SKH-1 Hairless Mouse Model Research Articles

Anti-skin aging activity of eggshell membrane administration and its underlying mechanism

BackgroundThere is active research on developing materials for improving skin function. Eggshell membrane (ESM) is one such raw material that is consumed as a functional food to support skin health. However, studies on the mechanism of improvement of skin function on ingestion of ESM are still lacking.ObjectivesTo explore this mechanism of action, we conducted an ultraviolet (UV) irradiation study on a SKH-1 hairless mouse model. Feeding ESM was found to improve skin moisture and reduce wrinkles during 12 weeks of UVB irradiation.ResultsOral administration of ESM restored moisture in the dorsal skin tissue of mice. In addition, oral ingestion of ESM also reversed the increased transepidermal water loss and reduction of mRNA expression of hyaluronic synthases induced by UVB irradiation. Furthermore, UVB irradiation-induced collagen degradation was inhibited, and the expression of the collagenase MMP was reduced in the ESM intake group compared to the control. These results confirmed that oral ingestion of the ESM has an anti-wrinkle effect. In addition, the mRNA expression of the antioxidant enzyme SOD1, which was reduced on UVB irradiation, was restored on ingestion of the ESM. Restoring the expression of antioxidant enzymes is a key strategy for improving skin function of the ESM.ConclusionTaken together, the findings from our study reveal the potential of ESM as a nutricosmetic material with anti-wrinkle and skin moisturizing properties.

Chamaejasmine Isolated from Wikstroemia dolichantha Diels Suppresses 2,4-Dinitrofluoro-benzene-Induced Atopic Dermatitis in SKH-1 Hairless Mice.

Plants of the genus Wikstroemia have long been used as traditional medicines to treat diseases like pneumonia, rheumatism, and bronchitis. This study was designed to determine the effect of chamaejasmine, a biflavonoid present in W. dolichantha, on atopic dermatitis (AD)-like skin lesions in a 2,4-dinitrochlorobenzene (DNCB)-induced murine model of AD. Initially, we examined the anti-allergic activities of ten flavonoids from W. dolichantha by measuring β-hexosaminidase release from RBL-2H3 cells. Subsequently, an SKH-1 hairless mouse model of AD was developed based on the topical application of DNCB. Chamaejasmine (0.5%) or pimecrolimus (1%, positive control) were applied to dorsal skins of DNCB-sensitized AD mice for two weeks. Serum IL-4 and IgE levels were determined using enzyme-linked immunosorbent assay kits and transepidermal water loss (TEWL) and skin hydration were measured using a Tewameter TM210 and a SKIN-O-MAT, respectively. Of the ten flavonoids isolated from W. dolichantha, chamaejasmine most potently inhibited DNP-specific IgE-induced degranulation in RBL-2H3 cells. Topical administration of chamaejasmine attenuated the clinical symptoms of DNCB-induced dermatitis (i.e., itching, dryness, erythema, and edema). Histological analyses demonstrated that dermal thickness and mast cell infiltration in dermis were significantly reduced by chamaejasmine. In addition, 0.5% chamaejasmine inhibited DNCB-induced increases in total IL-4 and IgE levels in serum, improved skin barrier function, and increased epidermis moisture. Our findings suggest chamaejasmine might be an effective therapeutic agent for the treatment of atopic diseases.

Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model.

Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity.

Chemoprotective Effects of Dietary Grape Powder on UVB Radiation-Mediated Skin Carcinogenesis in SKH-1 Hairless Mice

