Inhibitors Of Skin Tumor Promotion Research Articles

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.

Rapamycin Is a Potent Inhibitor of Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate

Aberrant activation of phosphoinositide-3-kinase (PI3K)/Akt signaling has been implicated in the development and progression of multiple human cancers. During the process of skin tumor promotion induced by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), activation of epidermal Akt occurs as well as several downstream effectors of Akt, including the activation of mTORC1. Rapamycin, an established mTORC1 inhibitor, was used to further explore the role of mTORC1 signaling in epithelial carcinogenesis, specifically during the tumor promotion stage. Rapamycin blocked TPA-induced activation of mTORC1 as well as several downstream targets. In addition, TPA-induced epidermal hyperproliferation and hyperplasia were inhibited in a dose-dependent manner with topical rapamycin treatments. Immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin also significantly decreased the number of infiltrating macrophages, T cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Using a two-stage skin carcinogenesis protocol with 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as the promoter, rapamycin (5-200 nmol per mouse given topically 30 minutes prior to TPA) exerted a powerful antipromoting effect, reducing both tumor incidence and tumor multiplicity. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited further growth. Overall, the data indicate that rapamycin is a potent inhibitor of skin tumor promotion and suggest that signaling through mTORC1 contributes significantly to the process of skin tumor promotion. The data also suggest that blocking this pathway either alone or in combination with other agents targeting additional pathways may be an effective strategy for prevention of epithelial carcinogenesis.

Abstract 1847: Effect of rapamycin and metformin on skin tumor promotion in mice overweight and obese

Abstract The prevalence of obesity in the US has risen drastically over the past few decades and is an established risk factor for several human cancers. Insights into the mechanisms underlying this link are urgently needed to develop strategies for treatment and prevention. Various studies have shown that calorie restriction (CR) during tumor promotion in the two-stage model of skin carcinogenesis causes a significant reduction in tumorigenesis. In addition, diet induced obesity (DIO) leads to insulin resistance, increased circulating levels of IGF-1 and increased susceptibility to tumor development. Current data indicate that signaling through the IGF-1R and EGFR as well as several downstream signaling pathways plays an important role in dietary energy balance effects on tumor promotion during epithelial carcinogenesis. The current study was designed to evaluate the ability of rapamycin (an established mTORC1 inhibitor) to inhibit or reverse the effects of overweight/obesity on skin tumor promotion in mice. In initial experiments using female FVB mice on an AIN76A diet, rapamycin (5-1000 nmol per mouse) was found to be a potent inhibitor of skin tumor promotion by TPA. In this regard, rapamycin reduced both tumor multiplicity and incidence in a dose dependent manner. A dose of 5 nmol rapamycin given 30 min before each TPA treatment reduced the number of papillomas per mouse by approximately 50%. Rapamycin treatment inhibited TPA-induced mTORC1 activation as well as downstream signaling proteins p70S6KT389, prS6S240/244 and p4EBP1T37/46. Multiple treatment experiments were performed to evaluate the effects of rapamycin (5-200 nmol per mouse) on TPA-induced epidermal hyperproliferation (as assessed by epidermal hyperplasia and epidermal labeling index). Rapamycin significantly inhibited TPA-induced epidermal hyperproliferation in a dose dependent manner. In addition, immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin significantly decreased the number of infiltrating macrophages, T-cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited their growth. Two-stage skin carcinogenesis studies are currently underway to further evaluate the impact of rapamycin treatment on mice made overweight (AIN76A) or obese (DIO diet; 60 Kcal % fat) via dietary energy balance manipulation. In addition, groups of mice are also being treated with metformin. These ongoing studies will clarify whether suppression of the mTORC1 signaling pathway is a viable strategy for reversing the effects of obesity on tumor development in this well established model of epithelial carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1847. doi:10.1158/1538-7445.AM2011-1847

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

Abstract 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.

