The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice.

Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin.

The possible separation of 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation and hyperplasia by compound A.

Activated glucocorticoid receptor (GR) acts via two different mechanisms: transcriptional regulation that requires DNA-binding, and protein-protein interaction between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1). It has been postulated that many important effects of glucocorticoids, including their anti-inflammatory properties, depend on GR's transrepressive effects on NF-κB and AP-1. In the present study, we have employed a TPA-induced model of skin inflammation and epidermal hyperplasia to determine whether partial activation of the glucocorticoid receptor by compound A (CpdA) is sufficient to reverse the effect of TPA treatment. CpdA is a nonsteroidal GR modulator with high binding affinity, is capable of partial activation of GR. Topical application of TPA twice per week for 2 wk results in inflammation and epidermal hyperplasia. TPA treatment also elevates levels of c-jun (AP-1 component), cyclooxygenase-2 (COX-2), p50 (NF-κB component), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in the skin. Fluocinolone acetonide (FA) (a full GR agonist) was able to completely reverse the above effects of TPA. When applied alone, CpdA increased the epidermal thickness and keratinocyte proliferation as well as levels of c-jun, COX-2, IL-6, and IFN-γ. However, CpdA treatment resulted in a decrease in the number of p50 positive cells induced by TPA, suggesting its role in inhibition of NF-κB. The level of metallothionein-1 mRNA, regulated by GR was also significantly decreased in skin samples treated with CpdA. Our results suggest that CpdA is able to inhibit GR transactivation and activate only some transrepression properties of GR.

Inhibition of murine skin carcinogenesis by freeze-dried grape powder and other grape-derived major antioxidants.

Overexposure of the skin to carcinogenic insults causes a variety of adverse effects, among them the development of skin carcinomas. Since there is a need to develop efficient chemopreventive agents based on nutrition, our goal was to determine antioxidant and anti-carcinogenic properties of grapes by evaluating grape powder developed by the California Table Grape Commission. In order to elucidate the mechanism(s) of action of grape powder, three of the major antioxidant components found in grapes-resveratrol, catechin, quercetin, and grape seed extract, containing a proanthocyanidin B-2-gallate-were evaluated for their abilities to inhibit oxidative stress and to protect the immune system. Tested antioxidants given topically and/or systemically strongly inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced epidermal hyperplasia, proliferation, and inflammation. The hydroxylation of 2'-deoxyguanosine was markedly inhibited by topical and dietary administration of test variables, i.e., by approximately 40-70%. Simultaneous dietary and topical treatment with antioxidants reduced these biomarkers, showing strong additive and in some combinations synergistic effects. DMBA-mediated Ha-ras mutations in codon 61 were reduced by up to 50% with topical applications, but much higher inhibition was observed in mice treated with different combinations. The results of the present study clearly show impressive effects of combined topical and dietary treatments with above grape-derived antioxidants.

Differential effects of several phytochemicals and their derivatives on murine keratinocytes in vitro and in vivo: implications for skin cancer prevention.

The purpose of our study was to investigate in vitro the potential cancer preventive properties of several phytochemicals, i.e. grape seed extract (GSE), resveratrol (RES), ursolic acid (URA), ellagic acid (ELA), lycopene and N-acetyl-L-cysteine (NAC) to define the mechanisms by which these compounds may inhibit murine skin carcinogenesis. We measured quenching of peroxyl, superoxide and hydroxyl radicals by these phytochemicals. We also used adenosine triphosphate (ATP) bioluminescence, Caspase-Glo 3/7 and P450-Glo (CYP1A1 and CYP1B1) assays to study antiproliferative, proapoptotic and CYP-inhibiting effects of the phytochemicals. We next determined their effects on a 4 week inflammatory hyperplasia assay using 7,12-dimethylbenz[a]anthracene-induced murine skin carcinogenesis model to further understand their mechanism of action. Three murine keratinocyte cell lines, i.e. non-tumorigenic (3PC), papilloma-derived (MT1/2) and squamous cell carcinoma-derived (Ca3/7) cell lines, were used in in vitro assays. We have found that GSE, ELA and RES are potent scavengers of peroxyl and superoxide radicals. Statistically significant effects on activities of caspase-3 and -7 were observed only after GSE and URA treatments. All tested compounds protected cells from hydrogen peroxide-induced DNA damage. Using a short-term complete carcinogenesis assay, we have found that all selected compounds caused marked decreases of epidermal thickness and (except RES) reduced percentages of mice with mutation in codon 61 of Ha-ras oncogene. In conclusion, differential effects of tested phytochemicals on events and processes critical for the growth inhibition of keratinocytes in vitro and in vivo indicate that combinations of tested compounds may, in the future, better counteract both tumor initiation and tumor promotion/progression.

