Potential inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, hyperproliferation, and hyperplasiogenic responses by celecoxib in mouse skin.

PURPOSE: Skin exposure to noxious agents leads to cutaneous lesion marked by an increase in inflammation, cellular proliferation, and hyperplasiogenic reactions. Studies have demonstrated that these damages breach the skin integrity resulting in the aetiology of various cutaneous disorders like atopic dermatitis, eczema, psoriasis, and development of non-melanoma skin cancer. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, is an effective treatment for a variety of inflammatory diseases. Its importance in the therapy of skin problems, however, remains under appreciated. METHODS: We tested efficacy of topically applied celecoxib in mitigating skin inflammation, cellular proliferation, and hyperplasia induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in Swiss albino mice. RESULTS: Celecoxib (5 and 10 µmol) markedly reduced TPA (10 nmol) induced prostaglandin E2 (PGE2) production, oedema formation, myeloperoxidase (MPO) activity, and levels of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6). It also resulted in a considerable decrease in ornithine decarboxylase (ODC) activity and the incorporation of [3H]-thymidine into DNA. In addition, there was a significant reduction in histoarchitectural abnormalities such as epidermal thickness, number of epidermal cell layers, neutrophil infiltration, intercellular oedema, and vasodilation. CONCLUSION: Our results demonstrate that topical celecoxib can reduce the inflammation, hyperproliferation, and hyperplasiogenic events of skin insults suggesting that it may prove to be a valuable management option for cutaneous lesion and associated illnesses such as atopic dermatitis, eczema, and psoriasis, as well as the emergence of non-melanoma cancer.

The Anti-Tumorigenic Role of Cannabinoid Receptor 2 in Non-Melanoma Skin Cancer.

Five million non-melanoma skin cancers occur globally each year, and it is one of the most common malignant cancers. The dysregulation of the endocannabinoid system, particularly cannabinoid receptor 2 (CB2), is implicated in skin cancer development, progression, and metastasis. Comparing wildtype (WT) to systemic CB2 knockout (CB2-/-) mice, we performed a spontaneous cancer study in one-year old mice, and subsequently used the multi-stage chemical carcinogenesis model, wherein cancer is initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that aging CB2-/- mice have an increased incidence of spontaneous cancerous and precancerous skin lesions compared to their WT counterparts. In the DMBA/TPA model, CB2-/- developed more and larger papillomas, had decreased spontaneous regression of papillomas, and displayed an altered systemic immune profile, including upregulated CD4+ T cells and dendritic cells, compared to WT mice. Immune cell infiltration in the tumor microenvironment was generally low for both genotypes, although a trend of higher myeloid-derived suppressor cells was observed in the CB2-/- mice. CB2 expression in carcinogen-exposed skin was significantly higher compared to naïve skin in WT mice, suggesting a role of CB2 on keratinocytes. Taken together, our data show that endogenous CB2 activation plays an anti-tumorigenic role in non-melanoma skin carcinogenesis, potentially via an immune-mediated response involving the alteration of T cells and myeloid cells coupled with the modulation of keratinocyte activity.

Targeting 14-3-3ε activates apoptotic signaling to prevent cutaneous squamous cell carcinoma.

More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions.

Tumor promoter TPA activates Wnt/ß-catenin signaling in a casein kinase 1-dependent manner.

The tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/ß-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/ß-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/ß-catenin signaling in a casein kinase 1 (CK1) ε/δ-dependent manner. TPA stabilized CK1ε and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1ε-LRP6-axin1 complex, leading to an increase in cytosolic ß-catenin. Moreover, TPA increased the association of ß-catenin with TCF4E in a CK1ε/δ-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1ε/δ inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/ß-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/ß-catenin signaling.

ß-Elemene inhibits 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin tumorigenesis through suppression of NF-κB-associated signaling events in the mouse skin model.

