Hairless Mouse Epidermis Research Articles

Comparison of global DNA methylation analysis by whole genome bisulfite sequencing and the Infinium Mouse Methylation BeadChip using fresh and fresh-frozen mouse epidermis

ABSTRACT Until recently, studying the murine methylome was restricted to sequencing-based methods. In this study we compared the global DNA methylation levels of hairless mouse epidermis using the recently released Infinium Mouse Methylation BeadChip from Illumina and whole genome bisulphite sequencing (WGBS). We also studied the effect of sample storage conditions by using fresh and fresh-frozen epidermis. The DNA methylation levels of 123,851 CpG sites covered by both the BeadChip and WGBS were compared. DNA methylation levels obtained with WGBS and the BeadChip were strongly correlated (Pearson correlation r = 0.984). We applied a threshold of 15 reads for the WGBS methylation analysis. Even at a threshold of 10 reads, we observed no substantial difference in DNA methylation levels compared with that obtained with the BeadChip. The DNA methylation levels from the fresh and the fresh-frozen samples were strongly correlated when analysed with both the BeadChip (r = 0.999) and WGBS (r = 0.994). We conclude that the two methods of analysis generally work equally well for studies of DNA methylation of mouse epidermis and find that fresh and fresh-frozen epidermis can generally be used equally well. The choice of method will depend on the specific study’s aims and the available resources in the laboratory.

Age-related decline in the taurine content of the skin in rodents

Taurine, a sulfur-containing amino acid, occurs at high concentrations in the skin, and plays a role in maintaining the homeostasis of the skin. We investigated the effects of aging on the content and localization of taurine in the skin of mice and rats. Taurine was extracted from the skin samples of hairless mice and Sprague Dawley rats, and the taurine content of the skin was determined by high-performance liquid chromatography (HPLC). The results of the investigation revealed that the taurine content in both the dermis and epidermis of hairless mice declined significantly with age. Similar age-related decline in the skin taurine content was also observed in rats. In contrast, the taurine content in the sole remained unchanged with age. An immunohistochemical analysis also revealed a decreased skin taurine content in aged animals compared with younger animals, although no significant differences in the localization of taurine were observed between the two age groups. Supplementation of the drinking water of aged mice with 3% (w/v) taurine for 4 weeks increased the taurine content of the epidermis, but not the dermis. The present study showed for the first time that the taurine content of the skin decreased with age in mice and rats, which may be related to the impairment of the skin homeostasis observed with aging. The decreased taurine content of the epidermis in aged animals was able to be rescued by taurine supplementation.

Dietary effect of green tea extract on epidermal levels of skin pH related factors, lactate dehydrogenase protein expression and activity in UV-irradiated hairless mice

Purpose: Skin pH, an indicator of skin health, is maintained by various organic factors, which include lactate, free amino acid (FAA), and free fatty acid (FFA). As skin ages or with illness, skin pH becomes less acidic, and functional food has been developed to maintain the acidic pH of skin. In this study, we determined the dietary effect of green tea extract (GTE) on skin pH of photo-aged mice, as measured by epidermal levels of lactate, FAA, and FFA. The protein expression and activity of lactate dehydrogenase (LDH), an enzyme of pyruvate reduction for lactate generation, was further determined. Methods: Albino hairless mice were fed a control diet (group UV+) or a diet with 1% GTE (group GTE) in parallel with UV irradiation for 10 weeks. A normal control group was fed a control diet without UV irradiation for 10 weeks (group UV-). Results: Skin pH was higher (less acidic) in group UV+ than in group UV-. In parallel, epidermal levels of lactate and FFA, as well as of LDH protein expression and activity, were reduced in group UV+. Dietary supplementation of GTE (group GTE) reduced skin pH to similar to the level of group UV-, and inversely increased epidermal levels of lactate, LDH protein expression and activity, but not of FFA. Although epidermal levels of FAA were similar in groups UV- and UV+, it was increased in group GTE to a level higher than in group UV-. In further analysis of major FFA, epidermal levels of palmitic acid [16:0], oleic acid [18:1(n-9)], and linoleic acid [18:2(n-6), but not of stearic acid [18:0] in group GTE were similar to or lower than those in group UV+. Conclusion: Dietary GTE normalized skin pH with increased levels of lactate and FAA, as well as with increased protein expression and activity of LDH in the epidermis of UVB irradiated hairless mice.

