| Background and objectives:Gastric cancer is a common digestive malignant tumor and the mortality rate ranks the forefront of cancer.Peritoneal metastasis is a major route after radical gastrectomy for the treatment of gastric cancer,which is the leading cause of death in patients with gastric cancer.Peritoneal metastasis is the result of interaction between gastric cancer cells and multiple cells in abdominal cavity,such as gastric cancer cells and immune cells,gastric cancer cells and peritoneal mesothelial cells,free gastric cancer cells and their own together,and the interaction between these cells is accompanied by intercellular communication,which is vital for the tumor phenotype modeling and malignant evolution.The direct intercellular communication is mediated by cell-cell junctions,and the most common cell-cell junctions are gap junction,desmosome junction and tight junction.Tight junction shows no intercellular communication,and little is know about Desmosome junction,whereas gap junction is recently concerned, gap junction is a type of specific channel formed with each adjacent cell membrane providing a connection body (connexon), which is constituted by six connexins(Cx ) based on the same family,and there are different types in different tissues and organs, Cx43 expression is found mostly in epithelial and mesenchymal tissues.Gap junction mediated by Cx43 plays important roles in cell growth,differentiation and intercellular coordination.Previous studies indicated that Cx43 gene expression decreased during the process of carcinogenesis,which was viewed as an vital event in carcinogenesis.however,recent studies showed that Cx43 gene was tightly related to cancer metastasis,and might play an important role in metastasis.It is unclear that the expression level of Cx43 in primary gastric cancer and metastatic tumor tissues,meanwhile the effect of Cx43 mediated gap junction on the biological behavior of gastric cancer cells in vitro and its role in breakthroughing the peritoneal mesothelial barrier for gastric cancer cells are still unclear.It shows great significance to reveal the role and mechanism of Cx43 mediated gap junction in prevention and treatment of peritoneal metastasis.Therefore,in this study,the expression of Cx43 in primary gastric cancer tissues,free gastric cancer cells,peripheral lymph nodes and peritoneal metastatic tissues were detected,and its relationship with clinical characters was analysed as well.Moreover,Cx43 expressing vector and Cx43T154A site mutation vector were constructed,then gastric cancer cells BGC-823 was infected by these vectors,and the effect on the biological behavior of gastric cancer cells in vitro was detected as well.The interaction model of gastric cancer cells and peritoneal mesothelial cells in vitro was established to study the effect of Cx43 medicated gap junction on the peritoneal metastasis of gastric cancer cells,thus offering an novel theoretical and experimental basis for clinical prevention and treatment.Methods:1. SP immunohistochemistry were used to evaluate the expressions of Cx43 and E-cadherin in 75 specimens of gastric cancer tissues and adjacent normal tissues.Meanwhile the relationships between Cx43, E-cadherin expression and clinicopathological parameters were investigated.The expression of Cx43 and E-cadherin in metastatic lymph nodes was detected.2. SP immunohistochemistry and immunofluorescence were used to evaluate the expressions of Cx43 in 26 specimens of matched gastric cancer tissues, abdominal free gastric cancer cells and metastatic peritoneal tissues in patients with stageâ…£gastric cancer.3. Lentivial expression vectors of human Cx43 and Cx43T154A were constructed respectively, and then transduced into gastric cancer cell line BGC-823, and the cell growth, proliferation, adhesion, cell cycle, invasion and migration were investigated by MTT, Matrigel adhesion experiments in vitro, FCM, Transwell invasion assay, Cell scratch test respectively.4. The interaction model between gastric cancer cells and peritoneal mesothelial layers in vitro was established, cell adhesion was detected by FCM, and GJIC was assessed by LASM and fluorescence redistribution after photobleaching, meanwhile breakthroughing peritoneal mesothelial layers for gastric cancer cells was observed by LSCM.Results:1. There was positive staining of Cx43 protein in adjacent normal gastric tissues,while decreased expression was detected in gastric cancer tissues(p<0.01).The positive staining rate of E-cadherin in gastric cancer tissues was significantly lower than that in corresponding adjacent tissues(p<0.01). As the degree of histologic differentiation of gastric cancer decreased,stage of TNM upgraded,positive staining rates of Cx43 and E-cadherin proteins in gastric cancer tissues significantly increased(p<0.01),indicating there were positive correlations between their