ß-Elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane), a natural sesquiterpene-derived curcumae radix, exhibits a variety of pharmacologic properties including anticancer. However, the molecular action of ß-elemene in chemical-induced skin carcinogenesis remains unclear. Therefore, the present study executes to investigate a possible effect of ß-elemene in the 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor model. The experimental mice were subjected to execute two-stage skin carcinogenesis and it has been initiated by the addition of DMBA on the dorsal portion of the mouse skin. One week after, for chemical carcinogen of mice, topical exposure of DMBA has been induced following with TPA (5 nmol) in acetone (200 µL) given weekly twice for 20 weeks respectively. After completion of the experimental period, we noticed that 100% of tumor incidence, histopathological changes, decreased lipid peroxidation (LPO), and decreased antioxidant levels in DMBA/TPA-promoted skin carcinogenesis. Furthermore, enhanced activity of inflammatory protein markers (nuclear factor [NF]-κB, tumor necrosis factor-α, interleukin-6, cyclooxygenase-2, and nitric oxide synthase) and cell-proliferative messenger RNA markers (PCNA, cyclin D1), and increased antiapoptotic protein Bcl-2; decreased proapoptotic protein marker events Bax and caspase 3 and 9 expressions were noticed in DMBA/TPA promoted skin tissue. In this study, we noticed that ß-elemene noticeably reversed the histopathological changes and antioxidant levels in tumor-bearing mice. Conversely, ß-elemene effectively inhibits inflammation, cell proliferation events, and enhances proapoptotic factors, by suppression of NF-κB transcriptional activation in DMBA/TPA animals. Thus, we concluded that ß-elemene prevents DMBA/TPA promoted skin carcinogenesis through its antioxidant and abate inflammation markers and cell-proliferative markers also activating proapoptotic molecules.

Perphenazine Attenuates the Pro-Inflammatory Responses in Mouse Models of Th2-Type Allergic Dermatitis.

Developing dermatitis therapeutics has been faced with challenges including adverse effects of topical steroid and high cost of new developing drugs. Here, we found the expression levels of dopamine receptor D2 is higher in skin biopsies of dermatitis patients and an oxazolone-induced animal model of dermatitis. We used perphenazine, an FDA-approved dopamine receptor antagonist to determine the therapeutic effect. Two different animal models including 12-o-tetradecanoylphorbol-13-acetate (TPA) and oxazolone (OXA)-induced dermatitis were employed. TPA and OXA-mediated ear swelling was attenuated by perphenazine. Moreover, perphenazine inhibited infiltrated mast cells into lesion area. We found levels of serum IgE, histamine and cytokines are decreased in mice cotreated with perphenazine and OXA compared to OXA-treated mice. Overall, this is a first study showing that the FDA-approved, anti-psychotic drug, perphenazine, alleviates animal models of dermatitis.

Molecular Targets of Natural Products for Chondroprotection in Destructive Joint Diseases.

Osteoarthritis (OA) is the most common type of arthritis that occurs in an aged population. It affects any joints in the body and degenerates the articular cartilage and the subchondral bone. Despite the pathophysiology of OA being different, cartilage resorption is still a symbol of osteoarthritis. Matrix metalloproteinases (MMPs) are important proteolytic enzymes that degrade extra-cellular matrix proteins (ECM) in the body. MMPs contribute to the turnover of cartilage and its break down; their levels have increased in the joint tissues of OA patients. Application of chondroprotective drugs neutralize the activities of MMPs. Natural products derived from herbs and plants developed as traditional medicine have been paid attention to, due to their potential biological effects. The therapeutic value of natural products in OA has increased in reputation due to their clinical impact and insignificant side effects. Several MMPs inhibitor have been used as therapeutic drugs, for a long time. Recently, different types of compounds were reviewed for their biological activities. In this review, we summarize numerous natural products for the development of MMPs inhibitors in arthritic diseases and describe the major signaling targets that were involved for the treatments of these destructive joint diseases.

Effect of Terpenoids and Flavonoids Isolated from Baccharis conferta Kunth on TPA-Induced Ear Edema in Mice.

