Dickkopf-related protein 1/cytoskeleton-associated protein 4 signaling activation by Helicobacter pylori-induced activator protein-1 promotes gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

BACKGROUND: Gastric cancer (GC) is a common malignant tumor with high incidence and mortality rates globally, especially in East Asian countries. Helicobacter pylori (H. pylori) infection is a significant and independent risk factor for GC. However, its underlying mechanism of action is not fully understood. Dickkopf-related protein (DKK) 1 is a Wnt signaling antagonist, and cytoskeleton-associated protein (CKAP) 4 is a newly identified DKK1 receptor. Recent studies found that the binding of DKK1 to CAKP4 mediated the procancer signaling of DKK1 inde-pendent of Wnt signaling. We hypothesize that H. pylori-induced activation of DKK1/CKAP4 signaling contributes to the initiation and progression of GC. AIM: To investigate the interaction of H. pylori infection, DKK1 and CAKP4 in GC, as well as the underlying molecular mechanisms. METHODS: RNA sequencing was used to identify differentially expressed genes (DEGs) between H. pylori-infected and uninfected primary GC cells. Gain- and loss-of-function experiments were performed to verify the H. pylori-induced upregulation of activator protein-1 (AP-1) in GC cells. A dual-luciferase reporter assay and co-immunoprecipitation were used to determine the binding of AP-1 to the DKK1 promoter and DKK1 to CKAP4. Western blotting and immunohistochemistry detected the expression of DKK1, CKAP4, and phos-phatidylinositol 3-kinase (PI3K) pathway-related proteins in GC cells and tissues. Functional experiments and tumorigenicity in nude mice detected malignant behavior of GC cells in vitro and in vivo. RESULTS: We identified 32 DEGs between primary GC cells with and without H. pylori infection, including JUN, fos-like antigen-1 (FOSL1), and DKK1, and confirmed that the three proteins and CKAP4 were highly expressed in H. pylori-infected GC cells, H. pylori-infected gerbil gastric tissues, and human GC tissues. JUN and FOSL1 form AP-1 to transcriptionally activate DKK1 expression by binding to the DKK1 promoter. Activated DKK1 bound to CKAP4, but not the most common Wnt coreceptor low-density lipoprotein receptor-related protein 5/6, to promote GC cell growth, colony formation, migration, invasion, and xenograft tumor growth in nude mice. All these effects were driven by activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway. Targeting the PI3K signaling pathway by LY294002 inhibited DKK1-mediated CKAP4/PI3K signaling activity and the malignant behavior of GC cells. CONCLUSION: H. pylori induces JUN and FOSL1 expression to form AP-1, which transcriptionally activates DKK1. Binding of DKK1 to KAKP4 contributes to gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

Downregulation of the circadian Period family genes is positively correlated with poor head and neck squamous cell carcinoma prognosis.

We investigated the relationship between head and neck squamous cell carcinoma (HNSCC) and the mRNA and protein expression levels of the circadian genes of the Period (Per) family, Per1, Per2 and Per3. Tissue sections of HNSCC and normal head and neck tissues from two patient cohorts from two different hospitals were collected to assess the mRNA and protein expressions of the three Per family genes using real-time quantitative PCR (RT-PCR) and immunohistochemistry (IHC). The clinicopathological features and disease prognosis for the latter cohort were analyzed through IHC and statistical methods. Protein positive expression levels of the three Per family genes in HNSCC tissues was found to be approximately two times lower than that in normal tissues (p < .01). Moreover, patients with locally advanced HNSCC showed significantly greater downregulation of Per1, Per2 and Per3 mRNA expression levels as compared to patients with early-stage cancer (p < .05). Immunohistochemical examination of HNSCC patient tissues revealed a positive correlation between the Per family protein expression and the clinical tumor staging (p < .05). In addition, the Per protein-positive expression group showed higher 3-year survival rates [overall survival (OS) and progression-free survival (PFS)] as assessed by Kaplan-Meier plots and statistical analysis (p < .05). Our findings confirm the positive correlation between Per family gene expression and survival outcomes and support their role as prognostic markers for HNSCC.

Changed expressions of N-methyl-d-aspartate receptors in the brains of rats and primary neurons exposed to high level of fluoride.

