Induced Mouse Skin Carcinogenesis Research Articles

A TRPV1 antagonist, PAC-14028 does not increase the risk of tumorigenesis in chemically induced mouse skin carcinogenesis

PAC-14028 (Asivatrep: C21H22F5N3O3S) cream is a novel, topical nonsteroidal, anti-inflammatory, and TRPV1 (transient receptor potential vanilloid subfamily, member 1) antagonist for the treatment of mild to moderate atopic dermatitis. Concerns about the risk of tumor development by TRPV1 blockade in the skin have been prompted, but these findings were proved to be indirect or are still controversial. This study was tested to determine whether TRPV1 selective antagonist, PAC-14028 cream is safe from the promotion of skin tumorigenesis in the two-stage carcinogenesis model. PAC-14028 cream, 0.25%, 0.5%, or 1.0% was applied once daily topically to mouse skin for up to 24 weeks in two-stage chemical carcinogenesis testing using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Morbidity/death, clinical signs, tumor formation, activity of EGFR/Akt/mTOR signaling, and systemic exposure to PAC-14028 were investigated. Daily dermal administration of PAC-14028, was not skin carcinogenic. There was also no evidence on the activation of EGFR/Akt/mTOR signaling pathway by the topical treatment of PAC-14028. On Day 169, 1.0% (20 mg/kg/day) of PAC-14028 in female mice resulted in a Cmax and AUC0-τ of 12916.0 ng/mL and 78962.9 ng‧hr/mL, respectively. PAC-14028 cream was well tolerated and did not increase the risk of skin tumorigenesis in two-stage carcinogenesis study.

P-397 - Marliolide inhibits skin carcinogenesis by activating NRF2/ARE to induce heme oxygenase-1

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to produce three anti-oxidant molecules: carbon monoxide (CO), ferrous ion (Fe 2+ ), and biliverdin. Induction of HO-1 is currently considered as a feasible strategy to treat oxidative stress-related diseases. Here, we report marliolide as a novel inducer of HO-1 in human normal keratinocyte HaCaT cells. Mechanism-based studies demonstrated that the induction of HO-1 by marliolide occurred through activation of NRF2/ARE via direct binding of marliolide to KEAP1. Structure-activity relationship revealed chemical moieties of marliolide critical for induction of HO-1, which renders a support for Michael reaction as a potential mechanism of action. Finally, we observed that marliolide significantly inhibited the papilloma formation in DMBA/TPA–induced mouse skin carcinogenesis model and this event was closely associated with lowering the formation of 8-OH-G and 4-HNE in vivo. Together, we provide the first evidence that marliolide might be effective against oxidative stress-related skin disorders.

Marliolide inhibits skin carcinogenesis by activating NRF2/ARE to induce heme oxygenase-1

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to produce three anti-oxidant molecules: carbon monoxide (CO), ferrous ion (Fe2+), and biliverdin. Induction of HO-1 is currently considered as a feasible strategy to treat oxidative stress-related diseases. In the present study, we identified marliolide as a novel inducer of HO-1 in human normal keratinocyte HaCaT cells. Mechanism-based studies demonstrated that the induction of HO-1 by marliolide occurred through activation of NRF2/ARE via direct binding of marliolide to KEAP1. Structure-activity relationship revealed chemical moieties of marliolide critical for induction of HO-1, which renders a support for Michael reaction as a potential mechanism of action. Finally, we observed that marliolide significantly inhibited the papilloma formation in DMBA/TPA–induced mouse skin carcinogenesis model and this event was closely associated with lowering the formation of 8-OH-G and 4-HNE in vivo. Together, our study provides the first evidence that marliolide might be effective against oxidative stress-related skin disorders.

Prophylaxis of Diallyl Disulfide on Skin Carcinogenic Model via p21-dependent Nrf2 stabilization.