Dietary effect of green tea extract on hydration improvement and metabolism of free amino acid generation in epidermis of UV-irradiated hairless mice

Purpose: Ultraviolet (UV) irradiation decreases epidermal hydration, which is maintained by reduction of natural moisturizing factors (NMFs). Among various NMFs, free amino acids (AA) are major constituents generated by filaggrin degradation. This experiment was conducted to determine whether or not dietary supplementation of green tea extract (GTE) in UV-irradiated mice can improve epidermal levels of hydration, filaggrin, free AAs, and peptidylarginine deiminase-3 (PAD3) expression (an enzyme involved in filaggrin degradation). Methods: Hairless mice were fed a diet of 1% GTE for 10 weeks in parallel with UV irradiation (group UV+1%GTE). As controls, hairless mice were fed a control diet in parallel with (group UV+) or without (group UV-) UV irradiation. Results: In group UV+, epidermal levels of hydration and filaggrin were lower than those in group UV-; these levels increased in group UV+1% GTE to levels similar to group UV-. Epidermal levels of PAD3 and major AAs of NMF, alanine, glycine and serine were similar in groups UV- and UV+, whereas these levels highly increased in group UV+1% GTE. Conclusion: Dietary GTE improves epidermal hydration by filaggrin generation and degradation into AAs.

Abstract B47: Curcumin C3 ® prevents ultraviolet B radiation-induced epidermal damage in JB6 cells and mouse skin via a Fibroblast growth factor-2-dependent manner

Abstract Background and Significance: Non-melanoma skin cancer (NMSC) is the most common skin cancer in the U.S., with over a million new cases anticipated annually. Cutaneous squamous cell carcinoma (SCC) is a more aggressive form of NMSC. Chronic exposure to solar ultraviolet (UVB) is the major etiologic factor causing skin cell damage, photoaging and malignant transformation leading to SCC. UVB is a carcinogen and our previous studies established oral administration of Curcumin C3 complex (C3), a natural occurring polyphenol compound mixture, delayed UVB-induced tumor onset and multiplicity, but not tumor progression. Accordingly, we sort to determine the preventative efficacy of C3® against UVB-induced photodamage using JB6 cells and SKH-1 hairless mouse epidermis on the earlier neoplastic events in the development of UVB-induced skin carcinogenesis. Methods: SKH-1 mice were either administered vehicle or C3 (100mg/kg/b.w) complex orally for 14 days followed by a single exposure to UVB radiation (180mj/cm2). Mice were euthanized 24 hours after UVB exposure and skin tissues and serum were collected. For in vitro studies, promoter–sensitive JB6 mouse epithelial cells were pre-treated with C3® for 2 h and the effects on UV-induced early molecular events were assessed. Results: C3® attenuated UVB-induced epidermal hyperplasia and significantly decreased epidermal and plasma levels of fibroblast growth factor-2 (FGF-2); an important mitogen implicated in proliferation and differentiation of skin keratinocytes. Interestingly, the effects of C3® complex on FGF-2 levels were accompanied with a significant reduction in UVB-induced Nuclear factor KappaB (NF-KB) activation; an important nuclear transcription factor for tumor promotion. Further, the decrease in NF-KB was associated with an inhibition of BCL2 expression to induce apoptosis. In vitro, pretreatment of JB6 cells with C3® significantly decreased UVB-induced proliferation with a corresponding decrease in FGF-2 mRNA and protein levels. Further, C3 complex inhibited NF-KB phosphorylation and increased Bax expression leading to cell apoptosis. Conclusion: Our findings underscore the importance of curcumin (C3) in preventing UVB-induced skin cancer. It provides new insight into curcumin's role through FGF-2 in modulating the pathogenesis of photo carcinogenesis. Citation Format: Alok R. Khandelwal, Arun Anandharaj, Oleksandr Ekshyyan, Lauren D. Saucier, Tara N. Moore-Medlin, Fleurette Abreo, Cherie-Ann O. Nathan. Curcumin C3 ® prevents ultraviolet B radiation-induced epidermal damage in JB6 cells and mouse skin via a Fibroblast growth factor-2-dependent manner. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B47.

Red Light Modulates Ultraviolet-Induced Gene Expression in the Epidermis of Hairless Mice.

