Epidermal Ornithine Decarboxylase Research Articles

Hairless and the polyamine putrescine form a negative regulatory loop in the epidermis

Hairless (HR) is a nuclear protein with corepressor activity that is highly expressed in the skin and hair follicle. Mutations in Hairless lead to hair loss accompanied by the appearance of papules (atrichia with papular lesions), and similar phenotypes appear when the key polyamine enzymes ornithine decarboxylase (ODC) and spermidine/spermine N(1) -acetyltransferase (SSAT) are overexpressed. Both ODC and SSAT transgenic mice have elevated epidermal levels of putrescine, leading us to investigate the mechanistic link between putrescine and HR. We show here that HR and putrescine form a negative regulatory network, as epidermal ODC expression is elevated when HR is decreased and vice versa. We also show that the regulation of ODC by HR is dependent on the MYC superfamily of proteins, in particular MYC, MXI1 and MXD3. Furthermore, we found that elevated levels of putrescine lead to decreased HR expression, but that the SSAT-TG phenotype is distinct from that found when HR is mutated. Transcriptional microarray analysis of putrescine-treated primary human keratinocytes demonstrated differential regulation of genes involved in protein-protein interactions, nucleotide binding and transcription factor activity, suggesting that the putrescine-HR negative regulatory loop may have a large impact on epidermal homeostasis and hair follicle cycling.

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.

Abstract 1522: Elevated polyamine levels generate an immunosuppressive oncogenic microenvironment

Abstract Increased suprabasal epidermal activity of ornithine decarboxylase (ODC), a key regulatory enzyme in polyamine biosynthesis, stimulates cellular proliferation and vascularization in the underlying dermis, and promotes skin tumorigenesis after treatment with a single, subthreshold dose of a carcinogen in ODCER transgenic mice. Interestingly, skin tumor formation is also induced in ODCER transgenic mice by wounding and does not require prior carcinogen treatment. This wound-induced tumor formation is dependent on the polyamine-induction of sustained recruitment of inflammatory cells to the dermal wound site. Moreover, when weakly transformed (Ras activation) primary keratinocytes (Ker/Ras) were injected subcutaneously in syngeneic ODCER transgenic mice, tumor growth occurred while it did not in normal littermates. These data suggest the generation of an oncogenic stromal microenvironment in mice with elevated epidermal polyamine levels. Polyamine-Based Therapy (PaBT: combination of the well-characterized ODC inhibitor, difluoromethylornithine (DFMO), and an inhibitor of cellular polyamine transport, AMXT 1501) significantly inhibited tumor growth in wildtype FVB mice that were orthotopically injected with either syngeneic Ker/Ras cells or neu02 mammary tumor cells. Flow cytometry analyses of tumor inflammatory infiltrates revealed that PaBT significantly increased the percentage of CD3+ cell population (includes T cells and NK cells) and decreased the percentage of both F4/80+ (macrophage) and Gr-1+ (neutrophil and monocyte) subpopulations of CD45+ cells compared to tumors in control, non-treated mice. When PaBT treatment was stopped and tumors were resected, we re-challenged two weeks later with a second injection of neu02 tumor cells. Mice that had been previously treated with PaBT were protected from secondary tumor growth (demonstrating no or very little tumor growth) compared to mice that never received PaBT. In order to examine whether elevated polyamine biosynthesis suppresses a T-cell mediated adaptive immune response, we examined the effect of ODC overexpression in keratinocytes on a classic contact hypersensitivity (CHS) response test using ODCER transgenic mice. These mice showed reduced ear swelling in comparison to normal littermates together with reduced neutrophil infiltration and decreased migration of FITC-loaded dendritic cells to draining lymph nodes following hapten elicitation of CHS. Collectively, these data suggest that the immunosuppression mediated by increased epidermal ODC activity may contribute to an environment conducive to tumor growth, and, conversely, that depletion of tumor polyamines will block tumorigenesis in part by normalizing the stromal microenvironment and restoring tumor immune surveillance. (Supported by NIH grant CA070739 and funding from Aminex Therapeutics Inc) 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 1522. doi:1538-7445.AM2012-1522

Elevated Epidermal Ornithine Decarboxylase Activity Suppresses Contact Hypersensitivity

