Abstract
BackgroundGastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. Here, we determined whether GKN1 is a potential theragnostic marker for gastric cancer.MethodsWe identified GKN1 binding proteins using the protein microarray assay and investigated whether GKN1 is one of the exosomal cargo proteins by western blot, immunoprecipitation, and immunofluorescent assays. Cell proliferation and apoptosis were analyzed by MTT, BrdU incorporation, flow cytometry, and western blot assays. We further validated the functional relevance of exosomal GKN1 in MKN1-injected xenograft mice. The possibility of serum GKN1 as a diagnostic marker for gastric cancer was determined by ELISA assay.ResultsIn protein microarray assay, GKN1 binding to 27 exosomal proteins was clearly observed. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Exosomes carrying GKN1 inhibited cell proliferation and induced apoptosis in both AGS and MKN1 cells, and exosomes carrying GKN1-treated nude mice-bearing MKN1 xenograft tumors exhibited significantly reduced tumor volume and tumor weight. Silencing of clathrin markedly down-regulated the internalization of exosomal GKN1. Interestingly, serum GKN1 concentrations in patients with gastric cancer were significantly lower than those in healthy individuals and patients with colorectal and hepatocellular carcinomas.ConclusionsThe GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment.
Highlights
Human gastrokine 1 (GKN1), made of 185 amino acid residues, is a stomach-specific protein which is produced by gastric mucus-secreting cells, stored in cytoplasmic granules, and secreted with mucus onto the apical cell surfaceGastrokine 1 protein is a potential theragnostic target for gastric cancer[1, 2]
Immunoprecipitated proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and processed for immunoblotting with the indicated antibodies. c transmission electron microscopy (TEM) image of exosomes isolated from supernatants of HFE-145 cells. d Representative western blots of GKN1, TSG-101, and CD81 in exosomes isolated from the HFE-145-immortalized gastric epithelial cells, AGS and MKN1 gastric cancer cells
We found no GKN1 expression in exosomes derived from AGS and MKN1 cells, but we detected GKN1 expression in exosomes derived from HFE-145 cells as well as AGS and MKN1 cells treated with recombinant GKN1 protein (rGKN1)
Summary
Human gastrokine 1 (GKN1), made of 185 amino acid residues, is a stomach-specific protein which is produced by gastric mucus-secreting cells, stored in cytoplasmic granules, and secreted with mucus onto the apical cell surfaceGastrokine 1 protein is a potential theragnostic target for gastric cancer[1, 2]. GKN1 plays important roles in maintaining mucosal integrity and homeostasis, and in regulating cell proliferation and differentiation [6,7,8], no study has yet investigated how GKN1 secretion and internalization take place in gastric mucosal epithelium. Gastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Conclusions The GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment
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