Tumor Promoter 12-O-tetradecanoylphorbol-13-acetate Research Articles

Sulforaphane inhibits phorbol ester-stimulated IKK-NF-κB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-κB activating kinase and ERK

Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK–IKKβ.

Building Bridges toward Invasion: Tumor Promoter Treatment Induces a Novel Protein Kinase C-Dependent Phenotype in MCF10A Mammary Cell Acini

The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters many cellular processes through activation of its receptor protein kinase C (PKC), including gene expression, cell cycle, and the regulation of cell morphology, raising an important question for developing targeted methods to prevent cancer: which effects of TPA are crucial for carcinogenesis? To address this question, we studied TPA action in the 3-dimensional (3D) MCF10A human breast epithelial cell system, which models important features of in vivo epithelial tissue including growth constraints, structural organization of cells, and establishment of a basement membrane. MCF10A cells, which are immortalized but nontumorigenic, form hollow, spheroid structures in 3D culture referred to as acini. The development of normal acini requires the tight spatiotemporal regulation of cellular proliferation, polarization, apoptosis, and growth arrest. Treatment of MCF10A acini with TPA caused the appearance of multi-acinar structures. Surprisingly, this phenotype did not involve an increase in cell number or major changes in cell death, and polarization. Instead, live cell and confocal microscopy revealed that TPA stimulates MCF10A acini to aggregate. TPA induces the PKC-dependent production of actin-based protrusions, which leads to the formation of cellular bridges between acini, the clustering of acini, and allows cells to move into adjacent acini. During this process, the integrity of the laminin V basement membrane is disrupted, while E-cadherin-based cell-cell contacts remain intact. Altogether, our results show that under the biochemical and structural constraints of epithelial tissue, as modeled by the 3D MCF10A system, TPA induces a novel PKC-dependent phenotype that resembles local invasion. Of the many effects caused by TPA, these studies highlight the aggressive production of actin-based cellular protrusions as a potentially important event along the pathway to carcinogenesis.

Targeted deletion of RasGRP1 impairs skin tumorigenesis

Ras is frequently activated in cutaneous squamous cell carcinoma, a prevalent form of skin cancer. However, the pathways that contribute to Ras-induced transformation have not been entirely elucidated. We have previously demonstrated that in transgenic mice, overexpression of the Ras activator RasGRP1 promotes the formation of spontaneous skin tumors and enhances malignant progression in the multistage carcinogenesis skin model that relies on the oncogenic activation of H-Ras. Utilizing a RasGRP1 knockout mouse model (RasGRP1 KO), we now show that lack of RasGRP1 reduced the susceptibility to skin tumorigenesis. The dependency on RasGRP1 was associated with a diminished response to the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Specifically, we found impairment of epidermal hyperplasia induced by TPA through keratinocyte proliferation. Using a keratinocyte cell line that carries a ras oncogenic mutation, we also demonstrated that RasGRP1 could further activate Ras in response to TPA. Thus, we propose that RasGRP1 upregulates signaling from Ras and contributes to epidermal tumorigenesis by increasing the total dosage of active Ras.

Chemoprevention of Skin Cancer: Effect of Lawsonia inermis L. (Henna) Leaf Powder and its Pigment Artifact, Lawsone in the Epstein- Barr Virus Early Antigen Activation Assay and in Two-Stage Mouse Skin Carcinogenesis Models

