植物性雌激素对细胞间隙连接通讯及间隙连接蛋白磷酸化的影响
|
摘要
间隙连接(gap junctions，GJ)是相临细胞间进行离子和小分子流通与信息交换的直捷通道，在细胞增殖和分化中起重要作用。大部分肿瘤细胞间隙连接通讯(gap junctional intercellular communication，GJIC)减弱甚至完全消失，且与肿瘤的恶性化程度相关，但是当这些细胞转染了编码间隙连接蛋白connexin(Cx)的基因以后，GJIC功能得以恢复。Connexin 43(Cx43)是Cx蛋白家族最主要的成员之一，它存在于大多数组织细胞中，其磷酸化状态直接影响GJIC的功能。有报道表明一些植物性雌激素能抑制细胞GJIC功能，并引起Cx的磷酸化状态的改变。
[目的] 利用激光扫描共聚焦显微镜(laser scanning confocal microscope，LSCM)的荧光漂白后恢复(fluorescence redistribution after photobleaching，FRAP)技术，观察植物/真菌性雌激素槲皮素(quercetin，QC)、肠内脂(enterolactone，ENL)、拟雌内醇(coumestrol，COM)、染料木黄酮(genistein，GEN)和玉米赤霉烯酮(zearalenone，ZEL)对HaCaT细胞GJIC的作用，并通过免疫印迹法观察最具代表性的植物性雌激素之一—染料木黄酮对NIH3T3细胞Cx43蛋白磷酸化的影响。
[方法]
1．植物性雌激素和/或TPA对GJIC的影响
HaCaT细胞培养于含10％胎牛血清的DMEM完全培养基中，在37℃、5％
CO。的培养箱中培养传代。细胞接种于直径为 35 mm培养M中，种植密度为
ZX10勺XI，随后将培养M移入37C、50COZ的培养箱中培养2天。细胞培养结
束前 24小时换液，并加入不同浓度的受试物＊．l、l、10、100 VM人继续培养
24小时后用LSCM进行观察。以空白作正常对照，阳性对照组在培养结束前1
刁时加入 5 ＂g／thl的 TPA。
GJIC功能测定时，加入终浓度为 10 pg／inl的 5，6－羟基荧光素乙酸乙酚盐
（5，6－carboxyfluorescein diacetate，5，6－CFDA），在 37℃、5％ CO。的培养箱中培养
约 15 min，洗掉细胞外多余的 5，6（FDA后，细胞用 Hanks液负载，置于激光扫
描共聚焦显微镜上，按照 Zeiss LSM sic release 2刀 1中 FKAP分析程序，在显微
镜h20倍物镜）下随机定义感兴趣区域kegions of interest，ROI入 选择其中
的细胞作为淬灭对象，荧光漂自程度为细胞初始荧光强度的30％～50％，由漂白
后间隔时间 lmin的各次扫描图像共 11帧，获取被漂白细胞荧光恢复以及相邻
的未漂白细胞荧光变化的数据。通过拟合漂白细胞以及相邻未漂白细胞荧光强度
差值的时间变化函数，计算荧光恢复率（habX100％，其中Im为淬灭后所恢复
的荧光强度，几为淬灭的荧光强度）及细胞间荧光传递速率（厂，min”‘人本实
验中漂白后扫描门次，共有 10个数据位点用于函数拟合。数据表经 Microsoft
Excel转换后，导入SPSS 10刀作曲线拟合和 t检验。
（Iu人）l（In。．I。）＝XXp（W）
其中几：相邻未漂白细胞在！时间的荧光强度；几。：1＝0时间相邻未漂白细胞的荧光强度；几：漂白细胞
在1时间的荧光强度：仙：t＝0时间漂白细胞的荧光强度；厂：细胞间荧光传递速率
二．免疫印迹
NIH3T3小鼠肺成纤维细胞用含10％胎牛血清的yMI 1640完全培养基培
养传代，在 37oC、5％COZ的培养箱中培养。细胞接种于 100 mlQ8 cmZ）培养
瓶中，密度为 4xlo勺ml，每瓶 10 ml，培养 4天。细胞培养结束前 24 ’J’时加入受
试物（GEN，100 pM人
提取细胞总蛋白，用 Bradford法定量后储存于－70OC。10％SDS－聚丙烯酚胺
凝胶电泳，起始电压 100 V，待样品在分离胶顶部浓缩成一直线后，加大电压至
150 V，电泳 65 min左右。用 Bio－Rad半干转移仪进行转膜，120 a转移 30 min
左右。转移完毕后，NC膜用封闭缓冲液（20 mM Tis·HCI，150 mM NaCI，0．l％
2
Tween20，5％脱脂奶粉，pH 7．5）室温封闭 1小时。加入用封闭液稀释的兔抗
Cx43多抗（Zymed co＊ Catalog No．71－0700，l：2 000稀释度）室温摇动温育 1
小时，TBST洗膜后加入辣根过氧化物酶（HRP）标记的二抗记ymed Laboratories
IncJ Catalog No．sl＊ 120，l：2 000稀释度），室温摇动温育 1’J’ 时，洗膜。将膜
暴露于电化学发光（ECL）检测试剂中作用3～5 min（A液＊液一100：l），弃去