In this study, we isolated from the aerial parts of Baccharis conferta Kunth (i) a new neoclerodane, denominated "bacchofertone"; (ii) four known terpenes: schensianol A, bacchofertin, kingidiol and oleanolic acid; and (iii) two flavonoids: cirsimaritin and hispidulin. All structures were identified by an exhaustive analysis of nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Extracts from aerial parts were screened for anti-inflammatory activity in the mice ear edema model of 12-O-tetradecanoylforbol-13-acetate mice. Dichloromethane extract (BcD) exhibited 78.5 ± 0.72% inhibition of edema, followed by the BcD2 and BcD3 fractions of 71.4% and 82.9% respectively, at a dose of 1 mg/ear. Kingidiol and cirsimaritin were the most potent compounds identified, with a median effective dose of 0.12 and 0.16 mg/ear, respectively. A histological analysis showed that the topical application of TPA promoted intense cell infiltration, and this inflammatory parameter was reduced with the topical application of isolated compounds.

XB130 deficiency enhances carcinogen-induced skin tumorigenesis.

XB130 is an adaptor protein that functions as a mediator of multiple tyrosine kinases important for regulating cell proliferation, survival, migration and invasion. Formerly predicted as an oncogene, alterations of its expression are documented in various human cancers. However, the exact role of XB130 in tumorigenesis is unknown. To address its function in skin tumorigenesis, a two-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA) study was performed on XB130 knockout (KO), heterozygous (HZ) and wild-type (WT) littermate mice. DMBA/TPA-treated XB130 KO and HZ males developed a significantly higher number of epidermal tumors that were notably larger in size than did WT mice. Interestingly, DMBA/TPA-treated female mice did not show any difference in tumor multiplicity regardless of the genotypes. The skin tumor lesions of XB130 KO males were more progressed with an increased frequency of keratoacanthoma. Deficiency of XB130 dramatically increased epidermal tumor cell proliferation. The responses to DMBA and TPA stimuli were also individually investigated to elucidate the mechanistic role of XB130 at different stages of tumorigenesis. DMBA-treated male XB130 KO mice showed compensatory p53-mediated stress response. TPA-treated XB130 KO males demonstrated more skin ulceration with more severe edema, enhanced cell proliferation, accumulation of infiltrating neutrophils and increased production of pro-inflammatory cytokine genes compared with WT mice. Enhanced activities of nuclear factor-kappa B pathway, increased protein expression of metalloproteinase-9 and ERK1/2 phosphorylation were found in these KO mice. These findings demonstrate that XB130 acts as a tumor suppressor in carcinogen-induced skin tumorigenesis that may be mediated through inhibiting inflammation.

Modulation of cancer signalling pathway(s) in two -stage mouse skin tumorigenesis by annonacin.

BACKGROUND: Annonacin, an annonaceous acetogenin isolated from Annona muricata has been reported to be strongly cytotoxic against various cell lines, in vitro. Nevertheless, its effect against in vivo tumor promoting activity has not been reported yet. Therefore, this study was aimed to investigate antitumor-promoting activity of annonacin via in vivo two-stage mouse skin tumorigenesis model and its molecular pathways involved. METHODS: Mice were initiated with single dose of 7,12-dimethylbenz[α]anthracene (DMBA) (390 nmol/100 µL) followed by, in subsequent week, repeated promotion (twice weekly; 22 weeks) with 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 µL). Annonacin (85 nM) and curcumin (10 mg/kg; reference) were, respectively, applied topically to DMBA/TPA-induced mice 30 min before each TPA application for 22 weeks. Upon termination, histopathological examination of skin, liver and kidney as well as genes and proteins expression analysis were conducted to elucidate the potential mechanism of annonacin. RESULTS: With comparison to the carcinogen control, Annonacin significantly increased the tumor latency period and reduced the tumor incidence, tumor burden and tumor volume, respectively. In addition, it also suppressed tumorigenesis manifested by significant reduction of hyperkeratosis, dermal papillae and number of keratin pearls on skin tissues. Annonacin also appeared to be non-toxic to liver and kidney. Significant modulation of both AKT, ERK, mTOR, p38, PTEN and Src genes and proteins were also observed in annonacin-targeted signaling pathway(s) against tumorigenesis. CONCLUSIONS: Collectively, results of this study indicate that annonacin is a potential therapeutic compound targeting tumor promoting stage in skin tumorigenesis by modulating multiple gene and protein in cancer signaling pathways without apparent toxicity.