In continuation of our studies with chemoprevention potential of plant-derived naphthoquinone derivatives, leaf powder of the medicinal plant Lawsonia inermis L, commonly known as 'henna', was evaluated by its inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Lawsone (2-hydroxy- 1,4-naphthoquinone), the reddish orange pigment artifact formed during the extraction or preparation of the dye from henna leaves and believed to be the active component, was also assessed in this in vitro assay. Both showed a profound inhibition (>88%) of EBV-EA activation. In the in vivo two-stage mouse skin carcinogenesis study using UV-B radiation for initiation and TPA for tumor promotion, oral feeding of henna (0.0025%) in drinking water ad libitum decreased tumor incidence by 66% and multiplicity by 40% when compared to the positive control at 10 weeks of treatment. Similarly, in the above mouse model, orally fed lawsone (0.0025%) decreased tumor incidence by 72% and multiplicity by 50%. The tumor inhibitory trend continued throughout the 20-week test period. Similar antitumor activities were observed when henna (0.5 mg/ml) was applied topically on the back skin in the UV-B initiated, TPA promoted and peroxynitrite initiated, TPA promoted mouse skin carcinogenesis models. Topically applied lawsone (0.015 mg/ml) also exhibited similar protection against tumor formation in the 7,12-dimtehylbenz(a)anthracene induced and TPA promoted skin cancer in mice. Also, there was a delay of 1 to 2 weeks in tumor appearance in both henna and lawsone treated groups compared to control in all three test models. This study ascertains the skin cancer chemopreventive activity of henna leaf powder and lawsone when administered by either oral (through drinking water) or topical (by application on the back skin) routes. Further, it emphasizes the need for the evaluation of these henna-derived green chemopreventive candidates in combination with currently used sunscreen agents for complementary anticancer potential against UV-induced skin carcinogenesis.

Naturally Occurring Phenolic Acids Modulate TPA-induced Activation of EGFR, AP-1, and STATs in Mouse Epidermis

Epidermal growth factor receptor (EGFR) plays an important role in epithelial carcinogenesis and appears to be involved in STATs activation. In this study we investigated the possible interference of naturally occurring phenolic acids with EGFR, activator protein-1 (AP-1), and signal transducers and activators of transcription (STATs) pathways activated by topical application of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Balb/c mice epidermis. Pretreatment with tannic or chlorogenic acid resulted in a significant decrease in the phosphorylation of EGFR Y-1068 and Y-1173 tyrosine residues, which was accompanied by reduced activation of AP-1. Tannic acid decreased also the c-Jun AP-1 subunit level and binding to TPA response element (TRE) (3- and 2-fold in comparison with TPA-treated group respectively). Simultaneous reduction of JNK activity might be responsible for reduced activation of AP-1. In contrast to these more complex phenolics, protocatechuic acid increased the activity of JNK and was also the most efficient inhibitor of STATs activation. These results indicate that naturally occurring phenolic acids, by decreasing EGFR, AP-1, and STATs activation, may modulate other elements both upstream and downstream in these pathways and thus inhibit the tumor development. Although more complex phenolics affect mainly the EGFR/AP-1 pathway, STATs seem to be the most important targets for simple compounds, such as protocatechuic acid.

The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice

Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin.

Abstract 1290: Investigations to evaluate the role of Twist1 during tumor promotion and epithelial carcinogenesis.

Abstract Recent studies have identified the involvement of receptor tyrosine kinases (RTKs) and their downstream signaling pathways, including MAPK, PI3K/Akt/mTOR, and Stat3, as key regulatory factors of epidermal proliferative capacity during skin carcinogenesis. Through development and study of transgenic mice with overexpression of a constitutively active form of Stat3 (Stat3C) in the basal cell layer, previous studies in the laboratory discovered that constitutive Stat3 activation accelerates tumor progression in the two-stage skin carcinogenesis model, which utilizes the tumor initiator 7,12-dimethylbenz[α]anthracene (DMBA) and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Twist1, a well-known inducer of EMT, has been shown to be regulated by Stat3 in a number of systems. Western blot analysis of epidermal protein lysates from Stat3C transgenic mice indicated a two-fold induction of Twist1 expression as compared to wild type mice. In addition, immunofluorescence staining analysis of tumors from wild type mice [papillomas and squamous cell carcinomas (SCCs)] and Stat3C mice (SCCs) showed nuclear co-localization of Twist1 and Stat3. In the present study, we investigated the activation and subsequent downstream signaling of Twist1 during TPA-induced epidermal hyperproliferation. Western blot analysis of epidermal lysates from mice treated topically with multiple applications of TPA showed an initial decrease (4-6 h) followed by a subsequent rebound of Twist1 protein at 18 hours post TPA treatment. This initial decrease and then rebound coincided with that of the cell cycle regulatory proteins cyclin D1, cyclin E1, E2F-1, and c-myc. Interestingly, basal levels of Twist1 were reduced in epidermis specific Stat3 deficient mice. Furthermore, Stat3 deficient mice treated with TPA failed to show a rebound in expression of Twist1 in the epidermis. Immunofluorescence experiments of TPA treated epidermis revealed that nuclear localization of Twist1 was restricted to the proliferative compartment. Collectively, these results, combined with other data to be presented, suggest that Stat3 regulates basal levels of Twist1. This protein is localized in the nucleus of cells found primarily in the proliferative compartment of hyperplastic epidermis and skin tumors. The current results further suggest that Twist1 may play a role in cell cycle progression in keratinocytes during tumor promotion. Research supported by CA76520. Citation Format: Jaya Srivastava, Everardo Macias, Dharanija Rao, Kaoru Kiguchi, John DiGiovanni. Investigations to evaluate the role of Twist1 during tumor promotion and epithelial carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1290. doi:10.1158/1538-7445.AM2013-1290