Previous reports have shown that elevated polyamine biosynthesis is sufficient to promote skin tumorigenesis in susceptible mouse strains. We hypothesized that increased activity of epidermal ornithine decarboxylase (ODC), a key regulatory enzyme in polyamine biosynthesis, may suppress the cutaneous immune response in addition to stimulating proliferation. Using an ODCER transgenic mouse model in which ODC is targeted to the epidermis, we examined the effect of ODC overexpression in keratinocytes on a classic contact hypersensitivity (CHS) response. Compared with normal littermate mice, ODCER transgenic mice showed reduced ear swelling, reduced neutrophil infiltration, and decreased migration of fluorescein isothiocyanate-loaded dendritic cells (DCs) to draining lymph nodes following hapten elicitation of CHS. In addition, elevated epidermal ODC activity suppressed the levels of cytokines keratinocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin-6 (IL-6), and IL-10. Adoptive transfer of lymphocytes from sensitized ODCER transgenic or normal littermate mice to naive ODCER transgenic or wild-type mice indicated that elevated epidermal ODC activity suppresses both the sensitization and elicitation phases of CHS. The specific ODC inhibitor, α-difluoromethylornithine, abrogated all suppressive effects of ODC in CHS reactions. Collectively, these data suggest that the immunosuppression promoted by increased epidermal ODC is mediated by a reduction in cytokine levels, which suppresses DC migration and reduces immune cell infiltration to the site of hapten application.

Abstract 2471: Immunosuppressive effect of elevated epidermal ornithine decarboxylase activity

Abstract Tumor promoting agents, such as phorbol esters, promote tumorigenesis via induction of inflammation and hyperplasia. However, the immunosuppressive/immunomodulating properties of tumor promoting agents may play a relevant role in tumor promotion as well. Because elevated polyamine biosynthesis is sufficient to promote skin tumorigenesis, we hypothesized that increased activity of epidermal ornithine decarboxylase (ODC), a key regulatory enzyme in polyamine biosynthesis, may suppress the cutaneous immune response in addition to stimulating proliferation. To investigate the role of ODC in the control of cutaneous inflammation, a well-known model of T cell mediated inflammation was used. A contact hypersensitivity (CHS) response was elicited in the ears of both ODC transgenic mice, in which ODC is targeted to the epidermis, and their normal littermates by topical sensitization and challenge with oxazolone. Unlike in normal littermate mice, ear swelling was dramatically reduced in ODC transgenic mice after elicitation of CHS. Flow cytometric and immunohistochemical analyses of the infiltrating immune cells revealed fewer infiltrating immune cells, especially neutrophils, in the elicited ears of ODC transgenic mice. The specific ODC inhibitor, α-difluoromethylornithine, abrogated the suppressive effects of ODC in CHS reactions. Adoptive transfer of sensitized lymphocytes from either ODC transgenic or normal littermate mice to naïve ODC transgenic or wild-type mice indicated that elevated epidermal ODC activity suppress both the sensitization and elicitation phases of CHS. Cytokines are central mediators of immune functions associated with inflammatory reactions. The cytokine profile of ex vivo stimulated lymphocytes isolated from draining lymph nodes showed reduced levels of IL-6 and IL-10 in sensitized and challenged ODC transgenic mice. In vivo labeling of dendritic cells by topical application of fluorescein isothiocyanate (FITC) showed fewer FITC-labeled dendritic cells in the draining lymph nodes of ODC transgenic mice compared to normal littermates. Vascular permeability was assessed using Evans blue extravasation assays during the elicitation phase of CHS. At 24 hours after induction of CHS, there was 45% less plasma extravasation of Evans blue dye into the ear skin of ODC transgenic mice compared to normal littermates. Collectively these data indicate elevated epidermal ODC activity can suppress a number of functions associated with cutaneous inflammatory reactions. In addition to promoting cellular proliferation, the immune suppression mediated by increased epidermal ODC activity may provide an environment conducive to tumor growth. This work was supported by NIH grant CA070739. 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 2471.

Modulatory effect of Morus indica against two-stage skin carcinogenesis in Swiss albino mice: possible mechanism by inhibiting aryl hydrocarbon hydroxylase.