Protein kinase C ϵ stabilizes ß-catenin and regulates its subcellular localization in podocytes.

Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of ß-catenin, which promotes its degradation. ß-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with ß-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower ß-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, ß-catenin showed a reversed subcellular localization pattern: Although ß-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates ß-catenin expression and stabilization in a glycogen synthase kinase 3ß-independent manner. Further, ß-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the ß-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in ß-catenin that are required for full ß-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and ß-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of ß-catenin levels and define PKCϵ as an important ß-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes.

Phorbol-12-myristate-13-acetate-mediated stabilization of leukemia inhibitory factor (lif) mRNA: involvement of Nucleolin and PCBP1.

Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human lif mRNA revealed several conserved sequences with conventional cis-acting elements. A 216 nucleotide containing proximal cis-element with two AUUUA pentamers and four poly-rC sequences demonstrated significant mRNA destabilizing potential, which, on treatment with PMA, showed stabilizing activity. Affinity chromatography followed by western blot and RNA co-immunoprecipitation of PMA-treated U937 extract identified Nucleolin and PCBP1 as two protein trans-factors interacting with lif mRNA, specifically to the proximal non-conventional AU-rich region. PMA induced nucleo-cytoplasmic translocation of both Nucleolin and PCBP1. RNA-dependent in vivo co-association of both these proteins with lif mRNA was demonstrated by decreased co-precipitation in the presence of RNase. Ectopic overexpression of Nucleolin showed stabilization of both intrinsic lif mRNA and gfp reporter, whereas knockdown of Nucleolin and PCBP1 demonstrated a significant decrease in both lif mRNA and protein levels. Collectively, this report establishes the stabilization of lif mRNA by PMA, mediated by the interactions of two RNA-binding proteins, Nucleolin and PCBP1 with a proximal cis-element.

Dietary flavones counteract phorbol 12-myristate 13-acetate-induced SREBP-2 processing in hepatic cells.

Consumption of fruits and vegetables is generally regarded as beneficial to plasma lipid profile. The mechanism by which the plant foods induce desirable lipid changes remains unclear. SREBP-2 is crucial in cholesterol metabolism, and it is a major regulator of the cholesterol biosynthesis enzyme HMGCR. Our lab has previously illustrated that apigenin and luteolin could attenuate the nuclear translocation of SREBP-2 through an AMPK-dependent pathway. In the present study, these two flavones were studied for their ability to deter the same in an AMPK-independent signaling route. The processing of SREBP-2 protein was promoted by phorbol 12-myristate 13-acetate (PMA) in the hepatic cells WRL and HepG2, and the increased processing was reversed by apigenin or luteolin co-administration. EMSA results demonstrated that the PMA-induced DNA-binding activity was weakened by the flavones. The increased amount of nuclear SREBP-2 in cells was attenuated by the flavonoid as shown by immunocytochemical imaging. Quantitative reverse transcriptase-polymerase chain reaction assay demonstrated that the transcription of HMGCR under both flavone treatments was reduced. However, apigenin appeared to be stronger than luteolin in restraining PMA-induced HMGCR mRNA expression. Since PMA is a diacylglycerol analog, these findings might have some physiological implications.

Dual-specificity phosphatase 5 regulates nuclear ERK activity and suppresses skin cancer by inhibiting mutant Harvey-Ras (HRasQ61L)-driven SerpinB2 expression.

