Search

Background: Pleckstrin-2 (PLEK2) is a crucial mediator of cytoskeletal reorganization. However, the potential roles and clinical significance of PLEK2 in gastric cancer are still unknown. Methods: We aimed to illustrate the function of PLEK2 in gastric cancer. Pearson's correlation analysis was utilized to test the clinical impacts of the levels of PLEK2 in gastric cancer patients. Loss-of-function approaches were used to estimate the potential roles played by PLEK2 in modulating gastric cancer proliferation, self-renewal, and tumourigenicity. Immunoblotting and bioinformatics approaches were used to monitor the effect of PLEK2 on epithelial-mesenchymal transition (EMT) signalling pathways. Results: In gastric cancer tissue samples examined by western blot and real-time PCR, there was an increased level of PLEK2 compared with that in matched non-tumour samples. Kaplan-Meier plotter analysis revealed that gastric cancer patients with higher PLEK2 levels had substantially poorer overall survival (p=0.0028) compared with gastric cancer patients with lower PLEK2 levels. The upregulation or downregulation of PLEK2 in gastric cancer cell lines effectively enhanced or inhibited cell proliferation and pro-invasive behaviour, respectively. Additionally, we also found that PLEK2 enhanced EMT concomitantly with a reduction in E-cadherin and an elevation in Vimentin. Conclusions: Our findings demonstrated that PLEK2 plays a potential role in gastric cancer and may act as a prognostic factor for gastric cancer. Citation Format: Bo Sun, Jian-Bin Xiang, Yi Xuan, Zong-You Chen, Xiao-Dong Gu, Da-Zhi Xu. Pleckstrin-2 as a prognostic factor and mediator of gastric cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 361.

Background There is no consensus of opinion about postoperative adjuvant chemotherapy after radical surgery for advanced gastric cancer. This is a meta-analysis of the published results of relevant randomized clinical trials (RCTs). Methods Electronic databases from January 1998 to December 2007 were searched and 12 RCTs were selected. These included a total of 3809 patients. The hazard ratio (HR) for overall survival was calculated. Results The pooled HR for overall survival was 0·78 (95 per cent confidence interval 0·71 to 0·85) in favour of chemotherapy. Subgroup analysis showed that the advantage of chemotherapy was not influenced by depth of tumour infiltration, status of lymph node metastasis, type of lymphadenectomy, geographical distribution of patients or route of drug administration. Conclusions Postoperative chemotherapy can improve overall survival after radical surgery for gastric cancer; there is no standardized chemotherapy regimen. Japanese-style D2 radical surgery plus oral 5-fluorouracil appears an effective treatment at present.

Despite extensive research, the mechanism of immature dendritic cells (DCs) induced immune hyporesponsiveness remains incompletely understood.Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin (IL)-2, IL-4, IL-10, and interferon (INF)-γ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining.There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups (P0.05), indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups (P0.05). There was no significant difference in IL-4 levels between the groups (P0.05). We also showed that CD80/86 low DCs loaded with alloantigen (1) stimulated low T cell proliferative responses via the indirect recognition pathway and (2) enhanced apoptotic activity (P0.05) in co-cultured T cells.Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinically applicable.

Background Nucleation of cholesterol monohydrate crystals following the aggregation and fusion of cholesterol-enriched vesicles is a critical procedure in the formation of cholesterol gallstone. Biliary proteins play important roles in the process. It is inefficient to screen pro-nucleating or anti-nucleating proteins with routine physiochemical techniques, by which we discovered several pro-nucleating proteins. Methodology/principal findings Based on comparative proteomic technologies, we investigated the differentially expressed proteins between the cholesterol gallstone and control groups, and between the vesicular phase and micellar phase. There are 401±75 spots detected on the cholesterol gallstone group and 389±94 spots on the control group gels, 120±24 spots detected on vesicular phase and 198±37 on micellar phase gels, and accordingly 22 and 8 differentially expressed proteins were identified successfully, respectively. Three of them, HSA, Profilin and Retinol Binding Protein, were validated by Western blot. Conclusion/significance Some of the identified proteins are in good agreement with proteins reported to be involved in the gallstone formation before. The information from this study might provide some important clues to uncover the key proteins involved in the formation of cholesterol gallstone.