Expressions of N-methyl-d-aspartic acid receptors (NMDARs) in the brains of rats and primary neurons exposed to high fluoride were investigated. Sprague-Dawley rats were divided randomly into a fluorosis group (50ppm fluoride in the drinking water for 6 months) and controls (<0.5ppm fluoride) and the offspring from these rats sacrificed on postnatal days 1, 7, 14, 21 and 28. The primary cultured neurons from the hippocampus of neonatal rats were treated with 5 and 50ppm fluoride for 48h. NMDAR subunits at protein or mRNA levels were quantified by Western blotting or real-time PCR. The phosphorylated calmodulin-protein kinase II (CaMKII) was determined by Western blotting, concentration of Ca2+ in neurons by laser confocal microscopy and apoptosis by flow cytometry. In the brains of adult rats and pups as well as in primary neurons exposed to high fluoride, the mRNAs encoding GluN1 and GluN2B subunits and the corresponding proteins were elevated, the GluN3A lowered and the GluN2A unchanged. In addition, the level of phosphor-CaMKII was reduced, and Ca2+ influx and apoptosis enhanced in the brains of rats and cultured neurons exposed to high fluoride. The results indicate that such modifications may involve brain damage induced by chronic fluorosis.

H. pylori modifies methylation of global genomic DNA and the gastrin gene promoter in gastric mucosal cells and gastric cancer cells.

AIMS: The aim of this study was to evaluate the correlation between H. pylori infection and global DNA methylation, as well as the methylation levels of the gastrin promoters. MATERIALS AND METHODS: We constructed a eukaryotic expression vector, pcDNA3.1::cagA, and transfected it into GES-1 gastric mucosal cells and SGC-7901 gastric cancer cells. Both cell lines were infected with the H. pylori/CagA+ strain NCTC11637. Then, we detected global DNA methylation by capture and detection antibodies, followed by colorimetric quantification. The methylation levels of the gastrin promoter were evaluated by base-specific cleavage and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: In H. pylori/CagA+-infected GES-1 and SGC-7901 cells, the methylation levels of genomic DNA decreased by 49.4% and 18.8%, and in GES-1 and SGC-7901 cells transfected with pcDNA3.1::cagA, the methylation levels of genomic DNA decreased by 17.05% and 25.6%, respectively. Among 24 methylation sites detected in the gastrin promoter region, the methylation levels of 9 CpG sites were significantly decreased in H. pylori/CagA+-infected and pcDNA3.1:: cagA-transfected cells in comparison to corresponding control cells. CONCLUSION: These results indicate that H. pylori/CagA+ decreases the methylation of the genome and the gastrin promoter at some CpG sites in gastric mucosal and gastric cancer cells.

[Polymorphisms of homocysteine metabolism enzyme-related genes MS and MSR in Buyi, Dong and Miao ethnics from Guizhou].

OBJECTIVE: To investigate polymorphisms of homocysteine metabolism enzyme-related genes methionine synthase (MS) and methionine synthase reductase (MSR) in Buyi, Dong, Miao ethnics from Guizhou. METHODS: Genotypes of MS and MSR genes of healthy individuals from the three ethnic groups were determined with a TaqMan-MGB probe genotyping method and compared. RESULTS: For Buyi, Dong and Miao ethnics from Guizhou, frequencies of MS gene 2756G allele were respectively 12.0%, 8.9% and 15.4%. However, no significant difference was found by statistics. Frequencies of MS A2756G alleles for the three ethnic groups are similar to those of Han Chinese from Beijing and Henan, Hui ethnics from Ningxia as well as European populations, but differ significantly from those of Japanese, Indians, Africans and Nigerians (P < 0.05). Frequencies of MSR gene 66 G allele were respectively 32.3%, 30.4% and 21.2% for Buyi, Dong and Miao ethnics. Miao is significantly lower than Buyi and Dong (P< 0.05). Frequencies of MSR gene A66G alleles for the three ethnic groups are similar to those of Han Chinese from Beijing and Guangdong, Japanese, Africans and Nigerians populations, but differ significantly from those of Indians and European (P< 0.05). CONCLUSION: The distributions of MS gene A2756G and MSR gene A66G polymorphisms have differed significantly between the three ethnic groups and individuals from various regions.

[Association between the expression and methylation of energy-related genes with Helicobacter pylori infection in gastric cancer].