The modulatory effect of the methanolic extract of Morus indica on 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced oxidative stress and 7,12-dimethylbenz(a)anthracene induced and croton oil (0.5% per mouse/0.2 mL acetone, v/v) promoted skin tumourigenesis in Swiss albino mice was studied. The efficacy of the M. indica extract was also evaluated in-vitro by studying the inhibition of the activity and level of aryl hydrocarbon hydroxylase, cytochrome P450, DNA sugar damage in calf thymus DNA and Fe(++)/ascorbate-induced lipid peroxidation in microsomes of mice. Significant increases in the activity of antioxidant enzymes (P <0.001) and a concomitant decrease (P <0.001) in the cutaneous malondialdehyde level were observed at three doses of plant extract (2.5, 5.0 and 7.5 mg kg(-1)). Application of M. indica 1 h before each application of croton oil showed inhibitory effects on tumour promotion in terms of a reduction in the number of tumours/mouse and percentage of mice with tumours. It was also accompanied by an extension of the tumour latency period. TPA, which resulted in a rapid and transient stimulation of mouse epidermal ornithine decarboxylase activity (P <0.001), was inhibited dose dependently by pre-treatment with M. indica extract (P <0.001). The results suggest that M. indica extract may be useful as a therapeutic agent for cancer control as it blocks or suppresses events associated with chemical carcinogenesis.

Guggulsterone modulates MAPK and NF-kappaB pathways and inhibits skin tumorigenesis in SENCAR mice.

Guggulsterone (GUG), a resin of the Commiphora mukul tree, has been used in ayurvedic medicine for centuries to treat a variety of ailments. Recent studies have suggested that GUG may also possess anticancer effects. In the present study, we show that GUG possesses antitumor-promoting effects in SENCAR mouse skin tumorigenesis model. We first determined the effect of topical application of GUG to mice against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of GUG (1.6 micromol per mouse) 30 min prior to TPA (3.2 nmol per mouse) application onto the skin of mice afforded significant inhibition against TPA-mediated increase in skin edema and hyperplasia. Topical application of GUG was also found to result in substantial inhibition against TPA-induced epidermal (i) ornithine decarboxylase (ODC) activity; (ii) ODC, cyclooxygenase-2 and inducible nitric oxide synthase protein expressions; (iii) phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinases and p38; (iv) activation of NF-kappaB/p65 and IKK alpha/beta and (v) phosphorylation and degradation of I kappaB alpha. We next assessed the effect of topically applied GUG on TPA-induced skin tumor promotion in 7,12-dimethyl benz[a]anthracene-initiated mice. Compared with non-GUG-pretreated mice, animals pretreated with GUG showed significantly reduced tumor incidence, lower tumor body burden and a significant delay in the latency period for tumor appearance from 5 to 11 weeks. These results provide the first evidence that GUG possesses anti-skin tumor-promoting effects in SENCAR mice and inhibits conventional as well as novel biomarkers of tumor promotion. In summary, GUG could be useful for delaying tumor growth in humans.

Chemopreventive effects of -santalol on ultraviolet B radiation-induced skin tumor development in SKH-1 hairless mice

Recent studies from our laboratory have shown the chemopreventive effects of alpha-santalol against 7,12-dimethylbenzanthracene (DMBA) initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted skin tumor development in mice. The objective of the present investigation was to study the effects of alpha-santalol on ultraviolet B (UVB) radiation-induced skin tumor development and UVB-caused increase in epidermal ornithine decarboxylase (ODC) activity in female hairless SKH-1 mice. For the tumor studies, 180 mice were divided into three groups of 60 mice each, and each group was divided into two subgroups of 30 mice. The first subgroup served as control and was treated topically on the dorsal skin with acetone. The second subgroup served as experimental and was treated topically on the dorsal skin with alpha-santalol (5%, w/v in acetone). The tumorigenesis in the first group was initiated with UVB radiation and promoted with TPA; in the second group it was initiated with DMBA and promoted with UVB radiation; and in the third group it was both initiated and promoted with UVB radiation. In each case, the study was terminated at 30 weeks. Topical application of alpha-santalol significantly (P<0.05) decreased tumor incidence and multiplicity in all the three protocols, suggesting its chemopreventive efficacy against UVB radiation-caused tumor initiation, tumor promotion and complete carcinogenesis. In a short-term biochemical study, topical application of alpha-santalol also significantly (P<0.05) inhibited UVB-induced epidermal ODC activity. Together, for the first time, our findings suggest that alpha-santalol could be a potential chemopreventive agent against UVB-induced skin tumor development and, therefore, warrants further investigations.