expression(r=0.912,p<0.01).The expression of Cx43 and E-cadherin in paired metastatic lymph nodes was higher than that in primary gastric cancer,with statistical difference(p<0.01).2. Cx43 expression in abdominal free gastric cancer cells and peritoneal metastatic tissues were significantly increased,and it shows significant difference compared to matched primary tumors in patients with stageâ…£(p<0.01).3. Cell proliferation in site mutation group and empty vector group show no difference from non-transfected group(p>0.05),whereas the cells in Cx43 expressing group were emerging reduced cell proliferation ability(p<0.05).which indicated that GJIC plays an important role in regulating cell growth and proliferation.The results from cell adhesion with matrigel in vitro indicated the cell adhesion ability in Cx43 expressing and Cx43T154A site mutation groups was stronger than the empty vector group and non-transfection group(p<0.05),whereas there was no significant difference in Cx43 expressing group and Cx43T154A site mutation group(p>0.05). The results based on flow cytometry detection confirmed that G0/G1 phase cells increased,while the S phase and G2 / M phase cells reduced in Cx43 expressing group compared with non-transfected group,and the difference was statistically significant(p<0.05),however,there were no significant difference in Cx43T154A site mutation group and empty vector group compared to non-transfected group(p>0.05).The cell invasion and migration ability in Cx43 expressing group and site mutation group were stronger than that in non-transfected group(p<0. 05),whereas there was no significant difference between Cx43 expressing group and site mutation group,which indicated that whether the GJIC is functioning or not has no effect on the cell invasion and migration ability. 4. Gap junction intercellular communication between gastric cancer cells and peritoneal mesothelial cells was detected by Laser scanning confocal microscopy combined with fluorescence recovery after photobleaching, and th results showed that GJIC couldn't be established by gastric cancer cells in non-transfected group, empty vector group and site-directed mutagenesis group with adjacent peritoneal mesothelial cell.whereas gastric cancer cells in Cx43-expression group achieved it, mesothelial cells were treated with laser bleaching,followed by decreased fluorescence optical density value, then Calcein-AM flowed from adjacent gastric cancer cells through the gap junction channels into the mesothelial cells, and its optical density increased gradually.The results due to gastric cancer cells and peritoneal mesothelial cells co-culture model in vitro showed that only gastric cancer cells in Cx43 expressing group could breakthrough the human peritoneal mesothelial barrier by means of paracellular migration,indicating it is necessary to forming Cx43-mediated GJIC between gastric cancer cells and peritoneal mesothelial cells when breakthroughing the peritoneal mesothelial barrier for gastric cancer cells.Conclusion:1. The expression of Cx43 and E-cadherin in gastric carcinoma and adjacent tissues was significantly correlated and concurrent, and it showed positive expression in the adjacent normal gastric tissues, whereas the expression of Cx43 and E-cadherin in the gastric cancer tissues was significantly reduced compared with the adjacent tissues, indicating that abnormal expression of Cx43 and E-cadherin Synergistically plays an important role in the occurrence and development of gastric cancer.The expression of Cx43 and E-cadherin was closely related with the clinicopathologic characteristics of gastric cancer,and both of them may acted as indicators for judging the differentiation degree,clinical staging. Expression of Cx43 and E-cadherin was increased in MLNs compared to primary gastric cancer tissues, indicating that these two proteins play a critical role in lymph nodes metastasis of gastric cancer.2. The abundance of Cx43 protein in different stages during the process of gastric cancer is different; the expression of Cx43 in abdominal free gastric cancer cells and metastatic peritoneal tissues was significantly enhanced and might play a positive role in tumor metastasis.3. There was a significant effect on the biological behavior of gastric cancer cells such as cell growth, proliferation, adhesion, cell cycle, invasion, migration and etc by Cx43 mediated gap junction or gap junction intercellular communication.4. The gap junction mediated by Cx43 plays an important role in peritoneal metastasis of gastric cancer cells, and it is necessary to establish GJIC between gastric cancer cells and peritoneal mesothelial cells in breakthroughing the mesothelial cell barrier for gastric cancer cells.The diapedesis of gastric cancer cells through the mesothelial cell barrier was by means of paracellular migarion.
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