The purpose of this study was to investigate whether low-level light therapy (LLLT) was capable of modulating expression of ultraviolet (UV) light-responsive genes in vivo. The effects of 670 nm light-emitting diode (LED) array irradiation were investigated in a hairless SHK-1 mouse epidermis model. Mice were given a single dose of UVA/UVB light, or three doses of red light (670 nm @ 8 mW/cm(2) x 312 sec, 2.5 J/cm(2) per session) spread over 24 h along with combinations of pre- and post-UV treatment with red light. Levels of 14 UV-responsive mRNAs were quantified 24 h after UV irradiation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The transcription of mRNAs encoding for cluster of differentiation molecule 11b (CD11b) (p < 0.05) and interferon (IFN)-γ (p < 0.012) increased after irradiation with red light alone, whereas expression level of cyclooxygenase (COX)-2 (p < 0.02) was downregulated. Genes unresponsive to UV did not change their expression levels after exposure to red light either. Pretreatment with red light significantly modified response of Fos to UV exposure (p < 0.01). A synergy of UV and post-treatment with red light in reducing the transcription levels of CD11b (p < 0.05) and inducible nitric oxide synthase (iNOS) (p < 0.05) was observed. This is an initial observation that in mouse red light LLLT more often than not causes opposite gene expression changes or reduces those caused by moderate UVA-UVB irradiation.

Abstract 4644: Curcumin C3 ® prevents ultraviolet B radiation-induced acute damage in JB6 keratinocytes and mouse skin via FGF-2/mTOR/NF-KB pathway

Abstract Background and Significance: Aggressive Cutaneous Squamous cell carcinoma (CSCC) of the face has become the second most common type of skin cancer in the USA due to ultraviolet B (UVB) exposure in tanning booths and sun exposure of our military personnel. UVB is a carcinogen and our previous studies established oral administration of Curcumin C3 complex (C3), a standardized preparation of three curcumonoids - Curcumin (76.07%), Demethoxycurcumin (20.28%) and Bisdemethoxycurcumin (3.63%), delayed UVB-induced tumor onset and multiplicity, but not tumor progression. Accordingly, we sort to determine the preventative efficacy of C3® against UVB-induced photodamage in promotion sensitive JB6 skin keratinocytes and SKH-1 hairless mouse epidermis on the early neoplastic events in the development of UVB-induced skin carcinogenesis. Methods: SKH-1 mice were either administered vehicle or C3 (100mg/kg/b.w) complex orally for 14 days followed by a single exposure to UVB radiation (180mj/cm2). Mice were euthanized 24 hours after UVB exposure and skin tissues and serum were collected. For in vitro studies, promoter-sensitive JB6 mouse epithelial cells were pre-treated with C3® for 2 h and the effects on UVB-induced early molecular signaling events were assessed. Results: C3® attenuated UVB-induced epidermal hyperplasia and significantly decreased epidermal and plasma levels of fibroblast growth factor-2 (FGF-2); an important mitogen implicated in proliferation and differentiation of skin keratinocytes. In vitro, C3®blocked UVB-dependent FGF-2 induction and proliferation of cultured keratinocytes via down regulation of nuclear factor kappa B (NF-KB) and Mammalian target of rapamycin (mTOR) signaling pathways. Conclusion: Our findings suggest C3® has the ability to protect mice from UVB-induced acute damage. Further the photo-preventive effects of C3® are mediated, at least in part, via modulation of FGF-2/mTOR/NF-KB signaling. Citation Format: Alok R. Khandelwal, Arun Anandharaj, Ekshyyan Oleksandr, Tara Moore-Medlin, Abreo Fleurette, Cherie-Ann O. Nathan. Curcumin C3 ® prevents ultraviolet B radiation-induced acute damage in JB6 keratinocytes and mouse skin via FGF-2/mTOR/NF-KB pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4644. doi:10.1158/1538-7445.AM2015-4644

Co‐treatment with retinyl retinoate and a PPARα agonist reduces retinoid dermatitis

