Heterodimeric interaction between GKN2 and TFF1 entails synergistic antiproliferative and pro-apoptotic effects on gastric cancer cells.
ABSTRACT: BACKGROUND:GKN2 and TFF1 form a heterodimer that is only generated in the mucus-secreting cells of the normal stomach. The formation of this heterodimer is frequently disrupted in gastric cancer. However, the precise roles of GKN2 alone and in the heterodimer with TFF1 as well as the contributions of GKN2 and the heterodimer to gastric carcinogenesis are poorly understood. METHODS:Cell viability, proliferation, and apoptosis were analyzed in AGS, MKN1, MKN28, and MKN45 gastric cancer cells transfected with GKN2 and/or TFF1 using MTT, BrdU incorporation, and apoptosis assays, respectively. In addition, cell viability was examined in HFE-145 non-neoplastic gastric epithelial cells after GKN2 and/or TFF1 silencing. Furthermore, the cell cycle and the expression of cell cycle and apoptosis related proteins were assessed. The interaction between GKN2 and TFF1 was confirmed by co-immunoprecipitation. Immunohistochemistry was employed to explore TFF1 expression in 169 gastric cancer tissues. RESULTS:Co-transfection with GKN2 and TFF1 significantly inhibited cell viability and proliferation by inducing G1/S cell cycle arrest and suppressing positive cell cycle regulators. Simultaneous knockdown of GKN2 and TFF1 in HFE-145 cells resulted in markedly increased cell viability. Moreover, the interaction of GKN2 and TFF1 promoted cell death by enhancing caspase-3/7 activity and upregulating pro-apoptotic proteins. At the mRNA level, GKN2 and TFF1 were found to be positively correlated in non-tumor and tumor samples. Immunohistochemistry revealed loss of TFF1 expression in 128 (75.73%) of 169 gastric cancers. There was a borderline-significant association between GKN2 and TFF1 protein expression in gastric cancers (P = 0.0598). CONCLUSION:Collectively, our data demonstrated that the interaction between GKN2 and TFF1 can have synergistic antiproliferative and pro-apoptotic effects on gastric cancer.
Project description:TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.
Project description:Helicobacter pylori (H. pylori) infection has been associated with non-cardia adenocarcinoma in the stomach, while its role in gastric cardia adenocarcinoma (GCA) remains controversial. In addition, the association between H. pylori and the protective factors trefoil factor 1 (TFF1) and gastrokine 2 (GKN2) in gastroesophageal adenocarcinomas has not been fully investigated. Therefore, the mRNA and protein expression levels of TFF1 and GKN2 in GCA and distal gastric adenocarcinoma (DGA) were analyzed using quantitative PCR (qPCR) and immunohistochemistry, and the association with H. pylori infection was investigated. In addition, the effects of TFF1 and GKN2 overexpression on H. pylori-induced cells were investigated using western blot and reverse transcription-qPCR analysis. The comparative analysis of 16S rRNA-positive mRNA expression between GCA and DGA showed no statistically significant difference. However, the rate of the H. pylori vacuolating toxin A (VacA) genotype was significantly higher in GCA (49.2%) compared with that in DGA (26.9%; P<0.05). H. pylori infection downregulated the mRNA and protein expression levels of TFF1 and GKN2 in gastric tumor tissues, and the mRNA expression level of TFF1 and GKN2 was also markedly decreased in vitro. Furthermore, the cell proliferation varied in H. pylori total protein treatment group with the different doses. Notably, treatment with 20 µg/ml H. pylori total protein for 24 h resulted in the highest cellular proliferation rate. In addition, TFF1 and GKN2 overexpression inversely inhibited H. pylori-induced cell proliferation and upregulated NF-?B, tumor necrosis factor-?, IL-1?, IL-2, IL-4 and IL-6. The results of the present study indicate that H. pylori, particularly the VacA+ strain, plays an important role in GCA pathogenesis in high-risk areas of China, while TFF1/GKN2 inhibits H. pylori-induced cell proliferation and inflammation in GCA and DGA.
Project description:Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.
Project description:To investigate whether YAP upregulates MYC via miRNA repression, we sequenced miRNAs from gastric epithelial cells (HFE-145) either expressed with vector or YAP-5SA. Overall design: Duplicates of HFE-145 cells either expressing vector or YAP-5SA were analyzed.
