Radiation Therapy Generates Release of Microvesicle Particles in Keratinocytes

Abstract

Ionizing radiation (IR) exerts both tissue and systemic effects. However, the exact mechanism by which radiation therapy to skin results in local and systemic effects is incompletely defined. Previously our group has reported that IR of tumors results in the generation of the lipid mediator Platelet-activating factor (PAF) which resulted in systemic immunosuppressive effects via activation of regulatory T cells. Since PAF acting on the PAF receptor (PAFR) has been demonstrated to generate high levels of subcellular microvesicle particles (100-1000nm; MVP), and MVP are known to be able to signal systemically, the current studies seek to define whether IR of keratinocyte cells generates MVP, and to define the role of the PAFR in this process. Thus, we examine MVP release in the human keratinocyte cell line HaCaT. Moreover, the PAFR-dependency of IR-generated MVP is assessed by use of PAFR-positive KBP and PAFR-negative KBM cells. HaCaT cells (human keratinocytes), KBM cells (PAFR-negative), and KBP cells (PAFR-positive) were grown in 10cm dishes and treated with IR at either no treatment 0 Gy (NT), 4 Gy and 10 Gy. IR was delivered utilizing a technology company's medical linear accelerator radiotherapy system set up with dosimetry verified by nanodot optical stimulated luminescence (Landauer, Glenwood, IL). Some cell lines were treated with PAFR agonist N-methyl carbamoyl PAF (CPAF) and a phorbol ester TPA, known inducers of MVP release. A set of cells were treated with only 90% DMSO/10% ethanol vehicle in HBSS with BSA. After treatments, cells were incubated for 4 hours prior to extraction of MVPs. MVPs were isolated through centrifuging at 2000G for 20, supernatant was collected, transferred into different tubes, and centrifuged at 20,000 G for 70 mins. MVP was detected using a NanoSight NS300 instrument. MVP concentrations were recorded, and the data was statistically analyzed using Student's t-test (JMP, Cary, NC). IR treatment of HaCaT cells at various fluences exhibited statistically significant increases in MVP generation when compared to NT. Of note, 4 Gy resulted in the most fluence for MVP release but was not significant. IR treatment of KB cells resulted in MVP release in both KBM and KBP cells at both 4 and 10Gy. Augmented levels of MVP release were noted in KBP over KBM cells with any IR dose suggesting that the presence of the PAFR is involved in MVP release. Testing of inhibitors of the MVP generating enzyme acid sphingomyelinase (aSMase) also revealed involvement of this lipid metabolizing enzyme. The present studies indicate that RT can generates MVP production in epithelial cells. The mechanism for IR-generated MVP appears to involve aSMase and the PAFR. Target cell MVP release may provide a mechanism for RT effects, including the release of cytokines that influence systemic and local inflammation. Elucidation of this novel pathway may provide insights into IR effects on skin along with new therapeutic strategies.