Clinical outcome and cyclo-oxygenase-2 expression in five dogs with solar dermatitis/actinic keratosis treated with firocoxib.

BACKGROUND: The conversion of arachidonic acid into prostaglandin is catalysed by the cyclo-oxygenases (COX-1/COX-2). Several studies indicate that COX-2 is overexpressed in actinic keratosis in humans and dogs. Firocoxib is a COX-2-selective inhibitor that blocks the biochemical activity of COX-2. HYPOTHESIS/OBJECTIVES: To evaluate the efficacy of firocoxib (5 mg/kg orally once daily) for the treatment of dogs with solar dermatitis/actinic keratosis. METHODS: Firocoxib 5 mg/kg was given orally once daily for 180 days to five dogs with clinical signs and histopathological lesions consistent with solar dermatitis/actinic keratosis. On days 0, 50 and 180, the severity of erythema, skin shine, induration and the number of comedones were evaluated by a clinical scoring system. On the same days, samples were collected for histopathology from 'target lesions' and COX-2 expression was evaluated by immunohistochemistry. RESULTS: The clinical follow-up showed that four of five dogs improved with the treatment; improvement in terms of histological findings was correlated with the regularization of the epidermal proliferation rather than the recovery of dermal changes. CONCLUSIONS AND CLINICAL IMPORTANCE: A role for COX-2 might thus be hypothesized in the pathogenesis of canine solar dermatitis.

Immunohistochemical analysis of TIMP-3 and MMP-9 in actinic keratosis, squamous cell carcinoma of the skin, and basal cell carcinoma.

The expression of metalloproteinases and their inhibitors has been related to different invasive and metastatic potentials in cancer. This study aims to investigate the immunohistochemical expression of TIMP-3 and MMP-9 in samples of basal cell carcinoma (BCC), squamous cell carcinoma of the skin (SCC), and actinic keratosis (AK). Immunohistochemistry was performed to evaluate the expression of TIMP-3 and MMP-9 in samples of BCC (n=22), SCC (n=10), and AK (n=15). Ten fields of both tumor parenchyma and tumor stroma were photographed and counted in image software. The ratio of positive cells to total cells was used to quantify the staining. A higher expression of MMP-9 was found in tumor stroma of SCC compared to BCC and AK. No significant differences in TIMP-3 expression were observed among the groups. Considering the well-described differences between these neoplasms, these results provide additional evidence of the role of MMP-9 in tumor invasiveness of keratinocyte-derived tumors.

Cathepsin K expression in basal cell carcinoma.

BACKGROUND: Cathepsin K is a cysteine protease with strong collagenolytic and elastolytic properties. Recently, cathepsin K expression in tumour cells of malignant melanoma and in the stromal cells of squamous cell carcinoma of the skin has been reported to play an important role in tumour progression. However, its expression profile in basal cell carcinoma (BCC) has not yet been clarified. OBJECTIVE: The aim of this study is to examine the expression profile of cathepsin K in both the tumour cells and the peritumoural stromal cells of BCC in comparison with its expression in normal skin. METHODS: Fifty consecutive operative cases of BCC, 10 cases of actinic keratosis, 10 cases of Bowen's disease and five normal skin tissues were assessed for cathepsin K expression by immunohistochemical methods. RESULTS: In normal skin, cathepsin K expression was observed in the stratum corneum, mature sebaceous cells and outer root sheath of the hair follicles. Cathepsin K was expressed in the tumour cells of all BCC cases, in which 90% showed diffuse expression (>51% of tumour cells), as well as in the peritumoural stromal cells in all BCC cases. Focal cathepsin K expression was observed in the tumour cells of Bowen's disease (2/10 cases), but not in any of actinic keratosis (0/10 cases). CONCLUSION: Cathepsin K expression may contribute to tumour invasion and peculiar histopathological features, such as fibromucinous stroma around the tumour nests by mediating extracellular matrix degradation in BCC.

