Mouse Skin Tumor Promotion Research Articles

Antimutagenic Activity of Cassia Auriculata Linn Fractions along with Anticancer Activity in Male Albino Mice

Background: In recent years, there has been a surge in interest in studying plant-derived materials and their impact on DNA. Herbal products include a number of natural substances that may help protect cells against mutagen-induced cell damage.
 Aim: The purpose of this research was to assess the genotoxic effects of Cassia Auriculata Linn flavonoids (CAF) and Cassia Auriculata Linn saponin (CAS) rich fractions on mouse bone marrow cells utilizing chromosomal aberration test and micronucleus assay.
 Methodology: The suppressive impact of CAF and CAS on 7, 12-dimethylbenz (α) anthracene (DMBA) and Croton oil induced skin tumor promotion in mice with topical administration twice weekly for 18 weeks is also investigated in this work. Three dosages of 100 and 200 mg/kg body weight were used. Single oral dosages of CAF and CAS Fraction at the three levels did not enhance the number of micronucleate polychromatic erythrocytes in the micronucleus experiment.
 Result: In mice bone marrow cells, a single oral treatment of CAF and CAS fraction revealed no significant alterations in mitotic indices or chromosomal aberration induction. The clastogenicity of CYP was considerably decreased by pretreatment with CAF and CAS fraction. As a result, it can be stated that CAF and CAS fraction had no genotoxic impact on mouse bone marrow cells.
 Conclusions: The portions of Cassia Auriculata have been shown to be non-genotoxic and non-clastogenic at the quantities utilized in this investigation. CAF and CAS Fraction might possibly be a promising skin tumor promotion reducing agent, according to this research.

Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin.

Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventative agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential combinatorial inhibition of skin tumor promotion using the mouse two-stage skin carcinogenesis model. In short-term experiments, the combination of UA + Curc given topically prior to 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly inhibited activation of epidermal EGFR, p70S6K, NF-κB p50, Src, c-Jun, Rb, and IκBα. Levels of c-Fos, c-Jun, and Cox-2 were also significantly reduced by the combination compared to the TPA treated group. The alterations in these signaling pathways by the combination of UA + Curc were associated with decreased epidermal proliferation as assessed by measuring BrdU incorporation. Significant effects were also seen with the combination on epidermal inflammatory gene expression and dermal inflammation, with the greatest effects on expression of IL-1β, IL-6, IL-22, and CXCL2. Furthermore, results from skin tumor experiments demonstrated that the combination of UA + Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival, tumor size, and tumor weight, although tumor incidence and multiplicity were also further reduced by the combination. These results demonstrate the potential cancer chemopreventive activity and mechanism(s) for the combination of UA + Curc.

Abstract 278: Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin

Abstract Despite the many advances that have been made in understanding cancer development and the discovery of new therapies, cancer still remains the second leading cause of death in the US. In addition, approximately two out of five people are expected to be diagnosed with an invasive cancer in their lifetime. Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventive agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc alone have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due primarily to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), the combination of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and Stat3 to a greater extent than either compound alone. Additionally, these compounds increased levels of programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Furthermore, results from skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) indicate that the combination of UA and Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival and on tumor size. These results suggests the potential for the combination of UA and Curc to be used topically for chemopreventive strategies in non-melanoma skin cancer. Citation Format: Lisa Tremmel, Okkyung Rho, John DiGiovanni. Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 278.

Cancer Chemopreventive Activity of Xanthones from Calophyllum elatum

In our continuing search for compounds with antitumor-promoting activity, we screened eight xanthones isolated from Calophyllum elatum Bedd. (Guttiferae) by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12- O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Several compounds tested in this study showed inhibitory activity against EBV, without showing any cytotoxicity. Isogarciniaxanthone E (2) showed more potent activity than any of the other compounds tested. Furthermore, isogarciniaxanthone E (2) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Bryostatin 1 causes attenuation of TPA‑mediated tumor promotion in mouse skin.