Retinoids have been used for the treatment of skin disorders such as acne, psoriasis, and photoaging. However, despite their beneficial effects, topical retinoids often cause severe local irritation called retinoid dermatitis. We previously developed a novel vitamin A derivative, retinyl retinoate, which induces less irritation and affords excellent tolerance. In this study, we examined whether co-treatment with topical peroxisome proliferator-activated receptor-α (PPARα) agonists (e.g. WY14643) reduce retinoid dermatitis in hairless mouse skin. The effect of concomitant treatment with a PPARα agonist on retinoid dermatitis in hairless mouse epidermis was evaluated by measuring transepidermal water loss, epidermal histology, and cytokine expression. Retinyl retinoate induced less severe retinoid dermatitis than retinoic acid. Topical application of a PPARα agonist improved the stratum corneum structure and function, reduced mRNA expression of interleukin (IL)-1α, tumor necrosis factor-α and IL-8, and inhibited ear edema induced by retinoic acid or retinyl retinoate. Our results indicate that PPARα agonists can potentially be used to improve retinoid dermatitis. We suggest that co-treatment with retinyl retinoate and a PPARα agonist may reduce or prevent detrimental alterations in retinoid-treated skin.

Abstract 1592: Silibinin inhibits UVB-induced DNA-damage and apoptosis by enhancing interleukin-12 expression in JB6 cells and SKH-1 mouse skin

Abstract Ultraviolet-B (UVB) radiation plays a pivotal role in non-melanoma human skin cancers (NMSCs) by inducing cellular DNA damage, mutations and immunosuppression. Decades of research has demonstrated silibinin efficacy against UVB-induced skin carcinogenesis by regulating different molecular pathways in various cell lines and animal models; however, the role of silibinin in regulating interleukin-12 (IL-12), an immunomodulatory cytokine with the capacity to reduce UVB-induced DNA damage and apoptosis, has not been studied. Here, we report that UVB irradiation caused a dose- and time-dependent caspase 3 and PARP cleavage as well as apoptosis in JB6 cells. Addition of recombinant IL-12 or silibinin immediately after UVB significantly protected UVB-induced apoptosis in JB6 cells, and antibody-mediated blocking of IL-12 activity compromised the protective effect of both IL-12 and silibinin. In addition, both silibinin and IL-12 accelerated the repair of UVB-caused cyclobutane-pyrimidine dimers (CPDs) in JB6 cells. Additional studies confirmed that indeed silibinin causes a significant increase in IL-12 levels in UVB-irradiated JB6 cells as well as in the SKH-1 hairless mouse epidermis, and that similar to in vitro findings, silibinin topical application immediately after UVB exposure causes a very strong protection against UVB-induced TUNEL positive cells in epidermis possibly through a significantly accelerated repair of UVB-caused CPDs in skin epidermis. Together, these findings for the first time identify an important insight regarding the pharmacological mechanism wherein silibinin induces endogenous IL-12 in its efficacy against UVB-caused skin damages. Considering the fact that millions of people get constantly exposed to solar UVB radiation and live with persistent UVB-induced DNA lesions, the post-damage use of silibinin as an inducer of endogenous IL-12 in UVB-damaged human skin for the rapid repair of DNA damage could be a more practical and translational approach in reducing sunlight-caused damages in human skin which eventually lead to NMSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1592. doi:1538-7445.AM2012-1592

Silibinin prevents ultraviolet B radiation-induced epidermal damages in JB6 cells and mouse skin in a p53-GADD45α-dependent manner

Better preventive strategies are required to reduce ultraviolet (UV)-caused photodamage, the primary etiological factor for non-melanoma skin cancer (NMSC). Accordingly, here we examined the preventive efficacy of silibinin against UVB-induced photodamage using mouse epidermal JB6 cells and SKH1 hairless mouse epidermis. In JB6 cells, silibinin pretreatment protected against apoptosis and accelerated the repair of cyclobutane pyrimidine dimers (CPD) induced by moderate dose of UVB (50 mJ/cm(2)), which we are at risk of daily exposure. Silibinin also reversed UVB-induced S phase arrest, reducing both active DNA synthesizing and inactive S phase populations. In mechanistic studies, UVB-irradiated cells showed a transient upregulation of both phosphorylated (Ser-15 and Ser-392) and total p53, whereas silibinin pretreatment led to a more sustained upregulation and stronger nuclear localization of p53. Silibinin also caused a marked upregulation of GADD45α, a downstream target of p53, implicated in DNA repair and cell cycle regulation. Importantly, under p53 and GADD45α knockdown conditions, cells were more susceptible to UVB-induced apoptosis without any significant S phase arrest, and protective effects of silibinin were compromised. Similar to the in vitro results, topical application of silibinin prior to or immediately after UVB irradiation resulted in sustained increase in p53 and GADD45α levels and accelerated CPD removal in the epidermis of SKH1 hairless mice. Together, our results show for the first time that p53-mediated GADD45α upregulation is the key mechanism by which silibinin protects against UVB-induced photodamage and provides a strong rationale to investigate silibinin in reducing the risk and/or preventing early onset of NMSC.