Efficacy of a photolyase-based device in the treatment of cancerization field in patients with actinic keratosis and non-melanoma skin cancer.

Eryfotona AK-NMSC (ISDIN Spain) is a film-forming medical device in cream or fluid formulation containing the DNA-repair enzyme photolyase and high-protection UV filters in liposomes (repairsomes) indicated in the treatment of cancerization field in patients with actinic keratosis (AK) or non-melanoma skin cancer (NMSC). Photolyase is an enzyme that recognizes and directly repairs UV-induced DNA damage. The most common UV-induced DNA damage is the formation of cyclobutane pyrimidine dimers (CPD). Clinical studies evaluating the histological and cellular effects of Eryfotona AK-NMSC have shown a potential benefit in the treatment of the cancerization field in AK patients. In particular the use of Eryfotona AK-NMSC improves the confocal microscopic appearance of skin at the cancerization field level. In addition, Eryfotona AK-NMSC improves the p53 gene expression at keratinocyte level. In this study we reported a series of 6 cases of patients with AK or NMSC lesions treated with Eryfotona AK-NMSC fluid, both as coadjuvant and as single treatment, applied twice daily in the affected area with photograph documentation. Clinical photographs of the skin lesions at baseline and after Eryfotona AK-NMSC treatment were taken in all cases using a high-definition digital camera. Six patients with multiple AK lesions of the scalp or face with or without NMSC were treated for a mean of 1-3 months with Eryfotona AK-NMSC fluid formulation. Image documentations before and after treatment of this clinical series show a great improvement in AK lesions count and of cancerization field. This clinical series supports the clinical efficacy of the use of photolyase and high-protection UV filters in the treatment of cancerization field and AK lesions in patients with actinic damage.

Significance of cyclooxygenase 2, EZH-2 polycomb group and p53 expression in actinic keratosis and squamous cell carcinomas of the skin.

The development and progression of squamous cell carcinoma (SCC) of the skin is characterized by an accumulation of molecular changes. The aim of this study was to investigate the association of the immunohistochemical expression of cyclooxygenase-2 (COX-2), enhancer of zeste homolog 2 (EZH-2), and p53 in actinic keratosis (AK) and SCC and detect any differences between invasive and preinvasive squamous epidermal lesions. Forty-three cases with AK, 38 with SCC, and 9 with SCC arising on AK (SCC/AK) were studied. For COX-2 immunostaining, weak or no reaction was associated with AK (58.10% of cases), whereas moderate or strong reaction with SCCs (34.2% and 39.5%, respectively). Furthermore, 88.9% of the "mixed" SCC/AK specimens demonstrated moderate reaction (χ2 = 29.924, P < 0.0001). For EZH-2 immunostaining, a weak or no reaction was observed in 62.8% of AK cases, whereas a moderate reaction was observed in 42.1% of SCCs and 77.8% of "mixed" SCC/AK cases (χ2 = 18.91, P = 0.001). Weak immunoreactivity of p53 was associated with AK (58.1%), moderate with SCC (44.7%), and strong with SCC/AK lesions (66.7%) (χ2 = 15.999, P = 0.003). COX-2, p53, but mainly EZH-2 immune expression seems to be strongly associated with the biological potential of squamous epidermal cells and seems to be differentiating SCC by comparison to AK of the skin. The value of the combined expression of these markers is worth being further investigated as an additional tool for diagnostic, prognostic, and possibly, therapeutic use.

The expression of p53 and COX-2 in basal cell carcinoma, squamous cell carcinoma and actinic keratosis cases.