The present study was designed to investigate the tumor inhibitory potential of bryostatin 1 in a 12‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑induced mouse model of skin cancer. The radical inhibition potential of various doses of bryostatin 1 was investigated against 2,2‑diphenyl‑1‑picrylhydrazyl (DPPH) bleach invitro. The DPPH radical potential was observed compared with treatment with 5, 10, 15, 20, 25 and 30µM doses of bryostatin1. Invivo, bryostatin1 prevented the TPA‑mediated increase in the level of H2O2 and myeloperoxidase in mouse epidermal tissue. Pretreatment of the mice with bryostatin 1 (30µM) followed by administration of TPA reduced the edema, as demonstrated via punched‑out mouse ear tissue, to 7.2mg, compared with 14mg in the TPA‑treated group. Treatment with bryostatin 1 prior to TPA administration markedly prevented the inflammation of the skin by inhibiting hyperplasia in the epidermal layer and the aggregation of inflammatory cells. The results demonstrated that treatment of mice with bryostatin1 at a 30µM dose prior to TPA administration significantly (P<0.005) inhibited the TPA‑mediated increase in the level of COX‑2. The activity of ornithine decarboxylase, increased by TPA, was additionally inhibited following pretreatment of the mice with bryostatin 1. In the mice treated with bryostatin 1 at 30µM doses prior to the administration of TPA, the appearance of papillomas was 20%, compared with 100% in the TPA group. Mice pretreated with bryostatin 1 at 30µM doses prior to TPA administration exhibited the appearance of 0.4 mean papillomas in each animal, compared with 5.2 in the TPA group. Therefore, the results of the present study demonstrated that bryostatin 1 inhibited the development and progression of tumors of skin in the mice, through the prevention of inflammation‑inducing processes and the quenching of radicals. Therefore, bryostatin1 maybe considered to be adrug of importance in the treatment of skin tumor.

Abstract 5262: The inhibitory effect of combinations of curcumin, ursolic acid, and metformin on skin tumor promotion by TPA

Abstract The purpose of this study was to examine the effects of combinations of ursolic acid (UA), curcumin (Curc), and metformin (Met) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. Met has also been shown to have anti-cancer activity. In retrospective studies, type II diabetes patients taking Met have been shown to have a decreased cancer incidence. Met has also been shown to inhibit mouse skin tumor promotion by TPA. Met activates AMP-activated protein kinase (AMPK), which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. mTORC1 is a key kinase involved in the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, one of the most commonly altered pathways in human tumors. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), combinations of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and JNK to a greater extent than either compound alone. Additionally, these compounds increased levels of AMPK and programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Further studies using dietary administration of a combination of UA and Curc, as well as combinations of UA or Curc (in diet) with Met administered in the drinking water were conducted. These combinations produced greater inhibition of TPA-induced activation of epidermal NFκB, Cox-2, and EGFR than the compounds given alone. These combinations also decreased epidermal proliferation, again shown by an inhibition of BrdU incorporation. In addition to these data, the results from ongoing skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) using both topical administration and dietary administration of the various combinations will be presented. The current results indicate that certain combinations of UA, Curc and Met may inhibit mouse skin tumor promotion by TPA to a greater extent than the compounds alone. The ability of the combinations to work when given both topically as well as in the diet suggests the potential for broad applications for chemopreventive strategies. Citation Format: Lisa Tremmel, Okkyung Rho, John DiGiovanni. The inhibitory effect of combinations of curcumin, ursolic acid, and metformin on skin tumor promotion by TPA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5262. doi:10.1158/1538-7445.AM2017-5262

Biological activity of the bryostatin analog Merle 23 on mouse epidermal cells and mouse skin.

Bryostatin 1, a complex macrocyclic lactone, is the subject of multiple clinical trials for cancer chemotherapy. Although bryostatin 1 biochemically functions like the classic mouse skin tumor promoter phorbol 12-myristate 13-acetate (PMA) to bind to and activate protein kinase C, paradoxically, it fails to induce many of the typical phorbol ester responses, including tumor promotion. Intense synthetic efforts are currently underway to develop simplified bryostatin analogs that preserve the critical functional features of bryostatin 1, including its lack of tumor promoting activity. The degree to which bryostatin analogs maintain the unique pattern of biological behavior of bryostatin 1 depends on the specific cellular system and the specific response. Merle 23 is a significantly simplified bryostatin analog that retains bryostatin like activity only to a limited extent. Here, we show that in mouse epidermal cells the activity of Merle 23 was either similar to bryostatin 1 or intermediate between bryostatin 1 and PMA, depending on the specific parameter examined. We then examined the hyperplastic and tumor promoting activity of Merle 23 on mouse skin. Merle 23 showed substantially reduced hyperplasia and was not tumor promoting at a dose comparable to that for PMA. These results suggest that there may be substantial flexibility in the design of bryostatin analogs that retain its lack of tumor promoting activity. © 2016 Wiley Periodicals, Inc.

