Activity In Mouse Skin Research Articles

Boric Acid Solution Inhibits Candida albicans Infections in Mouse Skin via the IL-23/Th17 Axis.

The purpose of this study was to investigate the effect and mechanism of 3% boric acid solution (BAS) against Candida albicans (CA) infection via the interleukin-23 (IL-23)/T helper 17 cell (Th17) axis. 36 female mice were randomly divided into 3 groups, and 2 injection sites on the back of the mice were chosen at random. Group N was injected with sterile water for injection (SWFI), and Group M and Group B were injected with CA mycelium suspension. After successful model verification, the remaining mice entered the following treatments 5 days later. Group B was treated with 3% BAS, Group M was treated with SWFI, and Group N was not treated. Levels of interleukin-17 (IL-17), IL-22, and IL-23 in mouse blood were measured on days 1, 3, 5, and 7 of treatment. On day 7, IL-17, IL-22, and IL-23 in mouse skin were detected. Serum levels of IL-17, IL-22, and IL-23 in Group M were higher than in Group N on the first day of treatment (p < 0.05). Expression levels of IL-17, IL-22, and IL-23 in the epidermis of the skin lesions in Group M were higher than in Group N on day 7 (p < 0.05). The serum level of IL-17 in Group B was higher than in Group M on days 5 and 7 (p < 0.05). Serum levels of IL-22 in Group B on days 1, 5, and 7 were higher than in Group M (p < 0.05). Serum levels of IL-23 in Group B were higher than in Group M on days 3, 5, and 7 (p < 0.05). IL-17 and IL-23 in Group B reached a peak on day 5, significantly different on days 1, 3, and 7 (p < 0.05). The expression intensity of IL-17, IL-22, and IL-23 in the skin lesions of Group B was higher than that of Group M on day 7 (p < 0.05). We conclude that IL-17, IL-22, and IL-23 are involved in the anti-CA activity in mouse skin, and 3% BAS increased IL-17, IL-22, and IL-23 to mediate these effects.

Antitumor-Promoting Effect of Ethanolic Extract from Leaves of Perilla frutescens var. crispa (Benth.) In Mouse Skin

This study aimed to investigate the antitumor-promoting effects of total ethanolic extract from Perilla leaf (PLE) in a two-stage chemical carcinogenesis protocol in mice. Quantitative analysis active compounds in PLE were performed by thin layer chromatography with normal phase of silica gel F254 and HPLC-PDA with reversed phase of C18 column at UV 300 nm. The cutaneous tumors were initiated by a single application by 7,12-dimethylbenz[a]anthracene (DMBA) on the dorsal shaved skin, then promoted by repeated applications of croton oil during 20 weeks. PLE was topically applied daily until the end of the experiment. The results observed from thin layer chromatogram highlighted the presence of rosmarinic acid and luteolin in PLE. By means of HPLC-PDA analysis, the total content of rosmarinic acid and luteolin were found in amounts of 1.178 ± 0.011 % (w/w) and 0.105 ± 0.001 % (w/w), respectively. The PLE-treated group prolonged the latency period of tumor appearance up to 2 weeks and also signifi reduced tumor volume in dorsal skin of mice as compared to untreated group. In conclusion, the total ethanolic extract from leaves of Perilla frutescens demonstrated promising antitumor-promotion activity in mouse skin.

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

P-75 - The protective effects of hispidulin and sulforaphane on UVA-induced photoaging through Nrf2 activation in mouse skin

P-75 - The protective effects of hispidulin and sulforaphane on UVA-induced photoaging through Nrf2 activation in mouse skin

Cytokine production in the skin is regulated by metabolites from the microbiome that influence histone acetylation.