Molecular Mechanisms Underlying Chemopreventive Activities of Glycyrrhizic Acid against UVB-Radiation-Induced Carcinogenesis in SKH-1 Hairless Mouse Epidermis

Glycyrrhizic acid has been shown to possess anti-inflammation, antiviral and chemoprotective activity against tumors. We evaluated the protective effects of glycyrrhizic acid in UVB-radiation-induced skin tumor formation in SKH-1 hairless mice and the early molecular biomarkers of these effects. Mice irradiated at 180 mJ/cm² twice per week showed 100% tumor incidence in 20 weeks. Feeding with glycyrrhizic acid prior to UVB irradiation caused delays in tumor appearance, multiplicity and size. Feeding with glycyrrhizic acid for 2 weeks before a single UVB irradiation (180 mJ/cm²) resulted in significant decrease in UVB-radiation-induced thymine dimer-positive cells, expression of proliferative cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and apoptotic sunburn cells together with an increase in p53- and p21/Cip1-positive cell populations in epidermis. Simultaneously, glycyrrhizic acid also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and nitric oxide (NO) levels. Thus glycyrrhizic acid ameliorates UVB-radiation-induced tumorigenesis via downregulation of cell proliferation controls involving thymine dimer, PCNA, apoptosis and transcription factor NF-κB and of inflammatory responses involving COX-2, PGE2 and NO while upregulating of p53 and p21/Cip1 to prevent DNA damage and facilitate DNA repair.

Abstract 1815: Stimulation of apoptosis by caffeine or exercise is associated with an increase in thymine dimer formation in the epidermis of UVB-treated SKH-1 mice

Abstract In early studies, we found that oral administration of caffeine or voluntary running wheel exercise (RW) for 1 or 2 weeks prior to UVB irradiation enhanced UVB-induced apoptosis in the epidermis of SKH-1 hairless mice. In additional studies, we found that oral administration of caffeine or RW during the course of irradiation with UVB inhibited UVB-induced skin carcinogenesis. In the present study, we explored the possible mechanism for the effect of caffeine administration or RW to stimulate UVB-induced apoptosis in the epidermis of SKH-1 mice. Female SKH-1 mice (5 mice per time point) were treated with: (1) oral water (control), (2) oral caffeine (0.1 mg/ml in the drinking water) (3) RW, or (4) 0.1 mg/ml oral caffeine plus RW for 2 weeks. The pretreated mice were then exposed to a single irradiation with UVB (30 mJ/cm2) and sacrificed at 0 (no UVB treatment), or at 5 min, 0.5h, 1h, 2h, 4h, 6h, 10h, 16h, 24h and 48h after UVB. The results showed that oral caffeine, RW, or caffeine plus RW for 2 weeks stimulated UVB-induced caspase 3 positive cells by 30%, 164% or 333%, respectively, at 6 hr after UVB (peak timepoint for UVB-induced apoptosis). These treatments had little inhibitory effect on cell proliferation. Unexpectedly, all of these treatments enhanced UVB-induced thymine dimer formation as measured by immunohistochemistry. Oral caffeine, RW, or the combination of caffeine and RW increased UVB-induced thymine dimer positive cells by 52%, 56% or 75%, respectively at 0.5 hr after UVB (peak for UVB-induced thymine dimer formation), when compared to the water control group. Thymine dimer positive cells were gone by 48 h post-UVB. The effect of caffeine and RW to increase the number of thymine dimer positive cells preceded the increase in apoptosis in the epidermis of UVB irradiated mice. Slot-blot analysis of isolated epidermal DNA from the mice treated with oral caffeine, RW or their combination for 2 weeks for thymine dimers confirmed the immunohistochemistry data. In a separate experiment, mice treated with instant coffee (10 mg coffee solids/ml) or caffeine (0.4 mg/ml) in the drinking fluid for one week prior to UVB irradiation showed enhanced UVB-induced increase in thymine dimer positive cells by 75-80% at 15-30 min post-UVB compared with water control mice irradiated with UVB. The coffee-induced increase in thymine dimer positive cells was followed by an increase in apoptosis (about 2-fold) at 6-10 hr post-UVB. Our results indicate that RW, oral administration of caffeine or coffee increase UVB-induced thymine dimers in DNA followed by an increase in apoptosis in the epidermis. We hypothesize that caffeine- or voluntary running wheel exercise-induced increase in thymine dimers may play a role in the ability of caffeine administration and RW to increase UVB-induced apoptosis and to inhibit UVB-induced carcinogenesis (Supported by NIH Grants RO1 CA128997 and RO1CA114442). 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 1815. doi:10.1158/1538-7445.AM2011-1815