Desipramine inhibits the growth of a mouse skin squamous cell carcinoma cell line and affects glucocorticoid receptor-mediated transcription.

The purpose of this study was to examine the effect of tricyclic antidepressant desipramine (DMI) on the growth inhibition and translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus in cancerous and noncancerous cell lines and the effect of DMI on GR-mediated transcription. Nontumorigenic, immortalized keratinocytes cell line (3PC), papilloma (MT1/2), and squamous cell carcinoma (Ca3/7) cell lines were initially used to study the cell growth inhibition by DMI. Although, the growth of all three cell lines was suppressed by DMI, it was more effective in Ca3/7 cells. Therefore, we next examined the effect of DMI on Ca3/7 cells, resistant to growth inhibition by the synthetic glucocorticoid fluocinolone acetonide (FA). DMI inhibited cell proliferation in a time-dependent manner. The translocation of GR was induced by FA alone, DMI alone, and combination of both agents. FA induced GR-mediated transcription in Ca3/7 cells transfected with a luciferase reporter gene under the control of glucocorticoid response element (GRE), but DMI alone did not affect GR-mediated transcription. However, DMI inhibited FA-induced, GR-mediated transcription when both agents were given together. Pretreatment with DMI followed by combination of DMI and FA decreased GR-mediated transcription more than pretreatment with FA. The expression of metallothionein-1 (Mt-1) gene, which is regulated by GR, was induced significantly by the combination of DMI and FA, and enhanced significantly by pretreatment with FA but not DMI. DMI is suggested to inhibit the growth of Ca3/7 cells and to affect GR-mediated transcription.

[The biological role of D-glucaric acid and its derivatives: potential use in medicine]. / Biologiczna rola kwasu D-glukarowego i jego pochodnych; potencjalne zastosowanie w medycynie.

D-glucaric acid is a natural non-toxic compound produced in small amounts by mammals, including humans. In mammals, D-glucaric acid and D-glucaro-l,4-lactone are end-products of the D-glucuronic acid pathway. The enzyme D-glucuronolactone dehydrogenase has been found to be responsible for the oxidation of the lactone of D-glucuronic acid to D-glucaro-l,4;6,3-dilactone. This dilactone hydrolyzes spontaneously in aqueous solution to D-glucaro-l,4-lactone, a potent beta-glucuronidase inhibitor. D-glucaric acid is also found in many fruits and vegetables, with the highest concentrations found in grapefruits, apples, oranges, and cruciferous vegetables. b-glucuronidase is present in the circulation and probably all vertebrate tissues and is capable of hydrolyzing glucuronide conjugates. This enzyme is also produced by colonic microflora. Elevated b-glucuronidase activity is associated with an increased risk for various cancers, particularly hormone-dependent cancers such as breast and prostate cancer. D-glucaro-l,4-lactone increases detoxification of carcinogens and tumor promoters by inhibiting b-glucuronidase and preventing the hydrolysis of their glucuronides. D-glucaro-l,4-lactone was found to be formed from supplemented D-glucarate salt in the stomach and it is absorbed from the intestinal track, transported with the blood to different internal organs, and excreted in urine and, to a lesser extent, in bile. D-glucaro-l,4-lactone and its precursors exert their anticancer action in part through alterations in steroidogenesis accompanied by changes in the hormonal environment and proliferative status of the target organs. D-glucarates not only suppress cell proliferation and inflammation, but also induce apoptosis. By supplementing D-glucarates, one can favor the body's natural defense mechanism for eliminating carcinogens and tumor promoters and their effects.

Triterpenoid electrophiles (avicins) activate the innate stress response by redox regulation of a gene battery.

