Introduction

1. Ultraviolet A exposure might increase metastasis of mouse melanoma: a pilot studyPastilla et al.Photodermatol. Photoimmunol Photomed 2005; 21: 183-190

2. Introduction • UVA radiation (320-400nm) somes from excessive sunbathing and tanning in solaria. • UVB (280-320) is a causative factor in carcinogenesis (alteration of DNA). • UVB is absorbed in the SC whereas up to 50% of incident UVA penetrates Caucasian skin deep into the dermis. • Nevertheless, UVB is considered more often as harmful, whereas UVA is (was) regarded as a low health hazard.

3. During tanning, people use sunscreens that absorb the UVB portion of the UV spectrum, which also increases the time to erythema (MED), leading to longer tanning periods. • Moreover, UVA radiation during tanning in solaria may emit 5-10times more UVA as compared with natural solar radiation. • UVA exposure induces characteristic DNA mutations in epidermal basal cells. Some of the radiation induces activation of protein kinase C, secretion of metalloproteinases, changes in the expression of various adhesion molecules; involved in cellular functions directly or indirectly involved in the regulation of tumor metastasis.

4. Considering the depth of UVA penetration and the effect of UVA on the expression of adhesion molecules, the authors hypothesized the same could be true for melanoma cells. • UVA radiation might favour metastasis by weakening the homotypic melanoma-melanoma adhesion via alering E- and N-cadherin expression (E to N) profile and by increasing the adhesion between UVA exposed melanoma cells and the non irradiated endothelium(Pastila, 2005).

5. Material and methods • Cells: C57BL6 mice derived melanoma cell lines B16-F1 (low metastatic potential)and B16-F10 (high metastatic potential) were cultured in RPMI-1640, supplemented with FBS, pen-strept, and L-glutamine. • UVA radiation source and dosimetry -Original Philips UVA facial tanner lamp and 5mm thick UVB glass filter.

6. -Irradiances were determined by a double holographic grating spectroradiometer. It was calibrated against a 1000W halogen standard lamp. -The UVA spectrum that reached the lamp was 310-400nm; reaching UVA spectrum was 99.9% and UVB was 0.1%.

7. Animal experiment:-C56BL/6 mice 8-10 weeks of age. Groups of 10 mice. -Hair on the abdomen shaved for UVA irradiation. -Suspension of 50000 B16-F1 or B16-F10 cells injected in the tail vein. -Immediate 8J/cm2 of UVA. Some animals were exposed to two more doses (UVA 8J/cm2) on two consecutive post-injection days. -A control group received the B16-F10 cells but not the UVA, a group only received saline. -Mice terminated 14 days post-injection, lungs extracted and fixed (Bouuin), then dissection with a dissecting microscope.

9. Immunohistochemistry: -After qualitative evaluation, paraffin, sections in 5microns, hematoxyline-eosin, Melan A, N-cadherin, E-cadherin.

10. Results • No melanoma metastases were found in mice injected with saline. • Control mice who received the melanoma cells B16-F10 put weren’t exposed has a total of 25 lung metastases. • Control mice who received B16-F1 (low metastatic potential) has a total of 2 metastases (12 fold increase) • Control mice who received B16-F1 and received UVA: 27 metastases with on dose. Exposing mice to three doses did not result in an increase in melanoma metastases, because an equal number of 27 metastases was detected in 10 animals.

12. Lung metastases were scored according to their size, color, growth pattern and uniformity (next slide). The scoring method revealed that there was only a slight difference in the metastatic index (score) between the B16-F1 injected, UVA-exposed mice group. • However (unexpected), the tumor score was lower in the mice receiving three doses than the ones receiving only one dose.

14. Histological evaluation of the HE stained tissue sections and the expression of Melan A confirmed that the metastatic nodules were of melanoma origin. • The expression levels of cadherin E and N showed that lung metastases were strongly N-cadherin positive, whereas E-cadherin staining was weaker.

15. Discussion • Using C57BL/6 mice and B16-F1 and B16-F10 melanoma cell lines, demonstration has been made that UVA irradiation of animals, which have been IV injected with melanoma cells, leads to the increased formation of melanoma metastases in lungs. • UVA radiation might alter the adhesive properties of tumor cells by altering the expression of cell surface adhesion molecules (Leszczynski, 1995 & 1996). The decline in E cadherin and simultaneous increase in N-cadherin observed in vitro, are such, that if occurring in vivo, they might help the melanoma cells to extravasate into the internal target organs.

16. Low metastatic (lungs)(B16-F1) potential cell lines were compared with high metastatic potential cell lines (B16-F10). Moreover the control mice were injected with the high metastatic potential cell lines. • Therefore low metastatic potential melanoma cell lines exposed to UVA are compared with high metastatic potential melanoma cells control (non exposed). • The animal study has demonstrated that a single , moderately high UVA exposure of animals with IV injected melanoma cells causes a dramatic, over 12 fold, increase in lung metastasis formation B16-F1 melanoma cells.

17. The metastatic index (scorring) declined significantly with the subsequent exposures. This suggests the possibility that the consecutive exposures might cause some kind of suppressive effect (?) on the development of the tumor, but not on the frequency of metastases. • The physiological mechanism that regulates UVA induced increase of metastasis is still unknown, • -indirect mechanism (UVA induced immunosuppression, release of cytokines, release of eicosanoids). • direct mechanisms (UVA induced change in adhesive properties of melanoma cells=E- to N-cadherin).

18. Conclusion • UVA irradiation of mice that have melanoma cells present in the blood circulation dramatically increases the formation of melanoma metastases to the lungs. The effect might be direct, indirect or a combination of both. • Whether similar UVA-induced pro-metastatic effects are occurring in people sunbathing or using solaria remains to be determined. • Considering the recent findings of UVA-induced DNA mutations (Agar, 2004), this study further supplements and supports the notion that UVA radiation exposures might be more harmful than previously suggested.