Abstract 2481: Rapamycin is a potent inhibitor of skin tumor promotion by TPA

Abstract The activation of Akt signaling occurs early during skin tumorigenesis. Data from our lab has demonstrated that the phorbol ester TPA leads to activation of Akt as well as several downstream effectors of Akt. Of particular interest is the Akt downstream signaling molecule, mTOR, which is elevated in the epidermis of wild-type mice following treatment with TPA. These studies suggest that activation of mTOR may contribute to skin tumor promotion. To further test this hypothesis, rapamycin, which is an established mTORC1 inhibitor, was used to further explore the role of mTOR signaling in epithelial carcinogenesis and specifically during tumor promotion. In a multiple treatment regimen, rapamycin was applied topically 30 min prior to TPA at doses of 1 umol, 100 nmol and 20 nmol. Rapamycin, in a dose dependent manner inhibited TPA induced mTOR activity as well as several downstream signaling proteins including p70S6KT389 and pS6S240/244 as well as p4EBP1T37/46, the FRAP/mTOR phosphorylation site of translational repressor protein 4EBP1. In addition, the highest dose of rapamycin caused a reduction in Akt-mediated phosphorylation of Akt downstream targets GSK3B as well as PRAS40 as assessed by western blotting using phosphospecific antibodies. These data suggest that multiple treatments with rapamycin at higher doses blocked both mTORC1 and mTORC2, while lower doses primarily affected mTORC1. Multiple treatment experiments evaluating the effects of rapamycin (5-200 nmol/mouse) on epidermal hyperplasia and epidermal labeling index, two short-term markers of tumor promotion by TPA, demonstrated a significant inhibition of both parameters in a dose dependent manner. Using a two-stage chemical skin carcinogenesis model with the standard DMBA/TPA protocol, the ability of rapamycin to block skin tumor promotion by TPA was evaluated. Rapamycin exerted a powerful anti-promoting effect reducing the average number of papillomas per mouse as well as the overall incidence of papillomas. In this regard, even a 5 nmol topical dose of rapamycin given 30 min prior to each TPA treatment produced approximately 50% inhibition of papilloma formation. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited growth. Skin tumors from this study were collected from TPA or TPA + rapamycin treated groups and are being evaluated for expression of proteins involved in cell cycle regulation as well as proteins involved in autophagy and apoptosis. The results of these experiments will be presented. Collectively, the current results indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion, and that blocking this pathway may be an effective strategy for prevention of epithelial carcinogenesis. Supported by NIH grant CA37111. 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 2481.

Cancer chemopreventive activity of synthetic colorants used in foods, pharmaceuticals and cosmetic preparations

In continuation with our studies to uncover cancer chemopreventive effects of non-toxic natural colorants and other products of biologic and synthetic origin, we tested several Food and Drug Administration-approved synthetic colorants for antitumor promoting potential by the in vitro Epstein–Barr virus early antigen activation in Raji cells in response to the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA). Among 29 such colorants used in foods, pharmaceuticals and cosmetics and evaluated in vitro, six of the 10 most effective had an azo group. Three structurally unrelated colorants tested in this assay were also studied in vivo for chemoprevention of 7,12-dimethylbenz[ a]anthracene (DMBA)-induced TPA-promoted mouse skin carcinogenesis. The results indicate that tartrazine, indigo carmine and erythrosine are potent inhibitors of skin tumor promotion in mice treated with DMBA and TPA.

Inhibition of the induction of cancer by antioxidants.

Many different antioxidants have been shown to inhibit the induction of cancer by a wide variety of chemical carcinogens and/or radiation at many target sites in mice, rats, and hamsters. Evidence is accumulating that suggests that free radicals are important in all stages of chemical carcinogenesis. Both carcinogens and tumor promoters have also been shown to decrease the cellular activity of superoxide dismutase and catalase. A number of antioxidants and related compounds were tested to determine if they would inhibit either skin tumor initiation, promotion, and/or progression. In terms of skin tumor initiation, BHT, vitamin E and C and CuDIPS have been found to inhibit DMBA skin tumor initiation by approximately 50%. The mechanism of action of these compounds appears to be related to their effect on the metabolism of DMBA, since BHT and CuDIPS do not inhibit the initiating activity of BP-diol-epoxide and MNNG. Although several antioxidants do inhibit skin tumor initiation by procarcinogens, antioxidants are in general much more effective inhibitors of skin tumor promotion. BHT, BHA, parahydroxyanisole, disulfiran, and vitamin E and C inhibit skin tumor promotion by TPA and benzoyl peroxide by greater than 90%. We also determined the effect of free radical scavengers on the progression process. Of the agents tested glutathione and N-acyl dehydroalamines were the most effective in reducing carcinoma incidence. Diethyl maleate, a chemical that reduces glutathione levels, was effective in enhancing progression. In addition overexpression of g-glutamylt-ranspeptidase (GGT) which leads to a reduction in cellular glutathione levels also enhances progression. These results suggest that GGT has a functional role in skin tumor progression, and that a number of antioxidants are either effective inhibitors of skin tumor initiation, promotion, and/or progression.

