Inhibit Skin Tumor Promotion Research Articles

Evaluation of pentacyclic triterpenes found in Perilla frutescens for inhibition of skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

A series of pentacyclic tritperpenes found in Perilla frutescens (P. frutescens), including ursolic acid (UA), oleanolic acid (OA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) were evaluated for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and compared to UA as the prototype compound. All compounds were given topically 30 min prior to each TPA application and significantly inhibited skin tumor promotion. 3-epiCA and MA were significantly more effective than UA at inhibiting tumor development. All of these compounds significantly inhibited epidermal proliferation induced by TPA, however, CA, 3-epiCA and MA were more effective than UA. All compounds also reduced skin inflammation (assessed by infiltration of mast cells and T-cells) and inflammatory gene expression induced by TPA, however, 3-epiCA and MA were again more effective than UA. The greater ability of 3-epiCA and MA to inhibit skin tumor promotion was associated with greater reduction of Cox-2 and Twist1 proteins and inhibition of activation (i.e., phosphorylation) of IGF-1R, STAT3 and Src. Further study of these compounds, especially 3-epiCA and MA, for chemopreventive activity in other cancer model systems is warranted.

Effect of Combined Treatment with Ursolic Acid and Resveratrol on Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate.

In this study, the effects of combining ursolic acid + resveratrol, for possible combined inhibitory effects on skin tumor promotion, were evaluated. Ursolic acid, resveratrol, and the combination of ursolic acid + resveratrol were applied topically prior to 12-O-tetracanoylphorbol-13-acetate (TPA) treatment on mouse skin to examine their effect on TPA-induced signaling pathways, epidermal hyperproliferation, skin inflammation, inflammatory gene expression, and skin tumor promotion. The combination of ursolic acid + resveratrol produced a greater inhibition of TPA-induced epidermal hyperproliferation. The combination of ursolic acid + resveratrol inhibited TPA-induced signaling pathways, including EGFR, STAT3, Src, Akt, Cox-2, Fas, NF-κB, p38 MAPK, c-Jun, and JNK1/2 while increasing levels of tumor suppressors, such as p21 and PDCD4, to a greater extent compared with the groups treated with the individual compounds. Ursolic acid + resveratrol also induced a dramatic increase of p-AMPK-α(Thr172). Combined treatment with ursolic acid + resveratrol resulted in a greater inhibition of expression of proinflammatory cytokines, including Il1a, Il1b, and Il22. Furthermore, NF-κB, Egr-1, and AP-1 DNA binding activities after TPA treatment were dramatically decreased by the combination of ursolic acid + resveratrol. Treatment with ursolic acid + resveratrol during skin tumor promotion with TPA produced greater inhibition of tumor multiplicity and tumor size than with either agent alone. Collectively, the greater ability of the combination of ursolic acid + resveratrol to inhibit skin tumor promotion was due to the greater inhibitory effects on growth factor and inflammatory signaling, skin inflammation, and epidermal hyperproliferation induced by TPA treatment.

Abstract 1899: Anti-skin tumor promoting effects of pentacyclic triterpenes found in Perilla frutescens