Chemopreventive effects of various concentrations of α-santalol on skin cancer development in CD-1 mice

Previous studies from this laboratory have indicated that alpha-santalol (5%) provides chemopreventive effects in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin cancer in CD-1 and SENCAR mice. Skin cancer development is associated with increased ornithine decarboxylase (ODC) activity, DNA synthesis and rapid proliferation of epidermal cells. The purpose of this investigation was to determine the effects of various concentrations (1.25% and 2.5%) of alpha-santalol on DMBA-initiated and TPA-promoted skin cancer development, TPA-induced ODC activity, and DNA synthesis in CD-1 mice. alpha-Santalol treatment at both concentrations (1.25% and 2.5%) prevented the skin cancer development. alpha-Santalol treatment (1.25% and 2.5%) resulted in a significant decrease in the TPA-induced ODC activity and incorporation of [3H]thymidine in DNA in the epidermis of CD-1 mice. There was no significant difference in the effects of 1.25% and 2.5% alpha-santalol on tumour incidence, multiplicity, epidermal TPA-induced ODC activity, or DNA synthesis in CD-1 mice.

Suprabasal Induction of Ornithine Decarboxylase in Adult Mouse Skin Is Sufficient to Activate Keratinocytes

To study the effects of de novo induction of ornithine decarboxylase (ODC) activity in adult, quiescent skin, we generated transgenic mice in which the suprabasal expression of an inducible form of the ODC protein fused to a modified estrogen receptor ligand-binding domain (ODCER) is driven by an involucrin promoter. After topical treatment with the inducing agent 4-hydroxytamoxifen (4OHT), ODC activity and putrescine levels were dramatically increased in the epidermis but not in the dermis of transgenic mice. 4OHT treatment stimulated both proliferation as measured by bromodeoxyuridine incorporation in basal epidermal cells and differentiation shown by increased expression of differentiation markers. Furthermore, induction of ODC activity did not rescue primary epidermal keratinocyte cultures isolated from ODCER2 mice from a calcium-triggered DNA synthesis block, as measured by [3H]thymidine incorporation. In vivo induction of epidermal ODC enzyme activity significantly stimulated the vascularization of ODCER transgenic skin. Increased expression of interleukin-1beta and keratin 6, markers of keratinocyte activation seen in wound healing, was also observed in 4OHT-treated transgenic skin. These results suggest that de novo suprabasal induction of ODC activity in adult mouse skin activates keratinocytes and stimulates vascularization in the dermal layer in a manner similar to skin undergoing wound healing.

Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-kappaB pathways and inhibits skin tumorigenesis in CD-1 mice.

Chemoprevention has come of age as an effective cancer control modality; however, the search for novel agent(s) for the armamentarium of cancer chemoprevention continues. We argue that agents capable of intervening at more than one critical pathway in the carcinogenesis process will have greater advantage over other single-target agents. Pomegranate fruit extract (PFE) derived from the tree Punica granatum possesses strong antioxidant and antiinflammatory properties. Pomegranate fruit was extracted with acetone and analyzed based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and found to contain anthocyanins, ellagitannins and hydrolyzable tannins. We evaluated whether PFE possesses antitumor-promoting effects. We first determined the effect of topical application of PFE to CD-1 mice against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of PFE (2 mg/mouse) 30 min prior to TPA (3.2 nmole/mouse) application on mouse skin afforded significant inhibition, in a time-dependent manner, against TPA-mediated increase in skin edema and hyperplasia, epidermal ornithine decarboxylase (ODC) activity and protein expression of ODC and cyclooxygenase-2. We also found that topical application of PFE resulted in inhibition of TPA-induced phosphorylation of ERK1/2, p38 and JNK1/2, as well as activation of NF-kappaB and IKKalpha and phosphorylation and degradation of IkappaBalpha. We next assessed the effect of skin application of PFE on TPA-induced skin tumor promotion in 7,12-dimethylbenz(a)anthracene-initiated CD-1 mouse. The animals pretreated with PFE showed substantially reduced tumor incidence and lower tumor body burden when assessed as total number of tumors per group, percent of mice with tumors and number of tumors per animal as compared to animals that did not receive PFE. In TPA-treated group, 100% of the mice developed tumors at 16 weeks on test, whereas at this time in PFE-treated group, only 30% mice exhibited tumors. Skin application of PFE prior to TPA application also resulted in a significant delay in latency period from 9 to 14 weeks and afforded protection when tumor data were considered in terms of tumor incidence and tumor multiplicity. The results of our study provide clear evidence that PFE possesses antiskin-tumor-promoting effects in CD-1 mouse. Because PFE is capable of inhibiting conventional as well as novel biomarkers of TPA-induced tumor promotion, it may possess chemopreventive activity in a wide range of tumor models. Thus, an in-depth study to define active agent(s) in PFE capable of affording antitumor-promoting effect is warranted.