Skin cancer is the most frequently diagnosed cancer in the United States, and solar UVR is an established causative factor for approximately 90% of these cases. Despite efforts aimed at UV protection, including use of sunscreen and clothing, annual cases of skin cancer continue to rise. Here, we report that dietary grape powder mitigates UVB-mediated skin carcinogenesis in an SKH-1 hairless mouse model. Using a UVB initiation-promotion protocol, whereby mice were exposed to 180 mJ/cm2 UVB two times per week for 28 weeks, we determined the effects of a grape powder-fortified diet (3% or 5%) on skin carcinogenesis. Grape powder consumption at both doses resulted in marked inhibition in tumor incidence, as well as a delay in onset of tumorigenesis. Molecular analyses of skin and tumor tissue showed that grape powder-mediated protective response against UVB-induced skin cancer was accompanied by enhanced DNA damage repair, reduced proliferation, increased apoptosis, and modulations in several oxidative stress markers specifically related to inhibition of oxidative stress and increased reactive oxygen species metabolism. NRF2, an activator of cellular antioxidant response, was decreased by grape powder feeding, suggesting a supportive role in tumor cell survival. Overall, our study suggested that dietary grape, containing several antioxidants in natural amalgamation, may protect against UVB-mediated skin carcinogenesis.

Abstract 5263: Chemopreventive effects of dietary grapes on skin cancer

Abstract Non-melanoma skin cancer (NMSC) is the most commonly diagnosed malignancy in the United States, affecting more than 3 million Americans each year. Ultraviolet (UV) radiation from the sun, particularly its UVB component (290-320 nm), is an established causative factor for ~90% of skin cancers. The two most common forms of skin cancer, basal- and squamous- cell carcinomas, are treatable if detected early. However, they can be difficult to treat and potentially fatal if left until late stages. Further, epidemiological studies have suggested an amplified risk of other deadly cancers in individuals with a history of skin cancer. Therefore, it is important to design novel approaches, especially focusing on prevention, for the management of skin cancers. Studies from our laboratory and by others have shown that topical application of the grape antioxidant resveratrol possesses promise in the prevention of skin cancer. However, ideally cancer preventive agents need to be orally administrable for ease of use and broader human acceptability. With this in mind, in this study, we determined the efficacy of dietary grape powder (containing resveratrol in natural amalgamation with catechins, anthocyanins, polyphenols and flavonols) against UVB-mediated skin tumorigenesis in the SKH-1 hairless mouse model, which is regarded to have relevance to human NMSC. We employed a UVB initiation-promotion protocol in which the mice were subjected to chronic UVB exposure (180 mJ/cm2; twice weekly, for 28 weeks). The animals received either AIN-76A or grape powder (GP) fortified diet (3% and 5% GP, obtained from the California Table Grape Commission), all of which were sugar-matched to the highest GP content. Our data demonstrated that the consumption of GP at both 3% and 5% resulted in a significant inhibition in skin tumor incidence and delay in the onset of tumorigenesis. The average consumption of feed per mouse was 3.5 g/day, corresponding to 105 and 174 mg GP/day in the 3% and 5% GP treatment groups. This dosing regimen seems to be easily achievable for human consumption, as it corresponds to 25.5 and 42.4 g/day, which are equivalent to 1.1 and 1.8 serving of fresh grapes, respectively. Our data also demonstrated that the observed skin cancer chemopreventive effects of grape powder were accompanied by significant i) decreases in cellular proliferation markers Ki67 and PCNA, ii) decreases in the oxidative stress marker 4-HNE, and iii) increases in the levels of cleaved caspase 7 and poly (ADP-ribose) polymerase (PARP). Interestingly, NRF2, an activator of cellular antioxidant response, was found to be downregulated in GP treated tumors, compared to UVB alone groups, suggesting a potential protective role of NRF2 in the survival of tumor cells. Overall, our study suggests a strong chemopreventive effect of dietary grape and provides a basis for future human studies. Citation Format: Chandra K. Singh, Mary A. Ndiaye, Charlotte A. Mintie, Gagan Chhabra, Nihal Ahmad. Chemopreventive effects of dietary grapes on skin cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5263. doi:10.1158/1538-7445.AM2017-5263

Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid.

Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2 and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca2+ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH2 and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH2-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH2 downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH2 in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis.

