Inhibitory effect of resveratrol on the proliferation of GH3 pituitary-adenoma cells and voltage-dependent potassium current

摘要

BACKGROUND:Recent researches indicate that activation of potassium channel is likely to cause many kinds of cells to proliferate and differentiate;using chemical to block the potassium channel can restrain the proliferation of small lung-cancer cells.breast cancer.prostate cancer and human lymphocyte,etc.Previous researches proved that resveratrol(RE),a selective estrogen receptor modulator(SERM).could inhibit growth of GH3 calls,induce apoptosis,and resist tumor through interfering K+ channel.OBJECTIVE:To investigate the effects of RE on Voltage-dependent K+ current [Ik(v)] and cell proliferation in GH3 pituitary-tumor cells.DESIGN:Observational contrast study.SETTING:Department of Neurosurgery.the First Clinical Hospital of Harbin Medical University;Department of Microbiology,Harbin Medical University;Department of Pharmacology,Harbin Medical University.MATERIALS: GH3 pituitary-tumor cell line of rats was purchased from the American Type Culture Collection (ATCC).RE and[3-(4,5-dimethylthiazo1-2-y1)-2.5-diphenyl-tetrazolium bromide](MTT)were obtained from Sigma Chemical CO,St Louis,USA;Ham's F-10 medium from Gibco BRL;Equine serum and fetal bovine serum from Hyclone Laboratories,Logan,UT;FACSCalibur flow cytometer from BD Company,USA.RE was dissolved in ethanol and stored at-20 ℃.It was diluted to different concentrations (10.50,100 μmol/L)with medium and extra cellular solution when needed.rhe final concentration of ethanol was Jess than 0.01%.METHODS:The experiment was carried out in the Department of Microbiology and Pharmacology of Harbin Medical University from March 2005 to January 2006.①Cell preparation:Proliferating indexes affected by 10.50 and 100 μmol/L RE were measured with MTT,respectively.0.0001 volume fraction of ethan ol was added into control group.Inhibitory rate of cellular growth was calculated as the following formula:Inhibitory rate (%)=(1-A value in experimental group/A value in control group)x100%.The experiments mentioned above were done for 3 times.②Cell cycle with flow cytometry:Cell cycle and apoptosis rate were measured in RE groups(affected by RE in various concentrations)and controI group (affected by 0.0001 volume fraction of ethanol)with flow cytometry.③Whole-cell patch clamp electrophysiological recording:The GH3 cells were used for experiment when adhesion was well to the bottom with continuously flush of Ca2+-free Tyrode's solution at 3 Ml/min after 48 hours incubation in 3.5 cm diameter plate.The potassium current and resting potential of the same call without RE and with different concentrations of RE were recorded with the whole cell voltage-clamp method and current-clamp method. Cells before adding medicines were regarded as in control group;otherwise in experimental group.④Statistical analysis:The differences between different treatment groups were determined by the analysis of variance and t test.MAIN OUTCOME MEASURES: Effect of RE in different concentrations on cell proliferation,distribution of cell cycle,and Ik(v) in GH3 call.RESULTS:①Influence of RE in different concentrations on the proliferation of GH3 call: MTT assay showed the cell growth suppression rate was 44.6%,52.3%,61.9%respectively at the dose of 10μmol/L,50μmol/L and 100 μmol/L and had a dose-effect relationship after 3 days incubation with RE.They were statistically significant in comparison with the calls without treatment of RE(P＜0.05-0.01).②Influenca of RE on GH3 cell proliferating cycle: Flow cytometry results revealed all cells with treatment of RE were blocked in the G0/G1,G1 stage of cell cycle.The cell proportion in the S and G2/M stage decreased significantly.There was positive cor relation existed between effects of RE and RE concentration.There was significant difference as compared with control group (P＜0.05-0.01).③Influence of RE on the voltage-dependent K+current and membrene resting Potential of GH3 cell:The voltage-dependent extroversion K+current [Ik(v)] was recorded in control group:apparently Ik(v) decrease in 50 μmol/L and 100 μmol/L RE groups;no changes of Ik(v) in 10 μmol/L RE group.The Ik(v) value changed from(1 250±62)Pa to(93±39)Pa(P＜0 05)when 50 μmol/L RE was added.CONCLUSION:The anti-proliferative activity of RE may be partially explained by blockade of voltage-dependent K+ channel present in GH3 cells;meanwhile,the effect is characterized by dosage dependence.