Study On The Mechanism Of Wnt / β-catenin Signaling Pathway Mediated By Proinflammatory And Probiotic Agents

OBJECTIVE:To maintain the remission of ulcerative colitis (UC) and to prevent carcinogenesis are focuses of clinical research. Clinical studies have proved that probiotics exert beneficial effects in the treatment of UC, whereas the mechanism is still unclear. IL-6 and TNFa are cytokines playing important roles in the pathogenesis of ulcerative colitis related colorectal cancer (UCRCC). The objectives of this study are:1) Stimulate normal colon epithelial cell and colon cancer cell with IL-6 or TNFα, then evaluate their influences on the expression of β-catenin, which is the key factor in Wnt signaling pathway; 2) Co-culture cell strains mentioned above with conditioned probiotic media, and detect its effect on the expression of P-catenin. Furthermore, explore the possible signaling pathway involved in the mechanism of UC carcinogenesis.METHODS:In this study, we use normal colon epithelial cell strain CCC-HIE-2, colon cancer cell strain Caco2 and colon cancer cell strain HCT 116. Expression of P-catenin was detected by western blot and real-time PCR in both cell lines with and without stimulation of TNF-α and IL-6. To evaluate β-catenin expression in transcriptional and translational levels, we assessed β-catenin combined with nuclear TCF4 by co-immunoprecipitation, assessed the transcriptional activity of β-catenin/TCF4 complex by dual luciferase reporter gene assay. NF-κB signaling pathway was blocked by specific inhibitor PDTC. Then added IL-6 and TNFα separately to repeat experiments listed above. Bifid triple viable powder (BIFICO-SINE) is composed by Bificobacterium, Lactobacillus acidophilus and Enterococcus faecalis. The probiotics were cultivated in MRS broth medium. Centrifuge and filter to obtain sterile conditioned probiotic media. Co-culture colon cancer cell line HCT116 with conditioned probiotic media, and then verify P-catenin in protein, translational and transcriptional levels.RESULTS:1. The optimal stimulation condition for Caco2 is IL-6 10ng/ml working for 6h and TNF-α 10ng/ml working for 6h. The optimal stimulation condition for CCC-HIE-2 is IL-6 10ng/ml working for 6h and TNF-α 10ng/ml working for 6h.2. Under optimal stimulations (IL-6 10ng/ml,6h; TNFa 10ng/ml,6h), β-catenin expression of Caco2 in both protein and mRNA levels were significantly elevated. The upregulated P-catenin entered nucleus to form complex with TCF4. The β-catenin/TCF4 complex could specifically activate downstream gene transcription, and enhanced Wnt signaling pathway.3. NF-κB signaling pathway was blocked by the specific inhibitor PDTC. PDTC (50uM,1h) suppressed the up-regulation effects of IL-6 (10ng/ml,6h) and TNFa(10ng/ml,6h) on Wnt signaling pathway。4. According to the growth curve, the conditioned probiotic media with optimal effect on cell strains was obtained by culturing 0.5g bifid triple viable powder in 100ml MRS broth medium. The inoculum was cultured until it reached the mid-exponential phase of growth (OD600 1.1±0.2).5. Stimulating HCT116 with IL-6(10ng/ml,6h) and TNFa(10ng/ml,6h), we noticed that β-catenin expression increased through up-regulation of Wnt signaling pathway. The influences IL-6(10ng/ml,6h) and TNFα(10ng/ml,6h) exerted on HCT116 was inhibited by PDTC (50uM,1h).6.1% conditioned probiotic media worked 0.5h on HCT 116, P-catenin expression in both protein and mRNA levels were significantly elevated. The upregulated β-catenin entered nucleus to form complex with TCF4. The β-catenin/TCF4 complex could specifically activate downstream gene transcription, and enhanced Wnt signaling pathway. The conditioned probiotic media partially reversed the inhibitory effects of PDTC.CONCLUSION:IL-6 and TNF-α stimulated expression of β-catenin in both protein and mRNA levels. This process was mediated by NF-κB signaling pathway. The elevated β-catenin entered nucleus to form β-catenin/TCF4 complex, which specifically stimulated Wnt signaling pathway and led to target gene transcription. IL-6 and TNF-α stimulated expression of β-catenin in both protein and mRNA levels. This process was mediated by NF-κB signaling pathway. The elevated β-catenin entered nucleus to form β-catenin/TCF4 complex, which specifically stimulated Wnt signaling pathway and led to target gene transcription. Co-culture HCT116 with conditioned probiotic media, we observed increase in β-catenin expression and up-regulation of Wnt signaling pathway. The conditioned probiotic media and cytokines (IL-6, TNFα) may regulate Wnt signaling pathway through multiple pathways besides NF-κB signaling pathway. This study suggested new thoughts for the mechanisms of ulcerative colitis carcinogenesis.OBJECTIVE:Gastric neuroendocrine neoplasms(GNENs)is kind of rare neoplasms, leading to the inevitability of medical misdiagnosis and mistreatment. To assess the clinical characteristics, diagnosis and therapeutic modalities for gastric neu-roendocrine neoplasms (GNENs) among the Chinese population in a single institution. The aim of this study is to increase awareness of GNENs in Chinese population.METHODS:A total of 57 patients with histologically confirmed GNENs, who were diagnosed between 1995 and 2015 at the Peking Union Medical College Hospital were retrospectively reviewed. Utilizing the 2012 guideline released by European Neuroendocrine Tumor Society and the World Health Organization (WHO) 2010 grading criteria, we conducted a retrospective study to assess the clinicopathological characteristics, diagnostic methods and therapeutic interventions for primary GNENs.RESULTS:A total of 57 patients were recruited in the study, the ratio of male/female is 1.7/1. Patients with GNENs mostly presented with non-specific symptoms, such as abdominal pain, abdominal distention, acid reflux and heartburn. Gastric body was the most common site of involvement, followed by cardia.25 patients were categorized as type 1 GNENs, more than 80% instances were seen in patients with chronic gastritis and 56% with anemia. All three type 2 patients were diagnosed with MEN1 or gastrinoma. The other 29 patients were classified as having type 3 GNENs, of whom 62.1% were complicated with lymphoinvasion and 41.4% complicated with hepatic metastasis. The choice of imaging modality depended on the tumor subtype. Chromogranin A (CgA) and synaptophysin (Syn) were indispensable immunohistochemical markers for diagnosis. Significant inter-group differences in the positivity rate of CD56 were observed among the three grades (G1, G2 and G3). Therapeutic modalities included endoscopic intervention, surgical resection and pharmacotherapy, which were largely guided by the tumor subtype and the presence or absence of distant metastasis or tumor recurrence.CONCLUSIONS:Routine endoscopic examination is recommended for the early diagnosis of GNENs. Histopathological examination can make the definite diagnosis of GNENs and clarify the nature of gastric polyps.A multidisciplinary approach is important in the management of patients with GNENs.