Of the 50 patients, 28 were male and 22 were female and the ages ranged between 42 and 65. methylation. Treatment with the drug, 5-aza-2-deoxycytidine, increased the expression of WIF-1 in the GBC-SD gallbladder cell line. In addition, a WIF-1-expression plasmid was transfected into GBC-SD cells, and it was found that cell proliferation, invasion and metastasis declined significantly, whereas the apoptotic rate increased. A nude mouse tumor transplantation experiment showed that the oncogenicity of the GBC-SD cells expressing WIF-1 was substantially SB 216763 lower, compared with that of the untransfected GBC-SD cells and of GBD-SD cells expressing the control plasmid. A fluorescent protein chip experiment showed that the restored expression of WIF-1 affected the expression of several cellular proteins. These alterations may explain the different biological behavior of the tumor cells expressing WIF-1. As an effective inhibitory factor of the Wnt signaling pathway, WIF-1 modulated the expression of proteins controlling the proliferation, apoptosis and metastasis Rabbit polyclonal to PGM1 of gallbladder tumor cells, thus suppressing the tumor. Therefore, WIF-1 may be an effective treatment target for gallbladder cancer. (10), reported a novel extracellular SB 216763 inhibitor protein, which can bind to Wnt proteins and affect their function. This was termed Wnt inhibitory factor 1 (WIF-1). At present, the gene sequence for WIF-1 has been determined, and its spatial structure has also been confirmed (11). WIF-1 belongs to the secreted Frizzled-related protein family and can inhibit the classical and non-classical Wnt signaling pathways (12,13). The SB 216763 abnormal expression of WIF-1 in certain types of tumor has also been confirmed (14C16). However, the expression of WIF-1 in gallbladder cancer, the effects of WIF-1 on the biological behavior of gallbladder cancer and the associated mechanisms remain to be fully elucidated In the present study, it was shown in gallbladder tumors and three gallbladder cancer cell lines that the expression levels of WIF-1 were low. This low expression was associated with methylation of the WIF-1 gene promoter. Following treatment of GBC-SD cells with 5-aza-2-deoxycytidine (5-Aza-dC), the expression of WIF-1 recovered. The current study aimed to elucidate the effects of WIF-1 on tumor growth, invasion and metastasis, thus a GBC-SD cell line was constructed, which stably expressed WIF-1, and the expression of proteins closely associated with the Wnt signaling pathway were analyzed. It was found that WIF-1 significantly inhibited tumor cell proliferation, migration and invasion, and increased the apoptotic rate of the tumor cells. Protein expression levels were also altered following transfection. These results showed that WIF-1 markedly inhibited tumor growth, invasion and metastasis. Therefore, WIF-1 may be an effective treatment target for gallbladder cancer. Materials and methods Case collection and immunohistochemistry A total of 40 gallbladder cancer specimens were collected from the Union Hospital of Fujian Medical University (Fujian, China), following surgical resection between 2004 and 2011. Of the 40 patients, 18 were male and 22 were female, 19 patients were 60 years old and 21 were 60 years old. All cases were confirmed by histopathological examinations. In addition, 50 chronic cholecystitis specimens were collected from the Union Hospital of Fujian Medical University following surgical resection in 2012, and were confirmed by histopathological examinations. Of the 50 patients, 28 were male and 22 were female and the ages ranged between 42 and 65. The current study was approved by the ethics committee of the Affiliated Union Hospital of Fujian Medical University (Fuzhou, China). The rabbit anti-human monoclonal WIF-1 antibody (#5502; 1:200; incubation at 4C for 8 h) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and a goat anti-rabbit secondary antibody kit (kt-9903; 1:50; incubation at 37C for 20 min) was purchased from Beijing ZhongShan Biotechnology Company (Beijing, China). The expression of WIF-1 was detected using routine En Vision two-step immunohistochemical staining (Fuzhou Maxim Biotech, Inc., Fuzhou, China). Selected paraffin blocks (4 experiments, 15 nude mice were successfully inoculated, and the tumors were excised from the mice in the different groups and were then weighed. No lymph node metastases were observed in the three groups of nude mice. There was a significant difference in tumor weight between the WGBC-SD group and the GBC-SD group, and between the WGBC-SD group and the NGBC-SD group (P 0.001). No significant difference in tumor weight was observed between the GBC-SD group and the NGBC-SD group (P 0.05). The results indicated that WIF-1 inhibited the oncogenicity of the GBC-SD cells and (Fig. 4D). Discussion (20), which reported that CPG island methylation of the