Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin

Abstract: Solar ultraviolet (UV) radiation, particularly its UVB (290–320 nm) component, is the primary cause of many adverse biological effects including photoageing and skin cancer. UVB radiation causes DNA damage, protein oxidation and induces matrix metalloproteinases (MMPs). Photochemoprevention via the use of botanical antioxidants in affording protection to human skin against UVB damage is receiving increasing attention. Pomegranate, from the tree Punica granatum, contains anthocyanins and hydrolysable tannins and possesses strong antioxidant and anti‐tumor‐promoting properties. In this study, we determined the effect of pomegranate‐derived products – POMx juice, POMx extract and pomegranate oil (POMo) – against UVB‐mediated damage using reconstituted human skin (EpiDerm^TM FT‐200). EpiDerm was treated with POMx juice (1–2 μl/0.1 ml/well), POMx extract (5–10 μg/0.1 ml/well) and POMo (1–2 μl/0.1 ml/well) for 1 h prior to UVB (60 mJ/cm²) irradiation and was harvested 12 h post‐UVB to assess protein oxidation, markers of DNA damage and photoageing by Western blot analysis and immunohistochemistry. Pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) cyclobutane pyrimidine dimers (CPD), (ii) 8‐dihydro‐2′‐deoxyguanosine (8‐OHdG), (iii) protein oxidation and (iv) proliferating cell nuclear antigen (PCNA) protein expression. We also found that pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) collagenase (MMP‐1), (ii) gelatinase (MMP‐2, MMP‐9), (iii) stromelysin (MMP‐3), (iv) marilysin (MMP‐7), (v) elastase (MMP‐12) and (vi) tropoelastin. Gelatin zymography revealed that pomegranate‐derived products inhibited UVB‐induced MMP‐2 and MMP‐9 activities. Pomegranate‐derived products also caused a decrease in UVB‐induced protein expression of c‐Fos and phosphorylation of c‐Jun. Collectively, these results suggest that all three pomegranate‐derived products may be useful against UVB‐induced damage to human skin.     

Evaluation of sunscreen products using a reconstructed skin model exposed to simulated daily ultraviolet radiation: relevance of filtration profile and SPF value for daily photoprotection

Background: The recent definition of a standard daily ultraviolet radiation (DUVR) has allowed us to reproduce non‐zenithal sun exposure conditions. Exposure to simulated DUVR induces biological damage in human skin, suggesting the need for an appropriate photoprotection. Methods: Sunscreen products were evaluated using human reconstructed skin in vitro. Two commercial sunscreens (A and B) having similar sun (burn) protection factor (SPF) values (～15) but different profiles of transmission over the UVA range were tested on skin models exposed to increasing doses of DUVR. Another pair of sunscreens was also tested. One (product C) had an SPF ～18 with a well‐balanced UVB–UVA profile and the other (product D) an SPF of ～27 with low UVA absorption. Biological parameters were assessed by (i) histology, (ii) vimentin immunostaining for dermal fibroblasts, and (iii) analysis of matrix metalloprotease (MMP)‐1 secretion. Results: Products A and C gave better protection from DUVR with regard to fibroblast alterations and MMP‐1 release compared with products B and D, respectively. Conclusion: To ensure an efficient daily photoprotection from DUVR, the filtration profile of the product should be well balanced with a sufficient level of UVA absorption. With regard to end points evaluated in this study, our data suggest that a higher SPF value does not compensate for low UVA filtration.     

Inhibition of nitric oxide and reactive oxygen species production improves the ability of a sunscreen to protect from sunburn, immunosuppression and photocarcinogenesis

BACKGROUND: More effective strategies are required for the prevention of skin cancer, which is caused by ultraviolet (UV) radiation in sunlight. Sunscreens containing UV filters or reflectors offer some protection from sunlight. Pharmacologically active compounds that reduce UV damage offer considerable potential for improving sunscreen formulations. However, few studies have investigated whether the addition of such biological modifiers are an improvement. OBJECTIVES: In this study we supplemented a 2-ethyl hexyl methoxycinnamate-based sunscreen with the nitric oxide (NO) inhibitor NG-monomethyl-L-arginine acetate, the iron chelator 2,2'-dipyridyl, which reduces reactive oxygen species (ROS) production, or both. This was to determine whether inhibition of NO, ROS, or both could improve photoprotection by a sunscreen. METHODS: These sunscreens were compared for photoprotection from sunburn, immunosuppression and skin carcinogenesis in mice. To observe additional photoprotection by the NO and ROS inhibitors, UV doses were used that exceeded the protective capacity of the sunscreen. RESULTS: The combined inhibition of both NO and ROS production, but neither alone, increased sunscreen protection from sunburn and immunosuppression. Similarly, inhibition of both NO and ROS but neither alone reduced tumour multiplicity and incidence, therefore improving sunscreen protection from photocarcinogenesis. CONCLUSIONS: Whether NO and ROS inhibition were independently improving sunscreen photoprotection, with both being required for an observable effect, or whether inhibition of an interaction between NO and ROS was responsible for improved photoprotection by the sunscreen is unknown. These studies show that supplementation of a sunscreen with inhibitors of NO and ROS production improves the ability of the sunscreen to protect from sunburn, immunosuppression and photocarcinogenesis. Such an approach may be useful for reducing skin cancer incidence in humans.     

