Kathleen L. Tober

Gender Differences in UVB-Induced Skin Carcinogenesis, Inflammation, and DNA Damage

The American Cancer Society reports the incidence of squamous cell carcinoma in males to be thrice the incidence in females. This increased squamous cell carcinoma incidence has been attributed to men accumulating more sun exposure and using less sun protection than women. To date, there have been no controlled studies examining the effect of gender on skin tumor development following equal doses of UVB. Gender differences in UVB-induced skin carcinogenesis were examined using the Skh-1 mouse model. After chronic exposure to equal doses of UVB, male mice developed tumors earlier and had more tumors than female mice; tumors in male mice tended to be larger, and the total tumor burden was greater than in females. In addition, tumors in males were of more advanced histologic grade compared with those of female mice. To evaluate the contribution of differences in inflammation and DNA damage to differences in skin carcinogenesis, male and female Skh-1 mice were exposed once to 2,240 J/m(2) UVB and examined 48 h after exposure. Surprisingly, male mice developed less of an inflammatory response, as determined by skin fold thickness and myeloperoxidase activity, compared with females. Interestingly, male mice showed more cutaneous oxidative DNA damage than the females and lower antioxidant levels. These results show a gender bias in skin carcinogenesis and suggest that the gender difference in tumor development is more influenced by the extent of oxidative DNA damage and antioxidant capacities than by inflammatory response.

The impact of cyclooxygenase-2 mediated inflammation on scarless fetal wound healing

Cyclooxygenase-2 (COX-2) and the prostaglandin products generated as a result of COX-2 activity mediate a variety of biological and pathological processes. Scarless healing occurs in fetal skin in the first and second trimesters of development. This scarless healing process is known to proceed without a significant inflammatory response, which appears to be important for the lack of scarring. Because the COX-2 pathway is an integral component of inflammation, we investigated its role in the fetal repair process using a mouse model of scarless fetal wound healing. COX-2 expression in scarless and fibrotic fetal wounds was examined. In addition, the ability of exogenous prostaglandin E(2) to alter scarless fetal healing was evaluated. The results suggest that the COX-2 pathway is involved in scar production in fetal skin and that targeting COX-2 may be useful for limiting scar formation in adult skin.

Clinically relevant immunosuppressants influence UVB-induced tumor size through effects on inflammation and angiogenesis.

Immunosuppressive therapies allow long‐term patient and transplant survival, but are associated with increased development of UV‐induced skin cancers, particularly squamous cell carcinomas. The mechanisms by which CsA, MMF, tacrolimus (TAC) or sirolimus (SRL), alone or in dual combinations, influence tumor development and progression are not completely understood. In the current study, chronically UV‐exposed mice treated with SRL alone or in combination with CsA or TAC developed more tumors than mice treated with vehicle or other immunosuppressants, but the tumors were significantly smaller and less advanced. Mice treated with CsA or TAC developed significantly larger tumors than vehicle‐treated mice, and a larger percentage in the CsA group were malignant. The addition of MMF to CsA, but not to TAC, significantly reduced tumor size. Immunosuppressant effects on UVB‐induced inflammation and tumor angiogenesis may explain these findings. CsA enhanced both UVB‐induced inflammation and tumor blood vessel density, while MMF reduced inflammation. Addition of MMF to CsA reduced tumor size and vascularity. SRL did not affect inflammation, but significantly reduced tumor vascularity. Thus the choice of immunosuppressants has important implications for tumor number, size and progression, likely due to the influence of immunosuppressants on UVB‐induced inflammation and angiogenesis.

Inhibition of ultraviolet light B-induced cutaneous inflammation by a specific cyclooxygenase-2 inhibitor.

Ultraviolet B (UVB) radiation is responsible for the majority of cutaneous damage following both acute and long-term exposure, and is believed to be the most important etiologic agent in human skin cancer. UVB carcinogenesis initially induces an inflammatory response characterized by edema, dermal infiltration of leukocytes, as well as the production and release of prostaglandins, which may be critical to the observed damaging effects of UVB light on skin. Recently, a specific cyclooxygenase-2 (COX-2) inhibitor, Celecoxib, was developed, which inhibits COX-2-induced inflammation without inhibiting the cytoprotective function of cyclooxygenase-1 (COX-1). Studies have demonstrated that oral administration of Celecoxib decreased the incidence of skin and colon tumors. Recently, the process of inflammation has been linked to tumor formation. The present study examined the effects of a topical application of Celecoxib on the acute UVB-induced cutaneous inflammatory response. We show that topical Celecoxib treatment effectively reduced many parameters of UVB-mediated inflammation, including edema, dermal myeloperoxidase activity, neutrophil infiltration,and prostaglandin E2 (PGE2) levels. By inhibiting this inflammatory response, topical Celecoxib treatment could ultimately be effective in preventing tumor development and progression in the skin, which is known to result from long-term UV exposure.