Abstract 2483: Molecular mechanisms underlying chemopreventive activities of glycyrrhizic acid against UVB-induced carcinogenesis in SKH-1 hairless mouse epidermis

Abstract Glycyrrhizic acid (GL) has been shown to possess anti-inflammation, antiviral, and chemoprotective activity against tumors. We assessed the protective effects of GL in UVB-induced skin tumor formation in SKH-1 hairless mice and the early molecular biomarkers. Mice irradiated at 180 mJ/cm2 twice per week elicited 100% tumor incidence in 20 weeks. Feeding of GL prior to UVB irradiation caused a strong protection against photocarcinogenesis in terms of delay in tumor appearance, multiplicity, and size. It took 24 weeks to get 100% tumor incidence in GL plus UVB treated-group; the first tumor appearance in UVB-treated animals occurred at 16th week, which was delayed by 3 weeks in GL plus UVB-treated group. Feeding of GL for 2 weeks before a single UVB (180mJ/cm2) irradiation resulted in a strong and significant decrease in UVB-induced thymine dimer-positive cells (p &amp;lt; 0.004), proliferative cell nuclear antigen PCNA (p &amp;lt; 0.01), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells (p &amp;lt; 0.01) together with an increase in p53 and p21/Cip1-positive cell population in epidermis (p &amp;lt; 0.02 ∼ 0.003). Simultaneously, GL also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and nitric oxide (NO) levels (p &amp;lt; 0.01-0.004). Taken together, these results suggest that GL causes a strong protective effect against photocarcinogenesis via down-regulation of cell proliferative controls, involving thymine dimer, PCNA, apoptosis, transcription factors NF-kB and of inflammatory responses involving COX-2, PGE2, and NO, while up-regulation of p53, p21/Cip1 to prevent DNA damage. These results provide a focus for the rational development of GL as a novel chemopreventive agent against UV-induced human skin cancer. 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 2483. doi:10.1158/1538-7445.AM2011-2483

P38 MAP kinase plays a functional role in UVB‐Induced mouse skin carcinogenesis

UVB irradiation of epidermal keratinocytes results in the activation of the p38 mitogen-activated protein kinase (MAPK) pathway and subsequently activator protein-1 (AP-1) transcription factor activation and cyclooxygenase-2 (COX-2) expression. AP-1 and COX-2 have been shown to play functional roles in UVB-induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH-1 hairless mice and assess UVB-induced AP-1 activation, COX-2 expression, and the skin carcinogenesis response in these mice compared to wild-type littermates. We observed a significant inhibition of UVB-induced AP-1 activation and COX-2 expression in p38DN transgenic mice, leading to a significant reduction of UVB-induced tumor number and growth compared to wild-type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and growth rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki-67 staining in p38DN mice compared to wild-type. Although we detected no difference in chronic apoptotic rates between transgenic and nontransgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB-induced apoptosis of keratinocytes. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV-induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX-2 expression and proliferation of UVB-irradiated cells.

Effect of Protein Malnutrition on the Skin Epidermis of Hairless Mice

The purpose of this study was to evaluate the effect of protein malnutrition on the skin epidermis of mice. A low protein diet induced thinning of the skin epidermis, a decrease of cell proliferative activity in epidermal cells and a decrease of stratum corneum hydration. Dityrosine was immunostained in the cytoplasm of epidermal cells in the low protein diet group. Plasma advanced oxidation protein product (AOPP) levels were significantly more increased in the low protein diet group than in the control diet group. These results suggest that protein malnutrition adversely affects the structure and water barrier and reservoir functions of the skin epidermis, and these pathological changes are associated with the expressions of protein oxidation markers, dityrosine and AOPP.

