Correlative Study Of The Fucosylated Oligosaccharide Lewis Y Antigen With Its Synthesis Enzyme Fucosyltransferase â…£ And Helicobacrter Pylori In Gastric Cancer

I. BackgroundGastric cancer is one of the most common malignant tumors in the world, representing the third leading cause for cancer-related death in men and the fifth leading cause in women. Approximately two-thirds of gastric cancer cases were diagnosed in developing countries, such as Japan, China, South America, Eastern Europe, and Middle East. China has high prevalence of gastric cancer and alone accounts42%of all gastric cancer cases worldwide. Helicobacter pylori (H. pylori), a major causative agent for the development of chronic gastritis, gastric ulcer and gastric cancer. It is gram-negative, microaerophillic, flagellated and spiral-shaped bacilli. More than50%of the world population is colonized by H. pylori, but few of them suffer from active disease. Developing countries had a prevalence rate of (80%) as compared to developed countries (20-50%). H. pylori cytotoxin associated antigen A (CagA) is a first bacterial oncoprotein, plays a significant role in the development of gastric cancer. It is128kDa protein encoded by a40kb cag-pathogenicity island (cag PAI) of H. pylori. It is translocated by the type IV secretion system into gastric epithelial cells, where it become phosphorylated and contributed to cell scattering and H. pylori-associated diseases.Gastric cancer cells have the characteristic of abnormal glycosylation at cell surface, such as changes observed in ABH and Lewis related histo-blood group antigens. Fucose is one of the constituents to participate in the synthesis of carbohydrate structure of some essential growth factor receptors, which play a critical function in the development of gastric cancer. Fucosylation has important roles in cancer biology, as increased fucosylation levels of glycoproteins and glycolipids have been reported in many cancers, including breast, prostate, lung, and gastric cancer. Fucosyltransferase Ⅳ (FUT4) is the key enzyme for the synthesis of Lewis Y (LeY) carried by the glycoproteins and glycolipids on the cell membrane. LeY is a difucosylated oligosaccharide, which is highly expressed in60-90%of human epithelial cancers cells. Glycosylation of EGFR plays an important function in binding of EGFR with its ligands, lead to stimulate cancer cell proliferation. H. pylori infection deregulates the epidermal growth factor receptor (EGFR) activation and modifies cell proliferation, differentiation and apoptosis of gastric epithelial cells.Gastric cancer has high mortality rate due to lack of precise and specific clinical biomarker for gastric cancer diagnosis and effective treatment. China has high incidence of gastric cancer with the leading cause of cancer deaths. Although the gastric cancer treatment has been improved in recent years, it still has high metastasis and death rate. It is the utmost important to identify potential new biomarker targets for diagnosis and effective treatment for H. pylori infection as well as prevention from gastric cancer. Celecoxib (COX-2inhibitor) inhibits gastric cancer cell proliferation and downregulated the cyclooxygenase-2(COX-2) expression. COX-2is highly expresses in different types of cancer, including gastric cancer. H. pylori deregulates the cell proliferation and apoptosis with the overexpression of COX-2in gastric cancer. The treatment efficacy of celecoxib is low, which limits their applications and effectiveness in gastric cancer treatment. It is important to develop a better strategy to improve the efficacy of celecoxib. Anti-LeY antibody is widely used to treat different types of cancer. Hence, Combinational therapy of celecoxib and anti-LeY antibody might be the better strategy to effectively treat gastric cancer.Ginsenoside Rg3is a Chinese herbal medicine that inhibits the cell proliferation and induces apoptotic in cancers cells. FUT4is regulated by the SP1and HSF1transcription factors, which is an essential enzyme that catalyzes the synthesis of LeY oligosaccharides. Based on our results gathered from previous study, we hypothesized that Rg3induced gastric cancer cell apoptosis with the downregulation of FUT4expression via SP1and HSF1regulation. The role of H. pylori CagA on fucosylation in gastric cancer has not been studied. In this study, we analyzed the role of CagA to promote the fucosylation with the stimulation of cell proliferation. Moreover, investigated the therapeutic role of celecoxib plus anti-LeY antibody and Rg3in the inhibition of cell proliferation, as well as induction of apoptosis in gastric cancer.