Cancer prevention through intake of biologically active natural products appears to be an accessible way to reduce the risk of cancer. Diallyl disulfide (DADS), a major garlic derivative, has exhibited potential role in cancer therapy. The study is aimed to evaluate the prophylactic effect of DADS in chemically induced mouse skin carcinogenesis and investigate the molecular targets mediated by DADS. Two-stage chemically induced carcinogenesis model by cutaneous application of DMBA and subsequent TPA was established to study the prophylactic effect of DADS. As a result, we observed that DADS dose-dependently attenuated skin tumor incidence and multiplicity in the model mice, which was related to the up-regulation of a bunch of antioxidant enzymes activities and the nuclear accumulation of Nrf2. Furthermore, we developed skin carcinogenesis in Nrf2 knockout mice which could reverse the activity of DADS. Finally, we uncovered the underlying mechanism that DADS promoted the endogenous interaction between p21 and Nrf2, which was critical for impairing the Keap1-mediated degradation of Nrf2. Based on the results, we concluded that DADS was a promising cancer chemoprevention agent and suggested a garlic-rich diet might be beneficial to reduce the cancer risk in our daily life.

A low toxic synthetic dendrimer conjugated podophyllotoxin nanodevice with potent antitumor activity against the DMBA/TPA induced mouse skin carcinogenesis model

D-PODO in tumor-bearing mice revealed a 50%–60% inhibition of skin tumor formation and reduced toxicity compared to PODO.

Chemopreventive effects of curcumin on chemically induced mouse skin carcinogenesis in BK5.insulin-like growth factor-1 transgenic mice.

The insulin-like growth factor-1 (IGF-1) signaling pathway is strongly associated with the risk of various cancers, and its inhibition has emerged as a potent anticancer strategy. Accumulating evidence from in vitro studies has shown that curcumin is a potent inhibitor of the IGF-1 signaling pathway. However, direct evidence that curcumin modulates IGF-1-induced tumorigenesis in a physiological system has not been reported. Therefore, in this study, we assessed the anticarcinogenic activity of curcumin on skin cancer by using BK5.IGF-1 transgenic (Tg) mice that overexpress IGF-1 in the skin epidermis. In 7,12-dimethylbenz(a)anthracene (DMBA)-tetradecanoyl phorbol-13-acetate (TPA) two-stage skin carcinogenesis, a curcumin diet (0.02% wt/wt) fed for 14 wk remarkably reduced mouse skin tumor multiplicity by 53%, epidermal hyperplasia and proliferation compared to the control diet group. TPA-induced phosphorylation of Akt, S6 kinase (S6K), and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) in mouse skin was lower in the curcumin group than in the control group. Curcumin treatment inhibited IGF-1-induced phosphorylation of the IGF-1 receptor, insulin receptor substrate-1, Akt, S6K, and 4EBP1 in the mouse keratinocyte cell line, C50 in a dose-dependent manner. Taken together, these data suggest that curcumin exerts significant anticarcinogenic activity in skin cancer through the inhibition of IGF-1 signaling.

5-Demethyltangeretin (5-DTAN) is more effective than tangeretin in 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin carcinogenesis

5-Demethyltangeretin (5-DTAN) is more effective than tangeretin in 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin carcinogenesis

Fermented Prunus mume with Probiotics Inhibits 7,12-Dimethylbenz[a]anthracene and 12-O-Tetradecanoyl phorbol-13-acetate Induced Skin Carcinogenesis through Alleviation of Oxidative Stress

Maesil (Prunus mume Siebold and Zucc.), a member of the genus Rosaceae, has been reported to have antioxidative effects, as well as anticancer influence in many cancer lines. Thus, this present study was designed to investigate the inhibitory effect of fermented Maesil with probiotics against 7,12-dimethylbenz[a]anthracene (DMBA), 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced mouse skin carcinogenesis via its antioxidative potential. Mice were fed a diet containing fermented Maesil, containing either 1% (1% FM fed group) or 2% (2% FM fed group) along with probiotics following DMBA and TPA exposure. Continuous ingestion of the experimental feed markedly inhibited skin carcinogenesis, as evidenced by a marked decrease in papilloma numbers and epidermal hyperplasia as well as cellular proliferation and the percentage of proliferating-cell nuclear antigen positive cells. Also, the FM fed group showed an increase of total antioxidant capacity as well as an increased level of phase II detoxifying enzymes such as superoxide dismutase, concurrent with a decreased lipid peroxidation activity level. Taken together, these results suggest that fermented Maesil has the ability to suppress the development of DMBA-TPA induced skin carcinogenesis, via the reduction of lipid peroxidation, enhancing total antioxidant capacity and phase II detoxifying enzyme.