Avicins are proapoptotic and anti-inflammatory triterpene electrophiles isolated from an Australian desert tree, Acacia victoriae. The presence of two alpha,beta unsaturated carbonyl groups (Michael reaction sites) in the side chain of the avicin molecule prompted us to study its effects on NF-E2-related factor 2 (Nrf2), a redox-regulated transcription factor that controls the expression of a battery of detoxification and antioxidant proteins via its binding to antioxidant response element (ARE). Avicin D-treated Hep G2 cells showed translocation of Nrf2 into the nucleus and a time-dependent increase in ARE activity. These properties were sensitive to DTT, suggesting that avicins affect one or more critical cysteine residues, probably on the Keap1 molecule. Downstream of ARE, an activation of a battery of stress-induced proteins occurred. The implications of these findings were evaluated in vivo in mouse skin exposed to an ancient stressor, UV light. Avicins inhibited epidermal hyperplasia, reduced p53 mutation, enhanced apoptosis, decreased generation of 8-hydroxy-2'-deoxyguanosine, and enhanced expression of NADPH:quinone oxidoreductase 1 and heme oxygenase-1. These data, combined with our earlier published work, demonstrate that avicins represent a new class of plant stress metabolites capable of activating stress adaptation and suppressing proinflammatory components of the innate immune system in human cells by redox regulation. The relevance for treatment of clinical diseases in which stress responses are dysfunctional or deficient is discussed.

Short-term biomarkers of cigarette smoke condensate tumor promoting potential in mouse skin.

Previous studies demonstrated that cigarette smoke condensates (CSCs) possessing significantly different tumorigenic potentials according to a standardized 30-week mouse skin tumor-promotion protocol could likewise be discriminated utilizing short-term indices of sustained hyperplasia and/or inflammation (G. M. Curtin et al., 2004, Toxicol. Sci. 81, 14-25). The current study employed a truncated initiation-promotion protocol to further evaluate CSC-induced hyperplasia, examining issues related to time course of induction, existence of a threshold and suitable dynamic range for detectable responses, and reversibility. Condensate application (9-36 mg "tar"/200-microl application, thrice-weekly for 3-15 weeks) induced treatment-related increases for epidermal thickness, proliferative index as assessed by 5-bromo-2'-deoxyuridine (BrdU) labeling, and ornithine decarboxylase (ODC) expression. Interestingly, observed increases for interfollicular BrdU labeling and ODC expression were partially reversed but still elevated upon cessation of promotion, while increases within the perifollicular epidermis remained elevated at a level similar to that observed during CSC application. In particular, assessments based on perifollicular ODC expression would appear to provide a greater opportunity for test article discrimination based on a rapid time to induction, a low threshold and expanded dynamic range of responses, and the potential to account for irreversible changes. These findings are particularly intriguing based on reports suggesting that ODC expression may be necessary for tumor promotion and that mouse skin tumors originate primarily within the perifollicular epidermis.

Changes in leukemic cell nuclei revealed by differential scanning calorimetry.

Using differential scanning calorimetry we analyzed the thermal profiles of nuclei from normal and B-cell chronic lymphocytic leukemia mononuclear cells. Intact nuclear fraction of normal mononuclear cells is characterized by four thermal transitions, i.e., at 60, 70, 83 and 103 degrees C. Leukemic nuclear samples revealed the transitions at 67 and 83 degrees C, however, in more aggressive stage of the disease additional thermal peaks at 76 and 93 degrees C were observed. Our very preliminary results revealed that mononuclear cell nuclear fraction from blood of patients responding to the used therapy, i.e., cladribine alone or its combination with mitoxantrone and cyclophosphamide indicates decrease (or even loss) of transition at 93 degrees C concomitant with increase of transition at 76 degrees C. A complementary study showed that in mononuclear cells of patients who appeared to be sensitive to chemotherapy the decrease of antiapoptotic Bcl-2 protein expression and signs of apoptotic morphology were observed.

Short-term in vitro and in vivo analyses for assessing the tumor-promoting potentials of cigarette smoke condensates.

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Expression of a 65 kDa oncofetal protein in human prostatic carcinoma.