13- cis Retinoic acid ameliorates benzoyl peroxide-induced oxidative stress and hyperproliferative response in murine skin: a chemopreventive study

The present paper assesses the chemopreventive potential of retinoic acid on benzoyl peroxide (BPO)-induced cutaneous tumor promotion response and oxidative stress in murine skin. In this study, we have shown the activities of cutaneous antioxidant enzymes and phase II metabolizing enzymes and the glutathione content were decreased while epidermal ornithine decarboxylase (ODC) activity and DNA synthesis were induced in benzoyl peroxide treated animals. Topical application of retinoic acid resulted in significant inhibition of benzoyl peroxide-induced epidermal ornithine decarboxylase activity and DNA synthesis. Application of retinoic acid at three different doses prior to the application of benzoyl peroxide recovered the depleted level of glutathione, inhibited activities of antioxidant and phase II metabolizing enzymes, thus resulting in significant inhibition of oxidative stress in dose dependent manner. Enhanced susceptibility of cutaneous microsomal lipid peroxidation and xanthine oxidase activity were significantly reduced ( P>0.05). The antimutagenic effect of retinoic acid was tested against benzoyl peroxide mediated mutagenicity in Salmonella typhimurium strain TA-98 and TA-100 using 3-methyl cholanthrene-induced murine skin (S9 fraction) as the metabolic activation system. Indeed, with the addition of various concentrations of retinoic acid there was significant reduction in the number of revertants per plate in concentration dependent manner. In summary, our data indicates that retinoic acid may exhibit cancer chemopreventive activity in skin tumorigenesis model.

Pharmacological effects of Daphne genkwa and Chinese medical prescription, "Jyu-So-To"

Daphne genkwa (Thymelaeaceae) has been used as a folk medicine in China. We investigated the effects of D. genkwa and Jyu-So-To on various pharmacologic models in mice including the azoxymethane (AOM)-induced colonic aberrant crypt focus formation assay, ornithine decarboxylase (ODC) activity assay, and two types of mouse ear swelling model. Administration of 236.3 ppm of Jyu-So-To in drinking water significantly suppressed AOM-induced colonic aberrant crypt focus formation (p < 0.05), with an inhibitory ratio of 46.7%. The effects of several extracts with organic solvents of D. genkwa on murine epidermal ODC activity were examined. In particular, the inhibitory ratio of the n-hexane extract was 30.8%. In the 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model in mice, the methanol extract resulted in 56.3% inhibition compared with the control. On the other hand, there are two peaks of responses at 1 h (immediate-phase reaction; IPR) and 24 h (late-phase reaction) in biphasic cutaneous reactions, which are enhanced in the dinitrofluorobenzene model (DNFB). The water extract of D. genkwa clearly inhibited the IPR ear swelling. These results suggest that D. genkwa and Jyu-So-To should be a promising source of antitumor, antiinflammatory, and antiallergy agents.

Lupeol modulates NF-kappaB and PI3K/Akt pathways and inhibits skin cancer in CD-1 mice.

