Identification And Function Analysis Of Gastric Cancer Associated Antigen-MG7

Gastric cancer is one of the most prevalent malignancies worldwide, and is the second cause of tumor-related death. In gastric cancer researches,early diagnosis and treatment is of the most importance. Few overt symptoms are shown at the early stage of gastric cancer, while most of the diagnosis are made at the advanced stage with poor prognosis. The low sensitivity and specificity of many available cancer markers, including carcinoembryonic antigen (CEA), CA 50,etc. have hampered their use in detecting gastric carcinoma. Therefore, it is vital to seek new gastric cancer antigen at the histological, cytological and serological levels for diagnosis and treatment of gastric cancer.A series of gastrointestinal cancer specific mAb named MG series were discovered in our lab by Prof. Fan Danming in 1980s. Among these specific antibodies, MG7 showed the highest specificity and sensitivity. Further studies of MG7, including the effects of MG7 in diagnosis, evaluation of prognosis, dynamic follow-up study and targeting therapy of gastric cancer, as well as preparation of gastric cancer specific nanovaccine, et al., have been carried out by medical experts in and out of our lab, with dozens of papers subscribed in both domestic and foreign journals.However, up to now, little is known about MG7-Ag, which has limited the international generalization of MG7-Ab and hindered further researches. The scientists in the Hong Kong Polytechnic University have identified MG7-Ag as hnRNPA2/B1, the characteritics of which are different from that of MG7-Ag in our prevous resrarch.In the present study we used immunoprecipitation, 2-D PAGE, mass spectrometry and other methodes to identify MG7-Ag. The discovery of new gastric cancer antigens or proteins may greatly facilitate the studies of molecular mechanisms of gastric cancer.Besides, attempts were also made in our study to establish a convenient serologic method for gastric cancer screening in the high risk population. The studies of MG7 mAb effect on gastric cancer cell apoptosis may help to find new methods in clinical pharmacal therapies.In conclusion, our study has deepgoing theoretical values and good clinical prospects.【Objectives】(1) To investigate the expression or distribution of MG7-Ag in different gastric mucosal tissues and the relationship between expression intensity of MG7-Ag with the clinicopathologic characteristics of gastric cancer; (2) To examine the expression difference between MG7-Ag and hnRNPA2/B1 protein; (3) To identify MG7-Ag; (4) To establish a convenient serologic method for early diagnosis of gastric cancer by detecting MG7-Ag;(5) To investigate the effects of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell line.【Methods】(1) MG7 hybridoma cells were resuscitated and cultured for MG7-Ab preparation. (2)The expression or distribution characteristics of MG7-Ag in different gastric mucosal tissues were determined by immunohistochemistry assay, and the relationship between MG7-Ag expression intensity with the clinicopathologic characteristics were eveluated. (3)The expression difference between MG7-Ag and hnRNPA2/B1 protein in gastric cancer tissue was investigated by immunohistochemistry assay. (4)The expression difference between MG7-Ag and hnRNPA2/B1 protein in gastric cancer cells was detected by immunocytochemistry assay and laser scanning confocal microscope. (5)The expression difference between MG7-Ag and hnRNPA2/B1 protein in gastric cancer cells was detected by Western blot. (6) MG7-Ag identification was carried out by Immunoprecipitation, Western blot,2-D PAGE and mass spectrometry.(7) Enzyme-linked immunoadsorption assay was used to establish a convenient serologic method for early diagnosis of gastric cancer by detecting MG7-Ag. (8) The convenient serologic method for early diagnosis of gastric cancer by detecting MG7-Ag was applied in detection of gastric cancer, precancerous lesion and other tumors. (9) The effects of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell line were evaluated by flow cytometry.【Results】1. The expression or distribution characteristics of MG7-Ag in different gastric mucosal tissues and the relationship between MG7-Ag expression intensity with the clinicopathologic characteristics of gastric cancer. We detected the expression and distribution of MG7-Ag by immunohistochemistry analysis in different gastric mucosal tissues(gastric cancer specimens,116; superficial gastritis,intestinal metaplasia, atypical hyperplasia and atrophic gastritis specimens, 84) and we found that the positive expression rates of MG7-Ag gradually increased in superficial gastritis, intestinal metaplasia,atypical hyperplasia and gastric cancer (P<0. 