Abstract Immune functions of the skin, including cytokine production from epidermal keratinocytes, are influenced by the resident microbiota. We hypothesized that a prominent member of the normal skin microbiome, Propionibacterium acnes (P. acnes), can affect cutaneous inflammation through the epigenetic mechanism of inhibiting histone deacetylase (HDAC) activity. GC-MS analysis revealed that P. acnes can generate high levels of the short-chain fatty acids (SCFAs) propionic and valeric acid. These molecules increased levels of histone acetylation as seen by Western blot analysis of H3K9ac and H3K27ac and ChIP-PCR of H3K9ac in the IL-6 promoter. Using qPCR, RNA-sequencing, and multiplex ELISA we found that HDAC inhibition alone had no effect on cytokine production from primary cultures of normal human keratinocytes (NHEK), but greatly increased the production of many cytokines and chemokines (i.e. IL-1β, IL-6, IL-8, TNFα, and CCL5) elicited by ligands of TLR2 or TLR3. In direct contrast, HDAC inhibition suppressed TLR-mediated inductions of these cytokines in monocytes and dendritic cells. The mechanism of inhibition in PBMCs and DCs was consistent with previous findings demonstrating increased expression of Mi-2β and activation of the NuRD co-repressor complex, events that were absent in NHEK. Inhibition of HDAC activity in mouse skin confirmed these responses in vivo. siRNA screening identified a novel mechanism involving HDAC8 and HDAC9 as responsible for these effects in keratinocytes. Together, these results show that the skin local environment and microbe-derived SCFAs influence inflammatory gene expression in a cell type-specific manner, thus explaining selective inflammation in diseases such as acne.

The NADPH‐Production Enzyme Isocitrate Dehydrogenase Maintains Quiescence in Hair Follicle Stem Cells

NADPH is a critical metabolite that is important for regenerating reduced glutathione from oxidized glutathione and eliminating reactive oxygen species (ROS). Metabolic flux and microarray experiments in fibroblasts demonstrated that NADPH‐producing enzymes, glucose‐6‐phosphate dehydrogenase (G6PD), isocitrate dehydrogenase (IDH), and the upstream transcription factor NRF2, are all activated in quiescent compared with proliferating fibroblasts. We demonstrated that G6PD and NRF2 are functionally important for quiescence by showing that inhibition of G6PD or NRF2 results in oxidative stress and apoptosis specifically in quiescent fibroblasts. Flow cytometry experiments demonstrated that ROS in quiescent cells can be derived from mitochondrial superoxide that results from increased mitochondrial activity in the serum‐starved compared with proliferating fibroblasts, as well as an increase in reactive nitrogen species that arise from peroxisomes. To understand the physiological role of NADPH‐production pathways, we examined their potential activity in mouse skin. Consistent with our findings in quiescent fibroblasts, in situ metabolic activity assays revealed higher potential activity for G6PD and IDH in non‐dividing cells. Of particular interest was the high IDH potential in quiescent hair follicle stem cells. Staining of live mouse skin with monochlorobimane showed higher reactivity, consistent with higher levels of reduced glutathione, in hair follicle stem cells. Inhibition of IDH activity in healthy mouse skin with two different inhibitors resulted in progression in the hair follicle cycle from a quiescent to proliferative state suggesting that the high IDH activity observed in hair follicle stem cells may contribute to the maintenance of these stem cells in a quiescent state. Going forward, our focus is on understanding the role of IDH in the maintenance of stem cells as opposed to proliferating, committed progenitor cells.Support or Funding InformationH.A.C. was the Milton E. Cassel scholar of the Rita Allen Foundation. This work was supported by NIGMS Center of Excellence grant P50 GM071508, two grants from the Cancer Institute of New Jersey, the New Jersey Commission on Cancer Research, National Cancer Institute 1RC1 CA147961‐01, a Focused Funding Grant to H.A.C. from the Johnson &amp; Johnson Foundation and a grant from the PhRMA Foundation to H.A.C, and XXXX to W.E.L. J.M.S. was supported by NIH training grant T32 HG003284. E.M.H. acknowledges a Bowen Fellowship from Princeton University and the New Jersey Commission on Cancer Research. E.J.S. acknowledges support from the National Science Foundation.

Topical application of a platelet activating factor receptor agonist suppresses phorbol ester-induced acute and chronic inflammation and has cancer chemopreventive activity in mouse skin.

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-Kit W-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-Kit W-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development.