Chemoprevention has become an effective cancer control modality; however, the search for novel agent(s) for the armamentarium of cancer chemoprevention continues. We argue that agents capable for inhibition of promotion stage of tumorigenesis with the ability to intervene at several critical pathways in the tumorigenesis process will have greater advantage over other single-target agents. Lupeol, a triterpene, is the principal constituent of common fruit plants such as olive, mango, fig and medicinal herbs that have been used to treat skin aliments. Lupeol has been reported to possess a wide range of medicinal properties that include strong antioxidant, antimutagenic, anti-inflammatory and antiarthritic effects. In the present study, we show that Lupeol possesses antitumor-promoting effects in a mouse skin tumorigenesis model. We first determined the effect of topical application of Lupeol to CD-1 mouse against 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of Lupeol (1-2 mg/mouse) 30 min prior to TPA (3.2 nmol/mouse) application onto the skin of CD-1 mice afforded significant inhibition, in a time- and dose-dependent manner, against TPA-mediated increase in (i) skin edema and hyperplasia, (ii) epidermal ornithine decarboxylase (ODC) activity, and (iii) protein expression of ODC, cyclo-oxygenase-2 and nitric oxide synthase. As of the role of nuclear factor kappa B (NF-kappaB) and phosphatidyl inositol 3-kinase (PI3K)/Akt signaling in tumor promotion, we next determined the effect of topical application of Lupeol to mouse skin against these signaling pathways. We found that Lupeol treatment to mouse skin resulted in the inhibition of TPA-induced (i) activation of PI3K, (ii) phosphorylation of Akt at Thr(308), (iii) activation of NF-kappaB and IKKalpha, and (iv) degradation and phosphorylation of IkappaBalpha. The animals pretreated with Lupeol showed significantly reduced tumor incidence, lower tumor body burden and a significant delay in the latency period for tumor appearance. At the termination of the experiment at 28 weeks, 100% of the animals in TPA-treated group exhibited seven to eight tumors/mouse, whereas only 53% of the mice receiving Lupeol prior to TPA treatment exhibited one to three tumors/mouse. These results for the first time provide evidence that Lupeol possesses antiskin tumor-promoting effects in CD-1 mouse and inhibits conventional as well as novel biomarkers of tumor promotion. We suggest that Lupeol is an attractive antitumor-promoting agent that must be evaluated in tumor models other than skin carcinogenesis.

Inhibition of 12- O-tetradecanoylphorbol-13-acetate-induced tumor promotion markers in CD-1 mouse skin by oleandrin

Oleandrin, derived from the leaves of Nerium oleander, has been shown to possess anti-inflammatory and tumor cell growth-inhibitory effects. Here, we provide evidence that oleandrin could possess anti-tumor promoting effects. We determined the effect of topical application of oleandrin to CD-1 mice against l2- O-tetradecanoylphorbol-13-acetate (TPA), a widely studied skin tumor promoter, -induced conventional and novel markers of skin tumor promotion. Topical application of oleandrin (2 mg per mouse) 30 min before TPA (3.2 nmol per mouse) application onto the skin afforded significant inhibition, in a time-dependent manner, against TPA-mediated increase in cutaneous edema and hyperplasia, epidermal ornithine decarboxylase (ODC) activity and ODC and cyclooxgenase-2 (COX-2) protein expression. In search for novel markers of skin tumor promotion, we found that TPA application to mouse skin resulted, as an early event, in an increased expression of phosphatidyinositol 3-kinase (PI3K), phosphorylation of Akt at threonine 308 and activation of nuclear factor kappa B (NF-κB). Topical application of oleandrin before TPA application to mouse skin resulted in significant reduction in TPA-induced expression of PI3K and phosphorylation of Akt, and inhibition of NF-κB activation. NF-κB is a eukaryotic transcription factor that is critically involved in regulating the expression of specific genes that participate in inflammation, apoptosis and cell proliferation. Employing Western blot analysis, we found that oleandrin application to mouse skin resulted in inhibition of TPA-induced activation of NF-κB, IKKα and phosphorylation and degradation of IκBα. Our data suggest that oleandrin could be a useful anti-tumor promoting agent because it inhibits several biomarkers of TPA-induced tumor promotion in an in vivo animal model. One might envision the use of chemopreventive agents such as oleandrin in an emollient or patch for chemoprevention or treatment of skin cancer.