Tamoxifen-loaded novel liposomal formulations: evaluation of anticancer activity on DMBA-TPA induced mouse skin carcinogenesis

Purpose: Tamoxifen (TAM) is a non-steroidal estrogen receptor modulator known for its anticancer activity. Apart from marked breast cancer activity, this drug has also shown potential in treating other types of cancers including skin cancers. TAM is reported to be associated with serious side effects primarily due to its systemic distribution. The localized delivery of this drug in this regard would be highly beneficial with respect to safety as well as efficacy.Methods: In the current studies, an endeavor has been made to investigate the efficacy of topically applied liposome-encapsulated TAM on skin cancer model. The drug was encapsulated in phospholipid-based vesicular systems viz. conventional liposomes and elastic liposomes. Incidence of papillomas and histopathological examination were employed to determine the efficacy of the tested formulations.Results: The results demonstrated carrier-dependent strong inhibition of skin carcinogenesis with encapsulated drug vis-à-vis drug in the solution form. The encouraging findings from the current work construe immense potential of the TAM-loaded liposomal systems in the management of skin cancer.

Pterostilbene, a natural analogue of resveratrol, potently inhibits 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin carcinogenesis

We reported previously that pterostilbene, a natural analogue of resveratrol from blueberries, strongly suppressed lipopolysaccharide-induced up-expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in murine macrophages. In this study, we further investigated pterostilbene's molecular mechanism of action and its anti-tumor properties. Pretreatment with pterostilbene has resulted in the reduction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced nuclear translocation of the nuclear factor-κB (NFκB) subunits. Pterostilbene also reduced TPA-induced phosphorylation of IκBα and p65 and caused subsequent degradation of IκBα. Moreover, pterostilbene markedly suppressed TPA-induced activation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK)1/2, phosphatidylinositol 3-kinase (PI3K) and Akt, which are upstream of NFκB and activator protein 1 (AP-1). Furthermore, pterostilbene significantly inhibited 7,12-dimethylbenz[a]anthracene (DMBA)/TPA-induced skin tumor formation measured by the tumor multiplicity of papillomas at 20 weeks. The presented data has, for the first time, revealed that pterostilbene is an effective anti-tumor agent that functions by downregulating inflammatory iNOS and COX-2 gene expression in mouse skin. It is suggested that pterostilbene is a novel functional agent capable of preventing inflammation-associated tumorigenesis.

Inhibitory effects of interferon-gamma plasmid DNA on DMBA-TPA induced mouse skin carcinogenesis

Interferon-gamma (IFN-γ) exhibits biological activities that are considered to have important roles in tumor suppression. Therefore, the IFN-γ gene is a potential candidate for in vivo cytokine gene therapy against skin cancer. The present study evaluated the efficacy of a hydrodynamics-based IFN-γ gene transfection for skin cancer treatment, in which the plasmid DNA encoding IFN-γ was administered into the tail vein of mice following 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis. Serum levels of IFN-γ were substantially elevated without liver toxicity. The mice injected with IFN-γ plasmid DNA displayed a marked reduction in papilloma numbers, suppressed proliferation of epidermal cells and induction of caspase-3-mediated apoptosis. These results suggest that the hydrodynamics-based transfection of IFN-γ plasmid DNA is a convenient and efficient means of skin cancer gene therapy.