Abstract 5238: The chemopreventive potential of silver nanoparticles against UVB-induced skin carcinogenesis in mouse model

Abstract Solar ultraviolet (UV) radiation, particularly its UVB component, is an established cause of human skin carcinogenesis due to its ability to cause DNA damage in skin cells. Although several sunscreen formulations have been developed and commercialized to limit or minimize UVB exposure to skin cells, incidence of skin cancer has continued to increase suggesting their limited or no efficacy in preventing UV-induced skin cancer occurrence. Recently, we revealed the potential of silver nanoparticles (AgNPs; ≤ 50 nm) against UVB radiation-induced DNA damage in human keratinocytes (HaCaT). Here we performed preclinical evaluation of chemopreventive efficacy of AgNPs against UVB-induced skin tumorigenesis in SKH-1 hairless mouse model. The different concentrations (20 and 40 mg/kg) of AgNPs mixed with hydrophilic cream base were uniformly applied topically onto the dorsal area of mouse skin prior to UVB-irradiation to assess chemopreventive efficacy for several weeks. The UVB treatment group showed tumor formation within 13 weeks, and had high incidence (93.3%) rate by the end of the study (29 weeks). Interestingly, the pretreatment of mice with AgNPs significantly increased the latency period (6-9 weeks) of UVB-induced tumor formation. Moreover, overall tumor incidence was found to be significantly decreased (50 and 78%; in 20 and 40 mg/kg respectively) as compared to AgNPs-untreated mice. Tumor multiplicity and average tumor volume/tumor-bearing mouse were also observed to be significantly reduced in AgNPs-treated mice. Additionally, AgNPs treatment alone for 29 weeks did not induce any apparent signs of toxicity and changes in the body weight suggesting the safety of AgNPs. Altogether, these findings suggest that AgNPs are potential chemopreventive agents against UVB radiation-induced skin carcinogenesis and thus opening the ways for human applications. Citation Format: Nikhil Tyagi, Sumit Arora, Sanjeev K. Srivastava, James E. Carter, Ajay P. Singh, Seema Singh. The chemopreventive potential of silver nanoparticles against UVB-induced skin carcinogenesis in mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5238.

Topical treatment with pterostilbene, a natural phytoalexin, effectively protects hairless mice against UVB radiation-induced skin damage and carcinogenesis

The aim of our study was to investigate in the SKH-1 hairless mouse model the effect of pterostilbene (Pter), a natural dimethoxy analog of resveratrol (Resv), against procarcinogenic ultraviolet B radiation (UVB)-induced skin damage. Pter prevented acute UVB (360mJ/cm2)-induced increase in skin fold, thickness, and redness, as well as photoaging-associated skin wrinkling and hyperplasia. Pter, but not Resv, effectively prevented chronic UVB (180mJ/cm2, three doses/week for 6 months)-induced skin carcinogenesis (90% of Pter-treated mice did not develop skin carcinomas, whereas a large number of tumors were observed in all controls). This anticarcinogenic effect was associated with (a) maintenance of skin antioxidant defenses (i.e., glutathione (GSH) levels, catalase, superoxide, and GSH peroxidase activities) close to control values (untreated mice) and (b) an inhibition of UVB-induced oxidative damage (using as biomarkers 8-hydroxy-2′-deoxyguanosine, protein carbonyls, and isoprostanes). The molecular mechanism underlying the photoprotective effect elicited by Pter was further evaluated using HaCaT immortalized human keratinocytes and was shown to involve potential modulation of the Nrf2-dependent antioxidant response.

Time course of lewisite‐induced skin lesions and inflammatory response in the SKH‐1 hairless mouse model

Data on the toxicity of lewisite (L), a vesicant chemical warfare agent, are scarce and conflicting, and the use of the specific antidote is not without drawbacks. This study was designed to evaluate if the SKH-1 hairless mouse model was suitable to study the L-induced skin injuries. We studied the progression of lesions following exposure to L vapors for 21 days using paraclinical parameters (color, transepidermal water loss (TEWL), and biomechanical measurements), histological assessments, and biochemical indexes of inflammation. Some data were also obtained over 27 weeks. The development of lesions was similar to that reported in other models. The TEWL parameter appeared to be the most appropriate index to follow their progression. Histological analysis showed inflammatory cell infiltration and microvesications at day 1 and a complete wound closure by day 21. Biochemical studies indicated a deregulation of the levels of several cytokines and receptors involved in inflammation. An increase in the quantity of pro-matrix metalloproteinases 2 and 9 was shown as observed in other models. This suggests that the SKH-1 mouse model is relevant for the investigation of the physiopathological process of skin lesions induced by L and to screen new treatment candidates.