Cancer Chemopreventive Retinoids: Validation and Analysis of &amp;lt;i&amp;gt;in Vivo&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Bioassay Results

Several natural and synthetic retinoids (vitamin-A derived analogies) were examined for their potential anti-cancer activity in both in vivo animal models and a novel in vitro human keratinocyte clonal growth bioassay system. The natural retinoids included all-trans-retinoic (RA), 13-cis-retinoic acid, 4-oxoretinoic acid, and retinol. Among the synthetic retinoids tested were all trans N-(4-hydroxy(phenyl)retinamide, 3-substituted oxoretinoic acids, and 13 cis-N-ethylretinamide. The animal models employed were: 1) vitamin A-deficient hamster tracheal organ assay (HTOC); 2) the benzo(α)pyrene-induced squamous metaplasia in a hamster tracheal organ system (BP-HTOC); 3) the mouse skin tumor promoter (TPA)-induced ornithine decarboxylase enzyme assay(ODC); 4) the mouse skin papilloma (MPA) assay; and 5) a novel retinoid bioassay in which retinoids display IC50 values to inhibit clonal growth of NHK. All-trans-RA, 4-oxoretinoic acid and retinol were consistently more active than any of the synthetic derivatives in all bioassays tested. A statistical model was developed and significant positive correlations were found between: 1) ED50 values in the HTOC system and reduction in TPA-induced ODC enzyme activity; 2) tumors per animal in the MPA bioassay and suppression of TPA-induced ODC activity; and 3) a positive correlation between suppression of tumors per animal in the MPA assay, and retinoid inhibition of keratinocyte clonal growth. Test retinoids, were tested for their capacity to inhibit the clonal growth of a squamous carcinoma cell line (SCC-25), which were found to be 2 - 3 logs less sensitive for each tested retinoid than the corresponding activity against NHK cells. Antineoplastic retinoid drugs were reviewed.

Low dose triterpene-quinone fraction from Ardisia crispa root precludes chemical-induced mouse skin tumor promotion.

BackgroundDrastic increment of skin cancer incidence has driven natural product-based chemoprevention as a promising approach in anticancer drug development. Apart from its traditional usages against various ailments, Ardisia crispa (Family: Myrsinaceae) specifically its triterpene-quinone fraction (TQF) which was isolated from the root hexane extract (ACRH) was recently reported to exert antitumor promoting activity in vitro. This study aimed at determining chemopreventive effect of TQF against chemically-induced mouse skin tumorigenesis as well as elucidating its possible pathway(s).MethodsMice (n = 10) were initiated with single dose of 7,12-dimethylbenz[α]anthracene (DMBA) (390 nmol/100 μl) followed by, a week later, repeated promotion (twice weekly; 20 weeks) with 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 μl). TQF (10, 30 and 100 mg/kg) and curcumin (10 mg/kg; reference) were, respectively, applied topically to DMBA/TPA-induced mice 30 min before each TPA application. Upon termination, histopathological and biochemical analysis, as well as Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and transcription factor enzyme-linked immunosorbent assay (ELISA) assays were performed to elucidate the potential mechanism of TQF.ResultsWith comparison to the carcinogen control, results revealed that lower dose of TQF (10 mg/kg) conferred antitumor promoting effect via significant (P < 0.05) suppression against lipid peroxidation (LPO), apoptotic index (cell death) and nuclear factor-kappa B (NF-κB), along with reduction of keratinocyte proliferation; whilst its higher dose (100 mg/kg) was found to promote tumorigenesis by significantly (P < 0.05) increasing LPO and apoptotic index, in addition to aggravating keratinocyte proliferation.ConclusionsThis study evidenced that TQF, particularly at its lower dosage (10 mg/kg), ameliorated DMBA/TPA-induced mouse skin tumorigenesis. Though, future investigations are warranted to determine the lowest possible therapeutic dose of TQF in subsequent in vivo chemopreventive studies.