Inhibitory effects of chlorophyllin, hemin and tetrakis(4-benzoic acid)porphyrin on oxidative DNA damage and mouse skin inflammation induced by 12- O-tetradecanoylphorbol-13-acetate as a possible anti-tumor promoting mechanism

Reactive oxygen species (ROS) from both endogenous and exogenous sources can cause oxidative DNA damage and dysregulated cell signaling, which are involved in the multistage process of carcinogenesis such as tumor initiation, promotion and progression. A number of structurally different anticarcinogenic agents inhibit inflammation and tumor promotion as they reduce ROS production and oxidative DNA damage. Evidence suggests that porphyrins can interfere with the actions of various carcinogens and mutagens by forming face-to-face complexes and their antimutagenic or antigenotoxic effects may also be attributed to their antioxidant activities. However, little is known regarding the anti-tumor promoting potential and mechanism of the porphyrin compounds. Based on our previous results on the inhibitory effects of chlorophyllin (CHL), hemin and tetrakis(4-benzoic acid)porphyrin (TBAP) against two-stage mouse skin carcinogenesis, we have investigated their anti-tumor promoting mechanisms. In the present work, CHL, hemin and TBAP reduced superoxide anion generation by 12- O-tetradecanoylphorbol-13-acetate (TPA) in differentiated HL-60 cells and the production of hydroxyl radicals by Fenton reaction. Porphyrins exert a dose-related inhibition of his + reversion in Salmonella typhimurium TA102 induced by tert-butylhydroperoxide ( t-BOOH). DNA strand breaks by ROS derived from H 2O 2/Cu(II) and the formation of 8-hydroxydeoxyguanosine (8-OH-dG) in calf thymus DNA treated with H 2O 2/UV also were inhibited markedly by porphyrins in a concentration-dependent manner. Furthermore, CHL, hemin and TBAP decreased myeloperoxidase (MPO) activity and H 2O 2 formation as well as epidermal ornithine decarboxylase (ODC) activity in mouse skin treated with TPA. These results demonstrate that the antioxidative properties of porphyrins are important for inhibiting TPA-induced tumor promotion.

Protective Effects of Lycopene Against Ultraviolet B-Induced Photodamage

Lycopene, an acyclic hydrocarbon carotenoid found in tomatoes and their products, is a well-established potent antioxidant, and its anticancer properties have been shown in cultured cells and animal models. We investigated the protective effects of two concentrations of topical lycopene against acute ultraviolet B (UVB)-induced photodamage. Application of lycopene dose dependently inhibited UVB-induced ornithine decarboxylase (P < 0.05) and myeloperoxidase (P < 0.05) and significantly reduced bifold skin thickness (P < 0.05). Immunohistochemical staining revealed increased active caspase-3 of apoptotic pathway in the UVB-exposed group compared with the unexposed control. Application of topical lycopene prevented the cleavage of caspase-3. UVB irradiation completely diminished proliferating cell nuclear antigen (PCNA), and the untreated skin maintained positively stained cells throughout the basal epidermis. Topical application of lycopene significantly reversed UVB-induced PCNA inhibition, and normal PCNA staining was restored in the lycopene-treated skin. Our results suggest that topical lycopene is able to exert its protective effects against acute UVB-induced photodamage. Furthermore, it may act as a preventative agent via inhibition of epidermal ornithine decarboxylase activity, reducing inflammatory responses, maintaining normal cell proliferation, and possibly preventing DNA damage as indicated by blocking the necessitating step of apoptosis following UVB injury.

Inhibition of benzoyl peroxide and ultraviolet-B radiation induced oxidative stress and tumor promotion markers by cycloartenol in murine skin

The chemopreventive potential of cycloartenol on benzoyl peroxide and UVB radiation-induced cutaneous tumor promotion markers and oxidative stress in murine skin is assessed. Benzoyl peroxide treatment (20 mg/animal/0.2 ml acetone) and UVB radiation (0.420 J/m2/s) caused a decrease in the activities of cutaneous antioxidant enzymes namely, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, phase II metabolizing enzyme such as glutathione-S-transferase and quinone reductase and depletion in the level of cutaneous glutathione. There was also enhancement in cutaneous microsomal lipid peroxidation, xanthine oxidase activity, [14C]-ornithine decarboxylase activity and [3H]-thymidine incorporation into cutaneous DNA. Cycloartenol was topically applied prior to the application of benzoyl peroxide at dose levels of 0.2 mg and 0.4 mg/kg body weight in acetone, which resulted in significant inhibition of epidermal ornithine decarboxylase activity and DNA synthesis (P < 0.001). There was also significant reduction of lipid peroxidation and xanthine oxidase activity (P < 0.001). In addition, the depleted levels of glutathione, inhibited activities of antioxidant and phase II metabolizing enzymes, were also recovered to a significant level (P < 0.001). The data indicate that cycloartenol is an effective chemopreventive agent in skin carcinogenesis.

