection against induction of mouse skin papillomas with low and high risk of conversion to malignancy by green tea polyphenols.

We earlier showed that a polyphenolic fraction isolated from green tea (GTP) affords protection against tumor promotion and tumor progression in SENCAR mouse skin. The present study was designed to further evaluate the protective effect of GTP against the induction and subsequent progression of papillomas to squamous cell carcinomas (SCCs) in experimental protocols where papillomas were developed with a low or high probability of their malignant conversion. Topical application of GTP (6 mg/animal) 30 min prior to that of 12-O-tetradecanoylphorbol-13-acetate (TPA) either once a week for 5 weeks (high risk TPA protocol) or once a week for 20 weeks (low risk TPA protocol) or mezerein (MEZ) twice a week for 20 weeks (high risk MEZ protocol) in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin resulted in significant protection against skin tumor promotion in terms of tumor incidence (32-60%), multiplicity (49-63%) and tumor volume/mouse (73-90%) at the termination of the experiment at 20 weeks. In three separate malignant progression experiments when papilloma yield in DMBA-initiated and TPA or MEZ promoted low and high risk protocols was stabilized at 20 weeks, animals were divided into two subgroups. These animals were either topically treated twice weekly with acetone (0.2 ml/animal, spontaneous malignant conversion group) or with GTP (6 mg/animal in 0.2 ml acetone) for an additional period of 31 weeks. During these treatment regimens, all suspected carcinomas were recorded and each one was verified histopathologically either at the time when tumor-bearing mouse died/moribund or at the termination of the experiment at 51 weeks. GTP resulted in significant protection against the malignant conversion of papillomas to SCC in all the protocols employed. At the termination of the experiment at 51 weeks, these protective effects were evident in terms of mice with carcinomas (35-41%), carcinomas per mouse (47-55%) and percent malignant conversion of papillomas to carcinomas (47-58%). The kinetics of malignant conversion suggest that a subset of papillomas formed in the early phase of tumor promotion in all the protocols had a higher probability of malignant conversion into SCCs because all the positive control groups (acetone treated) produced nearly the same number of carcinomas (33-38 in a group of 20 animals) at the end of the progression period. In the GTP-treated group of animals the number of carcinomas formed was less (14-20 in a group of 20 animals), which shows the ability of GTP to protect against the malignant conversion of papillomas of higher probability of malignant conversion to SCCs. The results of this study suggest that irrespective of the risk involved, GTP may be highly useful in affording protection against skin cancer risk.

Inhibitory effects of curcumin on tumorigenesis in mice

Curcumin (diferuloylmethane), the naturally occurring yellow pigment in turmeric and curry, is isolated from the rhizomes of the plant Curcuma longa Linn. Curcumin inhibits tumorigenesis during both initiation and promotion (post-initiation) periods in several experimental animal models. Topical application of curcumin inhibits benzo[a]pyrene (B[a]P)-mediated formation of DNA-B[a]P adducts in the epidermis. It also reduces 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced increases in skin inflammation, epidermal DNA synthesis, ornithine decarboxylase (ODC) mRNA level, ODC activity, hyperplasia, formation of c-Fos, and c-Jun proteins, hydrogen peroxide, and the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HmdU). Topical application of curcumin inhibits TPA-induced increases in the percent of epidermal cells in synthetic (S) phase of the cell cycle. Curcumin is a strong inhibitor of arachidonic acid-induced edema of mouse ears in vivo and epidermal cyclooxygenase and lipoxygenase activities in vitro. Commercial curcumin isolated from the rhizome of the plant Curcuma longa Linn contains 3 major curcuminoids (approximately 77% curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin). Commercial curcumin, pure curcumin, and demethoxycurcumin are about equipotent as inhibitors of TPA-induced tumor promotion in mouse skin, whereas bisdemethoxycurcumin is somewhat less active. Topical application of curcumin inhibits tumor initiation by B[a]P and tumor promotion by TPA in mouse skin. Dietary curcumin (commercial grade) inhibits B[a]P-induced forestomach carcinogenesis, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)-induced duodenal carcinogenesis, and azoxymethane (AOM)-induced colon carcinogenesis. Dietary curcumin had little or no effect on 4-(methylnitosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis and 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast carcinogenesis in mice. Poor circulating bioavailability of curcumin may account for the lack of lung and breast carcinogenesis inhibition.