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.

Isothiocyanate metabolism, distribution, and interconversion in mice following consumption of thermally processed broccoli sprouts or purified sulforaphane

SCOPE Broccoli sprouts are a rich source of glucosinolates, a group of phytochemicals that when hydrolyzed, are associated with cancer prevention. Our objectives were to investigate the metabolism, distribution, and interconversion of isothiocyanates (ITCs) in mice fed thermally processed broccoli sprout powders (BSPs) or the purified ITC sulforaphane. METHODS AND RESULTS For 1 wk, mice were fed a control diet (n = 20) or one of four treatment diets (n = 10 each) containing nonheated BSP, 60°C mildly heated BSP, 5-min steamed BSP, or 3 mmol purified sulforaphane. Sulforaphane and erucin metabolite concentrations in skin, liver, kidney, bladder, lung, and plasma were quantified using HPLC-MS/MS. Thermal intensity of BSP processing had disparate effects on ITC metabolite concentrations upon consumption. Mild heating generally resulted in the greatest ITC metabolite concentrations in vivo, followed by the nonheated and steamed BSP diets. We observed interconversion between sulforaphane and erucin species or metabolites, and report that erucin is the favored form in liver, kidney, and bladder, even when only sulforaphane is consumed. CONCLUSION ITC metabolites were distributed to all tissues analyzed, suggesting the potential for systemic benefits. We report for the first time tissue-dependent ratio of sulforaphane and erucin, though further investigation is warranted to assess biological activity of individual forms.

Preventative topical diclofenac treatment differentially decreases tumor burden in male and female Skh-1 mice in a model of UVB-induced cutaneous squamous cell carcinoma

Ultraviolet B (UVB) light is the major environmental carcinogen contributing to non-melanoma skin cancer (NMSC) development. There are over 3.5 million NMSC diagnoses in two million patients annually, with men having a 3-fold greater incidence of squamous cell carcinoma (SCC) compared with women. Chronic inflammation has been linked to tumorigenesis, with a key role for the cyclooxygenase-2 (COX-2) enzyme. Diclofenac, a COX-2 inhibitor and non-steroidal anti-inflammatory drug, currently is prescribed to patients as a short-term therapeutic agent to induce SCC precursor lesion regression. However, its efficacy as a preventative agent in patients without evidence of precursor lesions but with significant UVB-induced cutaneous damage has not been explored. We previously demonstrated in a murine model of UVB-induced skin carcinogenesis that when exposed to equivalent UVB doses, male mice had lower levels of inflammation but developed increased tumor multiplicity, burden and grade compared with female mice. Because of the discrepancy in the degree of inflammation between male and female skin, we sought to determine if topical treatment of previously damaged skin with an anti-inflammatory COX-2 inhibitor would decrease tumor burden and if it would be equally effective in the sexes. Our results demonstrated that despite observed sex differences in the inflammatory response, prolonged topical diclofenac treatment of chronically UVB-damaged skin effectively reduced tumor multiplicity in both sexes. Unexpectedly, tumor burden was significantly decreased only in male mice. Our data suggest a new therapeutic use for currently available topical diclofenac as a preventative intervention for patients predisposed to cutaneous SCC development before lesions appear.

