Role Of Gap Junction Composed With Cx43 In The Invasion And Metastasis Of Testicular Cancer Cells Resistanted To Cisplatin

Objective:To investigate the effect of gap junction composed with Cx43 on the invasion and metastasis of mice testicular cancer cells acquired resistance to cisplatin.Methods:Using the method of gradually increasing concentration induced resistant cell lines I-10/DDP. Cells were treated with increasing concentrations of cisplatin(0, 2, 4, 8, 16,32, 64, 128 μM) for 24 h, and the cell viability was assessed by MTT assay, and then resistance index(RI) was calculated.1. Western blotting assay was used to explore the drug resistance related protein expression leves(Multi-drug resistant protein 1, MDR1; P-glycoprotein, P-gp) in I-10 and I-10/DDP.2. Immunofluorescence assay was used to detect the expression changes of Cx43 on the membrane of I-10 and I-10/DDP cells.3. Parachute assay was used to detect the function of GJ by dye spread, and GJ function was modulated by the tool medicine.4. Molecular biology methods were used to upregulate and downregulate the expression of Cx43.5. Transwell method was used to detect the abilities of invasion and metastasis and explore the influence of GJ function on the abilities of invasion and metastasis after using tool medicine and molecular biology methods.Results:1. Establishing a CDDP-resistant I-10 cell line.IC50 of I-10/DDP cell was 84.171 μM and IC50 of I-10 cell was 21.451 μM.I-10/DDP cell line showed a 3.924-fold higher resistance to CDDP than the I-10 cell line. Western blotting showed that MDR1 and P-gp expression levels of I-10/DDP cells was significantly higher than I-10 cells.These results demonstrated that a cisplatin-resistance I-10 cell line was established.2. Cx43 expression on membrane.I-10/DDP cells showed a significantly decreased Cx43 expression on membrane,compared with I-10 cells.3. GJ function.I-10/DDP cells showed a remarkably decreased GJ function, compared with I-10cells(*P<0.05).4. Invasion and migration.I-10/DDP cells displayed increased potential for invasion and migration, compared with I-10 cells(**P<0.01).5. Effect of GJ function on invasion and migration.GJ function was enhanced by10 μM RA and in contrast, inhibited by 25 μM Oleamide significantly. Invasion and metastasis ability of I-10/DDP cells was receded by RA and heightened by Oleamide markedly(*P<0.05;**P<0.01).6. Effect of Cx43 expression modulated by molecular biology methods on invasion and migration.Invasion and metastasis ability of I-10/DDP cells knocking down Cx43 was heightened. On the contrary, invasion and metastasis ability was receded while Cx43 was over-expressed in I-10/DDP cells(*P<0.05).Conclusion:1. The decreased expression of Cx43 may be relevant to cisplatin acquired drug resistance of testicular cancer I-10;2. The gap junction composed with Cx43 inhibits ability of invasion and metastasis in I-10/DDP.