Abstract
Nearly 85% of radiotherapy patients develop acute radiation dermatitis, which is an inflammatory reaction of the skin at the treatment field and in the surrounding area. The aims of this study were to unravel the mechanisms of radiation-induced inflammatory responses after localized irradiation in a human 3D organotypic skin culture model. This could provide possible inflammatory targets for reduction of skin side effects. 3D organotypic skin cultures were set up and locally irradiated with 225 kVp X-rays, using a combination of full exposure and partial shielding (50%) of the cultures. The secretion of pro-inflammatory cytokines, the phenotype, and the differentiation markers expression of the cultures were assessed up to 10 days postirradiation. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX-2) pathways have been studied. The results showed fast activation of NF-κB, most likely triggered by DNA damage in the irradiated cells, followed by upregulation of p38 MAPK and COX-2 in the irradiated and surrounding, non-irradiated, areas of the 3D cultures. The application of the COX-2 inhibitor sc-236 was effective at reducing the COX-2 mRNA levels 4 h postirradiation. The same inhibitor also suppressed the PGE2 secretion significantly 72 h after the treatment. The expression of a pro-inflammatory phenotype and abnormal differentiation markers of the cultures were also reduced. However, the use of an NF-κB inhibitor (Bay 11-7085) did not have the predicted positive effect on the cultures phenotype postirradiation. Radiation-induced pro-inflammatory responses have been observed in the 3D skin model. The activated signaling pathways involved NF-κB transcription factor and its downstream target COX-2. Further experiments aiming to suppress the inflammatory response via specific inhibitors showed that COX-2 is a suitable target for reduction of the normal skin inflammatory responses at radiotherapy, while NF-κB inhibition had detrimental effects on the 3D skin model development.
Highlights
The skin is one of the most important and dose-limiting organs that is inevitably included in the exposed field during conventional radiotherapy
We focused on two main inflammation controlling molecules NF-κB and COX-2 and the cytokines involved in the signaling under their control
In addition to the morphological analysis, we investigated the effects on stratification of the 3D model after the COX-2 inhibitor treatment through following differentiation in the 3D cultures and immunofluorescence of paraffin-embedded sections for expression of the early differentiation marker K1 (Figures 3B,C)
Summary
The skin is one of the most important and dose-limiting organs that is inevitably included in the exposed field during conventional radiotherapy. Spreading out the dose over 6–8 weeks enables the skin to tolerate doses up to 60 Gy through stem cell repopulation [3, 4] After this period, the epidermis either heals or the changes progress to chronic wounds that might lead to necrosis [5]. The epidermis either heals or the changes progress to chronic wounds that might lead to necrosis [5] These effects develop at different levels during conventional radiotherapy. The late chronic reactions are reported to be permanent and progressive without complete treatment [6] This widely affects the quality of life of breast cancer patients [7, 8]. One of the main reasons is that mechanistic studies of skin reactions that involve all the stages at cellular, functional, and systemic level have been limited
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