Intake of high-fat diet stimulates the risk of ultraviolet radiation-induced skin tumors and malignant progression of papillomas to carcinoma in SKH-1 hairless mice

Previously, we showed that administration of a high-fat diet (HF-diet) to C57BL/6 mice exacerbates their response to short-term UVB radiation-induced inflammation in the skin. To explore the effects of an HF-diet on UVB-induced tumorigenesis, we have used the SKH-1 hairless mouse model in which the mice are exposed to UVB radiation (180mJ/cm2) three times a week for 24weeks. The development of UVB-induced skin tumors was rapid and the tumor multiplicity and tumor size were significantly higher (P<0.01–0.005) in the mice fed an HF-diet than the mice fed a control-diet (C-diet). Moreover, the malignant progression of UVB-induced papillomas to carcinomas was higher in HF-diet-fed mice. On analysis of tumors and tumor-uninvolved skin samples from the tumor-bearing mice, we found that administration of an HF-diet significantly enhanced the levels of UVB-induced expression of cyclooxygenase-2 (COX-2), prostaglandin E2 (P<0.01), and PGE2 receptors, and activation of NF-κB in the UVB-exposed skin as well as in tumors. In addition the HF-diet enhanced the expression of proinflammatory cytokines, including tumor necrosis factor-α (P<0.01), interleukin (IL)-1β (P<0.01) and IL-6 (P<0.05) in the UVB-exposed skin as well as in tumors. Western blot analysis revealed that HF-diet enhanced the levels of epidermal cell proliferation, phosphatidylinositol 3-kinase and phosphorylation of Akt at Ser473 in UVB-exposed skin and skin tumors. Collectively, these data demonstrate that the regular consumption of an HF-diet increases the risk of photocarcinogenesis in mice and that this is associated with enhanced expression of inflammatory mediators in the UVB-exposed skin and tumors.

Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1

Exposure of keratinocytes (KC) to ultraviolet (UV) radiation results in the initiation of apoptosis, a protective mechanism that eliminates cells harboring irreparable DNA damage. Hence, a manipulation of UV-induced apoptosis may significantly influence photocarcinogenesis. We have discovered that the aryl hydrocarbon receptor (AHR), a key regulator of drug metabolism and an UVB-sensitive transcription factor, serves an anti-apoptotic function in UVB-irradiated human KC. Chemical and shRNA-mediated inhibition of AHR signaling sensitized KC to UVB-induced apoptosis by decreasing the expression of E2F1 and its target gene checkpoint kinase 1 (CHK1). The decreased expression of these cell-cycle regulators was due to an enhanced expression of p27(KIP1) and an associated decrease in phosphorylation of both cyclin-dependent kinase 2 and its substrate molecule retinoblastoma protein. The subsequent inhibition of E2F1 autoregulation and downstream CHK1 expression resulted in an enhanced susceptibility of damaged cells to undergo apoptosis. Accordingly, ectopic overexpression of either E2F1 or CHK1 in AHR-knockdown KC attenuated the observed sensitization to UVB-induced apoptosis. Using an AHR-knockout SKH-1 hairless mouse model, we next demonstrated the physiological relevance of the anti-apoptotic function of AHR. In contrast to their AHR-proficient littermates, the constitutive expression of E2F1 and CHK1 was significantly reduced in the skin of AHR-knockout mice. Accordingly, a single exposure of the animals to UVB resulted in an enhanced cleavage of caspase-3 in the skin of AHR-knockout mice. These results identify for the first time the AHR-E2F1-CHK1 axis as a novel anti-apoptotic pathway in KC, which may represent a suitable target for chemoprevention of non-melanoma skin cancer.

Abstract 4860: Chemopreventive effects of α-santalol on ultraviolet B induced skin tumorigenesis by modulating cell cycle regulators.

