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Site Specific Targeting of PUMA Induced ROS Prevents Radiation Injury via a Smad3 Independent Mechanism
Karan Mehta, BS, Philip Lotfi, BS, Marc Soares, MD, Robert Dolitsky, BS, Piul Rabbani, PhD, April Ducksworth, MD, Nakul Rao, MD, Jessica Chang, BS, Amanda Hua, BS, Camilo Doig, BS, Camille Kim, BS, Pierre Saadeh, MD, Daniel Ceradini, MD.
NYU Department of Plastic Surgery, New York, NY, USA.
Purpose: Although radiation therapy is an instrumental tool in the treatment of numerous cancers, it is hampered by its detrimental effects on the skin. Namely, impaired wound healing, fibrosis and scarring often requiring surgical intervention. Previously, we have demonstrated that radiation induced PUMA expression and subsequent ROS overproduction is a critical factor in the development of cutaneous fibrosis. However, it is unclear whether this effect is dependent on SMAD3 expression, a central fibrosis pathway. Here, we investigate the downstream molecular mechanism for radiation protection with PUMA knock down in cutaneous radiation injury.
Methods: Fibroblasts treated with a single dose of radiation (15 Gy) following transfection with PUMA or nonsense siRNA were analyzed by rtPCR and Western blot for gene expression. PUMA or nonsense siRNA complexed with cationic lipid nanoparticles was applied to the skin of C57BL6 mice followed by cutaneous radiation injury. Clinical, histologic, and gene expression analysis for cutaneous fibrosis was performed. A SCAR index was calculated by staining with Sirius red and comparing the degree of pathologic dense red birefringence of collagen bundles to the yellow-green birefringence seen in uninjured tissue. Tensiometry was performed to assess the extent of fibrosis.
Results: Topical silencing of PUMA using a cationic lipid nanoparticle delivery system resulted in a significant reduction in radiation induced injury in mice at 1 month. Clinically, PUMA treated mice showed markedly reduced ulceration, thickening and scarring at the site of injury as compared to nonsense siRNA treated animals. Appreciable re-growth of hair was also noted at irradiated sites in PUMA treated mice. These gross findings were supported by histology. Dermal thickness was significantly decreased in irradiated PUMA treated skin vs. nonsense treated skin (440um PUMA treated vs. 1,004um nonsense treated, p<.001). Vessel wall thickness was significantly improved as well (18.9um nonsense treated vs. 9.3um PUMA treated, p<.001). Less subcutaneous fat was lost in irradiated PUMA treated skin vs. nonsense treated skin (1.25mm2 PUMA treated vs. .63mm2 nonsense treated, p<.005). An 8.5 fold decrease in SCAR index was demonstrated in PUMA treated mice as compared to nonsense treated mice indicating a drastic reduction in fibrosis. Reduced fibrosis was supported by tensiometric measurements which indicated a 58% reduction in stiffness in PUMA silenced skin compared to irradiated nonsense control mice (0.25 N/mm nonsense control vs. 0.10 N/mm PUMA treated p<.001). Interestingly, PUMA silencing resulted in no change in SMAD3 expression suggesting the potential for an independent fibrotic pathway.
Conclusion: Through the use of PUMA silencing, we have demonstrated a powerful and effective method of limiting the destructive fibrotic and ulcerative sequelae of radiation therapy. Mechanistically, our results indicate that a pathway independent of the well established TGF-beta/SMAD3 fibrotic cascade may be at play. The use of topical inhibition of PUMA presents a clinically relevant and readily applicable approach to limiting the harmful skin effects of radiation treatment.
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