Abstract 5551: Potentiation of PAH-induced cell transformation by phenolic fraction of tobacco smoke: A mechanistic insight

Abstract It is reported that the known content of carcinogenic polynuclear aromatic hydrocarbons (PAHs) in the tobacco smoke condensate (TSC) alone cannot account for the observed carcinogenicity of the TSC tested on mouse skin (Orris, L. et.al., 1958. J. Natl. Cancer Inst., 21: 557-561). The phenolic fraction of TSC (TSCPhFr) which is devoid of PAHs exhibits strong tumor-promoting activity in mouse skin initiated with PAHs (Roe et. al., 1959. Brit. J. Cancer., 13: 623-633). The mechanism(s) underlying the tumor promoting effect of TSCPhFr is not known. In the present communication we report that (±)-anti-BP-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogenic metabolite of tobacco smoke carcinogen benzo[a]pyrene, elicits both apoptosis induction and cell transformation in promotion sensitive mouse epidermal JB6 cell which has been extensively used for tumor promotion studies. We observed that TSCPhFr significantly potentiates BPDE-induced cell transformation associated with decreased apoptosis response indicating its tumor promoting potential. Increased cell transformation and decreased apoptosis by TSCPhFr was associated with attenuation of BPDE-induced p53 accumulation and NF-κB activation. We examined whether attenuation of p53 and NF-κB responses by TSCPhFr has any role in this regard. Chemical inhibition of p53 in JB6 cells using known p53 inhibitor pifithrin alpha showed potentiation of BPDE-induced p53 accumulation instead of down-regulation as expected. Since pifithrin alpha's role in p53 inhibition is controversial as reported in many studies, we used genomic inhibitor (p53 siRNA) to down-regulate p53 response. We observed that either JB6 cells transfected with p53 siRNA or p53 deficient HCT116 cells (HCT116p53-/−) shows significantly less apoptosis induction and more colony formation on soft agar by BPDE compared to control cells. In p53 impaired both cell lines (which are observed to have faster growth rate compared to normal cells) TSCPhFr has practically negligible effect on apoptosis induction and soft agar colony formation in response to BPDE. Interestingly treatment of cells with IKK-NBD peptide NF-κB-specific inhibitor) has variable effects on BPDE-induced apoptosis induction and cell transformation. Our observations indicate that attenuation of BPDE-induced p53 response by TSCPhFr has a role in apoptosis inhibition and increased cell transformation. These findings have implication with regard to the underlying mechanism of tumor promoting activity of TSCPhFr in PAH-induced carcinogenesis. Although p53 mediated NF-κB activation is involved in DNA damage-induced apoptosis induction as reported by others (Nature 2000, 404:892-897), the role of NF-κB in PAH-induced apoptosis and cell transformation is not clear (supported by NCI grant 1R15CA125630). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5551. doi:10.1158/1538-7445.AM2011-5551

7,3′,4′-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.

Ultraviolet B radiation activates NF‐κB and induces iNOS expression in HR‐1 hairless mouse skin: Role of IκB kinase‐β

Exposure to ultraviolet B (UVB) radiation is known to cause inflammatory tissue damage and skin cancer. One of the molecular links between inflammation and cancer is the eukaryotic transcription factor nuclear factor-kappaB (NF-κB), which is known to regulate expression of various pro-inflammatory genes including inducible nitric oxide synthase (iNOS). The present study was aimed at elucidating the molecular mechanisms underlying UVB-induced NF-κB activation and iNOS expression in hairless mouse skin. Irradiation of male HR-1 hairless mouse skin with UVB (5 kJ/m(2) ) resulted in increased degradation of IκBα, nuclear translocation of p65 and p50, and the DNA binding of NF-κB. Exposure to UVB radiation induced the phosphorylation and the catalytic activity of an upstream kinase IκB kinase-β (IKKβ). Pharmacological inhibition of IKKβ attenuated UVB-induced NF-κB activation in mouse skin. Irradiation of mouse skin with UVB also increased phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Pretreatment with SC-514, a specific inhibitor of IKKβ, attenuated UVB-induced phosphorylation of ERK and p38 MAP kinase. A kinetic study showed that UVB significantly increased the expression of iNOS in mouse skin at 6 h postirradiation, which was abrogated by pretreatment with SC-514. In conclusion, the upstream kinase IKKβ is involved in UVB-induced activation of MAP kinases and NF-κB, and expression of iNOS in mouse skin.