Abstract 4570: Genetic deletion of TNFα in mice inhibits UVR-induced development of cutaneous squamous cell carcinomas via suppression of its own receptors and associated signals to epidermal cell survival

Abstract Skin cancer is the second most common malignancy encountered in the U.S. with an expected diagnosis of 1.3 million new cases of non-melanoma skin cancer each year. Cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most common non-melanoma forms of human skin cancer. BCC is rarely life threatening because it is slow growing and is mostly localized. SCC, unlike BCC, invades the nearby tissue. Chronic exposure to ultraviolet radiation (UVR) is the most common etiologic factor linked to the development of cutaneous SCC. PKCϵ transgenic mice provide a unique model to investigate the molecular basis of the development of SCC. PKC is a major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). PKCϵ is among six isoforms (α, δ, ϵ, η, μ, ζ) expressed in the mouse skin. To determine the in vivo functional specificity of PKCϵ in mouse skin carcinogenesis, we generated FVB/N PKCϵ transgenic mouse lines 224 and 215 that overexpress approximately 8- and 18-fold respectively PKCϵ protein over endogenous levels in basal epidermal cells. We observed that epidermal PKCϵ level dictates the susceptibility of transgenic mice to the development of SCC elicited by either the repeated exposure to UVR or using the DMBA-TPA tumor promotion protocol. Our earlier studies to elucidate mechanisms of PKCϵ-mediated development of SCC indicated an elevated release of cytokine TNFα. To conclusively determine whether TNFα is essential for the development of SCC in PKCϵ transgenic mice, we generated TNFα-knockout//PKCϵ transgenic mice by crossbreeding TNFα knockout mice with PKCϵ transgenic mice. We now present that deletion of both TNFα alleles in PKCϵ transgenic mice significantly (p = 0.0023) inhibited the development of cutaneous SCC by repeated UVR exposures. PKCϵ overexpressing mice deficient in TNFα elicited both increase in SCC latency (21 weeks) and decrease in SCC incidence (55%). Inhibition of SCC development in TNFα-knockout//PKCϵ transgenic mice accompanied by: 1) suppression of the expression levels of TNFα receptors TNFRI and TNFRII, 2) inhibition of the activation of transcription factors Stat3 and NF-κB, and 3) decreased expression levels of cell proliferation marker ornithine decarboxylase and metastatic markers MMP9 and MMP7. In summary, our results indicate that deletion of TNFα in mice inhibits UVR-induced development of cutaneous SCC probably via suppression of its own receptors and associated signals to epidermal cell survival pathways. (Support: NIH Grant CA102431) Citation Format: Ashok Singh, Anupama Singh, Samuel J. Bauer, Ajit K. Verma. Genetic deletion of TNFα in mice inhibits UVR-induced development of cutaneous squamous cell carcinomas via suppression of its own receptors and associated signals to epidermal cell survival. [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 4570. doi:10.1158/1538-7445.AM2015-4570

Relevance of the mouse skin initiation–promotion model for the classification of carcinogenic substances encountered at the workplace

The Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission of the Deutsche Forschungsgemeinschaft) evaluates chemical substances using scientific criteria to prevent adverse effects on health at the work place. As part of this task there is a need to evaluate tumor promoting activity of chemicals (enhancement of formation of squamous cell carcinomas via premalignant papillomas) obtained from two-stage initiation/promotion experiments using the mouse skin model. In the present communication we address this issue by comparing responses seen in mouse skin with those in humans. We conclude that tumor promotional effects seen in such animal models be carefully analyzed on a case by case basis. Substances that elicit a rather non-specific effect that is restricted to the high dose range are considered to be irrelevant to humans and thus do not require classification as carcinogens. In contrast, substances that might have both a mode of action and a potency similar to the specific effects seen with TPA (12-O-tetradecanoylphorbol-13-acetate), the prototype tumor promoter in mouse skin, which triggers receptor-mediated signal cascades in the very low dose range, have to be classified in a category for carcinogens.