01). It indicated that the expression of MG7-Ag in gastric cancer mainly located in the upper part of kytoplasm or cell membrane in glandular epithelium cells and gastric gland intracavity. The MG7-Ag staining was positive in most of the basal epithelial cells of gastric mucosa gland, and the positive rate decreased gradually towards the top, but the MG7-Ag expression on the top cavosurface epithelial cells was mostly positive. The MG7-Ag expression was closely related with tissue differentiation and clinical phases, but not with age or sex.2. Significant difference exists between the expression of MG7-Ag and hnRNPA2/B1 protein in gastric cancer cellsImmunohistochemistry was carried out to determine the expression of MG7-Ag and hnRNPA2/B1 on the same paraffin-embedded gastric cancer section, and the results showed that MG7-Ag expressed mainly on the cell membrane, and little in the nucleus.In contrast, the expression of hnRNPA2/B1 mainly distributed in the cell nucleus. Immunocytochemistry and laser scanning confocal microscope were carried out to detect the expression of MG7-Ag and hnRNPA2/B1 in gastric cancer cell lines SGC7901 with MKN45.The results showed that the former mainly distributed on the cell membrane and little in the nucleus, while the latter mainly distributed in the cell nucleus. Western blot carried out on gastric cancer cell line MKN45 showed that two strips with Mr of 100 kDa and 120 kDa were detected by MG7 antibody, compared with another two strips with Mr of 34 kDa and 37 kDa detected by hnRNPA2/B1 antibody.3. The identification of MG7-AgMG7-Ab was crosslinked to gastric cancer cell line SGC7901, MKN45, and gastric cancer tissue respectively by immunoprecipitation technique, and 1-D or 2-D polyacrylamide gel electrophoresis was proceeded. Protein spots, which were recognized and matched to those detected on the Western blot, were excised from a sliver stained gel or a coomassie blue stained gel and transferred to microcentrifuge tubes for mass spectrometry analysis.The two strips with Mr of 100 kDa were excised from a sliver stained gel by 1-D polyacrylamide gel electrophoresis, which were immunoprecipitate of gastric cancer tissue and MG7-Ab, immunoprecipitate of gastric cancer cell line MKN45 and MG7-Ab, respectively. Both of the strips were identified as anti-colorectal carcinoma heavy chain by LC-MS/MS analysis. The two strips with Mr of 100 kDa were excised from a coomassie blue stained gel by 1-D polyacrylamide gel electrophoresis, which were immunoprecipitate of gastric cancer cell line MKN45 and MG7-Ab, immunoprecipitate of gastric cancer cell line SGC7901 and MG7-Ab, respectively. The both strips were identified as anti-human CD19 monoclonal antibody 4G7 immunoglobulin gamma1 heavy chain by ESI-MS/MS analysis.The strip with Mr of 100 kDa was excised from a coomassie blue stained gel by 2-D polyacrylamide gel electrophoresis, which was immunoprecipitate of gastric cancer tissue and MG7-Ab.The strip was identified as IGHG3 protein by MALDI-TOF/TOF analysis. MG7-Ag was detected using IgG antibody (anti-human) by Western blot, which indicated that MG7-Ag is Ig heavy chain- like molecular.4. The development application of the serologic method in early diagnosis of gastric cancer by detecting MG7-AgUsing proper coating condition and antibody concentration, indirect BA-ELISA (biotin-avidin ELISA) was carried out before and after the operation in patients with gastric cancer and patients with precancerous lesions or other tumor groups to detect MG7-Ag in sera. PBS (0.01M) was taken as the negative control. A sample was considered"positive"if its absorbance was 2.1-fold of or higher than the negative value. The positive rate of gastric cancer-related MG7-Ag determined by ELISA was 83.6% in preoperative gastric cancer patients (97 of 116),which is higher than that of postoperative patients(63, 47.6%), precancerous lesions patients(78,12.8%), and other tumor groups, respectively (P <0.01). MG7-Ag can also be detected in sera of patients with other tumors (45.2% for lung cancer, 45.5% for rectal cancer, 17.6% for colonic cancer, and 14.2% for breast cancer), but no MG7-Ag was detected in sera of patients with endometrial cancer, lymphoma, or acute leukemia. The expression level of circulating MG7-Ag was significantly associated with TNM stage (P < 0.01) and lymph node metastasis (N0, 30, 73.3%;≥N1, 86,95.3%,P < 0.01). The positive rate of circulating MG7-Ag in stageⅠ(6, 50%),Ⅱ(46, 84.8%),Ⅲ(62, 96.8%) andⅣ(2, 100%)increased gradually. The above results indicated that MG7-Ag was a relatively specific marker in gastric cancer, and the expression of MG7-Ag was positive gradually along with the advancement of gastric cancer. The indirect BA-ELISA (biotin-avidin ELISA) method carried out to detect MG7-Ag in sera of patients may be taken as a convenient method for early diagnosis of gastric cancer.5. The effect of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell lineFlow cytometry was applied to detect the effect of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell line KATOⅢ.The results showed that MG7 monoclonal antibody would enhance the early apoptosis in human gastric cancer cell(P<0.05) and induce cell cycle arrest at G1 phrase remarkably.【Conclusions】In conclusion, the expression of MG7-Ag was statistically correlated with the differentiation level (P <0.01) and pathological stage (P <0.01) of gastric cancer. It indicated that MG7-Ag may play an important role in gastric cancer carcinogenesis. There was significant difference between the expression of MG7-Ag and hnRNPA2/B1 protein in gastric cancer cells. It indicated that MG7-Ag and hnRNPA2/B1 were not the same. MG7-Ag may be Ig heavy chain- like molecular. A serologic method for early diagnosis of gastric cancer was established by enzyme-linked immunoadsordent assay, with promising values in clinical use. MG7 monoclonal antibody would enhance the early apoptosis in human gastric cancer cells (P<0.05) and may induce cell cycle arrest at G1 phrase remarkably.