TPA‐induced invasion of HT29 cells is mediated through p67phox‐dependent matrix metalloproteniase‐7 induction

Tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced ROS have been implicated in increased expression of matrix metalloproteniase‐7 (Matrilysin, MMP‐7), which is an important enzyme for cancer invasion and also a prognostic marker in colorectal cancer. However, the source of TPA‐induced ROS is still controversial whether it is NADPH oxidase or mitochondria. In the present study, we report that TPA‐induced ROS production and matrigel invasion of HT‐29 were inhibited by apocynin and diphenyleneiodinium (DPI), NADPH oxidase inhibitors, but not by other inhibitors for mitochondria or cytosolic enzymes. TPA increased p67phox expression in a time‐dependent manner without altering the level of other components of NADPH oxidase. In addition, TPA‐induced invasion of HT29 cells was significantly blocked by treatment with p67phox and Nox2 siRNAs but not with Nox1 siRNA. Moreover, TPA decreased mRNA and protein levels of ¥â‐catenin that is an integral part of adherens junction associated with other catenins and cadherins. The TPA‐induced ¥â‐catenin decrease and loss of cell‐cell adherence were also reversed by treatment with apocynin. Taken together, TPA induces HT29 cancer cell invasion by increasing MMP‐7 expression and by downregulating ¥â‐catenin through p67phox‐dependent ROS production.

3,3'-Diindolylmethane(DIM)이 Human Mammary Epithelial Cell에서 12-O-tetradecanoylphorbol-13-acetate에 의해 유도된 COX-2 발현에 미치는 영향

3,3"-Diindolylmethane(DIM)은 십자화과 채소에 포함되어 있는 indole-3-carbinol(I3C)이 동물의 산성 위액에서 중합되어 생성된 물질이다. 지금까지 DIM은 유방암, 전립선암 그리고 대장암 세포주에서 항암효과가 있다고 알려져 있으며 그 기작에 대한 연구도 다양하게 진행되고 있다. 그러나 정상세포에서 암화과정 중 암의 촉진과 진행과정의 주요한 항암 표적인 항염증에 대한 연구는 보고된 바 없다. 따라서 본 연구에서는 유방상피세포인 MCF-10A 세포에 12-Ο-tetradecanoylphorbol-13-acetate(TPA)로 염증반응을 유도한 후 DIM이 염증작용에 미치는 영향을 조사하였다. 그 결과 MCF-10A 세포에서 TPA에 의해 유도된 COX-2 단백질 및 mRNA 발현이 DIM 처리에 의해 현저히 감소하였다. 또한 TPA에 의해 유도된 IκB-α의 분해, p65의 핵으로의 이동, NF-κB DNA binding activity 역시 DIM의 처리에 의해 감소하였다. 이는 DIM이 인간의 유방상피세포인 MCF-10A 세포에서 TPA에 의해 유도된 NF-κB 신호전달체계의 활성을 억제함으로써 COX-2의 발현을 억제하여 염증반응을 조절함을 나타낸다. 그러므로 이러한 결과들은 DIM을 염증성 질환 예방제 또는 치료제로 개발할 수 있는 가능성을 시사한다.

PKM2 inhibitor shikonin suppresses TPA‐induced mitochondrial malfunction and proliferation of skin epidermal JB6 cells

Chemoprevention has been a pivotal and effective strategy during the skin cancer treatment. Using human skin normal and tumor samples, we demonstrated that both the expression and activity levels of pyruvate kinase M2 (PKM2) were higher in skin tumor tissues than normal tissues, suggesting that PKM2, one of important metabolic enzyme, might serve as a target for skin cancer prevention and/or therapy. Shikonin, a small-molecule active chemical, has been studied as an anti-cancer drug candidate in human cancer models. However, the mechanism of action and the chemopreventive potential of shikonin are unclear. Herein, we used the skin epidermal JB6 P+ cells and demonstrated that shikonin suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induced neoplastic cell transformation and PKM2 activation in the early stage of carcinogenesis. Mitochondrial functions were inhibited by TPA treatment, as indicated by reduced mitochondrial membrane potential and mitochondrial respiration, which were restored by shikonin. We also examined the levels of lactate as a glycolysis marker, and shikonin suppressed its increase caused by tumor promoter treatment. Modulation of cell metabolism by shikonin was associated with G2-M phase accumulation, and Fra-1 (a major subunit of activator protein 1 in skin tumorigenesis) downregulation. In addition, we demonstrated that AMP-activated protein kinase (AMPK), an energy sensor, which is inactivated by TPA, shikonin could reverse AMPK activity. These results suggest that shikonin bears chemopreventive potential for human skin cancers in which PKM2 is upregulated, which might be mediated by inhibiting oncogenic activation, PKM2 activation, and mitochondrial dysfunction.

Withaferin A suppresses tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate‐induced decreases in isocitrate dehydrogenase 1 activity and mitochondrial function in skin epidermal JB6 cells

Withaferin A (WA) is a bioactive compound derived from Withania somnifera. The antitumor activity of WA has been well studied in human cancer models; however, its chemopreventive potential is unclear. In the present study, we used the skin epidermal JB6 P+ cells, a well-established model for tumor promotion, and demonstrated that WA suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell transformation and cell proliferation. Interestingly, TPA inactivated isocitrate dehydrogenase 1 (IDH1), which was reversed by WA. Similar results were also observed in mouse skin tissue. Therefore, we focused on metabolism as the potential mechanism of action. We found that mitochondrial functions were downregulated by TPA treatment, as indicated by reduced mitochondrial membrane potential, complex I activity and mitochondrial respiration. However, all of these downregulations were inhibited by WA. In addition, we examined the levels of α-ketoglutarate, a product of IDH1, and WA blocked its reduction upon TPA treatment. Finally, we detected the lactate level as a glycolysis marker, and WA suppressed its elevation caused by tumor promoter treatment. Altogether, these results suggest that WA might exert its chemopreventive activity via inhibiting not only oncogenic activation, but also IDH1 inactivation and mitochondrial dysfunction in early tumorigenesis.

Evaluation of skin cancer chemoprevention potential of sunscreen agents using the Epstein–Barr virus early antigen activation in vitro assay

In our continuing search for novel cancer chemopreventive compounds of natural and synthetic origin, we have evaluated 14 commonly used ultraviolet (UV) sunscreen agents (designated UV-1 to UV-14) for their skin cancer chemoprevention potential. They belong to 8 different chemical categories: aminobenzoate (UV-5, UV-7, UV-8 and UV-14), benzophenone (UV-1, UV-2, UV-3 and UV-13), benzotriazole (UV-10), benzyloxyphenol (UV-9), cinnamate (UV-6), quinolone (UV-4), salicylate (UV-11) and xanthone (UV-12). In the in vitro assay employed, the sunscreens were assessed by their inhibition of the Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in human lymphoblastoid Raji cells. All sunscreens tested were found to exhibit anti-tumour promoting activity: listed in decreasing order, moderate (UV-11, UV-2, UV-7, UV-12, UV-3, UV-9 and UV-14) to weak (UV-1, UV-6, UV-8, UV-16, UV-5, UV-4 and UV-10) with octyl salicylate (UV-11) as the most potent and drometrizole (UV-10) as the least potent among the compounds evaluated. A plausible relationship between the antioxidant property of sunscreens and their ability to promote anti-tumour activity was noted. The results call for a comprehensive analysis of skin cancer chemoprevention potential of currently used UV sunscreen agents around the globe to identify those with the best clinical profile.

Polymeric black tea polyphenols modulate the localization and activity of 12-O-tetradecanoylphorbol-13-acetate-mediated kinases in mouse skin: Mechanisms of their anti-tumor-promoting action

Polymeric black tea polyphenols (PBPs) have been shown to possess anti-tumor-promoting effects in two-stage skin carcinogenesis. However, their mechanisms of action are not fully elucidated. In this study, mechanisms of PBP-mediated antipromoting effects were investigated in a mouse model employing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to controls, a single topical application of TPA to mouse skin increased the translocation of protein kinase C (PKC) from cytosol to membrane. Pretreatment with PBPs 1–3 decreased TPA-induced translocation of PKC isozymes (α, β, η, γ, ε) from cytosol to membrane, whereas PBPs 4 and 5 were less effective. The levels of PKCs δ and ζ in cytosol/membrane were similar in all the treatment groups. Complementary confocal microscopic evaluation showed a decrease in TPA-induced PKCα fluorescence in PBP-3-pretreated membranes, whereas pretreatment with PBP-5 did not show a similar decrease. Based on the experiments with specific enzyme inhibitors and phosphospecific antibodies, both PBP-3 and PBP-5 were observed to decrease TPA-induced level and/or activity of phosphatidylinositol 3-kinase (PI3K) and AKT1 (pS473). An additional ability of PBP-3 to inhibit site-specific phosphorylation of PKCα at all three positions responsible for its activation [PKCα (pT497), PKC PAN (βII pS660), PKCα/βII (pT638/641)] and AKT1 at the Thr308 position, along with a decrease in TPA-induced PDK1 protein level, correlated with the inhibition of translocation of PKC, which may impart relatively stronger chemoprotective activity to PBP-3 than to PBP-5. Altogether, PBP-mediated decrease in TPA-induced PKC phosphorylation correlated well with decreased TPA-induced NF-κB phosphorylation and downstream target proteins associated with proliferation, apoptosis, and inflammation in mouse skin. Results suggest that the antipromoting effects of PBPs are due to modulation of TPA-induced PI3K-mediated signal transduction.

Abstract 549: Role of the glucocorticoid receptor in skin carcinogenesis and stem cell maintenance: Implication for the use of selective GR activators (SEGRA) as anti-cancer drugs

Abstract Glucocorticoids are important physiological and pharmacological regulators of epidermal proliferation, differentiation and skin tumorigenesis. The glucocorticoid effects are mediated via the glucocorticoid receptor (GR), a transcription factor that regulates genes expression by DNA-binding dependent transactivation, and DNA-binding independent transrepression through negative interaction with other transcription factors. Our previous studies demonstrated that keratin5.GR transgenic animals are resistant to skin carcinogenesis. We also found that GR diminishes the number of follicular epithelial stem cells (SC), reduces their proliferative and survival potential and affects the expression of follicular SC “signature” genes. To further explore the tumor suppressor role of GR in skin carcinogenesis we generated skin-specific GR knockout animals by breeding of GRflox/flox animals with Keratin5.Cre recombinase animals in C57Bl genetic background. Skin-specific GR KO (Cre/GRfl/fl) mice do not have gross skin or hair phenotype. However, the detailed analysis of keratinocyte markers revealed decreased expression of some intermediate/late markers of differentiation including Keratin 10 and loricrin. We also found that Cre/GRfl/fl mice are more sensitive to the effects of UV irradiation and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The ongoing DMBA/TPA skin carcinogenesis experiment showed that Cre/GRfl/fl mice are more prone to skin carcinogenesis: they have higher tumor incidence and tumor multiplicity compared to GRfl/fl littermates. The analysis of global effect of the GR overexpression and GR silencing on gene expression in follicular epithelial SC, basal keratinocytes, and mouse skin tumors 1) revealed important GR targets in keratinocytes including STAT3 and REDD1 - the major regulator of mTOR; 2) provided an unexpected evidence that gene transrepression by GR plays critical role in the maintenance of SC and in inhibition of skin carcinogenesis. It is well understood that therapeutic effects of glucocorticoids are mediated by GR transreprssion, while many adverse metabolic effects are mediated by GR transactivation. Thus, our findings are important in light of drug discovery programs focused on the development of selective GR acivators (SEGRA) that preferentially induce GR transrepression. Some of SEGRA entered preclinical studies or clinical trials as anti-inflammatory GR ligands with reduced side effects. Our results suggest that SEGRA could be also used for skin cancer prevention/treatment. 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 549. doi:1538-7445.AM2012-549

Miz1 Is a Critical Repressor of cdkn1a during Skin Tumorigenesis

The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15Ink4), cdkn1a (p21Cip1), and cdkn1c (p57Kip2). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin.