Anti-tumor and Anti-oxidative Activity of Rosmarinus officinalis in 7, 12 Dimethyl Benz(a) Anthracene Induced Skin Carcinogenesis in Mice

The present investigation was undertaken to explore the antitumor-promoting activity of R.officinalis on 2-stage skin carcinogenesis, induced by a single topical application of 7,12-dimethylbenz(a)anthracene and promoted by repeated treatment of croton oil for 16 weeks in Swiss albino mice. Oral administration of R.officinalis leaves extract, during the peri-and post-initiation stage induced by 7,12dimethylbenz[a]anthracene (DMBA), reduced the tumor burden to 2.6 (positive control value: 5.16); cumulative number of papillomas to 13 (positive control value: 62) and percent incidence of mice bearing papillomas to 41.66 %, (positive control value: 100%). A significant (p<0.001) decreased was observed in Lipid peroxidation level by the administration of ROE. Reduced glutathione (GSH) and total proteins was found to be significantly elevated in liver and skin (p < 0.001) of mice in ROE treated group. Skin and liver of R.officinalis treated group animals showed a significant enhancement in antioxidant enzymes like superoxide dismutase (SOD) and Catalase, when compared with the carcinogen treated control. These studies indicate that R.officinalis could reduce the chemical induced tumor and oxidative stress during skin carcinogenesis.

Induction of apoptosis by tea polyphenols mediated through mitochondrial cell death pathway in mouse skin tumors

Many naturally occurring phytochemicals have shown cancer chemopreventive potential in a variety of bioassay systems. One such naturally occurring biologically active compound is tea (Camellia sinensis), which is the most consumed beverage in the world after water. The most abundant and active constituents of tea are polyphenols (epigallocatechin gallate and theaflavins). In the present study, cancer chemopreventive properties of both black tea polyphenols (BTP) and green tea polyphenols (GTP) on 7,12- dimethylbenz[a]anthracene (DMBA) induced mouse skin carcinogenesis were studied. BTP and GTP treatment showed delay in onset of tumorigenesis, reduction in cumulative number of tumors and increased tumor free survival. Both BTP and GTP were found to modulate the expression of proteins involved in apoptotic pathway. Tea polyphenols treatment along with DMBA exposure resulted in up-regulation of p53, and proapoptotic protein Bax, whereas enhanced expression of antiapoptotic proteins, Bcl-2 and survivin by DMBA were down-regulated. Further, we showed that tea polyphenols supplementation resulted in release of cytochrome c, caspases activation, and increase in apoptotic protease activating factor and poly (ADP-ribose) polymerase cleavage as mechanism of apoptosis induction. The results also provide strong evidence that BTP is a better chemopreventive agent against skin tumorigenesis as compared to GTP at the tested dose levels. Thus, we can conclude that the polyphenolic constituents present in black tea and green tea induce mitochondrial pathway of apoptosis and hence can be used as a potential chemopreventive agents against skin cancer.

Inhibition of 7,12‐Dimethylbenz[a]anthracene Induced Mouse Skin Carcinogenesis by Artemisia capillaris

Anticarcinogenic activity of medicinal herbs (Artemisia capillaris, Taxus cuspidata, Anthriscus sylveatris, and Curcuma longa) was examined for 7,12-dimethylbenz[a]anthracene (DMBA)-induced mouse skin carcinogenesis. Four types of solvent fractions (hexane, chloroform, ethyl acetate, and butanol) were prepared from the methanolic extract of medicinal herbs. The cytotoxicity and anticarcinogenic activities of solvent fractions were examined for mouse leukemia L1210 cancer cells and for female ICR mouse epidermal carcinogenesis induced by DMBA, respectively. The chloroform fraction of Artemisia capillaris, Taxus cuspidata, and Anthriscus sylveatris was more toxic to L1210 cells than other solvent fractions. The chloroform fraction of Artemisia capillaris markedly reduced the number of tumors/mouse and tumor incidence relative to that of other medicinal herbs tested. Major active chemical constituents in the chloroform fraction of Artemisia capillaries were found to be camphor, 1-borneol, coumarin, and achillin when analyzed by TLC and GC-MS. These results suggest that Artemisia capillaris was the most effective anticarcinogenic medicinal herb for DMBA-induced mouse epidermal carcinogenesis among 4 medicinal herbs tested, and the effect might be attributed to chemical compounds of camphor, 1-borneol, coumarin, and achillin.