Primary cancer prevention by green tea, and tertiary cancer prevention by the combination of green tea catechins and anticancer compounds.

Green tea is a daily beverage, a non-oxidized non-fermented product containing at least four green tea catechins. Considering our first results when repeated applications of (-)-epigallocatechin gallate (EGCG) prevented tumor promotion in mouse skin, we have continued to look at green tea as a possible cancer preventive agent. 1) The 10-year prospective cohort study by Drs. K. Nakachi and K. Imai revealed that drinking 10 Japanese-size cups (120 mL/cup) of green tea per day delayed cancer onset in humans by 7.3 years among females and by 3.2 years among males. The delay of cancer onset is of course significant evidence of primary cancer prevention in humans. 2) In collaboration with Dr. H. Moriwaki’s group we successfully presented a prototype of tertiary cancer prevention showing that 10 Japanese-size cups of green tea daily, supplemented with tablets of green tea extract (G.T.E), reduced recurrence of colorectal adenomas in polypectomy patients by 51.6% (from 31% to 15%). 3) In 1999, we first reported that the combination of green tea catechins and non-steroidal anti-inflammatory drugs showed synergistic anticancer effects in both in vitro and in vivo experiments, along with elucidation of the mechanism. 4) Further studies by other investigators have revealed that various combinations of EGCG or green tea extract and anticancer compounds inhibit tumor volume in xenograft mouse models implanted with various human cancer cell lines. Green tea is a cancer preventive, and green tea catechins act as synergists with anticancer compounds.

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

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Synthesis and Biological Activities of Simplified Analogs of the NaturalPKCLigands, Bryostatin‐1 and Aplysiatoxin

Protein kinase C (PKC) isozymes play central roles in signal transduction on the cell surface and could serve as promising therapeutic targets of intractable diseases like cancer, Alzheimer's disease, and acquired immunodeficiency syndrome (AIDS). Although natural PKC ligands like phorbol esters, ingenol esters, and teleocidins have the potential to become therapeutic leads, most of them are potent tumor promoters in mouse skin. By contrast, bryostatin-1 (bryo-1) isolated from marine bryozoan is a potent PKC activator with little tumor-promoting activity. Numerous investigations have suggested bryo-1 to be a promising therapeutic candidate for the above intractable diseases. However, there is a supply problem of bryo-1 both from natural sources and by organic synthesis. Recent approaches on the synthesis of bryo-1 have focused on its simplification, without decreasing the ability to activate PKC isozymes, to develop new medicinal leads. Another approach is to use the skeleton of natural PKC ligands to develop bryo-1 surrogates. We have recently identified 10-methyl-aplog-1 (26), a simplified analog of tumor-promoting aplysiatoxin (ATX), as a possible therapeutic lead for cancer. This review summarizes recent investigations on the simplification of natural PKC ligands, bryo-1 and ATX, to develop potential medicinal leads.

Inhibitory effects of Momordica grosvenori Swingle extracts on 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation and tumor promotion in mouse skin

Our previous data showed that the Momordica grosvenori Swingle extract (MSE) exhibited the anti-inflammatory effect through markedly suppressed LPS-induced up-regulation of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and ODC (ornithine decarboxylase) gene expression in RAW 264.7 cells. Regarding the link between inflammation and carcinogenesis, we further investigated the bio-molecular mechanisms of both anti-inflammatory and anti-tumor activities in vivo using a TPA (12-O-tetradecanoyl phorbol 13-acetate)-stimulated mouse skin model. Pretreatment with MSE in mouse skin has led to the reduction of TPA-induced nuclear translocation of the nuclear factor-κB (NFκB) subunits as well as phosphorylation of IκBα and p65 subsequent reduction of IκBα degradation. In addition, the MSE inhibitory effect on upstream of NFκB was found to involve the transcriptional effects of MAPK signaling as indicated by strong suppression on TPA-induced activation of extracellular signal regulate kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK)1/2, phosphatidylinositol 3-kinase (PI3K) and Akt. Moreover, MSE significantly inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-TPA-induced skin tumor formation in mice measured by the tumor multiplicity of papillomas at 20 weeks. The results suggested that MSE contained promising functional ingredients capable of preventing inflammation-associated tumorigenesis.