Anti-Tumor-Promoting Activity of Tibolone and its Metabolites

The aim of this study was to evaluate the cancer chemopreventive potential of the widely prescribed drug tibolone (17alpha-ethynyl-7alpha-methyl-5(10)-estren-3-one, CAS 5630-53-5) and its main metabolites, 17alpha-ethynyl-7alpha-methyl-4-estren-3-one (CAS 1162-60-3), 17alpha-ethynyl-7alpha-methyl-5(10)-estrene-3alpha,17beta-diol (CAS 100239-44-9) and 17alpha-ethynyl-7alpha-methyl-5(10)-estrene-3beta,17beta-diol (CAS 100239-45-0), by studying their anti-tumor-promoting activity. To this aim the test compounds were submitted to the short term in vitro assay for the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) as a primary screening for anti-tumor promoters. All the compounds showed high inhibitory activity and low cytotoxicity as compared to literature data. To extend the study to an animal model, tibolone and its 3alpha-hydroxy metabolite (CAS 100239-44-9) were also assayed in the in vivo two-stage on mouse skin carcinogenesis test, exhibiting significant inhibitory effects on TPA promoted mouse skin papillomas formation. A comparison with literature data indicated them as more potent compounds than other steroids previously studied such as digitoxigenin, cortisone, hydrocortisone, and prednisolone.

TPA-induced p21 expression augments G2/M arrest through a p53-independent mechanism in human breast cancer cells

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.

Abstract B59: A combination of resveratrol and ursolic acid produces synergistic inhibitory effects on markers of skin tumor promotion

Abstract Resveratrol (RES), found in grapes and Ursolic Acid (UA), found in rosemary and apples, delay the onset and reduce the number of experimentally-induced skin tumors in mice and inhibit the oncogenic Akt/mammalian target of rapamycin (Akt/mTOR) and Nuclear Factor kappa B (NF\#954;B) pathways. Based on their similar downstream effects via different proximal targets, we hypothesized that a combination treatment of RES and UA would synergize to produce a stronger inhibitory effect on skin tumor promotion. Methods: The dorsal skin of FVB mice (n=8) were shaved and treated topically twice per week for 2 weeks with sub-optimal doses of 2 μmol UA, 2 μmol RES, or a combination of both in 200 μL acetone. Each treatment was followed 30 minutes later with topical application of 3.4 nmol of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in 200 μL acetone. Animals were sacrificed either 6 h (Western blotting) or 48 h (histology) after the final TPA treatment. The skins of five mice per group were removed and the epidermis was collected by scraping for Western blotting. For immunohistochemical (IHC) analysis of epidermal proliferation three mice per group were i.p. injected with 100 mg/kg Bromodeoxyuridine (BrdU) in PBS 30 min. prior to sacrifice. Mouse skin from treated areas was fixed in formalin, paraffin embedded, and analyzed via IHC. Results: RES and UA in combination had a stronger effect on hallmarks of skin tumor promotion than was expected based on the sum of their individual inhibitory effects, indicating synergism between RES and UA. RES and UA in combination synergistically inhibited TPA-induced epidermal thickening (80.2% inhibition vs 66.1 % expected) and epidermal proliferation (100.0% inhibition vs 90.0% expected). Western blotting revealed that components of the NF\#954;B and Akt/mTOR pathways were synergistically downregulated by the RES + UA combination in mouse epidermis. TPA-induced Akt (thr308) phosphorylation (86.0% inhibition vs 26.6% expected) and NFkB subunit p65 (ser536) phosphorylation (100.0% inhibition vs 73.3% expected) were suppressed to a much stronger degree with the combination treatment than that predicted. Similar results were seen for other pathway components, such as p70S6K and ribosomal S6. The synergistic activities of RES and UA were also manifest in the HaCaT human keratinocyte cell line. Cells were treated with sub-optimal doses of RES, UA, or RES + UA and stimulated with 10 ng/ml human tumor necrosis factor alpha (TNFα) for 30 min. and cell lysates were analyzed via Western blotting. TNFα-mediated phosphorylation of p65 was not inhibited by 25 μM RES or 0.1 μM −1 μM UA, but combinations of RES + UA resulted in strong synergistic inhibition of p65 phosphorylation (>66% inhibition vs 0% expected). Also, when HaCaT cells were treated with NFkB- and Akt/mTOR-inhibitory doses of RES (150 μM-200 μM) and UA (5 μM-10 μM), a strong increase (5 fold) in phosphorylation/activation of the energy sensing AMP-activated protein kinase (AMPK) was observed by Western blotting. AMPK also inhibits NF\#954;B and Akt/mTOR activities in other systems, suggesting that AMPK activation may function as a mediator of the synergistic anti-tumor promoting effects of RES and UA. Conclusions: These results indicate that UA and RES synergize to inhibit markers of tumor promotion in both in vivo and in vitro skin models. Future studies will further elucidate the synergistic mechanism of RES and UA and also determine if RES and UA have a synergistic preventive effect on skin tumor promotion during two-stage skin carcinogenesis in vivo. Supported by NIH grants CA079065, supplemental CA079065-10S1 (ARRA), CA129409 and CA037111. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B59.