OBJECTIVE: The aim of this study was to investigate p53 and COX-2 expressions in basal cell carcinoma, squamous cell carcinoma and actinic keratoses, and to determine a possible relationship. MATERIAL AND METHOD: 50 basal cell carcinoma, 45 squamous cell carcinoma and 45 actinic keratosis cases were evaluated. The type of tumor in basal cell carcinoma and tumor differentiation in squamous cell carcinoma were noted and the paraffin block that best represented the tumor was chosen. Immunostaining by p53 and COX-2 was performed on sections of the paraffin blocks. RESULTS: p53 expression was observed in 98% of basal cell carcinoma, 88.9% of squamous cell carcinoma and all actinic keratosis cases. p53 expression was also noted in non-dysplastic appearing epithelium in actinic keratosis cases. COX-2 expression was seen in 90, 100 and 88.9% of the basal cell carcinoma, squamous cell carcinoma and actinic keratosis groups, respectively. Skin appendages, inflammatory cells and vascular structures were also stained by COX-2 besides tumor tissue. COX-2 expression increased by the p53 expression increase in basal cell carcinoma and squamous cell carcinoma. p53 and COX-2 expressions were not related in terms of tumor type in the BCC and were not related in terms of differentiation in SCC. CONCLUSION: The existence of p53 expression in actinic keratosis cases has supported the idea that p53 plays a role in the early steps of carcinogenesis in skin cancers. The fact that the expression of COX-2 increases in line with the increase of p53 expression in basal cell carcinoma and squamous cell carcinoma cases indicates that COX-2 expression may be affected by p53.

Expression of gelatinases (MMP-2, MMP-9) and gelatinase activator (MMP-14) in actinic keratosis and in in situ and invasive squamous cell carcinoma.

Given the established role of matrix metalloproteinases (MMPs) in physiological processes in the skin, we investigated the expression of MMP-2, MMP-9, and MMP-14 to evaluate their role in the grading and development of atypical epithelial lesions. Immunohistochemistry was performed using antibodies against these MMPs in actinic keratosis (AK; n = 24), squamous cell carcinoma (SCC) in situ (SCCIS; n = 27), SCC well differentiated (SCCWD; n = 28), and SCC moderately to poorly differentiated (SCCMPD; n = 20). Tumoral and stromal expression was assessed by intensity (SI) and percentage positivity (PC). The mean of the total score, calculated by adding intensity and percentage positivity, was used for statistical analyses. In AK, SCCIS, SCCWD, and SCCMPD, mean tumoral MMP-2 expression was 3.33, 4.07, 4.46, and 3.40, respectively (P = NS for all) and stromal expression was 1.42, 3.26, 3.07, and 1.55 respectively (P < 0.05 for AK vs. SCCIS/SCCWD and SCCMPD vs. SCCIS/SCCWD); mean tumoral MMP-9 expression was 4.33, 4.11, 4.46, and 3.35, respectively, and stromal expression was 4.29, 4.41, 4.75, and 4.60, respectively (P = NS for all) and, mean tumoral MMP-14 expression was 1.58, 2.41, 0.32, and 0.35, respectively (P < 0.05 AK vs. SCCWD and SCCIS vs. SCCWD/SCCMPD) and stromal expression was 3.04, 3.52, 0.46, and 0.60, respectively (P < 0.05 for AK vs. SCCWD/SCCMPD). Only MMP-14 showed a statistically significant linear trend with decreasing values for tumoral and stromal expression with invasion suggesting that it might be of use as a prognosticator. Enhanced stromal MMP-2 expression in SCCIS and SCCWD relative to AK suggests that it may be of relevance to disease progression.

Enhanced expression of retinoic acid-metabolizing enzyme CYP26A1 in sunlight-damaged human skin.

Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis. CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, has been shown to result in a state of functional VAD of the cell. Recently, we demonstrated that CYP26A1 efficiently promotes cell survival properties and eventually contributes to the carcinogenic process, implying roles as an oncogene. To clarify the possible association between VAD caused by CYP26A1 expression and the development of human epithelial neoplasia, we examined whether enhanced expression of CYP26A1 might be observed in various lesions of human skin. We report here that basal keratinocytes showed only weak positivity of CYP26A1 in sunlight-nonexposed areas, whereas strong positive staining was observed in skin from chronically sunexposed body areas and in epidermis that had the dysplastic changes known as actinic keratosis. However, we found no expression of constitutive CYP26A1 in skin malignancies such as squamous cell carcinomas. Our observation suggests an involvement of enhanced CYP26A1 expression causing a functional VAD state in skin that can potentially lead to neoplastic transformation of keratinocytes in an early phase during skin carcinogenesis.