Inhibition of skin tumor initiation, promotion, and progression by antioxidants and related compounds.

Antioxidants have been shown to inhibit the induction of cancer by a wide variety of chemical carcinogens and radiation at many target sites in mice, rats, hamsters, and man. Evidence is accumulating that suggests that free radicals are important in all stages of chemical carcinogenesis. Both carcinogens and tumor promoters have also been shown to decrease the cellular activity of superoxide dismutase and catalase. A number of antioxidants and related compounds were tested to determine if they would inhibit either skin tumor initiation, promotion, or progression. In terms of skin tumor initiation, compounds such as BHT, vitamins E and C, and CuDIPS have been found to inhibit DMBA skin tumor initiation. The mechanism of action of these compounds appears to be related to their effect on the metabolism of DMBA, as BHT and CuDIPS do not inhibit the initiating activity of BP-diol-epoxide and MNNG. Although several antioxidants do inhibit skin tumor initiation by procarcinogens, antioxidants are in general much more effective inhibitors of skin tumor promotion. BHT, BHA, parahydroxyanisole, disulfiran, and vitamins E and C as well as many other antioxidants are very effective inhibitors of skin tumor promotion. We also determined the effect of free radical scavengers on the progression process. Of the agents tested, glutathione and N-acyl dehydroalamines were the most effective in reducing carcinoma incidence. Diethyl maleate, a chemical that reduces glutathione levels, was effective in enhancing progression. In addition, overexpression of gamma-glutamyltranspeptidase (GGT), which leads to a reduction in cellular glutathione levels, also enhances progression.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of skin-tumor promotion. XIII. Inhibitory effects of euglobals and their related compounds on Epstein-Barr virus activation and on two-stage carcinogenesis of mouse skin tumors. (2).

One hundred and fifteen synthesized mono, di, and trihydroxybenzamide and thiobenzamide derivatives having structures related to euglobals were examined for their inhibitory effects on Epstein-Barr virus (EBV) activation by 12-O-tetradecanoylphorbol-13-acetate (TPA) as a primary screening test for anti-tumor-promoters. In general, 3-acyl-2,4,6-trihydroxybenzamide and 3-acyl-2,4,6-trihydroxythiobenzamide derivatives exhibited strong or moderate activities, and the latter compounds were less cytotoxic than the former. Meanwhile, little or no activity was observed with mono and dihydroxybenzamide and dihydroxythiobenzamide derivatives. Structural requirements for the activities of these compounds have been discussed in detail. Among the above compounds, compounds 36 and 73, which were significantly active on the inhibition of EBV activation, were investigated using a two-stage mouse skin carcinogenesis test induced by 7,12-dimethylbenz[a]anthracene (DMBA) and TPA. The results of the in vivo test showed that both compounds have a stronger inhibitory effect than that of the well-known anti-tumor-promoter, glycyrrhetic acid. These results suggested that the two compounds might be valuable as anti-tumor-promoters in chemical carcinogenesis.

Influences of sphingosine on two-stage skin tumorigenesis in Sencar mice

Sphingosine (SPH) was studied as an inhibitor of skin tumor promotion in skin cancer initiated by 7,12-dimethylbenz[ a]-anthracene (DMBA) and promoted by 12-0-tetradecanoylphorbol-13-acetate (TPA). Two tumorigenesis studies were conducted using female Sencar mice treated with 10 nmol DMBA in 0.2 ml acetone at 8 weeks of age and promoted beginning 1 week later with 3.2 nmol TPA applied twice per week. In the high-dose study, 10 μmol SPH was applied 30 min before each TPA treatment. The low-dose study used 0.5, 0.05, or 0.01 μmol treatments with SPH 30 min before TPA. In the high-dose study SPH treatment alone following initiation by DMBA, and SPH treatment preceding TPA in DMBA-initiated mice accelerated the development of papillomas in comparison with the DMBA/TPA-treated group. The low-dose experiment showed no consistent alteration of tumorigenesis by SPH in the DMBA/TPA-treated groups, and low doses of SPH following DMBA did not promote skin tumorigenesis. SPH treatment did not alter body weight in either experiment.

Studies on inhibitors of skin tumor promotion. XI. Inhibitory effects of flavonoids from Scutellaria baicalensis on Epstein-Barr virus activation and their anti-tumor-promoting activities.

To search for possible anti-tumor-promoters, fourteen flavones obtained from the root of Scutellaria baicalensis were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation by a short-term in vitro assay. Among these flavones, 5,7,2'-trihydroxy- and 5,7,2',3'-tetrahydroxyflavone showed remarkable inhibitory effects on the EBV-EA activation, and the effect of the latter on Raji cell cycle was also examined by flow cytometer. These two flavones exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Inhibitors of skin-tumor promotion. VIII. Inhibitory effects of euglobals and their related compounds on Epstein-Barr virus activation. (1).

Twelve euglobals from Eucalyptus globulus and their twenty-six related compounds were examined for their inhibitory effects on Epstein-Barr virus activation by a short-term in vitro assay. The results showed that most of the euglobals having monoterpene structures, and euglobal-III (8) had strong inhibitory activity. Grandinol (18), homograndinols (19 and 20), and compounds 26, 27, 28, and 32 showed stronger inhibitory effects. Based on the results, the structural requirements for the activity of these compounds were discussed.

Studies on Inhibitors of Skin-Tumor Promotion. Inhibitory Effects of Triterpenes from Cochlospermum tinctorium on Epstein-Barr Virus Activation

Arjunolic acid, an oleanene-type triterpene isolated from the rhizome of Cochlospermum tinctorium, its triacetate derivative, and their methyl esters were tested using the short-term in vitro assay on EBV-EA activation in Raji cells induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Their inhibitory effects on skin tumor promotors were found to be greater than those of previously studied natural products.

Effect of chemical constituents from plants on 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice.

The induction of edema in the mouse ear has been established as a reliable in vivo assay for tumor promoters. Therefore, inhibitors of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema are most likely to be inhibitors of skin tumor promotion. Besides the application for this assay for the screening of compounds, it also allows comparison of the activities of groups of related compounds such as flavonoids. Results obtained in this way showed that the double bond at C-2 and C-3 of the flavonoid structure is a prerequisite for anti-tumor-promoting activity, and indicated that activity in this screening assay for inhibitors of TPA-induced ear edema reflects the anti-tumor-promoting effect in two-stage carcinogenesis.

The mouse ear edema: A quantitatively evaluable assay for tumor promoting compounds and for inhibitors of tumor promotion

The induction of edema in the mouse ear has been established as a reliable in vivo assay for tumor promoting compounds and for inhibitors of tumor promotion. Apparently all skin tumor promoting compounds induce an edema, and all inhibitors of a tumor promoter-induced edema are most likely inhibitors of skin tumor promotion. No exception to this rule has been found as yet. Besides the application of this assay in the screening of compounds, the assay allows comparison of various compounds (e.g. complete and incomplete tumor promoters) with respect to their kinetics and dosage response in the induction of edema.

Inhibition of mouse skin tumor promotion by several inhibitors of arachidonic acid metabolism.

12-O-Tetradecanoylphorbol-13-acetate promotion of skin tumors in mice can be inhibited by topical application of either the phospholipase A2 inhibitor dibromoacetophenone or the cyclooxygenase-lipoxygenase inhibitors 5,8,11,14-eicosatetrayonic acid or 1-phenyl-3-pyrazolidinone. The phospholipase A2 inhibitors in particular appear to be among the most potent inhibitors of skin tumor promotion known. These results support the hypothesis that at least some of the products of arachidonic acid transformation are essential for tumor promotion.