Suppression of UVB-induced HIF-1α up-regulation by hyperoxia does not prevent wrinkle formation associated with increased MMPs activity in mouse skin

This study investigated whether the suppression of hypoxia-inducible factor (HIF)-1α up-regulation prevents high-dose and long-term UVB-induced wrinkle formation and angiogenesis associated with increased matrix metalloproteinase (MMP)-2 and MMP-9 activities. Twenty-four hairless mice were assigned to three groups: 1) control, 2) UVB-irradiation (UVB), and 3) UVB-irradiation followed by hyperoxia (UVB+HO). The backs of the mice were exposed to UVB irradiation three times a week for 10 weeks. To suppress UVB-induced cutaneous HIF-1α up-regulation, the mice were exposed to hyperoxia (90% oxygen) for 2 h immediately after each UVB irradiation. The UVB and UVB+HO groups had significantly increased degrees of wrinkle formation, dermal blood vessel density, and MMP-2 and MMP-9 activities compared with the control group. HIF-1α expression levels were significantly higher in the UVB group than in the control group whereas these levels remained unchanged in the UVB+HO groups. The activity of type 1 collagenase which digests collagen type 1 (a main component of the dermis), was similar in all groups; furthermore, the dermal soluble collagen content was similar in all groups. These results suggest that the suppression of increases in HIF-1α levels alone is insufficient to restrain wrinkle formation caused by higher doses and longer periods of the UVB irradiation that led to the up-regulation of MMP-2 and MMP-9 activities.

Distinct roles of JNK-1 and ERK-2 isoforms in permeability barrier repair and wound healing

c-Jun N-terminal kinases (JNKs, also called stress activated protein kinases) and the extra-cellular signal responsive kinases (ERKs) exert different functions in mitogenesis, maturation and differentiation of immune and epithelial cells. We investigated specific functions of individual JNK and ERK isoforms in skin permeability barrier repair and in wound healing. JNK1, but not JNK2 or JNK3, deficient mice revealed a delay in the permeability barrier repair after superficial injury to the skin (tape-stripping) as well as a delay in the healing of full skin thickness wounds. Skin barrier injury induced an increase in epidermal JNK1 enzyme activity in mouse skin in vivo, and JNK1 activity correlated with the degree of differentiation in organotypic keratinocyte cultures. Skin injury activated epidermal ERK2 enzyme activity with biphasic maxima after 30 min and 3 h, and the activity was independent from the differentiation state in keratinocyte culture. In summary, superficial and deep wound healing depends on the differential activity of MAP kinases such as JNK1 in epidermal differentiation and ERK2 in proliferation.

Comparative analysis of newborn and adult Bothrops jararaca snake venoms

Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercial antivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 5′-nucleotidase activities. However, newborn snake venom showed higher l-amino oxidase, hyaluronidase, platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities of B. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciously neutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.

3,3′‐diindolylmethane suppresses 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced inflammation and tumor promotion in mouse skin via the downregulation of inflammatory mediators

3,3'-Diindolylmethane (DIM) is a major acid-condensation product of indole-3-carbinol and is present in cruciferous vegetables. In this study, we evaluated the effects of DIM on antiinflammatory and antitumor promotion activity in mouse skin and explored the relevant mechanisms. When 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied topically to the mouse ear to induce inflammation, DIM pretreatment effectively inhibited TPA-induced ear edema formation. To evaluate the mechanisms underlying DIM's antiinflammatory effects, DIM was topically treated to the shaved backs of mice 30 min before TPA treatment. DIM inhibited the TPA-induced increases in the expression of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), chemokine (C-X-C motif) ligand (CXCL) 5, and interleukin (IL)-6 in mouse skin. DIM also inhibited nuclear factor-kappa B (NF-kappaB)'s DNA binding activity, the nuclear translocation of p65, and the degradation of inhibitor of kappaB (IkappaB) alpha in TPA-stimulated mouse skin. Furthermore, DIM reduced TPA-induced increases in the activity of extracellular signal regulated protein kinase (ERK)-1/2 and IkappaB kinase (IKK). When mouse skin papillomas were initiated via the topical application of 7,12-dimethylbenz[alpha]anthracene (DMBA) and promoted with repeated topical applications of TPA, repeated topical applications of DIM prior to each TPA treatment significantly suppressed the incidence and multiplicity of the papillomas. DIM also reduced the expression of COX-2 and iNOS, ERK phosphorylation, and the nuclear translocation of p65 in papillomas. Collectively, these results show that DIM exerts antiinflammatory and chemopreventive effects in mouse skin via the downregulation of COX-2, iNOS, CXCL5, and IL-6 expression, which may be mediated by reductions in NF-kappaB activation.

