The Primary Study Of The Relationship Between Tyrosine Protein Kinase And Salivary Adenoid Cystic Carcinoma

ObjectiveSalivary adenoid cystic carcinoma is one of the most common maxillofacial malignant tumors. It is 9.95% of all salivary tumors,24% of malignant salivary tumors, and the second of malignant salivary tumors. Adenoid cystic carcinoma is a kind of infiltrative tumor,the rate of distant metastasis is very high. Early metastasis can occur, and lung is the main part involved. It does harm to the health of patients. Recent years,research about tyrosine protein kinase(TPK) is a focus. Most growth factor receptors exhibit tyrosine kinase activities, they are auto-phosphorylated when combined with growth factor ligands, TPK is activated and growth stimulative signals is transducted into cells. TPK is not only involved in the transduction of extracellular signals such as hormone and growth factors, it is also involved in the cellular malignant transformation and proliferation.The mechanism of salivary adenoid cystic carcinoma carcinogenesis is still unknown now. In clinic , there are few potent drugs to treat it. In this experiment , we study the expressions of EGFR and ErbB - 2 in tissues and two cell lines of salivary adenoid cystic carcinoma using immunohistochemical and western blot methods, and discuss the relationship of EGFR, ErbB - 2 and adenoid cystic carcinoma carcinogenesis. In the experiment, in order to discuss the relationship of TPK and adenoid cystic carcinoma, genistein, an inhibitor of TPK, is used to treat SACC - 83 cells, and we investigate its antiproliferative effects in vitro and the influences of genistein to cell cycles, and measure the expressions of apoptosis — related genes and cyclins. These experiments provide new insights in mechanism of salivary adenoid cystic carcinoma carcinogenesis and its treat-ments in clinic.Method1. Cell cultureSACC -83 and SACC - LM cells were cultured with RPMI -1640 medium containing 10% fetal bovine serum in an incubator at 37℃ and a humidified 5% CO₂ atmosphere.2. MTT assayThe cells were seeded at a density of 0. 5 x 10⁴/well in a 96 well culture dish. After 24 h,the cells were treated with 0 - 220μmol/l genistein . The culture medium was replaced with fresh medium and genistein at third day of treatment. After cells were treated with genistein for 1 to 6 days,MTT(5mg/ml) was added into the dish with 20μl/well. The cells were incubated for 4 h with MTT followed by DMSO added,then the OD values were detected at 492nm.Survival rate = OD value of treatment group/ OD value of control group × 100%3. Phase contrast microscope was used to observe the status of cell growth and the morphological changes in each group.4. Flow cytometry for detecting cell cycleCells were serum - starved for 24 hours followed by treated with 220μmol/ 1 genistein for 24,48 and 72 hours(treated with 0. 2% DMSO for 72 hours as control). Cells were harvested by trypsinization, washed twice with PBS, then stained for 20 min at 4℃ with PI while protected from light. The samples were then analysed on a FACscan.5. AnnexinV/PI staining for detecting apoptosisCells were collected by centrifugation. Wash cells twice with cold PBS and then resuspend cells in 1 × binding buffer at a concentration of 1 × 10⁶ cells/ml. Transfer 100μl of the solution(1 × 10⁵ cells) to a 5ml culture tube. Add 5μl of Annexin V - FITC and 5μl of PI,gently vortex the cells and incubate for 15 min at room temperature in the dark. Add 400μl of 1 x binding buffer to each tube, analyze by flow cytometry within one hour.6. Western blot analysisThe protein concentration was determined using protein assay reagents. E-qual amounts of protein were separated by SDS - polyacrylamide gel electropho-resis. The gel was then transferred to a nitrocellulose membrane for 2 hours 50V, the membranes were blocked overnight at 4℃ with 5% BSA. The primary antibody (1:500) was applied to the membrane for 2 hours at room temperature, (a) after incubation with a HRP - labeled secondary antibody, immunocomplex was visualized by the ECL detection system. (b) after incubation with a alkaline phosphatase - labeled secondary antibody, immunocomplex was visualized with the staining agents.7. Immumohistochemical methodThere are 54 cases of SACC and 9 cases of normal salivary gland tissues. The samples were fixed by 10% formaldehyde solution,and 3μm paraffin sections were made. The sections were stained by streptavidin — biotin peroxidase method.Result一. The antiproliferative effect of TPK inhibitor genistein on salivary adenoid cystic carcinoma SACC - 83 cell line in vitro1. When treated with genistein of certain concentration for a certain timing , SACC - 83 cell growths