P53 tumor suppressor gene affects its transcriptional activity, numerous studies have shown that gene.The abnormal expression of WIF-1 in certain types of tumor has also been confirmed (14C16). compared with that of the untransfected GBC-SD cells and of GBD-SD cells expressing the control plasmid. A fluorescent protein chip experiment showed that the restored expression of WIF-1 affected the expression of several cellular proteins. These alterations may explain the different biological behavior of the tumor cells expressing WIF-1. As an effective inhibitory factor of the Wnt signaling pathway, WIF-1 modulated the expression of proteins controlling the proliferation, apoptosis and metastasis of gallbladder tumor cells, thus suppressing the tumor. Therefore, WIF-1 may be an effective treatment target for gallbladder cancer. (10), reported a novel extracellular inhibitor protein, which can bind to Wnt proteins and affect their function. This was termed Wnt inhibitory factor 1 (WIF-1). At present, the gene sequence for WIF-1 has been determined, and its spatial structure has also been confirmed (11). WIF-1 belongs to the secreted Frizzled-related protein family and can inhibit the classical and non-classical Wnt signaling pathways (12,13). The abnormal expression of WIF-1 in certain types of tumor has also been confirmed (14C16). However, the expression of WIF-1 in gallbladder cancer, the effects of WIF-1 on the biological behavior of gallbladder cancer and the associated mechanisms remain to be fully elucidated In the present study, it was shown in gallbladder tumors and three gallbladder cancer cell lines that the expression levels of WIF-1 were low. This low expression was associated with methylation of the WIF-1 gene promoter. Following treatment of GBC-SD cells with 5-aza-2-deoxycytidine (5-Aza-dC), the expression of WIF-1 recovered. The current study aimed to elucidate the effects of WIF-1 on tumor growth, invasion and metastasis, thus a GBC-SD cell line was constructed, which stably expressed WIF-1, and the expression of proteins closely associated with the Wnt signaling pathway were analyzed. It was found that WIF-1 significantly inhibited tumor cell proliferation, migration and invasion, and increased the apoptotic rate of the tumor cells. Protein expression levels were also altered following transfection. These results showed that WIF-1 markedly inhibited tumor growth, invasion and metastasis. Therefore, WIF-1 may be an effective treatment target for gallbladder cancer. Materials and methods Case collection and immunohistochemistry A total of 40 gallbladder malignancy specimens were collected from your Union Hospital of Fujian Medical University or college (Fujian, China), following medical resection between 2004 and 2011. Of the 40 individuals, 18 were male and 22 were female, 19 individuals were 60 years aged and 21 were 60 years aged. All cases were confirmed by histopathological examinations. In addition, 50 chronic cholecystitis specimens were collected from your Union Hospital of Fujian Medical University or college following medical resection in 2012, and were confirmed by histopathological examinations. Of the 50 individuals, 28 were male and 22 were female and the age groups ranged between 42 and 65. The current study was authorized by the ethics committee of the SB 216763 Affiliated Union Hospital of Fujian Medical University or college (Fuzhou, China). The rabbit anti-human monoclonal WIF-1 antibody (#5502; 1:200; incubation at 4C for 8 h) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and a goat anti-rabbit secondary antibody kit (kt-9903; 1:50; incubation at 37C for 20 min) was purchased from Beijing ZhongShan Biotechnology Organization (Beijing, China). The manifestation of WIF-1 was recognized using routine En Vision two-step immunohistochemical staining (Fuzhou Maxim Biotech, Inc., Fuzhou, China). Selected paraffin blocks (4 experiments, 15 nude mice were successfully inoculated, and the tumors were excised from your mice in the different groups and were then weighed. No lymph node metastases were observed in the three groups of nude mice. There was a significant difference in tumor excess weight between the WGBC-SD group and the GBC-SD group, and between the WGBC-SD group and the NGBC-SD group (P 0.001). No significant difference in tumor excess weight was observed between the GBC-SD group and the NGBC-SD group (P 0.05). The results indicated that WIF-1 inhibited the oncogenicity of the GBC-SD cells and (Fig. 4D). Conversation (20), which reported that CPG island methylation of the P53 tumor suppressor gene affects its transcriptional activity, several studies have shown that gene promoter hypermethylation is an important factor in gene manifestation (21C28). The methylation of tumor suppressor gene promoters often leads to the inactivation of tumor suppressor genes and tumorigenesis (22). The present study.