Abstract In this study, we conducted a comprehensive examination of a series of pentacyclic tritperpenes found in P. frutescens, including ursolic acid (UA) oleanolic acid (OA), augustic acid (AA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by TPA. For these experiments, female ICR mice (7-9 weeks of age) were treated with either acetone or 2 μmol of UA, OA, AA, CA, 3-epiCA, MA, or 3-epiMA 30 min prior to TPA (6.8 nmol) treatment The treatment protocol involved twice weekly treatments over a two week period. Mouse epidermis (6 hr time point) and whole skin (48 hr time point) were collected for Western blot analysis and histological analysis, respectively. Pretreatment with these compounds inhibited the activation of JNK1/2, Src, and Stat3 as well as the induction of Cox-2 protein. Pretreatment with the compounds also reversed the effect of TPA on PDCD4 and p27, and increased the activation of AMPK and LKB1. We also examined the effect of the compounds on TPA-induced epidermal hyperproliferation as assessed by epidermal thickness and epidermal labeling index (LI). All of the compounds examined significantly inhibited TPA-induced epidermal hyperproliferation (significant reductions in both epidermal thickness and LI). In addition, the effect of the triterpenes on TPA-induced infiltration of mast cells in dermis was evaluated. UA significantly inhibited the infiltration of dermal mast cells induced by TPA. Notably, OA, AA, CA, 3-epiCA, MA, and 3-epiMA produced a greater inhibitory effect than that seen with UA on the number of infiltrated mast cells. Thus, UA and a series of related triterpenes differentially inhibited multiple signaling pathways, epidermal hyperproliferation, and a marker of skin inflammation (infiltrated mast cells) induced by TPA. Several of the compounds including CA, 3-epiCA, MA, and 3-epiMA were particularly effective in these experiments. We are currently testing the ability of this group of compounds to inhibit skin tumor promotion by TPA in a two-stage skin carcinogenesis experiment. Collectively, the current data suggest that several pentacyclic triterpenes, in addition to UA, may have potent chemopreventive activity. Research supported by CA164159. Citation Format: Jiyoon Cho, Okkyung Rho, Jacob Junco, Thomas J. Slaga, Andrew M. Camelio, Dionicio Siegel, John DiGiovanni. Anti-skin tumor promoting effects of pentacyclic triterpenes found in Perilla frutescens. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1899. doi:10.1158/1538-7445.AM2015-1899

Abstract 5357: Targeting mTORC1 suppresses proliferation of keratinocyte stem cells and inhibits skin tumor promotion in mice

Abstract Considerable evidence indicates a crucial role of Akt and mTORC1 signaling in cancer growth and progression. Previously, we discovered that Akt and mTORC1 signaling pathways play an important role in epithelial multistage carcinogenesis using the mouse skin model, especially during the tumor promotion stage. Targeting mTORC1 with rapamycin effectively blocked mTORC1 downstream signaling, epidermal hyperproliferation induced by TPA in a dose-dependent manner in both wild-type and BK5.AktWT transgenic mice. Rapamycin effectively inhibited TPA skin tumor promotion in TPA-hypersensitive BK5.AktWT transgenic mice although higher doses were required to achieve similar levels of inhibition compared to wild-type mice. Elevated Akt expression led to a significant expansion of the label retaining cells (LRCs) as well as a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K and phospho-PRAS40 compared to wild-type mice. Furthermore, TPA-mediated proliferation and migration of LRCs to the interfollicular epidermis from the hair follicle were significantly inhibited by rapamycin. PRAS40 was originally identified as a novel Akt substrate through Akt-mediated phosphorylation at Thr246. Recently, PRAS40 has been identified as a negative regulator when bound to mTORC1. We have used the bovine keratin 5 (BK5) promoter to generate transgenic mice that overexpress a mutant form of PRAS40 (PRAS40T246A) to further examine the importance of mTORC1 signaling in epithelial carcinogenesis. BK5.PRAS40T246A transgenic mice were significantly less sensitive to TPA-induced epidermal hyperproliferation and hyperplasia measured by both epidermal thickness and labeling index. Overexpression of mutant PRAS40T246A in the epidermis led to a significant suppression of skin tumor development by 63% as well as reductions in tumor weight and tumor size compared to wild-type mice. Immunoprecipitation analysis showed that PRAS40T246A in BK5.PRAS40T246A mice remained bound to raptor even after TPA treatment. In addition, BK5.PRAS40T246A mice displayed reduced TPA-induced mTORC1 signaling and cell cycle progression, but increased autophagy signaling and elevated apoptotic cells in the epidermis following TPA treatment. Furthermore, TPA-induced proliferation and migration of LRCs was also effectively inhibited in BK5.PRAS40T246A mice. Collectively, the current data demonstrate that mTORC1 downstream signaling pathways contribute significantly to the process of skin tumor promotion leading to a potential target for chemoprevention of cancer. Supported by CA037111 and CA0129409. Citation Format: Okkyung Rho, Jiyoon Cho, John DiGiovanni. Targeting mTORC1 suppresses proliferation of keratinocyte stem cells and inhibits skin tumor promotion in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5357. doi:10.1158/1538-7445.AM2014-5357