Shikonin reduces oedema induced by phorbol ester by interfering with IκBα degradation thus inhibiting translocation of NF‐κB to the nucleus

In the present paper we studied the effect of shikonin on ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and determined the mechanisms through which shikonin might exert its topical anti-inflammatory action. Acute ear oedema was induced in mice by topical application of TPA. The in vitro assays used macrophages RAW 264.7 cells stimulated with lipopolysaccharide. Cyclooxygenase-2, inducible nitric oxide synthase, protein kinase Calpha, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (pERK), c-Jun N-terminal kinase (JNK), pJNK, p38, p-p38, p65, p-p65, inhibitor protein of nuclear factor-kappaB (NF-kappaB) (IkappaBalpha) and pIkappaBalpha were measured by Western blotting, activation and binding of NF-kappaB to DNA was detected by reporter gene and electrophoretic mobility shift assay, respectively, and NF-kappaB p65 localization was detected by immunocytochemistry. Shikonin reduced the oedema (inhibitory dose 50 = 1.0 mg per ear), the expression of cyclooxygenase-2 (70%) and of inducible nitric oxide synthase (100%) in vivo. It significantly decreased TPA-induced translocation of protein kinase Calpha, the phosphorylation and activation of ERK, the nuclear translocation of NF-kappaB and the TPA-induced NF-kappaB-DNA-binding activity in mouse skin. Moreover, in RAW 264.7 cells, shikonin significantly inhibited the binding of NF-kappaB to DNA in a dose-dependent manner and the nuclear translocation of p65. Shikonin exerted its topical anti-inflammatory action by interfering with the degradation of IkappaBalpha, thus inhibiting the activation of NF-kappaB.

Chondroitin Sulfate Extracted from Ascidian Tunic Inhibits Phorbol Ester-Induced Expression of Inflammatory Factors VCAM-1 and COX-2 by Blocking NF-κB Activation in Mouse Skin

Inflammatory factors are known to play a key role in promoting tumorigenesis; therefore, it is a promising strategy to inhibit the inflammation for cancer prevention. The current study was performed to investigate the potential effects of chondroitin sulfate (CS) extracted from ascidian tunic on the expression of inflammatory factors induced by treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA) and to elucidate the underlying molecular mechanism of CS action in mouse skin inflammation. TPA was topically applied to the shaven backs of ICR mice with or without CS (1 or 2 mg) for 4 h. The results demonstrated that CS suppressed TPA-induced edema and reduced the expression of cyclooxygenase-2, vascular cell adhesion molecule-1, and Akt signaling in mouse skin. These studies suggest that CS from ascidian tunic may be developed as an effective natural anti-inflammatory agent.

Hemorrhagic activity of the vascular apoptosis-inducing proteins VAP1 and VAP2 from Crotalus atrox

Vascular apoptosis-inducing proteins (VAPs) from hemorrhagic snake venom are apoptosis-inducing toxins that target vascular endothelial cells. We now show that VAP1 and VAP2 from Crotalus atrox have hemorrhagic activity in mouse skin following intradermal injection. Following intravenous injection, VAP2 induced hemorrhage in the lung, intestine and kidney. Although the hemorrhagic activity was relatively weak, these apoptosis toxins may play a role in the complex mechanism of snake venom-induced hemorrhage.

Antipsoriatic effects of avarol‐3′‐thiosalicylate are mediated by inhibition of TNF‐αgeneration and NF‐κB activation in mouse skin