Inhibitory effects of pentoxifylline on ultraviolet B light–induced cutaneous inflammation

It is now recognized that ultraviolet (UV) radiation is a potent environmental insult capable of interfering with immunity to skin cancers and modifying certain immunologic reactions within both locally irradiated skin and distant, unexposed sites. Exposure to UVB light (290–320 nm) induces a potent cutaneous inflammatory response that involves the infiltration of leukocytes into the dermis as well as the production of proinflammatory cytokines by both resident epidermal keratinocytes and dermal cells. Tumor necrosis factor‐α (TNF‐α) is a proinflammatory cytokine that has been shown to be a major mediator of UVB light effects on cutaneous immunity. Recent studies have demonstrated that pentoxifylline (PTX), a xanthine‐ derived phosphodiesterase inhibitor, has the ability to inhibit synthesis of TNF‐α. To examine the effects of PTX on UVB‐mediated cutaneous inflammation, Skh/hr hairless mice were injected intraperitoneally with either phosphate‐buffered saline or 50 μg/g PTX 1 h before exposure to 2240 J/m2 UVB. Reverse transcription–polymerase chain reaction and immunohistochemical techniques were used to demonstrate that 24 h to 1 wk after UVB‐light irradiation, PTX inhibited UVB‐induced TNF‐α gene expression, inhibited the increase in epidermal TNF‐α protein synthesis, blocked the increase in epidermal proliferation observed after exposure to UVB light, and decreased production of myeloperoxidase by neutrophils infiltrating into the dermis. These studies demonstrated that PTX modifies epidermal responses after acute UVB light exposure and suggest that PTX treatment may be used clinically to modulate the deleterious effects of long‐term UVB‐light irradiation. Mol. Carcinog. 22:16–25, 1998.

Differential Effects of Topical Vitamin E and C E Ferulic® Treatments on Ultraviolet Light B-Induced Cutaneous Tumor Development in Skh-1 Mice

Because of the ever-increasing incidence of ultraviolet light B (UVB)-induced skin cancer, considerable attention is being paid to prevention through the use of both sunscreens and after sun treatments, many of which contain antioxidants. Vitamin E is included as an antioxidant in many sunscreens and lotions currently on the market. Studies examining the efficacy of vitamin E as a topical preventative agent for UVB-induced skin cancer have yielded conflicting results. A likely contributor to differences in study outcome is the stability of vitamin E in the particular formulation being tested. In the current study we examined the effects of topical vitamin E alone as well as vitamin E combined with vitamin C and ferulic acid in a more stable topical formula (C E Ferulic®). Mice were exposed to UVB for 10 weeks in order to induce skin damage. Then, before the appearance of any cutaneous lesions, mice were treated for 15 weeks with a topical antioxidant, without any further UVB exposure. We found that topical C E Ferulic decreased tumor number and tumor burden and prevented the development of malignant skin tumors in female mice with chronically UVB-damaged skin. In contrast, female mice chronically exposed to UVB and treated topically with vitamin E alone showed a trend towards increased tumor growth rate and exhibited increased levels of overall DNA damage, cutaneous proliferation, and angiogenesis compared to vehicle-treated mice. Thus, we have demonstrated that topical 5% alpha tocopherol may actually promote carcinogenesis when applied on chronically UVB-damaged skin while treating with a more stable antioxidant compound may offer therapeutic benefits.

Possible cross-regulation of the E prostanoid receptors.

Exposure to UVB induces an inflammatory response in the skin that results in high levels of cyclooxygenase‐2 (COX‐2) and its enzymatic product, prostaglandin E2 (PGE2). PGE2 signals via one of four E prostanoid (EP) receptors, EP1–4, but the roles of each of these receptors in UVB‐mediated inflammation and skin carcinogenesis have not been fully defined. Topical application of ONO‐8713, an EP1 antagonist, reduced the acute inflammatory effects of UVB irradiation. This compound also reduced UVB‐induced tumor formation by approximately 50%, suggesting that signaling of PGE2 via the EP1 receptor may play a role in UVB‐mediated inflammation and carcinogenesis. Our laboratory has demonstrated that the EP1 receptor localized to the suprabasal layers of the epidermis and the EP3 receptor was found in the basal keratinocytes of unirradiated murine skin. While UVB exposure induced no change in the localization of the EP1 receptor, the EP3 receptor was detected in all layers of the epidermis in response to UVB. In mice that were topically treated with ONO‐8713, UVB‐induced changes in EP3 localization were prevented. This alteration in EP3 receptor localization was not seen following topical application of the anti‐inflammatory drug celecoxib, indicating that the effects of ONO‐8713 were not because of its anti‐inflammatory properties. These results suggest a previously undescribed interaction between the EP1 and EP3 receptors in the epidermis.