Modulation of biochemical parameters by Hemidesmus indicus in cumene hydroperoxide-induced murine skin: possible role in protection against free radicals-induced cutaneous oxidatve stress and tumor promotion

Hemidesmus indicus has been shown to possess significant activity against immunotoxicity and other pharmacological and physiological disorders. In this communication, we have shown the modulating effect of H. indicus on cumene hydroperoxide-mediated cutaneous oxidative stress and tumor promotion response in murine skin. Cumene hydroperoxide treatment (30 mg per animal) increased cutaneous microsomal lipid peroxidation and induction of xanthine oxidase activity which are accompanied by decrease in the activities of cutaneous antioxidant enzymes and depletion in the level of glutathione. Parallel to these changes a sharp decrease in the activities of phase II metabolizing enzymes was observed. Cumene hydroperoxide treatment also induced the ornithine decarboxylase activity and enhanced the [ 3 H ]-thymidine uptake in DNA synthesis in murine skin. Application of ethanolic extract of H. indicus at a dose level of 1.5 and 3.0 mg/kg body weight in acetone prior to that of cumene hydroperoxide treatment resulted in significant inhibition of cumene hydroperoxide-induced cutaneous oxidative stress, epidermal ornithine decarboxylase activity and enhanced DNA synthesis in a dose-dependent manner. Enhanced susceptibility of cutaneous microsomal membrane for lipid peroxidation and xanthine oxidase activity were significantly reduced ( P<0.01). In addition the depleted level of glutathione, inhibited activities of antioxidants and phase II metabolizing enzymes were recovered to significant level ( P<0.05). In summary, our data suggest that H. indicus is an effective chemopreventive agent in skin and capable of ameliorating hydroperoxide-induced cutaneous oxidative stress and tumor promotion.

Palm oil alleviates 12- O-tetradecanoyl-phorbol-13-acetate-induced tumor promotion response in murine skin

Palm oil is a rich source of vitamin E, carotenoids, tocotrienols and tocopherols which are natural antioxidants and act as scavengers of oxygen free radicals. 12- O-Tetradecanoyl-phorbol-13-acetate (TPA) is a known oxidant that promotes tumorigenesis in mouse skin through the elaboration of oxidative stress. In this study we therefore assessed the anti-tumor promoting potential of palm oil against TPA-mediated skin tumorigenesis in 7,12-dimethylbenz[ a]anthracene-initiated Swiss albino mice. Topical application of palm oil 1 h prior to application of TPA resulted in a significant protection against skin tumor promotion. The animals pre-treated with palm oil showed a decrease in both tumor incidence and tumor yield as compared to the TPA (alone)-treated group. Palm oil application also reduced the development of malignant tumors. Since TPA-induced epidermal ornithine decarboxylase (ODC) activity and [ 3H]thymidine incorporation are conventionally used markers of skin tumor promotion, we also assessed the effect of pre-application of palm oil on these parameters, and it was observed that the application of palm oil prior to the application of TPA alleviated both these TPA-induced markers of tumor promotion. The effect of pre-application of palm oil on TPA-mediated depletion in the non-enzymatic and enzymatic molecules was also assessed and it was observed that palm oil application prior to TPA application resulted in the recovery of TPA-mediated depletion in the levels of these molecules viz. glutathione, glutathione peroxidase, glutathione reductase, glutathione- S-transferase and catalase. Similarly, palm oil also exhibited a protective effect against Fe 2+-ascorbate-induced lipid peroxidation in the epidermal microsomes. The results of the present study thus suggest that palm oil possesses anti-skin tumor promoting effects, and that the mechanism of such effects may involve the inhibition of tumor promoter-induced epidermal ODC activity, [ 3H]thymidine incorporation and cutaneous oxidative stress.