To identify the proteins which play key roles during the formation of cholesterol gallstone, differential analysis was carried out that the proteome of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients.Vesicular and micellar phases were isolated by the density gradient ultracentrifugation method. Total proteins from the two phases were extracted, and the protein expressional profiles were established by two-dimensional electrophoresis respectively. The differentially expressed protein spots analyzed by ImageMaster two-dimensional electrophoresis analysis software were identified with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).The concentrations of proteins from vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were (1.5358 +/- 0.0682) mg/ml and (7.1222 +/- 0.2022) mg/ml (P0.01) respectively. The average matched protein spots were 120 +/- 24 and 198 +/- 37 in the two groups respectively. There were 72 +/- 16 matched spots in the two representative gels maps and the matched rate was 45.30%. Eight differentially expressed protein spots were identified from the two cholesterol-carrier phases. Among them, 6 were up-regulated with 2 down-regulated in vesicular phase compared with micellar phase. The abundance differentiation of RBP and HSA was confirmed by immunoblotting.The differential protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were established and 8 differential protein spots were identified successfully. The data may be a basis for further screening the key regulators of formation of cholesterol gallstone.

ObjectiveTo establish a rapid and precise detectiv e method of 33.5 kd vesicular protein and to screen an effective treatment of chol elithiasis. MethodsSpecific antibody of the biliary vesicular prot ein was obtained by immunizing rabbits and enzyme-linked immunosorbent assay (E LISA) kit was developed. The concentrations of 33.5 kd vesicular protein in seru m and bile of gallstone patients and control were examined respectively. The eff ects of Cholagogue Dry Syrup and Eulektrol Capsule on decreasing 33.5 kd vesicul ar protein were also studied by ELISA kit. ResultsOne-step ELISA equation was Y= 0.035 X (r=0.99).The vesicular protein concentrations in seru m and bile of cholesterol gallstone group (179.8±97.9 ) mg/L and (213.4±70. 1)mg/L respectively were significantly (P 0.05) higher than in the pigme nt stone group and control. Data showed that, with 2-week administration, Chola gogue Dry Syrup significantly decreased both biliary and serum 33.5 kd vesicular protein of cholesterol gallstone patients, while Eulekrol Capsule and control g roups didn’t have the same results. Conclusion The concentrations o f 33.5 kd protein are different in cholesterol gallstone patients and healthy gr oups which might be related to cholesterol nucleation process. Cholagogue Dry Sy rup is of cholagogic and litholytic effect by decreasing biliary lithogenesis.

AIM: The present study was undertaken to purify and partially characterize the 33.5-kilodalton (33.5 kDa) vesicular protein in human bile and to explore the possible molecular mechanisms of the initial crystal nucleation process. METHODS: The 33.5 kDa vesicular protein was isolated by ultracentrifugation and further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The purified 33.5 kDa vesicular protein was subjected to N-terminal amino acid sequencing and amino acid analysis. Cholesterol crystallization activity was detected by cholesterol crystal growth assay. The sugar chain of the 33.5 kDa vesicular protein was analyzed by dot-immunobinding assay of lectin coupled to a peroxidase (HRP-DSA, HRP-ConA, HRP-WGA) and was deglycosylated using two different enzymatic approaches (N-deglycosylation and O-deglycosylation) to determine the molecular weight of the protein component, the type of linkage between polypeptide and carbohydrate components. RESULTS: The 33.5 kDa vesicular protein with complicated glycan was an extensively glycosylated (37.3%) monomer and these sugar chains strongly bound to DSA, but did not bind to ConA. Amino acid sequencing indicated that the protein was unique. The 33.5 kDa vesicular protein exhibited potent cholesterol crystallization promoting activity in vitro with derived crystal growth curve indices It, Ig, Ic presented as 0.57, 1.52, and 1.63 respectively. Both enzymatic proteolysis and N-deglycosylation of the protein removed all activity. CONCLUSION: These data suggest the 33.5 kDa vesicular protein may be responsible for the pathogenesis of cholesterol gallstone disease, and the sugar chains play an important role in pro-nucleating process.