OBJECTIVE: To explore the association between the Helicobacter pylori (H. pylori) infection and the expression and methylation of energy-related genes in gastric cancer. METHODS: Real-time fluorescence quantitative reverse transcription (RT)-PCR was performed to quantify the expressions level of lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD) and Ran-specific GTPase-activating protein (RanGAP) genes in the samples of human gastric cancer (n = 30), metastatic lymph node (n = 30) and peri-cancerous tissues (n = 30) as confirmed by pathological examinations. Those patients were chosen of Affiliated Hospital of Guiyang Medical University, from January 2005 to December 2009. The relationship between the gene expression and H. pylori infection was analyzed. The methylation of LDH, DLD and RanGAP genes at promoter CpG island was measured by bisulfite sequencing (BSP). RESULTS: The relative gene expressions of LDH, DLD and RanGAP in peri-cancerous tissues, gastric cancer and metastatic lymph nodes were 1.0, 3.1, 3.0 and 1.0, 3.1, 2.8, and 1.0, 0.4, 0.5 respectively (all P < 0.05). The expression levels of LDH and DLD genes in H. pylori-positive gastric cancer was high than those in the negative group (2.3 vs 1.0, 3.0 vs 1.0, 2.6 vs 1.0, all P < 0.05). The demethylation of LDH and DLD genes at promoter -2325 and -1885 site as well as the over methylation of RanGAP gene at the promoter -570 and -170 sites respectively were detected in H. pylori infection and cagA-overexpressed cells. CONCLUSION: H. pylori infection may promote the development and progression of gastric cancer by inducing the aberrant methylation of LDH, DLD and RanGAP genes to up-regulate the gene expressions of LDH and DLD and down-regulate the gene expression of RanGAP.

[Identification of a TTR gene mutation in a family with hereditary vitreous amyloidosis].

OBJECTIVE: To study the disease gene in a family with hereditary vitreous amyloidosis. METHODS: A family with hereditary vitreous amyloidosis was investigated. Blood samples were collected from 4 members of this family including 3 patients and 1 asymptomatic individual. Genomic DNA was extracted from peripheral blood sample and subjected to amplification of 4 exons of transthyretin (TTR) gene. The PCR products were purified and subjected to direct sequencing. A total of 150 unrelated individuals were used as controls. RESULTS: A heterozygous mutation G to C at codon 103 in exon 3 of TTR gene (Gly103Arg) was detected in all 4 members of the family but not in the unrelated controls. CONCLUSION: The heterozygous Gly103Arg mutation of TTR gene may be related to the development of hereditary vitreous amyloidosis in this family.

[Neuron specific enolase gene silencing suppresses proliferation and promotes apoptosis of lung cancer cells in vitro].

OBJECTIVE: To study the effects of neuron specific enolase (NSE) gene silencing on the cell proliferation and apoptosis of lung cancer cells in vitro. METHODS: NSE protein expression was detected in human small cell lung cancer cell line NCI-H446 and non-small cell lung cancer cell line A549 by immunocytochemistry, and a small interference RNA (siRNA) was transfected into the cells to inhibit NSE gene expression. The changes in the cell cycle, apoptosis, Ki67 protein and caspase-3 activity in the transfected cells were observed by flow cytometry, Western blotting and colorimetric assay, respectively. RESULTS: Both A549 and NCI-H446 cells expressed NSE protein. Transfection of siRNA for NSE gene significantly inhibited the expression of NSE gene in the cells, resulting in an inhibition rate exceeding 90%. NSE gene silencing caused significantly decreased cell percentage in S phase and the expression of Ki67 protein, and increased the cell apoptotic rate and caspase-3 activity. CONCLUSION: NSE gene expression promotes the cell proliferation and inhibits the cell apoptosis in lung cancer cells with neuroendocrine differentiation, which might be a causative factor contributing to increased malignancy of the cells.

[Prognostic significance of hTERT mRNA quantitative expression in resected gastric carcinomas].

OBJECTIVE: By quantitative detection of telomerase expression, we investigated the relationship between telomerase expression and malignant behavior and prognosis in gastric carcinoma. METHODS: A real-time quantitative RT-PCR (RQ-PCR) was used to quantify the hTERT mRNA copy numbers in 89 samples of gastric carcinoma and corresponding non-cancerous tissues. The clinicopathological data of enrolled patients such as age, sex, tumor size, tumor site, pathologic type, histodifferentiation, infiltration depth, lymph node metastasis, stage and survival were obtained, and were made one factor analysis of variance and COX regression prognostic analysis with those above mentioned markers. Follow-up was completed as of February 28, 2010. The median follow-up was 24 months. RESULTS: hTERT from gastric carcinomas and corresponding non-cancerous tissues was 16.98 ± 3.56 and 11.37 ± 2.15, respectively (P < 0.05), the telomerase activity in gastric cancer was significantly higher than that in non-cancerous tissue (P < 0.05). Telomerase activity showed a positive correlation with depth of invasion, tumor differentiation and nodal metastasis (P < 0.01), and negative correlation with survival. CONCLUSIONS: Gastric cancer with high hTERT mRNA expression indicates a more malignant potential. Detection of hTERT mRNA in gastric cancer may be useful in a better understanding of invasion, metastasis, as well as prognosis of gastric cancer and provide a more efficient therapy. The quantitative expression of hTERT mRNA, infiltration depth and pTNM stage are significant afactors affecting the prognosis of patients with gastric carcinoma.

