Apoptosis-inducing effect of recombinant Caspase-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901

AIM: To investigate the apoptosis-inducing effect of Caspases-3 expressed by constructed eukaryotic vector on gastric cancer cell line SGC7901.METHODS: PCR was employed to amplify the sequences of both small and large subunits of Caspases-3. Its products were separately cloned into the Sma I site of pBluescript KS+ to generate both plasmids pBS/SS and pBS/LS. The small subunit fragment was excised from plasmid pBS/SS with BarrH- I and then inserted into the BarnH I site of plasmid pBS/LS preceding that of the large subunit to yield plasmid pBS/Rev-Caspase-3. Rev-Caspase-3 cDNA was excised with Kpn Ⅰ+Xba I and then subcloned into plasmid pcDNA3.1(+) to construct Rev-Caspase-3 eukaryotic expression vector pcDNA/Rev-Caspase-3, which was used to transiently transfect SGC7901 cell line. Cell count, MTT assay and electron microscopy were used to confirm the antiproliferation and apoptosis-inducing effect of Rev-Caspase-3 expression on gastric cancer cells.RESULTS: Plasmid pBS/Rev-Caspase-3 and eukaryotic expression vector pcDNA/Rev-Caspase-3 were successfully constructed. SGC7901 cells were transiently transfected by either pcDNA/Rev-Caspase-3 or pcDNA3.1 (+) for 24, 48,72, and 96 h respectively. Cell growth was measured by cell count and MTT assay. In cell count assay, the cell numbers were 1.S×106, 1.55×106, 2.0×106, and 3.1×106 in the experimental group and 2.5×106, 3.1×106, 4.0×106, and 5.7×106 in the control group at 24, 48, 72 and 96 h respectively.The growth of SGC7901 cells was suppressed by RevCaspase-3 in a time-dependent manner (P＜0.05). The results of MTT assay were similar to that of cell count (P＜0.05).The characteristics of apoptosis such as chromatin condensation, crescent formation and margination were seen and more obvious with time in the given-experimental period in the experimental group, but not easily observed in the control group.CONCLUSION: The expression of Rev-Caspase-3 by the constructed eukaryotic vector can significantly induce apoptosis of gastric cancer cell line SGC7901, which may exhibit a potential way in gastric cancer gene therapy.

Development of an oral DNA vaccine against MG7-Ag of gastric cancer using attenuated salmonella typhimurium as carrier

AIM: To develop an oral DNA vaccine against gastric cancer and evaluate its efficacy in mice.METHODS: The genes of the MG7-Ag mimotope and a universal Th epitope (Pan-DR epitope, PADRE) were included in the PCR primers. By PCR, the fusion gene of the two epitopes was amplified. The fusion gene was confirmed by sequencing and was then cloned into pcDNA3.1(+) plasmid. The pcDNA3.1 (+)-MG7/PADRE was used to transfect an attenuated Salrmonella typhimuriurm.C57BL/6 mice were orally immunized with 1x108 cfu Salrmonella transfectants. Salmonella harboring the empty pcDNA3.1(+) plasmid and phosphate buffer saline (PBS)were used as negative controls. At the 6th week, serum titer of MG7-Ag specific antibody was detected by ELtSA.At the 8th week cellular immunity was detected by an unprimed proliferation test of the spleenocytes by using a [3H]-thymidine incorporation assay. Ehrlich ascites carcinoma cells expressing MG7-Ag were used as a model in tumor challenge assay to evaluate the protective effect of the vaccine.RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than that in control groups (0.841 vs 0.347, P＜0.01; 0.841 vs 0.298,P＜0.01), while in vitro unprimed proliferation assay of the spleenocytes showed no statistical difference between those three groups. Two weeks after tumor challenge, 2 in 7 immunized mice were tumor free, while all the mice in the control groups showed tumor formation. CONCLUSION: Oral DNA vaccine against the MG7-Ag momitope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumor in mice, and the vaccine has partially protective effects.

Effect of Hewei-Decoction on chronic atrophic gastritis and eradication of Helicobacter pylori

AIM: To demonstrate the effect of Hewei-Decoction(Decoction for regulating the stomach) on chronic atrophic gastritis (CAG) and eradication of Helicobacter pylori.METHODS: Ninety patients with CAG entering the investigation were divided into six differentiation syndromes, based on their major symptoms and signs.Hewei-Decoction was taken by all the patients orally for 4or 8 wk. The efficacy was assessed by both the composite accumulation of reduced scores of major symptoms and the eradication of H pylori.χ2 test was used to compare the efficacy between Hpylori-positive and negative cases,and to disclose the relationship between efficacy and eradication of H pylori.REStULTS: In patients with six different syndrome types,the efficacy of Hewei-Decoction was 91.67% (11/12),92.86% (13/14), 97.22% (35/36), 87.50% (14/16),75.00% (6/8), 75.00% (3/4) respectively. The rate of highly efficacious was 58.33% (7/12), 50.00% (7/14),77.78% (28/36), 62.50% (10/16), 12.50% (1/8) and25.00% (1/4), respectively. The total efficacy was 91.11%(82/90), and the rate of highly efficacious was 60.00%(54/90). The eradication rate of H pyloriwas 67.86%(38/56). The therapeutic effect of Hewei-Decoction was better in H pylori positive cases than that in Hpylori-negative cases with the total effect of 96.43% vs82.35% (P＜0.05).In 56 H pylori positive cases, the therapeutic effect was better in H pylori eradicated cases than that in H pyloriexistent cases with the total effect of 97.37% vs 72.22%(P＜0.01).CONCLUSION: Hewei-Decoction is effective in most cases of all the syndrome types. The results indicate that eradication of H pylori is one of the important mechanisms for alleviation of symptoms and signs. Also, the decoction is efficacious in H pylori-negative cases.

