| 【Background】Gastric cancer is one of the most common cancers in our country, and thediagnosis and treatment of gastric cancer at the early stage is beneficial for theimprovement in survival rate. Tumor biomarkers are of great value in the earlydiagnosis of gastric cancer, but the specificity and sensitivity of most traditionalbiomarkers need to be further improved. MGd1is one of previously preparedmonoclonal antibodies against gastric cancer by our lab. In previous study, itsantigen, MGd1-Ag, was found to be expressed in gastric cancer at a relativelyhigher level. The serum level of MGd1-Ag was also proved to be helpful ingastric cancer diagnosis. Meanwhile, in this study, MGd1was showed to inhibitthe proliferation of gastric cancer cells by inducing apoptosis in vitro. Based onthe characteristics of MGd1-Ag in gastric cancer, we assumed that MGd1-Agcould serve as a novel candidate target in the diagnosis and treatment of gastriccancer. 【Objectives】This study was aimed to investigate the expression pattern and intensity ofMGd1-Ag in normal and neoplastic tissues of multiple organs. The expression ofMGd1-Ag was then analyzed combined with the clinical features of patients withgastric cancer. Furthermore, the nature of MGd1-Ag was to be identified throughproteomics researches and finally the effects of MGd1antibody on cancer cellswere to be studied in vitro.【Methods】1. The distribution of MGd1-Ag was studied by immunohistochemistry innormal and neoplastic tissues of multiple organs.2. The expression pattern and density of MGd1-Ag were observed in normalgastric mucosa, superficial gastritis, intestinal metaplasia, gastric cancer andlymph node metastatic foci, respectively.3. The correlation of MGd1-Ag and clinical parameters of gastric cancerpatients was analyzed, and the role of MGd1-Ag in predicting the prognosis ofgastric cancer was also studied.4. MGd1-Ag expression in gastric cancer cell lines was detected by Westernblot. Gastric cancer cell lines including SGC7901and KATOⅢwere selected tobe the model for identification of MGd1-Ag. The identification of MGd1-Ag iscarried out via immunoprecipitation and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.5. Laser scanning confocal microscope was used to confirm the localizationof MGd1-Ag in gastric cancer cells, and flow cytometry was used to analyze theratio of MGd1-Ag positive gastric cancer cells. The in vitro cytocidal effect ofMGd1to gastric cells was evaluated by MTT and the morphology changes of thecells after treated with MGd1were observed by microscope. The apoptosisinduced by MGd1was analyzed or detected by flow cytometry assay and High Contend Screening System, and the apoptosis-related molecules were evaluatedby Western blot.【Results】1. Among33normal tisses, Strong staining of MGd1-Ag was found only innormal lung tissue, and moderate expression of MGd1-Ag was observed inhematopoietic cells, esophageal squamous epithelial surface, small part of colonicepithelium, mucus glands of the salivary glands, basal cell of skin and fewglandular lumens of breast tissue. Among cancers from18organs, gastric,colorectal and lung cancers showed abundant distribution of MGd1-Ag.Moderate density of MGd1-Ag was also found in hepatobiliary cancer, pancreaticductal adenocarcinoma and uterine squamous cell carcinoma. Only weakexpression of MGd1-Ag was observed in liver cancer, sebaceous adenoma andseminoma.2. No immunohistochemical signal of MGd1-Ag was detected in normalgastric or superficial gastritis tissues, but it was positive in several cases ofintestinal metaplasia and atypical hyperplasia tissues. The expression density ofMGd1-Ag was significantly higher in gastric cancer of such situations includingpoor differentiation, advanced TNM staging and lymph node metastasis.However, no correlation was indicated between MGd1-Ag expression and genderor age of the patients. The total positive rate of MGd1-Ag was81.8%in gastriccancers. Kaplan-Meier analysis and Log-rank test showed that MGd1-Agexpression was correlated with prognosis of gastric cancer patients, and increasedMGd1-Ag expression in gastric cancer patients associated with poor outcomes.3. Western blot showed that MGd1-Ag was abundantly present in severalgastric cancer cell lines, but absent in GES, an immortalized gastric epithelialcellline. 4. MGd1-Ag was enriched by immunoprecipitation from SCG7901orKATOⅢ cell lysate. A95kD protein band was detected by silver staining andWestern blot analysis. After Coomassiae billiant blue G250staining, this bandwas cut for mass spectrometry analysis. The result indicated that Isoform1ofTNFRSF1B or ZNF185could be the candidate for MGd1-Ag.5. MGd1-Ag was mainly localized on the membrane of gastric cancer cellsdetermined by Laser scanning confocal microscope. And flow cytometry analysisshowed that the ratio of MGd1-Ag positive gastric cancer cells was more than95%. MGd1antibody inhibited the proliferation of SGC7901and AGS in vitrowith a concentration of100μg/ml or more. The inhibitory effect of MGd1togastric cells was dose-dependent with the peak at the concentration of400μg/ml.Flow cytometry and High Contend Screening assay showed that this phenomenonwas possibly due to the increased apoptosis induced by MGd1. and Western blotshowed up-regulation of Bcl-2and Cleaved capase3and down-regulation of Baxafter MGd1treatment.【Conclusion】MGd1-Ag was specifically expressed in tumors of gastrointestinal tract. Itwas increased in intestinal metaplasia and atypical hyperplasia, and, in gastriccancer, was correlated with grades of differentiation, TNM staging and lymphnode metastasis. MGd1-Ag expression was also correlated with prognosis ofgastric cancer patients, and increased MGd1-Ag expression in gastric cancerpatients associated with poor outcomes. Isoform1of TNFRSF1B or ZNF185might be MGd1-Ag molecule. MGd1dose-dependently inhibited the proliferationof gastric cancer cells in vitro by inducing apoptosis. Taken together, MGd1-Agis of great potential to be considered as a novel biomarker and therapeutic targetfor gastric cancer. |
PDF Full Text Request