| Part Ⅰ Effect of TRPC6 channels on ESCC cell lines cell cycleObjective To study the effect of TRPC6 channels on ESCC cell lines Eca109 cell cycle.Methods Cells harvested and fixed were washed and incubated with 0.2 mg/ml RNase and 10 μg/ml propidium iodide (PI). Cells were then assayed on FACS Calibur and cell cycle distributions were analyzed by CellQuest Pro software.Results Treatment with SKF96365 substantially increased the percentage of Ecal 09 cells in the G2/M phase and reduced that in G0/G1 phase in a time-dependent manner. Most of the cells (85.26%),24 hours after treatment were arrested in the G2/M phase. Moreover, the cell cycle profiles in Eca109 cells transfected with DNC6 against TRPC6 were also characterized by an increase in percentage of cells in G2/M phase and a decrease in numbers of cells in G0/G1 phase compared with those in control (p <0.01). The phosphorylation of Cdc2 at Tyr15 and the expression of cyclinB1 were markedly enhanced in ESCC cells in which TRPC6 was inhibited or down-regulated.Conclusion TRPC6 channels were essential for G2 phase transition in ESCC cells.Part II TRPC6 channel inhibitor is a potential radiosensitizerObjective To confirm whether TRPC6 channel is a candidate radiosensitivity.Method We chose Eca109 cell line which has TRPC6 channels expression. Cell cycle was investigated after TRPC6 channel inhibitor was treated. According to the result of cell cycle, radiation was applied at time of 8hrs SKF96365 with a 5μM concentration.Result TRPC6 channel inhibitor could block cell cycle at G2/M phase checkpoint. 4hrs after 5μM SKF96365 performed, G2/M phase rised 7.79%, when 31.65% and 49.95% after 6hrs and 7hrs, respectively. After radiation, we used CCK-8 to test Eca109 cell cytoactive and cell proliferation. So we have known that Eca109 cell arrest at G2/M phase by SKF96365, which received radiotherapy showed the worst cell proliferation.Conclusion TRPC6 channel inhibitor is a potential radiosensitizer.Part III TRPC6 channel inhibitor enhanced the effect of radiotherapy in vivoObjective To confirm whether TRPC6 channel is a candidate radiosensitivity by further study.Method Total of 20 male nude mice were randomly divided into four groups as follows:control, SKF96365, radio and combined radio-SKF96365. Each nude moce was injected with 7.5×105 Eca109 cells. In control group, nude mice were injected saline into the abdominal cavity at day 5-11. In SKF96365 group,20mg/kg 5μM SKF96365 was injected into the abdominal cavity of the nude mice at day 5-11. In radiation group, the nude mice received radiotherapy 2Gy per day at day 7-11. In combined radio-SKF96365 group,20mg/kg 5μM SKF96365 was injected into the abdominal cavity of the nude mice at day 5-11 and 2Gy radiotherapy was delivered to the tumor site at day 7-11. General state of health were observed, the tumor size in volume was measured with calipers two tomes every week, six weeks after seeding, mice were sacrificed by neck-break. The tumor size was measured n volume with caliper and in weigh with scale.Result At the end of observe, tumor size of control, SKF96365 radio and combined radio and SKF96365 groups were 307.15±21.35mm3,316.67±17.21mm3, 288.67±16.35mm3 and 166.67±23.12mm3.Conclusion TRPC6 is a potential novel esophageal squamous cell carcinoma radiosensitivity target. |
PDF Full Text Request