Ultraviolet A exposure might increase metastasis of mouse

melanoma: a pilot studyPastilla et al.

Photodermatol. Photoimmunol Photomed 2005; 21: 183-190

Introduction

UVA radiation (320-400nm) somes from UVA radiation (320-400nm) somes from excessive sunbathing and tanning in solaria.excessive sunbathing and tanning in solaria.

UVB (280-320) is a causative factor in UVB (280-320) is a causative factor in carcinogenesis (alteration of DNA).carcinogenesis (alteration of DNA).

UVB is absorbed in the SC whereas up to 50% of UVB is absorbed in the SC whereas up to 50% of incident UVA penetrates Caucasian skin deep into incident UVA penetrates Caucasian skin deep into the dermis.the dermis.

Nevertheless, UVB is considered more often as Nevertheless, UVB is considered more often as harmful, whereas UVA is (was) regarded as a low harmful, whereas UVA is (was) regarded as a low health hazard.health hazard.

During tanning, people use sunscreens that absorb the UVB During tanning, people use sunscreens that absorb the UVB portion of the UV spectrum, which also increases the time to portion of the UV spectrum, which also increases the time to erythema (MED), leading to longer tanning periods.erythema (MED), leading to longer tanning periods.

Moreover, UVA radiation during tanning in solaria may emit 5-Moreover, UVA radiation during tanning in solaria may emit 5-10times more UVA as compared with natural solar radiation.10times more UVA as compared with natural solar radiation.

UVA exposure induces characteristic DNA mutations in UVA exposure induces characteristic DNA mutations in epidermal basal cells. Some of the radiation induces activation of epidermal basal cells. Some of the radiation induces activation of protein kinase C, secretion of metalloproteinases, changes in the protein kinase C, secretion of metalloproteinases, changes in the expression of various adhesion molecules; involved in cellular expression of various adhesion molecules; involved in cellular functions directly or indirectly involved in the regulation of functions directly or indirectly involved in the regulation of tumor metastasis.tumor metastasis.

Considering the depth of UVA penetration and the Considering the depth of UVA penetration and the effect of UVA on the expression of adhesion effect of UVA on the expression of adhesion molecules, the authors hypothesized the same could molecules, the authors hypothesized the same could be true for melanoma cells.be true for melanoma cells.

UVA radiation might favour metastasis by weakening UVA radiation might favour metastasis by weakening the homotypic melanoma-melanoma adhesion via the homotypic melanoma-melanoma adhesion via alering E- and N-cadherin expression (E to N) profile alering E- and N-cadherin expression (E to N) profile and by increasing the adhesion between UVA and by increasing the adhesion between UVA exposed melanoma cells and the non irradiated exposed melanoma cells and the non irradiated endothelium(Pastila, 2005).endothelium(Pastila, 2005).

Material and methods

Cells: Cells: C57BL6 mice derived melanoma cell lines C57BL6 mice derived melanoma cell lines B16-F1 (low metastatic potential)and B16-F10 B16-F1 (low metastatic potential)and B16-F10 (high metastatic potential) were cultured in RPMI-(high metastatic potential) were cultured in RPMI-1640, supplemented with FBS, pen-strept, and L-1640, supplemented with FBS, pen-strept, and L-glutamine.glutamine.

UVA radiation source and dosimetryUVA radiation source and dosimetry-Original Philips UVA facial tanner lamp and -Original Philips UVA facial tanner lamp and 5mm thick UVB glass filter.5mm thick UVB glass filter.

-Irradiances were determined by a double -Irradiances were determined by a double holographic grating spectroradiometer. It holographic grating spectroradiometer. It was calibrated against a 1000W halogen was calibrated against a 1000W halogen standard lamp. standard lamp.

-The UVA spectrum that reached the lamp -The UVA spectrum that reached the lamp was 310-400nm; reaching UVA spectrum was 310-400nm; reaching UVA spectrum was 99.9% and UVB was 0.1%.was 99.9% and UVB was 0.1%.

Animal experimentAnimal experiment:-C56BL/6 mice 8-10 weeks of :-C56BL/6 mice 8-10 weeks of age. Groups of 10 mice.age. Groups of 10 mice.

-Hair on the abdomen shaved for UVA -Hair on the abdomen shaved for UVA irradiation.irradiation.

-Suspension of 50000 B16-F1 or B16-F10 -Suspension of 50000 B16-F1 or B16-F10 cells injected in the tail vein.cells injected in the tail vein.

-Immediate 8J/cm2 of UVA. Some animals -Immediate 8J/cm2 of UVA. Some animals were exposed to two more doses (UVA 8J/cm2) on were exposed to two more doses (UVA 8J/cm2) on two consecutive post-injection days.two consecutive post-injection days.

-A control group received the B16-F10 cells -A control group received the B16-F10 cells but not the UVA, a group only received saline.but not the UVA, a group only received saline.

-Mice terminated 14 days post-injection, -Mice terminated 14 days post-injection, lungs extracted and fixed (Bouuin), then dissection lungs extracted and fixed (Bouuin), then dissection with a dissecting microscope.with a dissecting microscope.

