The Role Of TRPC6 Channels In Human Esophageal Squamous Cell Carcinoma Development

Partâ… Effect of TRPC6 channels and T/L-VGCCs on ESCC cell lines growth and cell cycleObjective:To study the effect of TRPC6 channels and T/L-VGCCs on Eca109 cells proliferation and cell cycle.Methods:Eca109 cells were seeded at an initial density of 1-2×10⁵ per 35mm-dish.After 24 hours culture,the cells were treated with different interferences,including SKF96365,mibefradil and nifedipine.The cells were then harvested at the indicated time and counted by using the COULTER^TM.Cells were then assayed on FACSCalibur and cell cycle distributions were analyzed by CellQuest Pro software.Results:Twenty four hours after treatment with 10μM SKF96365,a nonspecific inhibitor for TRPC6 channels,the cell number of Eca109 was reduced by 24.3±8.0%compared to control groups treated with PBS,similar to the effect of 10μM mibefradil which reduced the cell number by 22.3±3.1%compared to control. Treatment with SKF96365 substantially increased the percentage of Eca109 cells in the G2/M phase and reduced that in G0/G1,mibefradil increased the percentage of Eca109 cells in the G0/G1 phase and reduced that in G2/M phase.Nifedipine has no effect on cell proliferation and cell cycle on Eca109 cells.Conclusion:These results suggest that TRPC6,but not T/L-type VGCCs,induced ESCC cells arrested in G2/M phase and subsequently suppressed the proliferation of these cells. Partâ…¡The mechanism of TRPC6 channels blocking induced G2/M phase arrest of ESCC cellsObjective:To study the mechanism of TRPC6 channels involved in G2/M phase transition on Eca109 cells.Methods:The GFP,DNC6(human) and WTC6(mouse) were constructed into an adenoviral vector with the CMV promoter.Both the DNC6 and WTC6 contained an internal ribosome entry site(IRES)-GFP tag for monitoring infection efficiency.Forty-eight hours after transfection,western blot or RT-PCR was used to detect protein or mRNA level of cdc25 phosphatases in Eca109 cells.Results:The transfection efficiency of adenovirus vector in Eca109 cells was about 95%.Western blot results showed that in Eca109 cells,cdc25C protein levels were dramatically reduced after DNC6 infection.In contrast,the expression levels of Cdc25A or B in Eca109 cells were not affected.The RT-PCR analysis further revealed that the cdc25C mRNA levels in Eca109 cells were greatly reduced after DNC6 infection,while the cdc25A and B mRNA levels remained unaffected.Conclusion:These results suggest that blockade of TRPC6 channels inhibited cdc2 activation via down-regulation of cdc25C..Partâ…¢Functional study of TRPC6 channels in cultured ESCC cell line Eca109 Objective:To study that Ca²⁺ influx through TRPC6 channels or T/L-VGCCs was critical for G2/M phase transition in ESCC cells.Methods:Calcium imaging was used to measure the change in intracellular Ca²⁺ concentration([Ca²⁺]_â…°).Nikon eclipse Te2000-e microscope was used to take pictures with dual excitations of Fura 2-AM at 340 nm and 380 nm.[Ca²⁺]_â…°was indicated by the 340/380 nm excitation ratio.The nystatin-perforated patch recording was performed at room temperature with an Axonpatch 700A amplifer at 5 kHz filtration. When the series resistance became＜30Mohm and stable for 2min,voltage ramps of 200-ms duration spanning the voltage range of-120 mV to 120 mV were delivered every 15 seconds while holding potential was -60mV.ALA-VM8 system was used to change the solution bathing the cells.Results:Mibefradil or nifedipine did not block UTP-induced Ca²⁺ elevation.The current/voltage dependence of this UTP-induced current displayed both inward and outward properties,with a reversal potential close to 0 mV,similar to the reported TRPC6-like current.This current also can not be suppressed by either mibefradil or nifedipine.Conclusion:Taken together,these results suggest that Ca²⁺ through TRPC6 channels rather than T/L-type VGCCs is important for the G2/M transition of human ESCC cells.Partâ…£The effect of TRPC6 channels in the growth of transplanted tumor in nude miceObjective:To provide direct evidence that TRPC6 channels are responsible for ESCC tumor development in vivo.Methods:Eighteen male nude mice were divided into three groups.The Eca109 cells were infected with GFP,WTC6 or DNC6 and 7.5×10⁵ cells were injected into the fight lank of mice.All nude mice were housed under standard laboratory conditions.Tumor size was measured every two days.The tumor volume was calculated by the formula V=0.5×L×W²(L,length;W,width).Four weeks later,the animals were sacrificed,and the tumors were removed and weighed.Results:Tumors formed by the cells transfected with DNC6 grew much slower compared to those formed by the cells transfected with GFP and WTC6.At the macroscopic observation,the difference in tumor size and volume among the three groups was indicated.Statistical analysis revealed that tumor mass in GFP,WTC6 or DNC6 group was 0.487±0.06,0.545±0.17 or 0.028±0.03(gram),respectively.Conclusion:These results suggest that TRPC6 channels were critical for the development of ESCC in vivo.