Mechanisms of Mycotoxin-induced Dermal Toxicity and Tumorigenesis Through Oxidative Stress-related Pathways.

Among the many mycotoxins, T-2 toxin, citrinin (CTN), patulin (PAT), aflatoxin B1 (AFB1) and ochratoxin A (OTA) are known to have the potential to induce dermal toxicity and/or tumorigenesis in rodent models. T-2 toxin, CTN, PAT and OTA induce apoptosis in mouse or rat skin. PAT, AFB1 and OTA have tumor initiating properties, and OTA is also a tumor promoter in mouse skin. This paper reviews the molecular mechanisms of dermal toxicity and tumorigenesis induced in rodent models by these mycotoxins especially from the viewpoint of oxidative stress-mediated pathways.

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.

Hexahydro-β-Acids Potently Inhibit 12-O-Tetradecanoylphorbol 13-Acetate-Induced Skin Inflammation and Tumor Promotion in Mice

We previously reported that hexahydro-beta-acids (HBAs), reduced derivatives of beta-acids (BA) from hop (Humulus lupulus L.), displayed more potent anti-inflammatory activity than BA in lipopolysaccharide-stimulated murine macrophages. In this study, we investigated the effects and underlying molecular mechanisms of hexahydro-β-acids (HBAs) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated mouse skin inflammation and in the two-stage carcinogenesis model. Female ICR mice pretreated with HBA at 1 and 10 μg significantly reduced ear edema, epidermal hyperplasia, and infiltration of inflammatory cells caused by TPA. Molecular analysis exhibited that HBA suppressed iNOS, COX-2, and ornithine decarboxylase (ODC) protein and gene expression through interfering with mitogen-activated protein kinases (MAPKs) and phosphatidylinositiol 3-kinase (PI3K)/Akt signaling as well as the activation of transcription factor NF-κB. Furthermore, application of HBA (1 and 10 μg) prior to each TPA treatment (17.2 ± 0.9 tumors/mouse) resulted in the significant reduction of tumor multiplicity (5.1 ± 1.2, P < 0.01 and 2.3 ± 1.2, P < 0.001, respectively) in 7,12-dimethyl-benzanthracene (DMBA)-initiated mouse skin. The tumor incidence was significantly lowered to 75% (P < 0.05) and 58.7% (P < 0.01) by HBA pretreatment, respectively, and significantly reduced the tumor weight (0.34 ± 0.14 g, P < 0.01 and 0.09 ± 0.10 g, P < 0.001, respectively) as compared to DMBA/TPA-induced tumors (0.76 ± 0.04 g).

The preventive role of breadfruit against inflammation‐associated epithelial carcinogenesis in mice

Artocarpus communis has been identified as a rich source of flavonoids and has been gaining attention for its potential chemopreventive abilities. In this study, methanol extracts from the fruit of A. communis (MEFA) and leaf of A. communis (MELA) were prepared, and their effects on inflammation-associated skin tumorigenesis were assessed using mouse models, including 12-O-tetradecanoylphorbol-13-acetate (TPA) induced cutaneous inflammation as well as 7,12-dimethylbenz[α]anthracene (DMBA) initiated and TPA-promoted skin tumorigenesis. According to the results, both MEFA and MELA decreased the intensity of leukocyte infiltration in mouse dorsal skin and cutaneous edema induced by TPA, which appeared to be mediated by inhibition of proinflammatory genes (inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), IL-1β, and IL-6) and proinflammatory mediators (TNF-α, IL-1β, and Prostaglandin E2 ). In addition, topical application with MEFA or MELA effectively attenuated tumor incidence, multiplicity, volume, malignancy as well as angiogenesis of TPA-stimulated skin tumor promotion in DMBA-initiated mice. Notably, immunohistochemical stain showed that MEFA and MELA attenuated COX-2 expression of both skin and tumor tissues in different animal tests, which may be closely related to the suppression of nuclear factor kappa B/activator protein signaling networks. These findings first demonstrate that flavonoid-rich A. communis may exert potent anti-inflammatory activity through modulation of COX-2 in TPA-activated skin and tumor tissues.