An analysis of cumulative lifetime solar ultraviolet radiation exposure and the benefits of daily sun protection

ABSTRACT:  Cumulative exposure to ultraviolet radiation (UVR) increases the risk of developing skin cancer, particularly squamous cell carcinoma and basal cell carcinoma. Thus, the need for protection from the sun is widely advocated, but consumers generally associate such protection with the occasional extreme exposure and tend to ignore the risk of long-term exposure. In fact, a sun exposure model predicts that over a lifetime, a person will receive tens of thousands of minimal erythema doses worth of UVR through normal, daily, incidental exposure. The cumulative effect of casual sun exposure over the years underscores the need for everyday basic UVR protection in which even low level (SPF 4–10) sunscreens are shown to offer significant benefit. Analysis shows that daily protection can reduce lifetime exposure by 50% or more.     

The density of epidermal p53 clones is higher adjacent to squamous cell carcinoma in comparison with basal cell carcinoma

BACKGROUND: It is well accepted that ultraviolet radiation from the sun can induce and promote growth of skin tumours. Skin cancer develops as a consequence of multiple genetic hits, where an initial, important step includes proliferation of cells susceptible to malignant transformation. Foci of morphologically normal epidermal keratinocytes overexpressing p53 protein are common in chronically sun-exposed skin. Such foci have previously been shown to represent expanding clones of p53-mutated keratinocytes. Although several characteristics concerning epidermal p53 clones remain to be resolved, an important role in skin carcinogenesis is anticipated. The density of epidermal p53 clones in human skin is largely unknown. OBJECTIVES: To compare the occurrence of epidermal p53 clones in skin surrounding cancers with that in skin surrounding benign melanocytic naevi. To assess the influence of age on frequency and size of epidermal p53 clones in human facial skin. METHODS: We have analysed the number and sizes of epidermal p53 clones in skin specimens from patients with squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and benign melanocytic naevi. Cases included normal facial skin from four different age groups. Tissue sections were immunohistochemically stained and the presence of p53 clones was recorded. Approximately 1.4 m of epidermis from a total of 112 biopsies was analysed. RESULTS: We found 128 epidermal p53 clones in biopsy specimens from 112 patients. The results showed that the number and size of p53 clones increase with age. In normal skin adjacent to SCC p53 clones were significantly more numerous and greater in size in comparison with those in normal skin both adjacent to benign naevi and adjacent to BCC. Interestingly, normal skin in the close vicinity of BCC and melanocytic naevi showed similar results regarding both number and size of epidermal p53 clones. CONCLUSIONS: Our findings suggest a connection between development of epidermal p53 clones and SCC.     

Immunophenotypic analysis of the p53 gene in non-melanoma skin cancer and correlation with apoptosis and cell proliferation

BACKGROUND: Sunlight precipitates a series of genetic events that lead to the development of skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The p53 tumour suppressor gene, which plays a pivotal role in cell division and apoptosis, is frequently found mutated in sunlight-induced skin tumours. OBJECTIVE: To investigate the immunoreactivity of the p53 gene in non-melanoma skin cancers and to correlate its expression with apoptotic and cell proliferation markers. METHODS: We analysed 35 non-melanoma tumours including 19 BCCs and 16 SCCs from sun-exposed skin areas. p53 protein expression was studied immunohistochemically using the DO7 monoclonal antibody against wild-type and mutant p53 forms. The percentage of p53-immunopositive nuclei was measured by image analysis. Cell proliferation and apoptosis were also assessed by image analysis following Ki-67 immunostaining and application of the TUNEL method on paraffin sections, respectively. RESULTS: The percentage of p53-expressing cells varied from 3.5 to 90 in BCCs (median value 54.4%) and from 3.7 to 94 in SCCs (median value 40.3%). The mean value of Ki-67-positive cells was comparable in both groups of tumours with a mean value of 40.6% in BCCs and 34.6% in SCCs. Conversely, the TUNEL assay showed sporadic staining of apoptotic cells within the tumours with a mean value of 1.12% in BCCs and 1.8% in SCCs. p53 protein expression was correlated positively with cell proliferation (r = 0.75, P = 0.000001) and negatively with apoptosis (r = -0.23, P = 0.05). CONCLUSION: p53 immunoreactivity was high in the majority of the skin carcinomas examined and correlated positively with cell proliferation and negatively with apoptosis. The p53 protein overexpression appears to be related to an inactivated protein resulting from mutations of the p53 gene or other unclear molecular mechanisms.