Project description:<h4>Background</h4>The GKN2 is a secretory protein, whose levels decrease in gastric cancer. The present study aimed to investigate the expression, function and mechanism of action of GKN2 in gastric cancer.<h4>Methods</h4>Molecular biology assays were performed to elucidate the function and underlying mechanisms of GKN2 in gastric cancer under stress-induced condition in vivo and in vitro. Clinical specimens were used to assess the correlation of GKN2 and prognosis.<h4>Results</h4>We found that overexpression of GKN2 significantly enhanced apoptosis and growth arrest in vitro. GKN2 expression increased in gastric cancer cells exposed to hydrogen peroxide and promoted reactive oxygen species-induced mitochondrial dysfunction and resulted in increased cell apoptosis via inhibition of NF-?B signaling pathway and activation of JNK signaling pathway through the direct interaction of GKN2 with Hsc70. Trefoil factor 1 might contribute to the tumor suppressing effects of GKN2. MiR-216a downregulated GKN2 expression. GKN2 also inhibited xenograft tumor growth and was an independent and significant prognostic factor for patients with gastric cancer treated with oxaliplatin.<h4>Conclusions</h4>Taken together, our data indicate that GKN2 may increase sensitivity of GC cells to the drugs which increase ROS levels in tumors. Inhibition of the interaction between GKN2 and Hsc70 could attenuate the effects induced by GKN2. GKN2 overexpression could be used to determine the subgroup of patients to obtain the more favorable outcome of oxaliplatin treatment and may be used as biomarker of the prognosis of this cancer.
Project description:The upregulation or mutation of C-MYC has been observed in gastric, colon, breast, and lung tumors and in Burkitt's lymphoma. However, little is known about the role C-MYC plays in gastric adenocarcinoma. In the present study, we intended to investigate the influence of C-MYC on the growth, proliferation, apoptosis, invasion, and cell cycle of the gastric cancer cell line SGC7901 and the gastric cell line HFE145. C-MYC cDNA was subcloned into a constitutive vector PCDNA3.1 followed by transfection in normal gastric cell line HFE145 by using liposome. Then stable transfectants were selected and appraised. Specific inhibition of C-MYC was achieved using a vector-based siRNA system which was transfected in gastric cancer cell line SGC7901. The apoptosis and cell cycles of these clones were analyzed by using flow cytometric assay. The growth and proliferation were analyzed by cell growth curves and colony-forming assay, respectively. The invasion of these clones was analyzed by using cell migration assay. The C-MYC stable expression clones (HFE-Myc) and C-MYC RNAi cells (SGC-MR) were detected and compared with their control groups, respectively. HFE-Myc grew faster than HFE145 and HFE-PC (HFE145 transfected with PCDNA3.1 vector). SGC-MR1, 2 grew slower than SGC7901 and SGC-MS1, 2 (SGC7901 transfected with scrambled control duplexes). The cell counts of HFE-Myc in the third, fourth, fifth, sixth, and seventh days were significantly more than those of control groups (P < 0.05). Those of SGC-MR1, 2 in the fourth, fifth, sixth, and seventh days were significantly fewer than those of control groups (P < 0.05). Cell cycle analysis showed that proportions of HFE-Myc and SGC-MR cells in G0-G1 and G2-M were different significantly with their control groups, respectively (P < 0.05). The apoptosis rate of HFE-Myc was significantly higher than those of control groups (P < 0.05). Results of colony-forming assay showed that the colony formation rate of HFE-Myc was higher than those of control groups; otherwise, the rate of SGC-MR was lower than those of their control groups (P < 0.05). The results of cell migration assay showed that there were no significant differences between experimental groups and control groups (P > 0.05). In conclusion, C-MYC can promote the growth and proliferation of normal gastric cells, and knockdown of C-MYC can restrain the growth and proliferation of gastric cancer cells. It can induce cell apoptosis and help tumor cell maintain malignant phenotype. But it can have not a detectable influence on the ability of invasion of gastric cancer cells.
