Magdalena C. Kowalczyk

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. Cancer Prev Res; 3(2); 170–8

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.

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.

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.

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. Mol. Carcinog.

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 GRs 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.

Effect of phosphodiesterase antagonists on glucocorticoid mediated growth inhibition in murine skin cell lines

The effects of two cyclic nucleotide phosphodiesterase type 4 (PDE4) inhibitors on proliferation of cell lines representing different stages of mouse skin tumorigenesis were studied. Skin papillomas and carcinomas become resistant to the growth inhibition by glucocorticoids. Their control of cellular functions is mediated by a well-known transcription factor, glucocorticoid receptor. The primary aim of the present study was to determine whether the PDE4 inhibitors, that raise intracellular cAMP levels, can increase the sensitivity of mouse skin papillomas and carcinomas to the glucocorticoids. We sought to establish the effect of cAMP signaling on the glucocorticoid receptor function using well-known model representing non-tumorigenic keratinocyte cell line (3PC), papilloma (MT1/2) and squamous cell carcinoma cell line (Ca3/7). These cells were treated with the glucocorticoid fluocinolone acetonide (FA) alone or in concert with PDE4 inhibitors--rolipram or YM976. Results of our study revealed that both PDE4 inhibitors may increase the sensitivity of transformed cell lines to the growth inhibitory effect of FA. In the transformed cell lines, changes in the viability of cells were accompanied by an increase in mRNA level of two negative regulators of the cell cycle--p21 and p27 proteins. Co-treatment with PDE4 inhibitors and FA caused inhibition of an endogenous glucocorticoid-responsive gene (MT-1) expression. Thus, the PDE4 inhibitors exerted a differential effect on non-transformed and transformed keratinocytes and on glucocorticoid receptor signal transduction. These findings warrant further studies to clarify the mechanism by which PDE4 inhibitors modulate glucocorticoid receptor signal transduction in transformed cells.

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.

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.

Abstract 3470: Effects of combined phytochemicals on skin tumorgenesis in SENCAR mice

The purpose of our study was to determine the effect of combined action of phytochemicals on early stages of skin tumorgenesis, i.e., initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (URA) 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 (6-7 weeks old) with shaved backs received one topical dose of DMBA (20 nmol), then after one month, two weekly doses (2 µg each) of TPA (tumor promoter) for 14 weeks until sacrifice. The phytochemicals RES (2.5 μmol) or URA (1 μmol) were applied topically 20 min prior to DMBA or TPA treatment and 2% dietary CG was given in the AIN-93G diet from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. We determined the tumor incidence (% of mice with tumors) and tumor multiplicity (number of tumors per mouse) and using RT-PCR and immunochistochemical analyses, we checked the expression of some cell proliferation and inflammation related genes. URA applied alone and in combination with CG during the promotion stage was a strong inhibitor of tumor multiplicity and tumor incidence. Combinations of URA with dietary CG appeared to show some synergistic effects. All compounds and their combinations reduced epidermal proliferation, but only URA and a combination CG/URA applied during promotion significantly reduced hyperplasia, as determined by epidermal thickness measurements taken at 14 weeks of TPA treatment. DMBA/TPA treatments resulted in epidermal hyperplasia, characterized by significant increase in transcription factor AP-1 components and nuclear factor kappa B (NFkB) expression levels with a simultaneously increased expression of the inflammation markers COX-2 and IL-6, compared to untreated controls. All anti-promotion treatments (either single compounds or their combinations) caused a marked decrease of inflammation related genes levels compared to the DMBA/TPA control. On the other hand, only URA and URA with dietary CG diminished the induced expression of proto-oncogenes c-jun and c-fos (components of AP-1) and the expression of NFkB protein p50. These results show that URA is a potent inhibitor of skin tumor promotion and it may be useful in the prevention of skin cancer and other epithelial cancers in humans. Supported by NIH grants CA 102747 and P30 CA 54174-16S1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3470.