The concentration of a novel 65 kDa oncofetal protein, p65, was measured in the sera of patients with prostatic malignancies using an enzyme-linked immunosorbent assay. The prostate cancer sera were positive for p65 in 40 out of 59 cases (68%), while only 7 of 79 normal sera (9%) and 12 of 61 sera from patients with benign diseases (20%) were positive. The detection system had an overall sensitivity of 68% and a specificity of 86%. Elevated p65 levels correlated positively with the pathologic stages of the prostate cancer. Using this marker in concert with PSA may increase our ability to evaluate treatment response and aid in early detection of prostate cancer.

Detoxifying cancer causing agents to prevent cancer.

Different vitamins and other micronutrients in vegetables, fruits, and other natural plant products may prevent cancer development (carcinogenesis) by interfering with detrimental actions of mutagens, carcinogens, and tumor promoters. The goal of current studies in cancer prevention is to determine the mechanisms of synergistic action of the natural source compounds known to inhibit one or more stages of carcinogenesis, that is, initiation and promotion/progression. Many natural cancer preventive agents are effective inhibitors of tumor initiation, promotion, and/or progression. The mechanism of action is related to their abilities to prevent critical carcinogen metabolism and to increase detoxification of carcinogens and tumor promoters. The authors review here the potential role of the detoxification system and, in particular, the roles of D-glucaric acid and the enzyme beta-glucuronidase in early detection and prevention of cancer. There is now growing evidence for the possible control of different stages of the cancer induction by inhibiting beta-glucuronidase with D-glucaric acid derivatives, especially with its salts (D-glucarates). D-Glucaric acid has been found in many vegetables and fruits. Therefore, the consumption of fruits and vegetables naturally rich in D-glucaric acid or self-medication with D-glucaric acid derivatives such as calcium D-glucarate offers a promising cancer prevention approach.

Effects of combined phytochemicals on skin tumorigenesis in SENCAR mice.

The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans.

Modulation of biomarkers related to tumor initiation and promotion in mouse skin by a natural ß-glucuronidase inhibitor and its precursors.

Carcinogen-mediated labilization of lysosomal enzymes such as ß-glucuronidase (ßG) is often associated with the general process of inflammation. Therefore, the primary goal of this study was to demonstrate that exposing the skin of SENCAR mice to the natural ßG inhibitor D-glucaro-1,4-lactone (1,4-GL) and its precursor D-glucuronic acid-γ-lactone (GUL), prior to and during 7,12-dimethylbenz[α]anthracene (DMBA) treatment inhibits not only epidermal hyperplasia but also inflammation in the mouse skin complete carcinogenesis model, i.e., the 4-week inflammatory-hyperplasia assay. Topical administration of 1,4-GL or GUL prior to repetitive, high-dose DMBA treatment markedly and in a dose-related manner inhibited DMBA-induced epidermal hyperplasia (i.e., up to 57%). DMBA-mediated Ha-ras mutations in codon 61 were reduced by up to 78% by 1,4-GL. DMBA-induced inflammation, as measured by dermal leukocyte counts and immunologically, was inhibited by up to 37% by topical 1,4-GL but not by GUL. The inhibition of cellular proliferation and inflammation coincided with the inhibition of ßG expression. Thus, the present study suggests that in the DMBA-induced complete skin carcinogenesis model, 1,4-GL when applied topically had both anti-proliferative properties as well as anti-inflammatory properties, whereas GUL had only anti-proliferative when applied topically. However, the number of inflammatory cells in the dermal portion of the skin of mice was significantly reduced by dietary treatment of GUL, whereas both topical and dietary treatments with 1,4-GL were very effective.

Dietary D-glucarate effects on the biomarkers of inflammation during early post-initiation stages of benzo[a]pyrene-induced lung tumorigenesis in A/J mice.