Ⅱ. Objectives1. We investigated the potential correlation of H. pylori CagA on EGFR activation with FUT4and LeY in gastritis, gastric ulcer, and gastric cancer tissue and serum samples. Furthermore, determine the stimulatory effect H. pylori CagA on the cell proliferation through FUT4fucosylation and Lewis blood group antigens in gastric cancer.2. We analyzed the H. pylori correlation with cyclooxygenase-2(COX-2), LeY and gastric markers (CA724, and GRN) in gastric patient’s tissue and serum samples. Moreover, we determine the combinational therapeutic effect of celecoxib with anti-LeY antibody to inhibit the gastric cancer cell proliferation.3. The therapeutic effect of Rg3were determined to induce apoptosis by inhibiting FUT4expression through SP1and HSF1regulation in CagA treated gastric cancer cells.Ⅲ. MethodsThe potential correlation of CagA with pEGFR, FUT4, LeY and H. pylori with cyclooxygenase-2(COX-2), LeY, gastric markers (CA724, and GRN) gastritis, gastric ulcer, and gastric cancer tissue and serum samples were determined by immunohistochemistry and (IHC) and enzyme-linked immunosorbent assay (ELISA). Furthermore, the effect of H. pylori CagA on the gastric cell proliferation was studied by using gastric cancer cell line (SGC-7901) and analyzed by Western blot, Immunostaining, ELISA and flow cytometry assay. The therapeutic effect of celecoxib and its combination with anti-LeY antibody on gastric cancer cell proliferation were determined by using primary gastric cancer cells. We developed the primary gastric cancer cells culture by using fresh surgical tissues and confirm its pure epithelial growth by PAS staining and immunocyto staining of cytokeratin-18, cytokeratin-19markers. The primary gastric cancer cells were treated with celecoxib, anti-LeY antibody, or the combination, and analyzed their antitumor efficacy on gastric cancer cell proliferation along with the expression level of COX-2, CA724, granulin (GRN) by Western blot, flow cytometry and ELISA. To analyze the Rg3mechanism to induce gastric cancer cells apoptosis, we treated the gastric cancer cells with H. pylori CagA and followed by the Rg3(50μg/ml), and analyzed their ability to induce apoptosis by evaluating the role of FUT4, as well as SP1and HSF1expression by Western blot, flow cytometry and ELISA.IV. Results(a) Effect of H. pylori CagA on cell proliferation and fucosylation. We analyzed the potential correlation among gastritis, gastric ulcer and gastric cancer by IHC and ELISA. We found a weak expression of CagA (0.644), p-EGFR (0.856), FUT4(0.949) and LeY (0.687) chronic gastritis by Newman-Keuls multiple test. However, gastric cancer showed significantly high expression levels of CagA (1.790), p-EGFR (1.533), FUT4(2.146) and LeY (1.695)(P<0.0001) as compared to gastritis and gastric ulcer by IHC. Moreover, H. pylori CagA showed significant correlation with p-EGFR (R—0.624), FUT4(R—0.814) and LeY(R—0.822)(P<0.0001). Serological analysis of gastric blood sample by ELISA demonstrated the consistent results with high expression of CagA (1.660), p-EGFR (1.587), FUT4(1.662) and LeY (1.610) in gastric cancer. The effect of H. pylori CagA on gastric cell proliferation showed significant increase in cell proliferation in a dose-dependent manner by trypan blue exclusion and CCK8cell proliferation assay (P<0.01). We found high expression of PCNA in CagA treated cells by semi-quantitative RT-PCR, Western blot, flow cytometry assay and immunocytochemical staining (P<0.01). The elevated EGFR phosphorylation was found in CagA treated cells as compared with that of the untreated cells. Moreover, treatment of cells with EGFR inhibitor (Tyrphostin AG1478,10-4M) followed by CagA treatment, inhibited the CagA-activated EGFR phosphorylation. The phosphorylation of (Extracellular signal-regulated kinases) ERK1/2and P38was found to be activated in CagA treated cells. Cells were pretreated with an ERK inhibitor (10"5M PD98059) and p38inhibitor (10-6M SB203580) followed by CagA were resulted in the inhibition of CagA activated phosphorylation of ERK1/and P38. The CagA effect on FuT4fucosylation showed the significant high expression of FUT4(P<0.01) in a dose-dependent manner in CagA treated cells. In FUT4siRNA transfection, results showed the significant decreased expression of FUT4in CagA treated cells. The LeY level was found to be significantly up regulated in a dose-dependent manner with the treatment of H. pylori CagA (P<0.01). However, FUT4siRNA transfection was results in the significant decreased of LeY level in CagA treated cells. We also found significant effect of CagA on LeB, LeX and sLeX as compared to LeA expression level.