Ectopic expression of dual-specificity phosphatase 5 (DUSP5), an inducible mitogen-activated protein (MAP) kinase phosphatase, specifically inactivates and anchors extracellular signal-regulated kinase (ERK)1/2 in the nucleus. However, the role of endogenous DUSP5 in regulating the outcome of Ras/ERK kinase signaling under normal and pathological conditions is unknown. Here we report that mice lacking DUSP5 show a greatly increased sensitivity to mutant Harvey-Ras (HRas(Q61L))-driven papilloma formation in the 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) model of skin carcinogenesis. Furthermore, mouse embryo fibroblasts (MEFs) from DUSP5(-/-) mice show increased levels of nuclear phospho-ERK immediately after TPA stimulation and fail to accumulate total ERK in the nucleus compared with DUSP5(+/+) cells. Surprisingly, a microarray analysis reveals that only a small number of Ras/ERK-dependent TPA-responsive transcripts are up-regulated on deletion of DUSP5 in MEFs and mouse skin. The most up-regulated gene on DUSP5 loss encodes SerpinB2, an inhibitor of extracellular urokinase plasminogen activator and deletion of DUSP5 acts synergistically with mutant HRas(Q61L) and TPA to activate ERK-dependent SerpinB2 expression at the transcriptional level. SerpinB2 has previously been implicated as a mediator of DMBA/TPA-induced skin carcinogenesis. By analyzing DUSP5(-/-), SerpinB2(-/-) double knockout mice, we demonstrate that deletion of SerpinB2 abrogates the increased sensitivity to papilloma formation seen on DUSP5 deletion. We conclude that DUSP5 performs a key nonredundant role in regulating nuclear ERK activation, localization, and gene expression. Furthermore, our results suggest an in vivo role for DUSP5 as a tumor suppressor by modulating the oncogenic potential of activated Ras in the epidermis.

Wild carrot pentane-based fractions suppress proliferation of human HaCaT keratinocytes and protect against chemically-induced skin cancer.

BACKGROUND: Previous studies in our laboratory showed that the Lebanese Daucus carota ssp. carota (wild carrot) oil extract possesses in vitro and in vivo anticancer activities. The present study aims to examine the cytotoxic effect of Daucus carota oil fractions on human epidermal keratinocytes and evaluate the chemopreventive activity of the pentane diethyl ether fraction on DMBA/TPA induced skin carcinogenesis in mice. METHODS: Wild carrot oil extract was chromatographed to yield four fractions (F1, 100% pentane; F2, 50:50 pentane:diethyl ether; F3, 100% diethyl ether; F4 93:7 chloroform:methanol). The cytotoxic effect of fractions (10, 25, 50 and 100 µg/mL) was tested on human epidermal keratinocytes (non-tumorigenic HaCaT cells and tumorigenic HaCaT-ras variants) using WST a ssay. Cell cycle phase distribution of tumorigenic HaCaT-ras variants was determined by flow cytometry post-treatment with F2 fraction. Apoptosis related proteins were also assessed using western blot. The antitumor activity of F2 fraction was also evaluated using a DMBA/TPA induced skin carcinoma in Balb/c mice. RESULTS: All fractions exhibited significant cytotoxicity, with HaCaT cells being 2.4-3 times less sensitive than HaCaT-ras A5 (benign tumorigenic), and HaCaT-ras II4 (malignant) cells. GC-MS analysis revealed the presence of a major compound (around 60%) in the pentane/diethylether fraction (F2), identified as 2-himachalen-6-ol. Treatment of HaCaT-ras A5 and HaCaT-ras II4 cells with F2 fraction resulted in the accumulation of cells in the sub-G1 apoptotic phase and decreased the population of cells in the S and G2/M phases. Additionally, F2 fraction treatment caused an up-regulation of the expression of pro-apoptotic (Bax) and down-regulation of the expression of anti-apoptotic (Bcl2) proteins. A decrease in the phosphorylation of AKT and ERK was also observed. Intraperitoneal treatment with F2 fraction (50 or 200 mg/kg) in the DMBA/TPA skin carcinogenesis mouse model showed a significant inhibition of papilloma incidence (mice with papilloma), yield (number of papilloma/mouse) and volume (tumor relative size) at weeks 15, 18 and 21. CONCLUSION: The present data reveal that F2 fraction has a remarkable antitumor activity against DMBA/TPA-induced skin carcinogenesis, an effect that may be mediated through inhibition of the MAPK/ERK and PI3K/AKT pathways.