A microarray-based gastric carcinoma prewarning system

AIM: To develop a microarray-based prewarning system consisting of gastric cancer chip, prewarning data and analysissoftware for early detection of gastric cancer and pre-cancerous lesions.METHODS: Two high-density chips with 8 464 human cDNA sites were used to primarily identify potential genes specific for normal gastric mucosa, pre-cancerous lesion and gastric cancer. The low-density chips, composed of selected genes associated with normal gastric mucosa,precancerous lesion and gastric cancer, were fabricated and used to screen 150 specimens including 60 specimens of gastric cancer, 60 of pre-cancerous tissues and 30 of normal gastric mucosa. CAD software was used to screen out the relevant genes and their critical threshold values of expression levels distinguishing normal mucosa from pre-cancerous lesion and cancer. All data were stored in a computer database to establish a prewarning data library for gastric cancer. Two potential markers brcaa1 and ndr1were identified by Western blot and immunohistochemistry.RESULTS: A total of 412 genes associated with three stages of gastric cancer development were identified.There were 216 genes displaying higher expression in gastric cancer, 85 genes displaying higher expression in pre-cancerous lesion and 88 genes displaying higher expression in normal gastric mucosa. Also 15 genes associated with metastasis of gastric cancer and 8 genes associated with risk factors were screened out for target genes of diagnosis chip of early gastric cancer. The threshold values of 412 selected genes to distinguish gastric cancer, pre-cancerous lesion from normal gastric mucosa were defined as 6.01±2.40, 4.86±1.94 and 5.42±2.17, respectively. These selected 412 genes and critical threshold values were compiled into an analysis software, which can automatically provide reports by analyzing the results of 412 genes obtained by examining gastric tissues. All data were compiled into a prewarning database for gastric cancer by CGO software. Northern blot and immunohistochemistry analysis confirmed that gene and protein of brcaa1 displayed lower expression in normal gastric mucosa and higher expression in gastric cancer tissues, conversely, ndr1 displayed lower expression in gastric cancer and higher expression in normal gastric mucosa.CONCLUSION: The microarray-based prewarning system for gastric cancer was developed. This system consisted of gastric cancer-associated gene chip, prewarning data and analysis software, which has a high potential for applications in the early detection of gastric cancer. The two potential markers brcaa1 and ndr1 identified may be used to distinguish cancer status fand non-cancer status.

Regulation of apoptosis by the papillomavirus E6 oncogene

Infection with human papillomaviruses is strongly associated with the development of multiple cancers including esophageal squamous cell carcinoma. The HPV E6 gene is essential for the oncogenic potential of HPV.The recgulation of apoptosis by oncogene has been relatel to carcinogenesis closely; therefore, the modulation of E6 on cellular apoptosis has become a hot research topic recently. Inactivation of the pro-apoptotic tumor suppressor p53 by E6 is an important mechanism by which E6promotes cell growth; it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis,numerous studies showed that E6 could in fact sensitize cells to apoptosis. The molecular basis for apoptosis modulation by E6 is poorly understood. In this article, we will present an overview of observations and current understanding of molecular basis for E6-induced apoptosis.

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGbl to screen a phage-displayed random peptide library fused with coat protein plII in order to get some information on mimotopes.lV^37BODS: Through affinity enrichment and EUSA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGbl-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGbl-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0±1.6) %to (39.0±2.7) %.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope

AIM:To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori ( H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of Hpyloriflagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K^2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum Hpylori-specific IgA and IgGl increased significantly in immunized group, while IgG2a only had an insignificant change. Hpylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.

Chinese literature associated with diagnosis of Helicobacter pylori

AIM:To synthetically analyze and probe into the diagnosis of H pyloriinfection,we followed the principles of evidencebased medicine.METHODS:A total of 22 papers of prevalence survey and case-control studies were selected for studying about diadynamic methods.Using meta-analysis, we analyzed the different diadynamic methods of H pyloriin China.RESULTS: Through meta-analysis,among the five diadynamic methods,the accuracy of polymerase chain reaction (PCR) was the highest (98.47%) and PCR was the most sensitive method (Sp:99.03%).CONCLUSION:Among the five diadynamic methods, the accuracy of PCR is the highest and PCR is the most sensitive method to diagnose the infection of Hpylori.