Metformin Inhibits Skin Tumor Promotion in Overweight and Obese Mice

In the present study, the ability of metformin to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was analyzed in mice maintained on either an overweight control diet or an obesity-inducing diet. Rapamycin was included for comparison, and a combination of metformin and rapamycin was also evaluated. Metformin (given in the drinking water) and rapamycin (given topically) inhibited development of both papillomas and squamous cell carcinomas in overweight and obese mice in a dose-dependent manner. A low-dose combination of these two compounds displayed an additive inhibitory effect on tumor development. Metformin treatment also reduced the size of papillomas. Interestingly, all treatments seemed to be at least as effective for inhibiting tumor formation in obese mice, and both metformin and rapamycin were more effective at reducing tumor size in obese mice compared with overweight control mice. The effect of metformin on skin tumor development was associated with a significant reduction in TPA-induced epidermal hyperproliferation. Furthermore, treatment with metformin led to activation of epidermal AMP-activated protein kinase (AMPK) and attenuated signaling through mTOR complex (mTORC)-1 and p70S6K. Combinations of metformin and rapamycin were more effective at blocking epidermal mTORC1 signaling induced by TPA consistent with the greater inhibitory effect on skin tumor promotion. Collectively, the current data demonstrate that metformin given in the drinking water effectively inhibited skin tumor promotion in both overweight and obese mice and that the mechanism involves activation of epidermal AMPK and attenuated signaling downstream of mTORC1.

Abstract 1010: Effect of dietary energy balance modulation on the ability of metformin to inhibit skin tumor promotion

Abstract The prevalence of obesity has drastically increased over the last decade and is associated with increased risk for several cancers. In contrast, calorie restriction (CR) universally inhibits cancer. Plausible mechanisms for the energy balance-cancer link include differential signaling through Akt/mTOR via the IGF-1R as well as other growth factor receptors. Previous data from our lab, using topical administration of rapamycin, support the hypothesis that pharmacological disruption of mTORC1 signaling mimics some of the effects of CR on skin tumor promotion. To further explore the role of mTORC1 signaling in tumor promotion we have conducted studies using metformin. In the present study, metformin was administered in the drinking water at doses of 250 mg/kg and 50 mg/kg body weight per day to female FVB/N mice, to assess its ability to reverse the effects of overweight/obesity on skin tumor promotion during two-stage skin carcinogenesis. Metformin treatment during skin tumor promotion significantly inhibited tumor multiplicity in a dose dependent manner in mice on an overweight control diet (AIN76A). There was a 70% reduction in papillomas per mouse at the 250 mg/kg dose and a 35% reduction at the 50 mg/kg dose. Short term mechanistic experiments with these same doses revealed significant decreases in TPA-induced epidermal hyperproliferation (as measured by epidermal hyperplasia and labeling index) after oral administration of metformin. Metformin also activated AMP Kinase in the epidermis as measured by pAMPKαT172 at both doses used as well as attenuated signaling through mTORC1 at the highest dose as measured by downstream signaling proteins p70S6KT389 and prS6S235/236. Treatment with the higher dose of metformin also prevented TPA-induced mTORC1 mediated degradation of translational repressor PDCD4. Current experiments are assessing the ability of metformin to suppress skin tumor promotion in mice maintained on an obesity inducing diet (60kcal% fat) and these studies will also be presented. Overall, the current data support the hypothesis that selectively targeting mTORC1, either directly (rapamycin) or indirectly via AMPK activation, may be an effective strategy for reversing the effects of overweight and obesity on tumor development during epithelial carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1010. doi:1538-7445.AM2012-1010

Dual inhibition of both the epidermal growth factor receptor and erbB2 effectively inhibits the promotion of skin tumors during two-stage carcinogenesis.

The erbB family of receptor tyrosine kinases are known to play important roles in normal epithelial development and epithelial neoplasia. Considerable evidence also suggests that signaling through the epidermal growth factor receptor (EGFR) plays an important role in multistage skin carcinogenesis in mice; however, less is known about the role of erbB2. In this study, to further examine the role of both erbB2 and EGFR in epithelial carcinogenesis, we examined the effect of a dual erbB2/EGFR tyrosine kinase inhibitor, GW2974, given in the diet on skin tumor promotion during two-stage carcinogenesis in wild-type and BK5.erbB2 mice. In BK5.erbB2 mice, erbB2 is overexpressed in the basal layer of epidermis and leads to heightened sensitivity to skin tumor development. GW2974 effectively inhibited skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in wild-type and BK5.erbB2 mice, although a more marked effect was seen in BK5.erbB2 mice. In addition, this inhibitory effect was reversible when GW2974 treatment was withdrawn. GW2974 inhibited 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation, which correlated with reduced activation of both the EGFR and erbB2. These results support the hypothesis that both the EGFR and erbB2 play an important role in the development of skin tumors during two-stage skin carcinogenesis, especially during the tumor promotion stage. Furthermore, the marked sensitivity of BK5.erbB2 mice to the inhibitory effects of GW2974 during tumor promotion suggest greater efficacy for this compound when erbB2 is overexpressed or amplified as an early event in the carcinogenic process.

