Abstract
Ceramide-induced endothelial cell apoptosis boosts intestinal stem cell radiosensitivity. However, the molecular connection between these two cellular compartments has not been clearly elucidated. Here we report that ceramide and its related enzyme acid sphingomyelinase (ASM) are secreted by irradiated endothelial cells and act as bystander factors to enhance the radiotoxicity of intestinal epithelium. Ceramide and the two isoforms of ASM were acutely secreted in the blood serum of wild-type mice after 15 Gy radiation dose, inducing a gastrointestinal syndrome. Interestingly, serum ceramide was not enhanced in irradiated ASMKO mice, which are unable to develop intestinal failure injury. Because ASM/ceramide were secreted by primary endothelial cells, their contribution was studied in intestinal epithelium dysfunction using coculture of primary endothelial cells and intestinal T84 cells. Adding exogenous ASM or ceramide enhanced epithelial cell growth arrest and death. Conversely, blocking their secretion by endothelial cells using genetic, pharmacologic, or immunologic approaches abolished intestinal T84 cell radiosensitivity. Use of enteroid models revealed ASM and ceramide-mediated deleterious mode-of-action: when ceramide reduced the number of intestinal crypt-forming enteroids without affecting their structure, ASM induced a significant decrease of enteroid growth without affecting their number. Identification of specific and different roles for ceramide and ASM secreted by irradiated endothelial cells opens new perspectives in the understanding of intestinal epithelial dysfunction after radiation and defines a new class of potential therapeutic radiomitigators. SIGNIFICANCE: This study identifies secreted ASM and ceramide as paracrine factors enhancing intestinal epithelial dysfunction, revealing a previously unknown class of mediators of radiosensitivity.
Highlights
Despite advances in treatment delivery techniques, normal tissue toxicity remains a major side-effect of radiotherapy [1]
Enhancement of acid sphingomyelinase (ASM) activities was concomitant with a sustained enrichment of ceramide concentration in the serum of irradiated WT mice starting as early as 30 minutes and remaining elevated for at least 96 hours (1.6-fold 96 hours after 15 Gy vs. 0 Gy; Fig. 1E)
Activities of S- and lysosomal acid sphingomyelinase (L-ASM) from intestinal mucosa of WT mice were only transiently enhanced 24 hours after irradiation and did not correlate with the time-dependent enrichment of intestinal ceramide observed after irradiation in both WT and ASMKO mice (Supplementary Fig. S2C–S2E)
Summary
Despite advances in treatment delivery techniques, normal tissue toxicity remains a major side-effect of radiotherapy [1]. Patients with abdominal or pelvic malignancies treated by high doses of ionizing radiation (IR) can develop radiation gastrointestinal (GI) syndrome, an acute deleterious radiotoxicity causing lethal damage to the GI tract. This syndrome has been considered to be strictly dependent on dysfunction of intestinal stem cells or clonogens [2]. When the death of those clonogens induces the collapse of crypt/villus unit, the persistence of some clonogens is sufficient to repair damaged gut epithelium. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Estephan contributed as co-first authors of this article
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