Human polymerase alpha inhibitors for skin tumors. Part 2. Modeling, synthesis and influence on normal and transformed keratinocytes of new thymidine and purine derivatives.

Recently, the three-dimensional structure of the active site of human DNA polymerase alpha (pol alpha) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol alpha in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

Expression levels of the microRNA processing enzymes Drosha and dicer in epithelial skin cancer.

BACKGROUND: Dysregulation of microRNA (miRNA) metabolism has been observed in a variety of human cancers. In this pilot study, we investigated expression profiles of the two most important enzymes of the miRNA machinery, Drosha and Dicer, in relation to epithelial skin cancer and its premalignant stage. METHODS: Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional) as well as from sites of healthy skin (intraindividual controls). Skin samples (n = 14) were also obtained from healthy subjects for additional controls. Dicer and Drosha mRNA levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: Drosha expression levels were significantly upregulated in both the BCC and SCC groups compared to those in the healthy controls (p < .01), while Dicer expression levels in the BCC group were significantly lower (p < .05). Dicer expression in the SCC group was significantly higher compared to intraindividual controls (p < .05), while Dicer expression levels in both the SCC and AK groups were not significantly different from healthy control samples (p > .05). In the premalignant AK group, we could not observe any significant difference in Drosha or Dicer expression levels compared to either healthy or intraindividual controls (p > .05). CONCLUSIONS: We observed dysregulation of Drosha and Dicer expression in epithelial tumors when compared to healthy control samples. Therefore, we favor the hypothesis that miRNAs are involved in the carcinogenesis of epithelial skin cancer.

Topical treatment of actinic keratoses with piroxicam 1% gel: a preliminary open-label study utilizing a new clinical score.

BACKGROUND: The cyclo-oxygenase enzymes 1 and 2 (COX-1 and COX-2) are both involved in skin tumorigenesis, causing inhibition of apoptosis, angiogenesis, invasiveness, recruitment of growth factors, immunosuppression, and production of carcinogens. Piroxicam is a nonselective nonsteroidal anti-inflammatory drug that blocks the activity of COX-1 and COX-2. OBJECTIVE: To evaluate the efficacy and tolerability of piroxicam 1% gel in the treatment of actinic keratoses. METHODS: Piroxicam 1% gel was applied twice daily for 12 weeks to 31 actinic keratoses. The lesions were evaluated clinically and by means of dermoscopy at an initial baseline visit, at intermediate visits, and after 90 days. Changes were evaluated using a new scoring system (AKESA), based on the clinical presence of erythema, scale, and atrophy on a target lesion. In our experience, the use of piroxicam 1% gel for 90 days induced complete regression in 48% of evaluated actinic keratoses, corresponding to keratotic and verrucous clinical variants. In these lesions, the AKESA score was markedly reduced after treatment. Adverse effects were pruritus, mild erythema, dry skin, and, rarely, rash. Our preliminary trial shows that piroxicam exerts anti-tumorigenic effects and may play a useful role in the chemoprevention of skin cancers.

Field treatment of actinic keratoses - focus on COX-2-inhibitors.

Actinic keratoses (AK) represent the most common carcinoma in situ of the skin and show continuously increasing incidences worldwide. Clinically, AK occur as multiple lesions in sun-exposed areas, which has been referred to as field cancerization. Novel treatment modalities for actinic field cancerization include 3 % diclofenac in 2.5 % hyaluronic acid (HA). Recent investigations have gained insights in the mode of action of diclofenac in HA, showing that the induction of apoptosis is the major mode of action of this treatment. Herein, we give an overview about actinic keratosis focusing on treatment with the COX-2 inhibitor diclofenac 3 % gel and summarize current concepts of its antineoplastic mode of action.