Abstract 2475: Effect of mTOR inhibition by rapamycin on promotion of skin tumors in BK5. Aktwt transgenic mice

Abstract Akt is a serine/threonine kinase involved in a variety of cellular responses including cell survival and proliferation. It has been shown that PI3K/Akt signaling is frequently altered (upregulated) in human cancers. mTOR is one of the Akt downstream signaling pathways that regulates protein synthesis, cell growth and proliferation. mTOR exists in two complexes, mTORC1 and mTORC2. Our laboratory previously reported that tumor promoters including TPA, okadaic acid (OA), and chrysarobin (CHRY) activate epidermal Akt after topical treatment of mouse epidermis and mouse primary cultured keratinocytes. Activation of Akt during tumor promoter treatment leads to enhanced downstream signaling including mTORC1 pathways. BK5. Aktwt transgenic mice where Akt is overexpressed in basal epidermis were significantly more sensitive to two-stage carcinogenesis, showing tumors with a much shorter latency, considerably larger in size, and enhanced progression of papillomas to SCCs. Importantly, Akt transgenic mice showed significant elevations in several downstream signaling pathways both in the absence and presence of TPA, including mTORC1 activity. Collectively, the studies with transgenic mice overexpressing Akt in epidermis (BK5.Aktwt) have suggested that activation of mTORC1 signaling may contribute to the process of skin tumor promotion. In this study, we have examined the ability of rapamycin, a known inhibitor of mTOR, on mTORC1 signaling in relation to its effect on TPA-induced epidermal cell proliferation and skin tumor promotion. Rapamycin given at doses of 1 μmol, 100 nmol, 20 nmol, and 5 nmol 30 min prior to 6.8 nmol TPA treatment inhibited mTORC1 activity in a dose-dependent manner. Notably, in a multiple treatment regimen (4 applications of rapamycin and TPA over a two week period) high doses of rapamycin significantly inhibited TPA-induced Akt phosphorylation at serine 473 as well as mTORC1 signaling in a dose dependent way, suggesting mTORC2 inhibition induced by prolonged high dose rapamycin treatment. The ability of rapamycin (at doses of 100, 20, and 5 nmol per mouse) to inhibit skin tumor promotion by TPA in BK5. Aktwt transgenic mice was compared with wild-type mice. Rapamycin significantly reduced TPA promotion in BK5.Aktwt mice, however, it was less effective compared to similar doses used in wild-type mice. The data demonstrate that rapamycin, although less effective, still retained significant inhibitory activity toward TPA promotion even when Akt and mTOR activities were elevated. These data demonstrate an important role for mTOR signaling in skin tumor promotion of TPA. 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 2475.

Dihydrolipoic acid inhibits skin tumor promotion through anti-inflammation and anti-oxidation

α-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several diseases, including hepatic disorder and diabetic polyneuropathy. However, the effects of LA or its reduced form, dihydrolipoic acid (DHLA), on cancer chemoprevention has never been reported. In the present study, we examined the effects of DHLA/LA on the production of nitric oxide (NO) by inducible NO synthase (iNOS) and the formation of prostaglandin E2 (PGE 2) by cyclooxygenase-2 (COX-2), two important mediators associated with inflammation. DHLA/LA significantly inhibited lipopolysaccharide (LPS)-induced NO and PGE 2 formation in RAW 264.7 cells. Meanwhile, treatment with DHLA/LA suppressed the expression of iNOS protein but, unexpectedly, did not affect or increase the expression of COX-2 protein. The in vivo anti-inflammatory and antitumor-promoting activities were evaluated by a topical 12- O-tetradecanoylphorbol 13-acetate (TPA) application to mouse skin with measurement of edema formation, epidermal thickness and hydrogen peroxide production. DHLA significantly inhibited the priming and activation stages of skin inflammation induced by a double TPA application, by decreasing the inflammatory parameters. Furthermore, DHLA inhibited DMBA (0.3 μmol)/TPA (2.0 nmol)-induced skin tumor formation by reducing the tumor incidence and tumor multiplicity. When applied topically onto the shaven backs of mice prior to TPA, DHLA markedly inhibited the expression of iNOS protein. DHLA also strongly and directly inhibited COX-2 activity. These results suggest that DHLA can be a possible chemopreventive agent in inflammation-associated tumorigenesis.