Project description:BACKGROUND:Genipin is a compound derived from gardenia fruit extract. Although Genipin has anti-tumor effects in various cancers, its effect and mechanism in gastric cancer remain unclear. Here, we investigated the relationship between the anticancer effect of Genipin and signal transducer and activator of transcription (Stat3)/myeloid cell leukemia-1 (Mcl-1) in human gastric cancers. METHODS:MTT assays were performed to determine the cell viability of gastric cancer and gastric epithelial cell lines (AGS, MKN45, SNU638, MKN74, HFE-145). A TUNEL assay and Western blotting were carried out to investigate apoptosis. Stat3 activity was measured by proteome profiler phospho kinase array, immunofluorescence and immunoblotting. Mitochondria function was monitored with an XF24 analyzer and by flow cytometry, confocal microscopy using fluorescent probes for general mitochondrial membrane potential (MMP). RESULTS:Genipin induced apoptosis in gastric cancer cells, including AGS and MKN45 cells. Genipin also reduced Mcl-1 mRNA and protein levels. Furthermore, we found that phosphorylation of Stat3 is regulated by Genipin. Additionally, the protein level of phospho Janus kinase 2 (JAK2) was decreased by Genipin treatment, indicating that the Stat3/JAK2/Mcl-1 pathway is suppressed by Genipin treatment in gastric cancer cells. Mcl-1 is closely related to mitochondrial function. These findings suggest that Genipin contributes to the collapse of mitochondrial functions like MMP. CONCLUSIONS:Genipin induced apoptosis by suppressing the Stat3/Mcl-1 pathway and led to mitochondrial dysfunction. Our results reveal a novel mechanism for the anti-cancer effect of Genipin in gastric cancer.
Project description:In this study, we investigated the role of Trefoil factor 1 (TFF1) in regulating cell proliferation and tumour development through ?-catenin signalling using in vivo and in vitro models of gastric tumorigenesis.Tff1-knockout (Tff1-KO) mice, immunohistochemistry, luciferase reporter, qRT-PCR, immunoblot, and phosphatase assays were used to examine the role of TFF1 on ?-catenin signalling pathway.Nuclear localisation of ?-catenin with transcriptional upregulation of its target genes, c-Myc and Ccnd1, was detected in hyperplastic tissue at an early age of 4-6?weeks and maintained during all stages of gastric tumorigenesis in the Tff1-KO mice. The reconstitution of TFF1 or TFF1 conditioned media significantly inhibited the ?-catenin/T-cell factor (TCF) transcription activity in MKN28 gastric cancer cells. In agreement with these results, we detected a reduction in the levels of nuclear ?-catenin with downregulation of c-MYC and CCND1 mRNA. Analysis of signalling molecules upstream of ?-catenin revealed a decrease in phosphorylated glycogen synthase kinase 3? (p-GSK3?) (Ser9) and p-AKT (Ser473) protein levels following the reconstitution of TFF1 expression; this was consistent with the increase of p-?-catenin (Ser33/37/Thr41) and decrease of p-?-catenin (Ser552). This TFF1-induced reduction in phosphorylation of GSK3?, and AKT was dependent on protein phosphatase 2A (PP2A) activity. The treatment with okadaic acid or knockdown of PP2A abrogated these effects. Consistent with the mouse data, we observed loss of TFF1 and an increase in nuclear localisation of ?-catenin in stages of human gastric tumorigenesis.Our data indicate that loss of TFF1 promotes ?-catenin activation and gastric tumorigenesis through regulation of PP2A, a major regulator of AKT-GSK3? signalling.
Project description:TFF1, a mucin-associated secreted peptide of gastric mucous cells, is known as a protective agent for stomach epithelium under different stimuli, but its role upon Helicobacter infection is still not clear. In this paper we characterized TFFs expression, with particular attention to TFF1, under Helicobacter infection in gastric cell lines. A mouse model was used to distinguish TFF1 mRNA expression between acute and chronic stages of Helicobacter infection. Our results show that TFF1 expression is induced in infected cells; in addition, the inflammatory response upon Helicobacter infection is inversely associated to pre-existing TFF1 protein levels. In infected mice, TFF1 is initially upregulated in gastric antrum in the acute phase of infection, along with IL-1? and IL-6. Then, expression of TFF1 is gradually silenced when the infection becomes chronic and IFN-?, CXCL5, and CXCL15 reach higher levels. Our data suggest that TFF1 might help cells to counteract bacteria colonization and the development of a chronic inflammation.