Cucurbitane-Type Triterpenoids from the Fruits of Momordica charantia and Their Cancer Chemopreventive Effects

Thirteen cucurbitane-type triterpene glycosides, including eight new compounds named charantosides I (6), II (7), III (10), IV (11), V (12), VI (13), VII (16), and VIII (17), and five known compounds, 8, 9, 14, 15, and 18, were isolated from a methanol extract of the fruits of Japanese Momordica charantia. The structures of the new compounds were determined on the basis of spectroscopic methods. On evaluation of these triterpene glycosides and five other cucurbitane-type triterpenes, 1-5, also isolated from the extract of M. charantia fruits, for their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, these compounds showed inhibitory effects on EBV-EA induction with IC(50) values of 200-409 mol ratio/32 pmol TPA. In addition, upon evaluation of compounds 1-5 for inhibitory effects against activation of (+/-)-(E)-methyl-2[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitrogen oxide (NO) donor, compounds 1-3 showed moderate inhibitory effects. Compounds 1 and 2 exhibited marked inhibitory effects in both 7,12-dimethylbenz[a]anthracene (DMBA)- and peroxynitrite (ONOO-; PN)-induced mouse skin carcinogenesis tests.

Chemopreventive and Antilipidperoxidative Potential of Clerodendron inerme (L) Gaertn in 7,12-dimethylbenz(a)anthracene Indcued Skin Carcinogenesis in Swiss Albino Mic

The present study has investigated the chemopreventive and antilipidperoxidative effects of the ethanolic extract of Clerodendron inerme leaves (CiELet) in DMBA induced skin carcinogenesis in Swiss albino mice. The skin squamous cell carcinoma was induced in the shaved back of mice, by painting with DMBA (25 microg 0.1 mL(-1) acetone) twice weekly for 8 weeks. We have observed 100% tumor formation in the fifteenth week of experimental period. Elevated lipid peroxidation and decline in enzymatic and non-enzymatic antioxidants status was observed in tumor bearing mice. Oral administration of CiELet (300 mg kg(-1) bw) for 25 weeks significantly prevented the tumor incidence, volume and burden of the tumor. The CiELet also showed potent antilipidperoxidative effect as well as enhanced the antioxidant defense mechanisms in DMBA painted mice. The present study thus demonstrated the chemopreventive and antilipidperoxidative efficacy of CiELet in DMBA induced mouse skin carcinogenesis.

Prostaglandin receptor EP2 is responsible for cyclooxygenase-2 induction by prostaglandin E2 in mouse skin

The EP2 prostanoid receptor is one of the four subtypes of receptors for prostaglandin E2 (PGE2). We previously reported that deletion of EP2 led to resistance to chemically induced mouse skin carcinogenesis, whereas overexpression of EP2 resulted in enhanced tumor development. The purpose of this study was to investigate the underlying molecular mechanisms. We found that EP2 knockout mice had reduced cyclooxygenase-2 (COX-2) expression after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment compared with wild-type (WT) mice. Further, primary keratinocytes from EP2 transgenic mice had increased COX-2 expression after either TPA or PGE2 treatment and COX-2 expression was blocked by 10 microM SQ 22,536, an adenylate cyclase inhibitor. EP2 knockout mice had significantly decreased, whereas EP2 transgenic mice had significantly increased PGE2 production in response to a single treatment of TPA. Cyclic AMP response element-binding protein (CREB) phosphorylation was elevated to a greater extent in keratinocytes from EP2 transgenic mice compared with those of WT mice following PGE2 treatment. A protein kinase A (PKA) inhibitor reduced PGE2-mediated CREB phosphorylation in keratinocytes from EP2 transgenic mice. Furthermore, we found that there was no CREB phosphorylation in EP2 knockout mice following PGE2 treatment. PGE2-induced DNA synthesis (cell proliferation) was significantly decreased in keratinocytes from EP2 knockout mice following pretreatment with 10 microM SQ 22,536. Taken together, EP2 activation of the PKA/CREB-signaling pathway is responsible for keratinocyte proliferation and our findings reveal a positive feedback loop between COX-2 and PGE2 that is mediated by the EP2 receptor.