Oral Feeding of Pomegranate Fruit Extract Inhibits Early Biomarkers of UVB Radiation‐induced Carcinogenesis in SKH‐1 Hairless Mouse Epidermis

Pomegranate from the plant Punica granatum L. possesses strong antioxidant and anti-inflammatory properties. Recently, we have demonstrated that treatment of normal human epidermal keratinocytes with pomegranate fruit extract (PFE) inhibited UVB-mediated activation of nuclear factor kappa B (NF-κB) and mitogen activated protein kinases pathways. Here, we evaluated the effect of PFE on early biomarkers of photocarcinogenesis employing SKH-1 hairless mice. PFE was provided in drinking water (0.2%, wt/vol) to SKH-1 hairless mice for 14 days before a single UVB (180 mJ cm(-2)) irradiation. We found that oral feeding of PFE inhibited UVB-induced: (1) skin edema; (2) hyperplasia; (3) infiltration of leukocytes; (4) lipid peroxidation; (5) hydrogen peroxide generation; (6) ornithine decarboxylase (ODC) activity; and (7) ODC, cyclooxygenase-2 and proliferating cell nuclear antigen protein expression. Oral feeding of PFE enhanced repair of UVB-mediated formation of cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Importantly, PFE treatment further enhanced UVB-mediated increase in tumor suppressor p53 and cyclin kinase inhibitor p21. Furthermore, oral feeding of PFE inhibited UVB-mediated: (1) nuclear translocation of NF-κB; (2) activation of IKKα; and (3) phosphorylation and degradation of IκBα. Taken together, we provide evidence that oral feeding of PFE to mice affords substantial protection from the adverse effects of UVB radiation via modulation in early biomarkers of photocarcinogenesis and provide suggestion for its photochemopreventive potential.

Photoprotective effect of Pothomorphe umbellata on UVB radiation-induced biomarkers involved in carcinogenesis of hairless mouse epidermis

In this study we assessed the protective effect of topical application of Pothomorphe umbellata extract on ultraviolet B (UVB)-induced skin lesion parameters in hairless mouse epidermis. A single dose of UVB irradiation (0.23 kJ/m2) resulted in a significant decrease in thymine dimer-positive cells and apoptotic sunburn cells, with an increase in p53 and proliferating cell nuclear antigen-positive cells in the epidermis. After 5 weeks (total dose 13.17 kJ/m2) and 15 weeks (total dose 55.51 kJ/m2) of irradiation, P. umbellata treatment inhibited the hyperplasic response and induced an increase in p53-positive cells. These findings suggest that P. umbellata extract affords protection against UVB-induced skin lesions.

Cytokine cascade after cessation of long-term topical glucocorticosteroid on hairless mouse epidermis

Cytokine cascade after cessation of long-term topical glucocorticosteroid on hairless mouse epidermis

K6PC-5, a Direct Activator of Sphingosine Kinase 1, Promotes Epidermal Differentiation Through Intracellular Ca2+ Signaling

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(2+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in HaCaT cells and Ca(2+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(2+)](i). The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca(2+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.

Radiation Sources Providing Increased UVA/UVB Ratios Attenuate the Apoptotic Effects of the UVB Waveband UVA-Dose-Dependently in Hairless Mouse Skin

UV radiation-induced epidermal apoptotic sunburn cells provide a mechanism for eliminating cells with irreparable DNA damage. The UVB (290-320 nm) waveband is mainly responsible, but the role of UVA (320-400 nm) is less clear, and possible waveband interactions have not been examined. Recent studies in mice reveal a protective role for UVA against UVB-induced inflammation and immunosuppression, mediated via cutaneous heme oxygenase (HO). As HO has antiapoptotic properties in other tissues, this study examines the effect of UVA/UVB waveband interaction on apoptosis in the Skh:hr-1 hairless mouse epidermis. Apoptosis was assessed by sunburn cell number, caspase-3-positive cell number, and degree of DNA fragmentation, in mice exposed to radiation sources providing a constant UVB dose with increasing proportions of UVA. The results indicated that as the UVA/UVB ratio was increased, both the sunburn cell and caspase-3-positive cell number decreased, and the degree of DNA fragmentation was reduced. Treatment of mice with the HO inhibitor, tin protoporphyrin-IX, markedly reduced the UVA antiapoptotic effect, confirming a major role for HO. The observations suggest that UVA reduces UVB-induced DNA damage, and may therefore have anti-photocarcinogenic properties that could be harnessed for better photoprotection in humans.