were significantly inhibited. With enlargement of concentration of genistein and elongation of action time, the inhibitory effects increase2. With enlargement of concentration of genistein and elongation of action time, SACC - 83 cells treated with genistein showed changes in morphology. The volume of cells was decreased, and suspended cells increased.3. Compared with the control group,the percentage of G₀/G₁ phase of cells treated with genistein decreased and that of S phase and G₂/M phase increased. Treated with 220μmol/l genistein for 72 h, SACC - 83 cell growths were arrested in G₂/M phase( P <0.01).4. Genistein induced SACC - 83 cell apoptosis. With elongation of actiontime,the totle apoptosis rates increased(P < 0. 01 ). Treated with 220μmol/l genistein for 72 h, the totle apoptosis rate was 51.62%.二. The growth inhibitory mechanism of TPK inhibitor genistein in SACC -83 cell lines1. The results of Western blot analysis indicated that , compared with the control group, the expression of survivin in groups treated with 220μmol/l genistein for 24 h,48 h and 72 h decreased,it is respectively 81% ,56% and 35% of control(P<0.01).2. The results of Western blot analysis indicated that , compared with the control group, the expression of bcl - 2 in groups treated with 220μmol/l genistein for 24 h,48 h and 72 h decreased,it is respectively 87% ,77% and 85% of control( P <0. 05 or P <0. 01).3. The results of Western blot analysis indicated that , compared with the control group,the expression of bax in groups treated with 220μmol/l genistein for 24 h,48 h and 72 h increased,it is respectively 1.72,2. 61 and 3.43 folds of control(P<0.05 or P <0.01).4. The results of Western blot analysis indicated that , compared with the control group, the expression of cyclinBl in groups treated with 220μmol/l genistein for 24 h,48 h and 72 h decreased,it is respectively 77% ,74% and 58% of control(P<0.01).5. The results of Western blot analysis indicated that , compared with the control group,the expression of Cdkl in groups treated with 220μmol/l genistein for 48 h and 72 h decreased,it is respectively 72% and 64% of control(P <0. 01).6. The results of Western blot analysis indicated that , compared with the control group, the expression of cyclinDl in groups treated with 220μmol/l genistein for 48 h and 72 h decreased,it is respectively 81% and 77% of control ( P < 0. 05 , P < 0. 01 respectively).7. The results of Western blot analysis indicated that , compared with the control group,the expression of Cdk4 in groups treated with 220μmol/l genistein for 24 h,48 h and 72 h decreased,it is respectively 76% ,65% and 54% of con-trol(P<0.01).三. Expression of EGFR and C - erbB - 2 in SACC tissues and two cell lines1. Expression of EGFR in SACC and normal salivary gland tissuesThere is positive expression of EGFR in cytoplasm and membrane of SACC cells. There is no expression of EGFR in alveolar cells of normal salivary gland , but in cytoplasm and membrane of ductal cells. In 54 cases of SACC, there are 35 positive cases and 19 negative cases; in 9 cases of normal salivary gland tissues , there are 3 positive cases and 6 negative cases. Through x² est,there is no significant difference between them( P > 0.05 ).2. Expression of C - erbB - 2 in SACC and normal salivary gland tissues There is positive expression of C - erbB - 2 in cytoplasm and membrane ofSACC cells. There is no expression of C - erbB - 2 in alveolar cells of normal salivary gland ,but in cytoplasm of ductal cells. In 54 cases of SACC,there are 39 positive cases and 15 negative cases;in 9 cases of normal salivary gland tissues, there are 2 positive cases and 7 negative cases. Through x² test, there is significant difference between them( P < 0. 05 ) , Expression of C - erbB - 2 is high in SACC.3. Relationship between expression of EGFR and C - erbB - 2 in SACC and pathologic typingThe positive rate of EGFR in three types (adenoid type, tubular type and parenchymal type) is 67% ,64% and 50%. Through x² test,there is no significant difference among them(P >0. 05). Positive expression of EGFR is not related to pathologic typing of SACC.The positive rate of C - erbB - 2 in three types (adenoid type, tubular type and parenchymal type) is 73% ,68% and 100%. Through x² test, there is no significant difference among them( P > 0.05 ). Positive expression of C - erbB -2 is not related to pathologic typing of SACC.4. Expression of EGFR and C - erbB - 2 in SACC - 83 and SACC - LM cell linesThe results of Western blot analysis indicated that , there is no significantdifference of EGFR and C - erbB - 2 expression between SACC - 83 and SACC- LM cell lines( P > 0. 05 ). However, C - erbB - 2 expression in membrane of...