(b) Combinational therapeutic effects of celecoxib and anti-Lewis Y antibody to inhibit cell proliferation and FUT4fucosylationThe established primary gastric cell was found to be epithelial and mucin secreting cells growth. The characterization of gastric cancer cells from gastric normal cells were demonstrated considerable variation between normal and cancer cells by CA724and GRN expression. PCNA expression showed evidence of high cell proliferation in gastric cancer culture cells. We analyzed the expression level of H. pylori, COX-2, CA724and GRN by IHC and ELISA in human gastric tissue and blood samples of gastritis, ulcer and gastric cancer. The comparative study of human gastric tissue were showed high expression of H. pylori (1.598), COX-2(1.498), CA724(1.653) and GRN (2.022) in gastric cancer as compared to gastritis and gastric ulcer by Newman-Keuls multiple test (ANOVA). Moreover, we found a significant correlation of H. pylori with LeY (R—0.861), COX-2(R—0.552), CA724(R—0.714) and GRN (R—0.664)(P<0.0001). Serological titer values of blood samples also showed the high expression of H. pylori (1.890), COX-2(1.763), CA724(1,734) and GRN (1.913) in gastric cancer blood samples. We found significant inhibitory effect of the celecoxib on FUT1, FUT4and LeY antigen level by Western blot and flow cytometry assay (P<0.01). In contrast, the cells treated with anti-LeY antibody showed low expression of H. pylori and their virulent proteins, heat shock protein (HSP), urease A (UreA), urease B (UreB), cytotoxin associated antigen (CagA) and vacuolating cytotoxin A (VacA), as compared to untreated cells (P<0.01). The combinational therapy of celecoxib and anti-LeY antibody showed significant low COX-2expression through MAPKs/COX-2signaling pathway (P<0.01) as compared to cells treated alone with celecoxib or anti-LeY antibody (P<0.05). The phosphorylation of ERK1/2and p38were found to be significantly inhibited in combinational treated cells as compared to the cells treated alone (P<0.01). Combinational treated cell together with an ERK inhibitor (10-5M PD98059) and p38inhibitor (10-6M SB203580) were resulted in the significant inhibition of ERK1/2and p38phosphorylation, as compared to cells treated alone (P<0.01). Additionally, the combination therapy leads to impressive inhibition of gastric cancer cell proliferation, with decreased expression of CA724and GRN (P<0.01).(c) Therapeutic effects of Rg3to induce apoptosis with the FUT4downregulation in gastric cancerThe therapeutic effect of Rg3showed significant stimulation of apoptosis in CagA treated gastric cancer cells by Western blotting assays, Annexin V-PI staining and nucleus condensation Hoechst staining (P<0.01). Additionally, Rg3-induced apoptosis with the activation of caspase-3,-8,-9and the PARP cleavage. Rg3effects on fucosylation showed significant downregulation of FUT4expression in gastric cancer cells (P<0.01). The expression level of SP1and HSFl transcription factor was examined in gastric cancer cells by Western blot, flow cytometry assay and immunofluorescent staining. The results showed the significant inhibitory and stimulatory effect of H. pylori CagA on SP1and HSF1expressions in gastric cancer cells, respectively. To analyze the therapeutic effect of Rg3on SP1and HSF1transcription factors, we treated gastric cancer cells with Rg3, and analyzed by Western blot. We found that, the Rg3significantly stimulate the SP1expression in CagA treated gastric cancer cells treated, as compare to CagA untreated cells (P<0.01), while the HSF1expression was significantly reduced by Rg3in CagA treated cancer cells (P<0.01). Moreover, cells were pretreated with an SP1inhibitor (10-10M Mithramycin A), HSFl inhibitor (10-6M KRIBB11) and FUT4siRNA, followed by the Rg3treatment. Treatments with the SP1inhibitor were resulted in the up-regulation of FUT4expression, while HSF1and FUT4inhibitors treatment showed low FUT4expression in CagA treated cells (P<0.01). The mechanism involved in Rg3-induced apoptosis showed that Rg3significantly induce the apoptosis in gastric cancer cells. Furthermore, treatments with the SP1inhibitor were resulted in the inactivation of caspase-3,-8,-9and the PARP, while HSF1and FUT4inhibitors treatment showed high caspase-3,-8,-9and the cleavage-PARP activation in CagA and untreated cells (P<0.01).(V) Conclusionsa) We suggested that gastric cancer have high expression of H. pylori CagA, pEGFR, FUT4and LeY compared to gastritis and ulcer, as well as significant correlation between them. Moreover, H. pylori CagA plays a key role in gastric cancer with overexpression of FUT4, LeY through EGFR/MAPKs signaling pathway.b) Anti-LeY antibody enhances the antitumor activity of celecoxib to inhibit gastric cancer cell proliferation by targeting COX-2and LeY antigen through downregulation of MAPKs/COX-2signal pathway, which might be a new feasible way for gastric cancer therapy.c) Rg3significantly induces the gastric cancer cell apoptosis by inhibiting FUT4expression via SP1and HSF1transcription regulation. Hence, SP1and HSF1could be the specific therapeutic target to treat gastric cancer.d) We proposed the FUT4/LeY, COX-2/LeY and SP1/HSF1are significant source of correlative gastric biomarkers for the diagnosis and treatment of gastric cancer.