Abstract Previous studies from our laboratory have shown chemopreventive effects of 5% α-santalol, a sequiterpene isolated from the sandalwood oil in ultraviolet-B induced skin tumor multiplicity in SKH-1 hairless mice. However, this dosage did not have significant effect on tumor incidence. The objective of this study was to investigate the effects of α-santalol (10%, w/v in acetone) on UVB-induced skin tumor incidence in SKH-1 hairless mouse model and to identify the molecular targets in α-santalol mediated protection. Tumor initiation and promotion were carried out by UVB radiation (30 mJ/cm2/day for 5 days a week up to 30 week) that is in the range of human exposure to sunlight that can cause skin cancer. Topical treatment of mice with α-santalol causes a significant reduction in tumor multiplicity, tumor volume and tumor incidence, and also delayed tumor development. Histopathological analysis of hematoxylin-eosin stained skin sections showed that α-santalol pretreatment strongly inhibited UVB-induced epidermal hyperplasia and total thickness of the epidermis. The proliferation potential of skin and tumor tissue sections from UVB-induced carcinogenesis protocol was evaluated by immunohistochemical expression of proliferation marker PCNA. Both intensity of brown staining as well as the number of positively stained nuclei were reduced in skin tumors and adjacent skin tissues from α-santalol treated group when compared to control group. Western blot analysis of skin lysates showed that α-santalol decreases the level of different cyclin-dependent kinases and associated cyclins, Cdc25 phosphatases (Cdc25B and Cdc25C) together with an up-regulation of CDK inhibitor Cip1/p21. However, the level of Kip1/p27 and p53 did not show a significant difference. Our data suggested that α-santalol (10%, topical) is a safer and promising skin cancer chemopreventive agent through targeting different cell cycle regulators. (This work is supported by a Translational Cancer Center Research Grant, funded by the State of South Dakota). Citation Format: Sreevidya Santha, Chandradhar Dwivedi. Chemopreventive effects of α-santalol on ultraviolet B induced skin tumorigenesis by modulating cell cycle regulators. [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 4860. doi:10.1158/1538-7445.AM2013-4860

Src kinase is a direct target of apigenin against UVB-induced skin inflammation

Apigenin, a flavonoid abundant in various vegetables and fruits, including parsley and onions, has been reported to possess anticarcinogenic effects. However, the direct molecular target of apigenin and its chemopreventive effect on ultraviolet (UV)-induced skin inflammation are not understood fully. Herein, we examined the anti-inflammatory effect of apigenin and its associated mechanisms in JB6 P+ cell line and SKH-1 hairless mouse model. Apigenin inhibited UVB-induced cyclooxygenase-2 (COX-2) expression, which is a well-known key mediator of inflammation and cancer, and restored the upstream stimulatory factor level in JB6 P+ cells. Immunoblot and kinase assay data demonstrate that Src activity was attenuated by apigenin, and this led to subsequent inhibition of UVB-induced phosphorylation of epidermal growth factor receptor, mitogen-activated protein kinases and Akt signaling. Inhibitory effects of apigenin on UVB-induced signaling were also confirmed in HaCaT human keratinocytes. In addition, in vitro pull-down assays revealed that apigenin binds Src in an adenosine triphosphate-competitive manner. Results using in vivo skin model indicate apigenin significantly inhibits UVB-induced ear edema development, COX-2 expression and Src kinase activity in SKH-1 hairless mice. Collectively, these findings suggest that apigenin exerts potent chemopreventive activity against UVB-induced skin inflammation primarily by targeting Src.

UV Light B–Mediated Inhibition of Skin Catalase Activity Promotes Gr-1+CD11b+ Myeloid Cell Expansion

Skin cancer incidence and mortality are higher in men compared to women, but the causes of this sex discrepancy remain largely unknown. Ultraviolet light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1+CD11b+ myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present within skin, has been associated with skin carcinogenesis. We utilized the outbred, immune competent Skh-1 hairless mouse model of ultraviolet light B (UVB)-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1+CD11b+ myeloid cell percentage. Acute UVB exposure induced Gr-1+CD11b+ myeloid cell skin infiltration, which was inhibited to a greater extent in males by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1+CD11b+ myeloid cells was 55% higher in male tumor-bearing mice compared to their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced Gr-1+CD11b+ myeloid cells and subsequent skin carcinogenesis.