[Helicobacter pylori cytotoxin associated protein CagA regulates gastrin gene promoter activity].

OBJECTIVE: To study the regulatory effect of Helicobacter pylori CagA protein on gastrin promoter and the related signaling pathways as to further elucidate the mechanism of the development and progression of human gastric carcinoma. METHODS: After pcDNA3.1ZEO(-)/CagAand PGL/GP were identified by double restriction enzyme digestion, PCR and sequencing, the gastric cancer cell lines AGS and SGC-7901 cells were co-transfected with pcDNA3.1ZEO(-)/CagA and PGL/GP for 48 h. Alternatively, AGS and SGC-7901 cells were transfected by PGL/GP for 36 h later, and infected with Helicobacter pylori for additional 12 h. Meanwhile, the transfected and infected cells were treated using the JAK2 signaling pathway inhibitor AG490 and the ERK signaling pathway inhibitor U0126. The untreated cells and empty-vector-transfected cells were used as the control. Finally, luciferase activity was detected using the luciferase reporter assay system in transfected and infected cells. The levels of gastrin mRNA was determined by TaqMan® real-time quantitative PCR. RESULTS: After co-transfection with pcDNA3.1ZEO(-)/CagA and PGL/GP, the activities of luciferase were increased by 251.3, 106.1 and 2.4 times in AGS cells and 35.8, 22.7 and 13.4 times in SGC-7901 cells, respectively, as compared with that of the control, pcDNA3.1 ZEO(-)/CagA + PGL3/Basic and pcDNA3.1 ZEO(-) + PGL/GP groups. The activities of luciferase in PGL/GP transfection and HP infection group were also increased by 1673.2, 33.5, 1.4 times in AGS cells and 1180.2, 72.2 and 1.5 times in SGC-7901 cells, respectively, as compared with that of the control, PGL3/Basic + HP and PGL/GP groups. There were statistically significant differences between them (P < 0.05), which suggested that the transcription activity of gastrin promoter increased significantly. But after adding the inhibitor AG490 and U0126, respectively, the activities of luciferase were significantly decreased by 95.7% (U0126) and 33.0% (AG490) in co-transfected AGS cells and 94.8% (U0126) and 86.2% (AG490) in co-transfected SGC-7901 cells with pcDNA3.1ZEO(-)/CagA and PGL/GP (P < 0.05). In the PGL/GP transfection and HP infection group, the activities of luciferase were significantly decreased by 24.6% (U0126) and 25.8% (AG490) in AGS cells and 57.3% (U0126) and 14.1% (AG490) after adding the inhibitor AG490 and U0126, respectively (P < 0.05). The results showed that the gastrin promoter activities were significantly inhibited. The gastrin mRNA levels were 3.0 and 4.5 times higher in HP-infected AGS and SGC-7901 cells, respectively, than that in the control groups. In the cells transfected with pcDNA3.1ZEO(-)/CagA, the gastrin mRNA levels were raised 10.8 and 2.3 times (AGS cells) and 10.9 and 16.2 times (SGC-7901 cells), respectively, as compared with that of control and pcDNA3.1ZEO(-) groups. All of the differences were statistically significant (P < 0.05). CONCLUSION: These results suggest that CagA may activate the gastrin promoter and up-regulate the expression of gastrin gene, and CagA is one of the important proteins in regulating gastrin gene expression. The ERK/MAPK and JAK/STAT signaling pathways may be involved in the controlling of gastrin gene expression by CagA.

Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro.

