2010;116:4262C4273. of type 2 diabetes with glycemia and hyperglucagonemia. The present observations provide experimental evidence that hyperglucagonemia Becampanel in type 2 diabetes promotes colon cancer progression via GCGR-mediated regulation of AMPK and MAPK pathways. . However, the mechanism of how glucagon promotes colon cancer cell proliferation has not yet been decided. Taken together with this background on glucagon, we hypothesized that hyperglucagonemia is usually one factor that increases the risk of colorectal cancer in diabetes patients. In this study, the direct effect of glucagon on colon cancer was investigated, and the glucagon mechanism of action as an activator of colon cancer cell proliferation was elucidated. Moreover, it was exhibited that hyperglucagonemia promotes colon cancer cell growth using a syngeneic mouse cancer model with type 2 diabetes. RESULTS GCGR is expressed in human colon cancer cell tissue and colon cancer cell lines The expression of GCGR was evaluated in human colon cancer tissues obtained from patients. Immunohistochemical staining showed that GCGR was expressed in colon cancer tissue (Physique ?(Figure1A).1A). Real-time PCR Becampanel and western blot analysis exhibited that GCGR was expressed in seven human malignancy cell lines including HT29, SW480, HCT116, CaCO2, T84, WiDr, and COLO205 (Physique 1B, 1C). Open in a separate window Physique 1 GCGR expression Expression of GCGR in human colon cancer tissue and colon cancer cell lines(A) Representative images of hematoxylin and eosin (HE) and GCGR staining of human colon cancer tissue obtained from patients with colon cancer. Scale bar, 100m. (B) Expressions of GCGR mRNA in seven human colon cancer cell lines evaluated by RT-PCR. Normal human liver mRNA is used as a positive control. cDNA replaced with water is used as unfavorable control (NC). (C) Expressions of GCGR protein in seven human colon cancer cell lines evaluated by western blot analysis. Normal human liver protein is used as a positive control. Glucagon promotes human colon cancer proliferation via activation of GCGR The effect of glucagon on cell proliferation was investigated using the MTT assay. Glucagon stimulation was performed by supplying 1.0 nM glucagon onto culture media every 24 h to maintain the concentration of glucagon, Becampanel because glucagon was not stable Gpr20 in culture media (Supplementary Determine 1). Glucagon significantly promoted the proliferation of HT29 and SW480 at 72 h after incubation with culture medium supplemented with 1.0 nM of glucagon every 24 h (Determine ?(Figure2A).2A). To investigate whether glucagon enhances cell growth through activation of GCGR, knockdown of GCGR in HT29 and SW480 was performed by siRNA, and GCGR knockdown cells were then incubated with or without glucagon. Western blotting showed that an approximately 70% reduction of GCGR expression was induced by siRNA in both HT29 and SW480 (Supplementary Physique 2). As shown in Figure ?Physique2B,2B, suppression of GCGR by siRNA abolished the promoting effect of glucagon on cell proliferation in both H29 and SW480 at 72 h after incubation with glucagon. These results indicated that glucagon directly stimulated colon cancer cell growth through binding to GCGR. Open in a separate window Physique 2 Tumor growth with glucagon Glucagon promotes proliferation of human colon cancer cell lines via GCGR activation(A) HT29 and SW480 cells were incubated in recommended culture media with or without stimulation by 1.0 nM of glucagon every 24 h. After 24, 48, and 72 h, cell viability was measured using an MTT assay. (B) HT29 and SW480 cells transfected with siRNA for control or GCGR were incubated in recommended culture media with or without stimulation by 1.0 nM glucagon every 24 h. After 24, 48, and 72 h, cell viability was measured using an MTT assay. Data are presented as means SD, studies using human colon cancer cell lines. Table 1 Characteristics of C57BL/6-DIO mice and studies. Next, AMPK signaling regulated by glucagon in colon cancer cells was investigated. AMPK plays a key Becampanel role as a grasp regulator of.