Pramipexole reduces inflammation in the experimental animal models of inflammation.

Pramipexole is a dopamine (DA) agonist (D2 subfamily receptors) that widely use in the treatment of Parkinson's diseases. Some epidemiological and genetic studies propose a role of inflammation in the pathophysiology of Parkinson's disease. To our knowledge, there is no study regarding the anti-inflammatory activity of pramipexol. Therefore, the aim of the study was to investigate anti-inflammatory effect of pramipexol. Anti-inflammatory effects of pramipexole were studied in three well-characterized animal models of inflammation, including carrageenan- or formalin-induced paw inflammation in rats, and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. The animals received pramipexol (0.25, 0.5 and 1 mg/kg, I.P.) 30 min before subplantar injection of carrageenan or formalin. Pramipexol (0.5 and 1 mg/kg) was also injected 30 min before topical application of TPA on the ear mice. Serum malondialdehyde (MDA) levels were evaluated in the carrageenan test. Finally, pathological examination of the inflamed tissues was carried out. Pramipexole significantly inhibited paw inflammation 1, 2, 3 and 4 h after carrageenan challenge compared with the control group (p < .001). Pramipexol also showed considerable anti-inflammatory activity against formalin-evoked paw edema over a period of 24 h (p < .001). TPA-induced ear edema was markedly decreased by pramipexol (p < .001). The pathological evaluation of the paws and ears revealed that pramipexole reduced tissue injury, neutrophil infiltration, and subcutaneous edema. Pramipexole did not alter the increased serum levels of MDA due to carrageenan injection. These data clearly indicate that pramipexol possesses significant anti-inflammatory activity. It seems that its antioxidants do not play an important role in these effects.

Possible Involvement of the Inhibition of NF-κB Factor in Anti-Inflammatory Actions That Melatonin Exerts on Mast Cells.

Melatonin is a molecule endogenously produced in a wide variety of immune cells, including mast cells (RBL-2H3). It exhibits immunomodulatory, anti-inflammatory and anti-apoptotic properties. The physiologic mechanisms underlying these activities of melatonin have not been clarified in mast cells. This work is designed to determine the anti-inflammatory effect and mechanism of action of melatonin on activated mast cells. RBL-2H3 were pre-treated with exogenous melatonin (MELx) at physiological (100nM) and pharmacological (1 mM) doses for 30 min, washed and activated with PMACI (phorbol 12-myristate 13-acetate plus calcium ionophore A23187) for 2 h and 12 h. The data shows that pre-treatment of MELx in stimulated mast cells, significantly reduced the levels of endogenous melatonin production (MELn), TNF-α and IL-6. These effects are directly related with the MELx concentration used. MELx also inhibited IKK/NF-κB signal transduction pathway in stimulated mast cells. These results indicate a molecular basis for the ability of melatonin to prevent inflammation and for the treatment of allergic inflammatory diseases through the down-regulation of mast cell activation. J. Cell. Biochem. 117: 1926-1933, 2016. © 2016 Wiley Periodicals, Inc.

Hematopoietic plakophilin-3 regulates acute tissue-specific and systemic inflammation in mice.

Plakophilin-3 (PKP3) is a member of the armadillo protein family, which is important in cell-cell contacts and signaling during development and tumorigenesis. In conventional facilities, PKP3-deficient mice (PKP3(-/-)) develop spontaneous dermatitis, indicating a possible involvement of PKP3 in inflammatory responses. Here, we show that PKP3 deficiency sensitizes mice to irritant contact dermatitis induced by phorbol myristate acetate (PMA). This sensitization occurred in mice with PKP3 deficiency in the hematopoietic system (PKP3(-/-hem)), but not if the deficiency was specific to skin keratinocytes (PKP3(-/-ker)). In a model of dextran sulfate sodium induced colitis, ubiquitous PKP3 deletion, but not intestinal epithelial PKP3 deficiency (PKP3(-/-IEC)), impaired survival from disease. Interestingly, PKP3(-/-hem) mice also displayed increased sensitivity to dextran sulfate sodium induced colitis. Finally, PKP3(-/-) mice were more sensitive to the lethality of lipopolysaccharide (LPS) injection than wild-type (WT) mice, and this phenotype was associated with increased intestinal permeability. PKP3(-/-IEC) mice did not reproduce the enhanced endotoxin reactivity of PKP3(-/-) mice, in contrast to PKP3(-/-hem) mice. Finally, in vitro stimulation of WT neutrophils with LPS or PMA increased Pkp3 expression. In conclusion, our data highlight a novel role for hematopoietic PKP3 in the regulation of both locally and systemically induced immune responses. Nonetheless, further research is needed to unravel the underlying mechanism.

Protective effects of ginsenoside F2 on 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation in mice.

Topical use of ginsenosides, the major bioactive substances in Panax ginseng, has been used for the treatment of irritated skin complaints. However, the protective mechanisms of ginsenosides remain unclear. In the present study, we investigated the anti-inflammatory role of ginsenoside F2 (GF2) on the skin inflammation. To induce irritant dermatitis, 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied on the surface of the mouse ears with or without treatments of GF2 and dexamethasone for 24 h. Protective effects of GF2 on edema and inflammation were assessed by measuring ear thickness, weights of skin punch, and inflammatory responses. In gross findings, treatments with GF2 significantly decreased skin thickness and weight compared to those of TPA-treated groups, which was comparable with the protective effects of dexamethasone. In addition, expression of inflammatory mediators was remarkably reduced in GF2-treated ears compared to that of vehicle-treated ears of mice. Interestingly, immunohistochemistry and flow cytometry analyses revealed that TPA treatment significantly increased infiltration of interleukin-17 (IL-17) producing dermal γδ T cells, while frequencies of γδ T cells was decreased by GF2 treatment, subsequently ameliorating inflammation in skin. Concomitantly, TPA-mediated skin inflammation was significantly ameliorated in IL-17A knock out mice. Furthermore, GF2 treatment inhibited infiltration and generation of reactive oxygen species (ROS) of neutrophils in damaged ears compared with vehicle-treated mice. These results clearly suggest that GF2 treatment ameliorates TPA-induced dermal inflammation by inhibiting production of IL-17 and ROS in γδ T cells and neutrophils, respectively. Therefore, as a natural compound, application of GF2 may be a novel therapeutic approach for treating skin inflammation.

Twist1 regulates keratinocyte proliferation and skin tumor promotion.

In the present study, we evaluated the effect of deleting Twist1 on keratinocyte proliferation and on skin tumor development using the two-stage chemical carcinogenesis model. BK5.Cre × Twist1(flox/flox) mice, which have a keratinocyte-specific Twist1 knockout (Twist1 KO), developed significantly reduced numbers of papilloma (70% reduction) and squamous cell carcinoma (75% reduction) as well as delayed tumor latency compared to wild-type (WT) mice. Interestingly, knockdown of Twist1 in primary keratinocytes impeded cell cycle progression at the G1/S transition that coincided with reduced levels of the cell cycle proteins c-Myc, Cyclin E1, and E2F1 and increased levels of p53 and p21. Furthermore, ChIP analyses revealed that Twist1 bound to the promoter regions of Cyclin E1, E2F1, and c-Myc at the canonical E-box binding motif suggesting a direct transcriptional regulation. Further analyses of Twist1 KO mice revealed a significant reduction in the number of label-retaining cells as well as the number of α6-integrin(+) /CD34(+) cells in the hair follicles of untreated mice compared to WT mice. These mice also exhibited significantly reduced epidermal proliferation in response to TPA treatment that again correlated with reduced levels of cell cycle regulators and increased levels of p53 and p21. Finally, Twist1 deficiency in keratinocytes led to an upregulation of p53 via its stabilization and nuclear localization, which is responsible for the increased expression of p21 in these cells. Collectively, these findings indicate that Twist1 has a novel role in epithelial carcinogenesis by regulating proliferation of keratinocytes, including keratinocyte stem cells during tumor promotion.