Molecular mechanism underlying anti-apoptotic and anti-inflammatory effects of Mamao ( Antidesma thwaitesianum Müll. Arg.) polyphenolics in human breast epithelial cells

There has been increasing interest in finding natural antioxidants to prevent free radical damage and retard the progress of chronic inflammatory diseases. Our previous data demonstrated the strong antioxidant properties of polyphenolics in Mamao seed (MS) and Mamao marc (MM) extracts. In this study we further investigated the effect of MS and MM polyphenolics on hydrogen peroxide (H 2O 2)-induced apoptosis and tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, using human breast epithelial (MCF10A) cells. MS and MM extracts conferred dose-dependent protection against H 2O 2-induced apoptosis by inhibiting PARP/caspase-3 cleavage, inducing anti-apoptotic Bcl-2 expression, and down-regulating pro-apoptotic Bax. Moreover, MS and MM polyphenolics inhibited TPA-induced COX-2 and NF-κB activation by blocking the degradation of cytoplasmic IκBα, as well as subsequent nuclear translocation of p65 and attenuation of the activation of ERK, but not JNK and p38. These data establish the molecular mechanism for the anti-apoptotic and anti-inflammatory effects of MS and MM polyphenolics.

The potential use of protein kinase D inhibitors for prevention/treatment of epidermal tumors

The serine/threonine kinase protein kinase D (PKD) has been proposed to be a pro-proliferative, anti-differentiative signal in epidermal keratinocytes. Indeed, the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induces biphasic PKD activation, which mirrors the biphasic response of initial differentiation followed by proliferation and tumor promotion seen in TPA-treated keratinocytes in vitro and epidermis in vivo. Our objective was to test the idea that PKD's pro-proliferative and/or anti-differentiative effects in keratinocytes contribute to TPA-induced tumorigenesis. Using western analysis and assays of keratinocyte proliferation and differentiation, we investigated the effect of inhibitors of PKD on keratinocyte function. We found that overexpression of a constitutively active PKD mutant increased, and of a dominant-negative PKD mutant decreased, keratinocyte proliferation. A recently described selective PKD inhibitor showed low potency to inhibit keratinocyte proliferation or PKD activation. Therefore, we tested the ability of known only relatively selective PKD inhibitors on keratinocyte function and protein kinase activation. H89 {N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline-sulfonamide}, a reported inhibitor of PKD and cAMP-dependent protein kinase, enhanced the effect of a differentiating agent on a marker of keratinocyte differentiation. Another reported non-selective PKD inhibitor, resveratrol stimulated differentiation and inhibited proliferation. The protein kinase C/PKD inhibitor Gö6976 blocked the increase in proliferation (as measured by DNA specific activity) induced by chronic TPA without affecting the initial TPA-elicited differentiation. Our results support the idea that relatively selective PKD inhibitors, such as Gö6976, H89 and resveratrol, might be useful for preventing/treating epidermal tumorigenesis without affecting keratinocyte differentiation.