Gastrin and cholecystokinin-B receptor (CCK-B) were co-expressed in human gastric carcinoma tissues, suggesting that a functional autocrine loop, the gastrin and CCK-B receptor loop, may be presented in gastric cancer cells and play an important role in the pathogenesis and progression of gastric carcinomas. The present study was aimed at studying the effects of blocking the gastrin and CCK-B receptor loop on cell proliferation and apoptosis in gastric cancer cell line SGC-7901 cells (SGC-7901 cells). First, the expression of gastrin and CCK-B receptor mRNAs and gastrin protein in SGC-7901 cells were measured by RT-PCR and immunocytochemistry, respectively. Radioimmunoassay (RIA) was used to detect the concentrations of gastrin in culture medium. The gastrin-CCK-B receptor axis was blocked by using a specific neutralizing antibody against human gastrin and siRNA specifically targeting human CCK-B receptors, respectively. Flow cytometry was used to measure the cell cycle and apoptotic cells, and western blotting was used to measure the expression of CCK-B receptor, caspase-3, and matrix metalloproteinase-2 (MMP-2) in cells. The results showed that SGC-7901 cells not only coexpressed gastrin and CCK-B receptor mRNAs, but also endogenously secreted gastrin protein into the culture medium, thus forming gastrin-CCK-B receptor autocrine loop. Biologically, disrupting gastrin-CCK-B receptor autocrine loop by neutralizing the endogenous gastrin or by knocking down CCK-B receptor expression significantly inhibited the cell proliferation and decreased the percentage of cells residing in the S-phase of the cell cycle, and meanwhile promoted cell apoptosis and increased caspase-3 expression as well as decreased MMP-2 expression. An autocrine loop between endogenously secreted gastrin and CCK-B receptors may play a key role in the regulation of cell proliferation and apoptosis in SGC-7901 cells.

[Blocking the gastrin/cholecystokinin-B receptor autocrine loop influences the growth and apoptosis of human gastric cancer cells].

OBJECTIVE: To study whether gastrin/cholecystokinin-B (CCK-B) receptor autocrine loop exists in human gastric cancer cells and the effects of gastrin/CCK-B receptor autocrine loop on the growth and apoptosis of human gastric cancer cells. METHODS: Human gastric cancer cells of the line SGC-7901 were cultured. The expression of gastrin and CCK-B was detected by RT-PCR, immunocytochemistry and radioimmunoassay. Antibody against gastrin was added so as to block the autocrine loop to observe the growth rate, cell cycle and apoptosis by 3-(4.5-dimethylthiazol-z-yl) -2, 5-diphenyl tertrazolium blue (MMT) colorimetric assay and flow cytometry (FCM). RESULTS: The SGC-7901 cells co-expressed gastrin and CCK-B receptor mRNAs, confirmed by sequencing, thus forming gastrin/the CCK-B receptor autocrine loop. After the blocking of the autocrine loop by antibody against gastrin, the cell growth rate and the number of cells residing in the S-phase of the cell cycle decreased and the apoptotic rate increased in an antibody concentration-dependent manner. CONCLUSION: Human gastric cancer SGC-7901 cells possess the gastrin/the CCK-B receptor autocrine loop. Blocking the autocrine loop inhibits the cell proliferation and promotes the cell apoptosis.

Coexpression of cholecystokinin-B/gastrin receptor and gastrin gene in human gastric tissues and gastric cancer cell line.

AIM: To compare the expression patterns of cholecystokinin-B (CCK-B)/gastrin receptor genes in matched human gastric carcinoma and adjacent non-neoplastic mucosa of patients with gastric cancer, inflammatory gastric mucosa from patients with gastritis, normal stomachs from 2 autopsied patients and a gastric carcinoma cell line (SGC-7901), and to explore their relationship with progression to malignancy of human gastric carcinomas. METHODS: RT-PCR and sequencing were employed to detect the mRNA expression levels of CCK-B receptor and gastrin gene in specimens from 30 patients with gastric carcinoma and healthy bordering non-cancerous mucosa, 10 gastritis patients and normal stomachs from 2 autopsied patients as well as SGC-7901. The results were semi-quantified by normalizing it to the mRNA level of beta-actin gene using Lab Image software. The sequences were analyzed by BLAST program. RESULTS: CCK-B receptor transcripts were detected in all of human gastric tissues in this study, including normal, inflammatory and malignant tissues and SGC-7901. However, the expression levels of CCK-B receptor in normal gastric tissues were higher than those in other groups (P<0.05), and its expressions did not correlate with the differentiation and metastasis of gastric cancer (P>0.05). On the other hand, gastrin mRNA was detected in SGC-7901 and in specimens obtained from gastric cancer patients (22/30) but not in other gastric tissues, and its expression was highly correlated with the metastases of gastric cancer (P<0.05). CONCLUSION: Human gastric carcinomas and gastric cancer cell line SGC-7901 cells coexpress CCK-B receptor and gastrin mRNA. Gastrin/CCK-B receptor autocrine or paracrine pathway may possibly play an important role in the progression of gastric cancer.