Abstract
Background
Development of ulcerative colitis is associated with epithelial apoptosis mediated by p53-apoptotic pathway through the activation B-cell lymphoma 2 (Bcl-2), Bcl-2 associated-X protein (BAX) and Bcl-2 antagonist/killer-1 (BAK1) proteins. Chromofungin (CHR), a chromogranin-A derived peptide expressing a cell penetrating peptide motif, decreased the severity of colitis via the suppression of mucosal and pro-inflammatory macrophages-related p53-dependent apoptosis.
Aims
We aimed to investigate a) whether the gene profile expression of apoptosis could be extended to other p53-associated genes; b) whether the gene expression of some of the p53-apoptosis marker could be confirmed by protein analysis; and c) whether due to the cell penetrating peptide motif, CHR could enter into peritoneal macrophages.
Methods
UC-related colitis was induced in C57BL/6 mice (7 weeks) by administering dextran sulfate sodium (DSS) (5%, 5 days). Preventive CHR (2.5 mg/kg/day) or vehicle treatment started 1-day before colitis induction and lasted for 5-days. Profiler™ PCR Array was performed to screen a panel of 84 genes representative of the p53 signal pathway in colitic whole mucosa distal colonic samples treated or not with CHR. Western blot analysis was performed to confirm individual protein changes. Naïve macrophages were plated overnight and nonadherent cells were removed the next day. Cells were incubated with rhodamined CHR (4 ul) for 5, 10, 20, 30 min before washing and fixing them, detection was made via confocal microscopy.
Results
In colitic conditions, an up regulation of 26 genes associated to the p53-dependent apoptosis pathway were detected including Apaf1, Bax, Bbc3, Bcl2, Cradd, Fadd, Cul9, Pmaip1, Tnfrsf10b. In vivo CHR treatment decreased significantly the colitis and was associated with a significant downregulation of 19 genes including the 9 aforementioned when compared with biopsies from colitic groups. Compared to untreated groups, colitic mice treated with CHR demonstrated a significant decrease of BAX and BAK protein and the apoptotic ER stress inducer marker, X-Binding Protein 1. A large number of peritoneal macrophages displayed rhodamine within the all intracellular compartment. The presence of the peptide inside the cell can be visible as early as 5 min and the signal gradually increases.
Conclusions
CHR decreases the inflammatory process via the suppression of a large number of p53-related apoptotic proteins. CHR quickly enters the macrophage but the exact mechanism of entrance needs to be further defined. Targeting functional analysis of CHR may lead in the future to novel therapeutics for UC.
Funding Agencies
CCCNSERC
Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide