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

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

Activation of AP-1 by okadaic acid in mouse keratinocytes associated with hyperphosphorylation of c-jun.

Okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, is also a potent mouse skin tumor promoter. The effects of OA on regulation of c-jun/activator protein-1 (AP-1) transcriptional activation were investigated in mouse keratinocytes. AP-1 DNA binding to the jun 12-O-tetradecanoylphorbol-13-acetate-response element (TGACATCA) as determined by gel shift analysis was strongly induced by OA (100 ng/mL) at 6 and 12 h. Preincubation of nuclear extracts with anti-c-jun antibody demonstrated that c-jun was a major component of the DNA-bound AP-1 complex induced by OA in 308 cells. Transfection of a c-jun promoter-reporter construct demonstrated that AP-1 transactivation was induced by OA. The mRNA level of the c-jun proto-oncogene was dramatically increased by 6 and 12 h of OA treatment. Furthermore, a significant induction of c-jun protein was stimulated by 6 and 12 h of OA treatment. Upon further analysis, it was found that OA induced a significant accumulation of Ser 73-phosphorylated c-jun protein in 308 cells. In summary, our data suggest that skin tumor promotion by OA is due at least in part to increased AP-1 DNA binding and transactivation mediated by c-jun hyperphosphorylation.

Okadaic acid treatment induces DNA adduct formation in BHK21 C13 fibroblasts and HESV keratinocytes

Okadaic acid (OA), a toxin involved in diarrhetic shellfish poisoning (DSP), has been shown to be a potent tumor promoter in mouse skin and glandular stomach. However, more recent studies tended to show that OA can also act as a genotoxic. In this study, using the 32P-postlabelling method, DNA adduct formation was obtained in two cell lines (BHK21 C13 fibroblasts and HESV keratinocytes) after treatment by OA for 24 h with a dose range between 0.01 and 5 nM. Nineteen adducts were observed with BHK21 C13 cells and 15 with HESV ones. Low doses did not show adduct formation. Intermediate doses have given the most important number of adducts and with higher doses, the number of adducts decreased dose dependently. Ten adducts were similar in the two strains while 9 were specific of BHK21 C13 cell line and 5 of HESV one. The highest total DNA adduct level from origin parts was estimated at 95.6 adducts/10 9 nucleotides for BHK21 C13 fibroblasts (1 nM OA treatment) and 31.1 adducts/10 9 nucleotides for HESV keratinocytes (0.5 nM OA treatment). In this case, the major adduct (number 3) represented 20% for the fibroblastic cell line and 30% for the keratinocytic strain. The genotoxic effect of OA showed in this study should lead to a more careful survey of DSP outbreaks.

Ability of tannins extracted from the leaves of various trees and shrubs to inhibit the biomarkers of tumor promotion in mouse skin in vivo

Eight heterogeneous tannin samples (HTSs) extracted from various tree/shrub leaves of African and Himalayan origin were tested topically for their ability to inhibit the biomarkers of tumor promotion in mouse skin in vivo. HTS2 (from Dichostachys cinerea) and HTS6 (from Cassia sieberiana) consistently inhibit tumor promoter-stimulated ornithine decarboxylase activity, DNA synthesis, hydroperoxide production, and edema formation almost as much as loblolly pine bark condensed tannin (LPB-CT), which is known to inhibit skin tumor promotion. The other HTSs tested have lesser or only partial inhibitory effects. The ability of HTSs to inhibit the biomarkers of tumor promotion may be related to their reducing power but there is no apparent correlation between their inhibitory effects and their proanthocyanidin contents expressed as absorbance units, protein precipitation activities, and relative degrees of polymerization. HTS6 is effective against a wide spectrum of tumor-promoting agents unrelated to 12-O-tetradecanoyl-phorbol-13-acetate. The antioxidant effects of HTS6 and LPB-CT are similar but do not resemble that of tannic acid. HTS6 and LPB-CT both fail to alter the covalent binding of a tumor-initiating dose of 7,12-dimethylbenz[a] anthracene to DNA but inhibit the stimulation of DNA synthesis caused by a carcinogenic dose of this compound. Some foliage tannins, therefore, have potent antioxidant and anti-inflammatory activities and may inhibit hyperplasia and tumor promotion but their efficacy may vary considerably depending on their origin, chemical composition, and biological properties.