Immunohistochemistry:Immunohistochemistry:

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

Results No melanoma metastases were found in mice injected No melanoma metastases were found in mice injected

with saline.with saline. Control mice who received the melanoma cells B16-F10 Control mice who received the melanoma cells B16-F10

put weren’t exposed has a total of 25 lung metastases.put weren’t exposed has a total of 25 lung metastases. Control mice who received B16-F1 (low metastatic Control mice who received B16-F1 (low metastatic

potential) has a total of 2 metastases (12 fold increase)potential) has a total of 2 metastases (12 fold increase) Control mice who received B16-F1 and received UVA: Control mice who received B16-F1 and received UVA:

27 metastases with on dose. Exposing mice to three 27 metastases with on dose. Exposing mice to three doses did not result in an increase in melanoma doses did not result in an increase in melanoma metastases, because an equal number of 27 metastases metastases, because an equal number of 27 metastases was detected in 10 animals.was detected in 10 animals.

Lung metastases were scored according to their Lung metastases were scored according to their size, color, growth pattern and uniformity (next size, color, growth pattern and uniformity (next slide). The scoring method revealed that there was slide). The scoring method revealed that there was only a slight difference in the metastatic index only a slight difference in the metastatic index (score) between the B16-F1 injected, UVA-(score) between the B16-F1 injected, UVA-exposed mice group.exposed mice group.

However (unexpected), the tumor score was lower However (unexpected), the tumor score was lower in the mice receiving three doses than the ones in the mice receiving three doses than the ones receiving only one dose.receiving only one dose.

Histological evaluation of the HE stained tissue Histological evaluation of the HE stained tissue sections and the expression of Melan A confirmed sections and the expression of Melan A confirmed that the metastatic nodules were of melanoma that the metastatic nodules were of melanoma origin.origin.

The expression levels of cadherin E and N showed The expression levels of cadherin E and N showed that lung metastases were strongly N-cadherin that lung metastases were strongly N-cadherin positive, whereas E-cadherin staining was weaker.positive, whereas E-cadherin staining was weaker.

Discussion Using C57BL/6 mice and B16-F1 and B16-F10 melanoma Using C57BL/6 mice and B16-F1 and B16-F10 melanoma

cell lines, demonstration has been made that UVA irradiation cell lines, demonstration has been made that UVA irradiation of animals, which have been IV injected with melanoma cells, of animals, which have been IV injected with melanoma cells, leads to the increased formation of melanoma metastases in leads to the increased formation of melanoma metastases in lungs.lungs.

UVA radiation might alter the adhesive properties of tumor UVA radiation might alter the adhesive properties of tumor cells by altering the expression of cell surface adhesion cells by altering the expression of cell surface adhesion molecules (Leszczynski, 1995 & 1996). The decline in E molecules (Leszczynski, 1995 & 1996). The decline in E cadherin and simultaneous increase in N-cadherin observed in cadherin and simultaneous increase in N-cadherin observed in vitro, are such, that if occurring in vivo, they might help the vitro, are such, that if occurring in vivo, they might help the melanoma cells to extravasate into the internal target organs.melanoma cells to extravasate into the internal target organs.

Low metastatic (lungs)(B16-F1) potential cell lines were Low metastatic (lungs)(B16-F1) potential cell lines were compared with high metastatic potential cell lines (B16-compared with high metastatic potential cell lines (B16-F10). Moreover the control mice were injected with the F10). Moreover the control mice were injected with the high metastatic potential cell lines.high metastatic potential cell lines.

Therefore low metastatic potential melanoma cell lines Therefore low metastatic potential melanoma cell lines exposed to UVA are compared with high metastatic exposed to UVA are compared with high metastatic potential melanoma cells control (non exposed).potential melanoma cells control (non exposed).

The animal study has demonstrated that a single , The animal study has demonstrated that a single , moderately high UVA exposure of animals with IV moderately high UVA exposure of animals with IV injected melanoma cells causes a dramatic, over 12 fold, injected melanoma cells causes a dramatic, over 12 fold, increase in lung metastasis formation B16-F1 melanoma increase in lung metastasis formation B16-F1 melanoma cells.cells.

The metastatic index (scorring) declined significantly The metastatic index (scorring) declined significantly with the subsequent exposures. This suggests the with the subsequent exposures. This suggests the possibility that the consecutive exposures might cause possibility that the consecutive exposures might cause some kind of suppressive effect (?) on the some kind of suppressive effect (?) on the development of the tumor, but not on the frequency of development of the tumor, but not on the frequency of metastases.metastases.

The physiological mechanism that regulates UVA The physiological mechanism that regulates UVA induced increase of metastasis is still unknown, induced increase of metastasis is still unknown, -indirect mechanism (UVA induced -indirect mechanism (UVA induced

immunosuppression, release of cytokines, release of immunosuppression, release of cytokines, release of eicosanoids).eicosanoids).

direct mechanisms (UVA induced change in direct mechanisms (UVA induced change in adhesive properties of melanoma cells=E- to N-adhesive properties of melanoma cells=E- to N-cadherin).cadherin).

Conclusion UVA irradiation of mice that have melanoma cells UVA irradiation of mice that have melanoma cells

present in the blood circulation dramatically increases present in the blood circulation dramatically increases the formation of melanoma metastases to the lungs. The the formation of melanoma metastases to the lungs. The effect might be direct, indirect or a combination of both.effect might be direct, indirect or a combination of both.

Whether similar UVA-induced pro-metastatic effects Whether similar UVA-induced pro-metastatic effects are occurring in people sunbathing or using solaria are occurring in people sunbathing or using solaria remains to be determined.remains to be determined.

Considering the recent findings of UVA-induced DNA Considering the recent findings of UVA-induced DNA mutations (Agar, 2004), this study further supplements mutations (Agar, 2004), this study further supplements and supports the notion that UVA radiation exposures and supports the notion that UVA radiation exposures might be more harmful than previously suggested.might be more harmful than previously suggested.