Jung Hwan Yoon1,2, In-Hye Ham3,4, Olga Kim1, Hassan Ashktorab5, Duane T Smoot6, Suk Woo Nam1,2, Jung Young Lee1,2, Hoon Hur7,8, Won Sang Park9,10. 1. Department of Pathology, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea. 2. Functional RNomics Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea. 3. Department of Surgery, Ajou University School of Medicine, Suwon, 16499, South Korea. 4. Brain Korea 21 Plus Research Center for Biomedical Science, Ajou University, Suwon, 16499, South Korea. 5. Department of Medicine, Howard University, Washington, DC, 20060, USA. 6. Department of Medicine, Meharry Medical Center, Nashville, TN, 37208, USA. 7. Department of Surgery, Ajou University School of Medicine, Suwon, 16499, South Korea. hhcmc75@ajou.ac.kr. 8. Brain Korea 21 Plus Research Center for Biomedical Science, Ajou University, Suwon, 16499, South Korea. hhcmc75@ajou.ac.kr. 9. Department of Pathology, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea. wonsang@catholic.ac.kr. 10. Functional RNomics Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea. wonsang@catholic.ac.kr.
Abstract
BACKGROUND: Gastrokine 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. METHODS: We 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. RESULTS: In 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. 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.
BACKGROUND:Gastrokine 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. METHODS: We 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. RESULTS: In 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. 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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