Crocetin from Saffron: An Active Component of an Ancient Spice

The known properties of saffron (Crocus sativus, L.) and its components have been examined. Recently, hormone like effects in green algae and the anti-cancerogenic and anti-toxic effects, have been observed. In particular, the effects of crocetin, a carotenoids (8,8′-diapo-8,8′-carotenoic acid) present in saffron and characterized by a diterpenic and symmetrical structure with seven double bonds and four methyl groups, have been taken into consideration. It has been found that this compound enhances the oxygen diffusivity through liquids, such as plasma. As a consequence of this property, it has been observed that crocetin increases alveolar oxygen transport and enhances pulmonary oxygenation. It improves cerebral oxygenation in hemorrhaged rats and positively acts in the atherosclerosis and arthritis treatment. It inhibits skin tumor promotion in mice (i.e., with benzo(a)pyrene); it has an inhibitory effect on intracellular nucleic acid and protein synthesis in malignant cells, as well as on protein-kinase-C and prorooncogene in INNIH/3T3 cells. This is most likely due to its anti-oxidant activity. Furthermore, crocetin protects against oxidative damage in rat primary hepatocytes. It also suppresses aflatoxin B1-induced hepatotoxic lesions and has a modulatory effect on aflatoxin, B1 cytotoxicity, and DNA adduct formation on C3H10/T1/2 fibroblast cells. It also has a protective effect on the bladder toxicity, induced by cyclophosphamide. The experiments reported in the scientific literature and the interesting results obtained have been carried out in vitro or on laboratory animals, but not yet on man.

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.

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.

Characterization of the antitumor-promoting activity of camptothecin in SENCAR mouse skin.

(+)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S phase cells, was tested topically for its ability to inhibit skin tumor initiation by 7,12-dimethylbenz[a]anthracene (DMBA) and complete tumor promotion by 12-0-tetradecanoylphorbol-13-acetate (TPA) in SENCAR mice. Even though CPT does not prevent the covalent binding of a subcarcinogenic dose of DMBA to DNA, it enhances early inhibition of DNA synthesis caused by this initiator and may decrease the essential role of DNA replication in tumor initiation. Indeed, CPT (400 nmol) applied 4 h before or 1 h after DMBA inhibits the yield, but not the incidence, of skin tumors initiated by this compound. Moreover, because it inhibits TPA-stimulated DNA synthesis at 16 h when applied 12 h after the tumor promoter, CPT partially decreases tumor initiation when DMBA is applied 16 h after a TPA pretreatment. CPT (400 nmol) applied 1 h before or 4, 12, 24 or 48 h after each promotion treatment with TPA remarkably inhibits the incidence and yield of skin tumors promoted by this agent. CPT delays and inhibits promotion of skin tumors the most when applied 12-24 h after each TPA treatment, at times when it can block the stimulation of DNA synthesis that follows the period of early inhibition caused by TPA. The ability of post-treatments with 25, 100 and 400 nmol CPT to inhibit skin tumor promotion is dose dependent. In the TPA (stage I)-mezerein (stage 2) protocol CPT (400 nmol) post-treatment inhibits both the first and second stages of tumor promotion, related to its ability to decrease the DNA and ornithine decarboxylase responses required for stages 1 and 2 respectively. The classic model of multistage skin carcinogenesis, therefore, may be valuable to determine if novel CPT analogs are more effective than their parent compound at inhibiting tumor initiation, promotion and progression.

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.