Previous studies showed that dietary calcium D-glucarate (CG) inhibited benzo[a]pyrene (B[a]P)-induced A/J mouse lung tumorigenesis, suppressing cell proliferation and chronic inflammation and inducing apoptosis during late post-initiation stages. The present study aimed to investigate changes in the homeostasis of cytokines in blood serum, as well as alterations in biomarkers of inflammation and apoptosis in lung tissue caused by dietary CG during early post-initiation stages of B[a]P-induced lung tumorigenesis. Two doses of 3 mg of B[a]P were given intragastrically to A/J mice 2 weeks apart. CG administration in the AIN-93G diet (2 and 4%, w/w) commenced at 2 weeks following the second dose of B[a]P. The levels of interleukin (IL)-6, IL-10 and tumor necrosis factor α (TNFα) in blood serum were investigated by FCAP array analysis. Two weeks after the second dose of B[a]P, approximately 8- and 28-fold increases of TNFα and IL-6, respectively, occurred in the blood serum and an approximately 16% decrease of IL-10 levels compared to the untreated control group was noted. At 4 weeks after the second dose of B[a]P and after 2 weeks of CG administration in the diet, the 2 and 4% CG diets significantly reduced the levels of IL-6 and TNFα (by 70 and 33%, respectively). In a dose-related manner, the diets also increased the level of anti-inflammatory cytokine IL-10 compared to the B[a]P group. At 6 weeks after the second dose of B[a]P, the cytokine levels in the serum continued to show a decrease in the CG-treated groups. These events are accompanied by an increased level of cleaved caspase-9 product with a molecular weight of 37 kDa. In conclusion, dietary D-glucarate decreases the level of proinflammatory cytokines, increases the level of the anti-inflammatory cytokine IL-10 during early post-initiation stages of B[a]P-induced lung tumorigenesis in A/J mice and affects apoptotic induction.

Effects of desipramine on the cell cycle and apoptosis in Ca3/7 mouse skin squamous carcinoma cells.

Desipramine (DMI) has been reported to induce glucocorticoid receptor-mediated signal transduction in recent studies. It has been suggested that a non-glucocorticoid receptor signaling pathway might play an important role in skin squamous carcinoma Ca3/7 cells. The aim of this study was to investigate the growth inhibitory effects of DMI on Ca3/7 cells by evaluating the mRNA expression of genes related to apoptosis and cell cycle progression. Hoechst nuclear staining and DNA fragmentation assays were used to detect apoptosis, and the cell cycle was analyzed by flow cytometry. Apoptotic bodies in the nuclei of cells and DNA fragmentation were observed when the Ca3/7 cells were treated with 20 microM DMI for 24 h. Quantitative RT-PCR (reverse transcriptional-polymerase chain reaction) showed that DMI caused a decrease in Bcl-2 and survivin but not Bcl-xL gene expression and an increase in the expression of Bax, Apaf-1, caspase-3 and caspase-7 in a dose- and time-dependent manner. DMI also caused translocation of the apoptosis-inducing factor from the cytoplasm to the nucleus as well as cell cycle arrest in the Ca3/7 cells. Quantitative RT-PCR revealed that DMI decreased the expression of the PCNA gene and caused an increase in the expression of the p21 and p27 genes in the Ca3/7 cells. Our results showed that DMI inhibited the growth of Ca3/7 cells by inducing both apoptosis and cell cycle arrest.

Synergistic effects of combined phytochemicals and skin cancer prevention in SENCAR mice.

The purpose of our study was to determine the inhibitory effect of combined phytochemicals on chemically induced murine skin tumorigenesis. Our hypothesis was that concurrent topical and dietary treatment with selected compounds would lead to more efficient prevention of skin cancer. We tested ellagic acid and calcium D-glucarate as components of diets, while resveratrol was applied topically; grape seed extract was applied topically or in the diet. The 4-week inflammatory-hyperplasia assay based on the 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis model in SENCAR mice was used. We have found that all the selected combinations caused a marked decrease of epidermal thickness compared with the DMBA-treated group and also with groups treated with a single compound and DMBA. All combinations of resveratrol with other compounds showed a synergistic effect on hyperplasia and Ha-ras mutations. Skin tissue of mice receiving the combinations showed decreased cell proliferation and Bcl2 expression; decreased p21, a regulator of cell cycle; and decreased marker of inflammation cyclooxygenase-2. All the selected combinations diminished the DMBA-induced mRNA expression of the CYP1B1 level, and also caused a marked decrease of proto-oncogenes c-jun and c-fos, components of transcription factor activator protein. In conclusion, all combinations showed either additive or synergistic effects and their joint actions allowed for decreasing the doses of the compounds. Especially, resveratrol combinations with ellagic acid, grape seed extract, and other phytochemicals are very potent inhibitors of skin tumorgenesis, based on the suppression of epidermal hyperplasia as well as on the modulation of intermediate biomarkers of cell proliferation, cell survival, inflammation, oncogene mutation, and apoptosis.