P048. Chemopreventive activity of natural compounds on nitric oxide donors induced mouse skin carcinogenesis

P048. Chemopreventive activity of natural compounds on nitric oxide donors induced mouse skin carcinogenesis

Autofluorescence characterization for the early diagnosis of neoplastic changes in DMBA/TPA‐induced mouse skin carcinogenesis

Squamous cell carcinoma (SCC), the second most common skin cancer, usually remains confined to the epidermis for some time but eventually penetrates the underlying tissues, if left untreated. The non-invasive early detection of the SCC is important for appropriate therapeutic strategies. In this study, we aim to characterize the tissue transformation in DMBA/TPA induced mouse skin tumor model using autofluorescence excitation emission matrix (EEM) in conjunction with a multivariate statistical method for early detection of the neoplastic changes. The fluorescence EEM from experimental group (n = 40; DMBA/TPA application), control group (n = 6; acetone application), and the blank group (n = 6; no application of DMBA/TPA or acetone) were measured every week using a spectrofluorometer coupled with a fiber optic bundle. The EEM was recorded at excitation wavelengths from 280 to 460 nm at 10 nm intervals and the fluorescence emission was scanned from 300 to 750 nm. The fluorescence emission characteristics corresponding to different fluorophores were extracted from the EEM and the spectral data were used in a multiple/linear discriminant statistical algorithm. The changes in the fluorescence emission intensity were observed as early as the 1st week of tumor initiation by DMBA. Morphological changes as well as differences in the gross appearance of the skin surface were observed during the entire tumor initiation and promotion period of 15 weeks. The statistical analysis was performed for each excitation wavelength in the EEM and better classification accuracy was obtained for 280 and 410 nm excitations, corresponding to tryptophan and endogenous porphyrins, respectively. The statistical analysis of the combination wavelengths resulted in 11.6% increase in the overall classification accuracy when compared to the highest classification accuracy obtained with single wavelength analysis. The intensity ratio mapping using the combination of emission intensities of key fluorophores such as tryptophan, collagen, NADH, and endogenous porphyrins from the measured EEM in conjunction with a simple multivariate statistical analysis can be used as a potential tool for the discrimination of early neoplastic changes with improved classification accuracy. Tryptophan and endogenous porphyrins may be used as biomarkers for the discrimination of early neoplastic changes when single wavelength excitations are used.

Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development

Chemically induced mouse skin carcinogenesis represents the most extensively utilized animal model to unravel the multistage nature of tumour development and to design novel therapeutic concepts of human epithelial neoplasia. We combined this tumour model with comprehensive gene expression analysis and could identify a large set of novel tumour-associated genes that have not been associated with epithelial skin cancer development yet. Expression data of selected genes were confirmed by semiquantitative and quantitative RT-PCR as well as in situ hybridization and immunofluorescence analysis on mouse tumour sections. Enhanced expression of genes identified in our screen was also demonstrated in mouse keratinocyte cell lines that form tumours in vivo. Self-organizing map clustering was performed to identify different kinetics of gene expression and coregulation during skin cancer progression. Detailed analysis of differential expressed genes according to their functional annotation confirmed the involvement of several biological processes, such as regulation of cell cycle, apoptosis, extracellular proteolysis and cell adhesion, during skin malignancy. Finally, we detected high transcript levels of ANXA1, LCN2 and S100A8 as well as reduced levels for NDR2 protein in human skin tumour specimens demonstrating that tumour-associated genes identified in the chemically induced tumour model might be of great relevance for the understanding of human epithelial malignancies as well.