Modulation of chrysarobin skin tumor promotion

The present study examined the effect of several prototypic inhibitors of phorbol ester skin tumor promotion on skin tumor promotion by chrysarobin, an anthrone tumor promoter. Retinoic acid (RA) inhibited skin tumor promotion by chrysarobin; however, the degree of inhibition was dependent on the treatment protocol. When RA (10 micrograms/mouse) was given 1 h after each twice-weekly application of chrysarobin (220 nmol/mouse), a marked inhibition of papilloma formation was observed (78%). In additional experiments, using a once-weekly application of chrysarobin, RA also inhibited skin tumor promotion but the magnitude of inhibition was less. Interestingly, RA (10 micrograms/mouse), given 1 or 6 h after the promoter, did not inhibit the induction of epidermal ornithine decarboxylase (ODC) activity induced by a single topical application of chrysarobin (220 nmol). Fluocinolone acetonide (1 microgram/mouse), given 5 min before each twice-weekly application of chrysarobin (220 nmol/mouse) effectively inhibited skin tumor promotion (88%). A 0.5 or 0.25% supplement of alpha-difluoromethylornithine (alpha-DFMO) in the drinking water inhibited the induction of epidermal ODC following chrysarobin (220 nmol/mouse) treatment by 85 or 70%, respectively. Supplements of both 0.25 and 0.5% of alpha-DFMO also led to a 50 and 61% inhibition, respectively, in the number of papillomas per mouse after 25 weeks of promotion with chrysarobin. Interestingly, 0.25% alpha-DFMO in the drinking water did not reduce the number of papillomas per mouse after 20 weeks of promotion with 1.7 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the number of papillomas per mouse that were greater than or equal to 4 mm in diameter was significantly reduced in both chrysarobin- and TPA-treated mice. The data indicate that RA, FA and alpha-DFMO may be general inhibitors of tumor promoter regardless of the chemical class of tumor promoter. The ability of these inhibitors of phorbol ester promotion to inhibit anthrone promotion indicates that some common biochemical pathways may exist for both classes of skin tumor promoters.

Antipromotional activity of dietary N-(4-hydroxyphenyl)retinamide in two-stage skin tumorigenesis in CD-1 and SENCAR mice

The activity of the synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR), as a promoter and as an inhibitor of tumor promotion in mouse skin was investigated using CD-1 and SENCAR mice. Dietary administration of 4-HPR inhibited skin tumor promotion by 12- O-tetradecanoylphorbol-13-acetate (TPA) in both mouse strains, although protective activity was observed only at high TPA doses. Dietary 4-HPR had no promoting activity in mice receiving initiation and no TPA promotion. These data suggest that retinoid promotion of skin tumorigenesis may be specific to retinoic acid, and is not necessarily characteristic of the entire chemical class.

Decreased ratio of reduced/oxidized glutathione in mouse epidermal cells treated with tumor promoters.

Cellular pro-oxidant states appear to play role in the promotion phase, presumably because tumor promoter-treated cells overproduce activated forms of oxygen and/or deficient in their ability to destroy them. Since one of the earliest responses to the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) may be the generation of reactive oxygen species, we have determined the effects of this compound on the natural glutathione-dependent antioxidant protective system of the epidermal cells. Here we report that eight (chemically different) tumor promoters (including the phorbol esters, hydrogen peroxide, benzoyl peroxide, anthralin and mezerein) decreases to various degrees the intracellular ratio of reduced (GSH)/oxidized (GSSG) glutathione in isolated mouse epidermal cells. TPA leads to a rapid, transient increase in GSH peroxidase activity within 20 min, concomitant with a marked decrease in the ratio of GSH/GSSG. Beyond 1 h, while the GSH/GSSG ratio remains low, the GSH peroxidase activity declines below the control level in TPA-treated epidermal cells. This sequence suggests that the GSH-dependent detoxifying system of the cell is initially turned on but then rapidly overwhelmed by the oxidative challenge linked to the tumor-promoting activity of TPA. Since free radical scavengers, GSH level-raising agents and selenium-containing compounds all inhibit the effects of TPA on both GSH metabolism and tumor promotion, it is proposed that the enhancement of the GSH-dependent antioxidant protective system of the epidermal cells during TPA treatment might inhibit skin tumor promotion.

Inhibition of tumor promotion by a lecanoric acid analogue.

3',5'-Dichloro-2,4'-dihydroxybenzanilide, an inhibitor of histidine decarboxylase, inhibited skin tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate in mice.