Avarol is a marine sesquiterpenoid hydroquinone with anti-inflammatory and antipsoriatic properties. The aim of this study was to evaluate the in vitro and in vivo pharmacological behaviour of the derivative avarol-3'-thiosalicylate (TA) on some inflammatory parameters related to the pathogenesis of psoriasis. Human neutrophils and monocytes as well as the human keratinocyte cell line HaCaT were used to study the effect of TA on oxidative stress, the arachidonic acid pathway, tumour necrosis factor-alpha (TNF-alpha) release and nuclear factor-kappaB (NF-kappaB) activation. All these parameters were also determined in vivo using the zymosan induced mouse air pouch model and the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced mouse epidermal hyperplasia model. TA showed antioxidant properties in human neutrophils and in the hypoxanthine/xanthine oxidase assay. This compound reduced, in a concentration-dependent manner, leukotriene B(4), prostaglandin E(2) and TNF-alpha production in activated leukocytes. Oral and intrapouch administration of TA in the mouse air pouch model produced a dose-dependent reduction of all these inflammatory mediators. TA also inhibited secretory phospholipase A(2) activity and NF-kappaB DNA-binding in HaCaT keratinocytes. In TPA-induced mouse epidermal hyperplasia, topical administration of TA reduced oedema, leukocyte infiltration, eicosanoid levels and TNF-alpha in skin. In addition, interleukin (IL)-1beta and IL-2 production were also inhibited. Finally, TA was also capable of suppressing NF-kappaB nuclear translocation in vivo. TA inhibited several key biomarkers up-regulated in the inflammatory response of psoriatic skin and this compound could be a promising antipsoriatic agent.

Jaceosidin Induces Apoptosis in ras‐Transformed Human Breast Epithelial Cells through Generation of Reactive Oxygen Species

Extracts of Artemisia plants possess anti-inflammatory and antioxidative activities. Eupatilin (5,7-dihydroxy-3',4',6-tri-methoxy-flavone), a pharmacologically active flavone derived from Artemisia asiatica, was shown to inhibit phorbol ester-induced cyclooxygenase-2 expression and NF-kappaB activation in mouse skin, and also to induce cell cycle arrest in ras-transformed human mammary epithelial (MCF10A-ras) cells. In this article, we examined the ability of jaceosidin (4',5,7-trihydroxy-3',6-dimethoxyflavone) isolated from Artemisia argyi to inhibit the proliferation of MCF10A-ras cells. Jaceosidin reduced the viability of MCF10A-ras cells to a greater extent than eupatilin. Jaceosidin treatment resulted in increased intracellular accumulation of reactive oxygen species (ROS) in MCF10A-ras cells, which was blocked by the antioxidant N-acetylcysteine (NAC). NAC attenuated jaceosidin-induced cytotoxicity. To better assess the proapoptotic effects of jaceosidin, we analyzed the treated cells by the flow cytometry. MCF10A-ras cells treated with jaceosidin (100 microM) exhibited the increased proportion of hypodiploid or apoptotic cells (48.72% as composed to 7.78% in control cells). Jaceosidin treatment also increased the ratio of proapoptotic Bax to the antiapoptotic Bcl-2 and induced the cleavage of caspase-3 and poly(ADP-ribose)polymerase (PARP). Moreover, jaceosidin elevated the expression of p53 and p21, while the compound inhibited the activation of ERK1/2 that is an important component of cell survival signaling.

Resveratrol inhibits phorbol ester-induced expression of COX-2 and activation of NF-κB in mouse skin by blocking IκB kinase activity

Aberrant expression of cyclooxygenase-2 (COX-2) has been implicated in tumor promotion. Resveratrol, a phytoalexin present in grapes, was reported to inhibit multistage mouse skin carcinogenesis. In the present study, we found that topically applied resveratrol significantly inhibited COX-2 expression induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Resveratrol-suppressed phosphorylation and subsequent degradation of IkappaBalpha, thereby inhibiting activation of nuclear factor-kappaB (NF-kappaB) in TPA-stimulated mouse skin. Pretreatment with resveratrol also suppressed TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Resveratrol blunted TPA-induced phosphorylation of p65 and its interaction with CBP/p300, rendering NF-kappaB transcriptionally inactive. To get further insights into the molecular basis of NF-kappaB inactivation by resveratrol, we examined the role of IkappaB kinase (IKK) in mediating TPA-induced activation of NF-kappaB and COX-2 expression. TPA treatment led to rapid induction of IKK activity in mouse skin, which was abolished either by resveratrol or an IKK inhibitor Bay 11-7082. Topical application of Bay 11-7082 also abrogated TPA-induced NF-kappaB activation and COX-2 expression, supporting the involvement of IKK in TPA-induced COX-2 expression. Taken together, the above findings suggest that resveratrol targets IKK in blocking TPA-induced NF-kappaB activation and COX-2 expression in mouse skin in vivo.