| Gap junctions (GJ) is a direct signaling pathway for neighboring cells. They contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. They are thought to play an important role in cell proliferation and differentiation. Most tumor cells have lost or reduced capacity of gap junctional intercellular communication (GJIC), but it can be recovered when they were transfected with connexin (Cx) genes, which encode the protein in gap junctions. Connexin43 (Cx43) is one of the most important members in the Cx protein family, which exist in common cells and tissues. The phosphorylation state of Cx43 may affect the function of GJIC directly. It was reported that some phytoestrogens could inhibit GJIC and promote the phosphorylation of Cx43. [Objectives] To observe the effects of phytoestrogens quercetin(QC), enterolactone(ENL), coumestrol(COM), genistein(GEN), and zearalenone(ZEL) on GJIC in HaCaT cells with different concentrations(0.1, 10, 100 μM)determined by fluorescence redistribution after photobleaching(FRAP)using a laser scanning confocal microscope(LSCM). Furthermore, the phosphorylation of Cx43 by genistein was investigated with Western Blotting analysis in NIH3T3 cells. [Methods] 1. Effects of natural estrogens and/or TPA on GJICHaCaT cells were cultured in DMEM containing 10% fetal bovine serum, 2 mMglutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin, and were maintained in a humidified incubator (37℃> 5% CO2). Cells were plated in 35 mm dishes at a density of 2×104/ml for 2 days. Phytoestrogens with different concentrations (0.1,1, 10, 100 μM) were added on the second day. TPA(5ng/ml) was added into dishes of positive group 1 h prior to the end of testing. Cells were then incubated with 5,6-carboxyfluorescein diacetate (5,6-CFDA) for 15 min. FRAP analysis was performed with a LSCM. The cells were rinsed with Hanks to remove extracellular dye and covered with Hanks for FRAP analysis.Cells in regions of interest (ROI) were randomly selected under the microscope with 20×objective lens according to FRAP protocol in Zeiss LSM 510 release 2.01 and photobleached to 30%~50% of their initial fluorescence intensity. They were then examined for recovery of fluorescence by scanning at interval of 1 min for 11 subsquent scanning, and the maximum intensity of recovered fluorescence (7m) were obtained. The intensity of recovery was expressed as: Im/ Ib×100%, where Ib is the intensity of the photobleached fluorescence. Fluorescence recovery was corrected by fluorescence loss of unbleached controls. The rate of fluorescence transfer (K, min-1) were also calculated ?The data were analyzed with SPSS 10.0. Analysis of variance was used to compare the means of the percentage of fluorescence recovery.(Iu-Ib) / (Iu0-Ib0) =exp (-Kt)Iu: fluorescence intensity of unbleached cells at time t; Iu0: fluorescence intensity of unbleached cells at time=0; Ib: fluorescence intensity of bleached cells at time t; Ib0: fluorescence intensity of bleached cells at time=02. Western blotting analysisNIH3T3 cells were cultured in RPMI 1640 (containing 10% FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin and 100 ug/ml streptomycin) and maintained in a humidified incubator (37℃ , 5% CO2). Before test, cells were plated in 100 ml (28 cm2) culture bottles with a density of 4×104/ml for 4 days. Genistein (100 μM) were added at the end of third day. TPA (5 ng/ml) was added into dishes of positive 1 h prior to the testing. The total protein was extracted and measured by Bradford method, and then stored at -70℃. Protein was separated by 10 % SDS-polyacrylamide gel electrophoresis (PAGE) for 65 min at 150 V, and transferredto nitrocellulose (NC) membrane for 30 min at 120 mA. The membrane was blocked for 1 hour at room temperature with blocking buffer(20 mM Tris HCl, 150 mM NaCl, 0.1% Tween-20, 5% nonfat dry milk, pH 7.5), and then incubated with rabbit anti-connexin43 po...
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