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.

Honokiol, a phytochemical from the Magnolia plant, inhibits photocarcinogenesis by targeting UVB-induced inflammatory mediators and cell cycle regulators: development of topical formulation

To develop newer and more effective chemopreventive agents for skin cancer, we assessed the effect of honokiol, a phytochemical from the Magnolia plant, on ultraviolet (UV) radiation-induced skin tumorigenesis using the SKH-1 hairless mouse model. Topical treatment of mice with honokiol in a hydrophilic cream-based topical formulation before or after UVB (180 mJ/cm(2)) irradiation resulted in a significant protection against photocarcinogenesis in terms of tumor multiplicity (28-60%, P < 0.05 to <0.001) and tumor volume per tumor-bearing mouse (33-80%, P < 0.05 to 0.001, n = 20). Honokiol also inhibited and delayed the malignant progression of papillomas to carcinomas. To investigate the in vivo molecular targets of honokiol efficacy, tumors and tumor-uninvolved skin samples from the tumor-bearing mice were analyzed for inflammatory mediators, cell cycle regulators and survival signals using immunostaining, western blotting and enzyme-linked immunosorbent assay. Treatment with honokiol significantly inhibited UVB-induced expression of cyclooxygenase-2, prostaglandin E(2) (P < 0.001), proliferating cell nuclear antigen and proinflammatory cytokines, such as tumor necrosis factor-α (P < 0.001), interleukin (IL)-1β (P < 0.01) and IL-6 (P < 0.001) in the skin as well as in skin tumors. Western blot analysis revealed that honokiol: (i) inhibited the levels of cyclins D1, D2 and E and associated cyclin-dependent kinases (CDKs)2, CDK4 and CDK6, (ii) upregulated Cip/p21 and Kip/p27 and (iii) inhibited the levels of phosphatidylinositol 3-kinase and the phosphorylation of Akt at Ser(473) in UVB-induced skin tumors. Together, our results indicate that honokiol holds promise for the prevention of UVB-induced skin cancer by targeting inflammatory mediators, cell cycle regulators and cell survival signals in UVB-exposed skin.

Abstract 1871: Honokiol, a natural plant product, inhibits ultraviolet radiation-induced skin tumorigenesis in mice through inhibition of inflammation and inflammatory mediators

Abstract Chronic exposure of solar ultraviolet (UV) radiation is the major etiologic agent for over one million new cases of non-melanoma skin cancers in the US each year. These cutaneous malignancies thus have a tremendous impact on public health and healthcare expenditures. The use of natural plant products has received considerable interest to protect skin from adverse biological effects of UV radiation since the protective clothing and use of sunscreens are not adequate for skin photoprotection. The photoprotective effect of honokiol, a plant product from Magnolia species, therefore assessed using SKH-1 hairless mouse model. To determine the skin cancer chemopreventive effect and associated mechanism of honokiol, a hydrophilic cream based topical formulation was developed. Different groups of mice (n=20) treated with or without honokiol were subjected to photocarcinogenesis protocol. Mice were irradiated to UVB (180 mJ/cm2) three times a week. At the termination of the experiment at 24th week, we found that topical application of honokiol (1mg &amp; 3mg/mouse) inhibited UVB-induced skin tumor development in terms of tumor incidence (10-20%), tumor multiplicity (40-60%) and tumor size (50-75%). Since UV-induced inflammation plays a crucial role in the development of skin tumors, we examined the effect of honokiol on UVB-induced biomarkers of inflammation, such as the expression levels of cyclooxygenase-2 (COX-2), prostaglandins (PG), proliferating cell nuclear antigen (PCNA), infiltration of inflammatory leukocytes, development of edema and epidermal hyperplastic response using immunostaining, western blotting and RT-PCR. Our data revealed that topical application of honokiol before each exposure of UVB radiation resulted in a dose-dependent inhibition of UVB-induced expression of COX-2, PGE2, developments of edema and infiltration of leukocytes at the UVB-irradiated skin sites. Honokiol also inhibited the levels of PCNA (hyperplastic potential) in epidermal keratinocytes of UVB-exposed skin. The inhibitory effects of honokiol on inflammatory mediators were also found in skin tumor samples. The results of this study suggest that honokiol has the ability to inhibit UVB radiation-induced skin tumor development in mice and it is associated with the inhibition of UVB-induced inflammation and inflammatory mediators in mouse skin and skin tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1871.

Luteolin Inhibits Protein Kinase Cϵ and c-Src Activities and UVB-Induced Skin Cancer

Luteolin, a flavonoid present in various vegetables including onion and broccoli, has been reported to possess anticarcinogenic effects. However, its chemopreventive effect on UV-induced skin cancer and its mechanism are not fully understood. Herein, we examined the chemopreventive effect and associated mechanisms of luteolin in the JB6 P+ cell line and the SKH-1 hairless mouse model. Luteolin suppressed UVB-induced cyclooxygenase-2 expression and activator protein-1 and nuclear factor-kappaB activity in JB6 P+ cells. Immunoblot and kinase assay data showed that luteolin attenuated protein kinase C(epsilon) (PKC(epsilon)) and Src kinase activities and subsequently inhibited UVB-induced phosphorylation of mitogen-activated protein kinases and the Akt signaling pathway. In addition, pull-down assays revealed that luteolin binds directly to PKC(epsilon) and Src in an ATP-competitive manner. Importantly, luteolin suppressed tumor incidence, multiplicity, and overall size in SKH-1 hairless mice. Analysis of the skin by immunohistochemistry and immunoblotting showed that luteolin-treated groups had a substantial reduction in the levels of cyclooxygenase-2, tumor necrosis factor-alpha, and proliferating cell nuclear antigen compared with groups treated with only UVB. Further analysis using skin lysates showed that luteolin inhibited PKC(epsilon) and Src kinase activity. Together, these data suggest that luteolin exerts potent chemopreventive activity against UVB-induced skin cancer mainly by targeting PKC(epsilon)and Src.

Inflammatory Biomarkers of Sulfur Mustard Analog 2-Chloroethyl Ethyl Sulfide–Induced Skin Injury in SKH-1 Hairless Mice

Sulfur mustard (HD) is an alkylating and cytotoxic chemical warfare agent, which inflicts severe skin toxicity and an inflammatory response. Effective medical countermeasures against HD-caused skin toxicity are lacking due to limited knowledge of related mechanisms, which is mainly attributed to the requirement of more applicable and efficient animal skin toxicity models. Using a less toxic analog of HD, chloroethyl ethyl sulfide (CEES), we identified quantifiable inflammatory biomarkers of CEES-induced skin injury in dose- (0.05-2 mg) and time- (3-168 h) response experiments, and developed a CEES-induced skin toxicity SKH-1 hairless mouse model. Topical CEES treatment at high doses caused a significant dose-dependent increase in skin bi-fold thickness indicating edema. Histopathological evaluation of CEES-treated skin sections revealed increases in epidermal and dermal thickness, number of pyknotic basal keratinocytes, dermal capillaries, neutrophils, macrophages, mast cells, and desquamation of epidermis. CEES-induced dose-dependent increases in epidermal cell apoptosis and basal cell proliferation were demonstrated by the terminal deoxynucleotidyl transferase (tdt)-mediated dUTP-biotin nick end labeling and proliferative cell nuclear antigen stainings, respectively. Following an increase in the mast cells, myeloperoxidase activity in the inflamed skin peaked at 24 h after CEES exposure coinciding with neutrophil infiltration. F4/80 staining of skin integuments revealed an increase in the number of macrophages after 24 h of CEES exposure. In conclusion, these results establish CEES-induced quantifiable inflammatory biomarkers in a more applicable and efficient SKH-1 hairless mouse model, which could be valuable for agent efficacy studies to develop potential prophylactic and therapeutic interventions for HD-induced skin toxicity.

Photoprotective Effects of Bucillamine Against UV‐induced Damage in an SKH‐1 Hairless Mouse Model†

UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2-methylpropionyl)-l-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm(-2)) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg(-1) of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.