ECL，膜吸干水份后用保鲜膜包好，暗室中在X光胶片上曝光3—30 min不等，
显影液中显影 5 min，在清水中漂洗后放入定影液中，至胶片透明。
【结果IQC在0．1～mpM浓度对GnC无明显影响，反而能桔抗T助引起的GnC
抑制，但在 100 pM时能抑制 GJIC功能。其它受试的 4种植物性雌激素均能不
同程度地抑制细胞 GJIC的功能，ENL在 0二叫 pM范围内都有显著性抑制，但
缺乏明确的剂量－依赖关系；ZEL和 GEN在 0．lpM对 GJIC没有影响，但在 l叫
pM都能显著性地抑制GJIC，并呈明确的剂量－依赖关系；COM的抑制作用不是
简单的线性关系，在0．lpM就有显著性抑制，但lpM的抑制作用没有统计学
显著性，而 10 pM以上能显著性抑制
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 GJIC
HaCaT cells were cultured in DMEM containing 10% fetal bovine serum, 2 mM
glutamine, 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=0
2. Western blotting analysis
NIH3T3 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 transferred
to 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
[目的] 利用激光扫描共聚焦显微镜(laser scanning confocal microscope，LSCM)的荧光漂白后恢复(fluorescence redistribution after photobleaching，FRAP)技术，观察植物/真菌性雌激素槲皮素(quercetin，QC)、肠内脂(enterolactone，ENL)、拟雌内醇(coumestrol，COM)、染料木黄酮(genistein，GEN)和玉米赤霉烯酮(zearalenone，ZEL)对HaCaT细胞GJIC的作用，并通过免疫印迹法观察最具代表性的植物性雌激素之一—染料木黄酮对NIH3T3细胞Cx43蛋白磷酸化的影响。
[方法]
1．植物性雌激素和/或TPA对GJIC的影响
HaCaT细胞培养于含10％胎牛血清的DMEM完全培养基中，在37℃、5％
CO。的培养箱中培养传代。细胞接种于直径为 35 mm培养M中，种植密度为
ZX10勺XI，随后将培养M移入37C、50COZ的培养箱中培养2天。细胞培养结
束前 24小时换液，并加入不同浓度的受试物＊．l、l、10、100 VM人继续培养
24小时后用LSCM进行观察。以空白作正常对照，阳性对照组在培养结束前1
刁时加入 5 ＂g／thl的 TPA。
GJIC功能测定时，加入终浓度为 10 pg／inl的 5，6－羟基荧光素乙酸乙酚盐
（5，6－carboxyfluorescein diacetate，5，6－CFDA），在 37℃、5％ CO。的培养箱中培养
约 15 min，洗掉细胞外多余的 5，6（FDA后，细胞用 Hanks液负载，置于激光扫
描共聚焦显微镜上，按照 Zeiss LSM sic release 2刀 1中 FKAP分析程序，在显微
镜h20倍物镜）下随机定义感兴趣区域kegions of interest，ROI入 选择其中
的细胞作为淬灭对象，荧光漂自程度为细胞初始荧光强度的30％～50％，由漂白
后间隔时间 lmin的各次扫描图像共 11帧，获取被漂白细胞荧光恢复以及相邻
的未漂白细胞荧光变化的数据。通过拟合漂白细胞以及相邻未漂白细胞荧光强度
差值的时间变化函数，计算荧光恢复率（habX100％，其中Im为淬灭后所恢复
的荧光强度，几为淬灭的荧光强度）及细胞间荧光传递速率（厂，min”‘人本实
验中漂白后扫描门次，共有 10个数据位点用于函数拟合。数据表经 Microsoft
Excel转换后，导入SPSS 10刀作曲线拟合和 t检验。
（Iu人）l（In。．I。）＝XXp（W）
其中几：相邻未漂白细胞在！时间的荧光强度；几。：1＝0时间相邻未漂白细胞的荧光强度；几：漂白细胞
在1时间的荧光强度：仙：t＝0时间漂白细胞的荧光强度；厂：细胞间荧光传递速率
二．免疫印迹
NIH3T3小鼠肺成纤维细胞用含10％胎牛血清的yMI 1640完全培养基培
养传代，在 37oC、5％COZ的培养箱中培养。细胞接种于 100 mlQ8 cmZ）培养
瓶中，密度为 4xlo勺ml，每瓶 10 ml，培养 4天。细胞培养结束前 24 ’J’时加入受
试物（GEN，100 pM人
提取细胞总蛋白，用 Bradford法定量后储存于－70OC。10％SDS－聚丙烯酚胺
凝胶电泳，起始电压 100 V，待样品在分离胶顶部浓缩成一直线后，加大电压至
150 V，电泳 65 min左右。用 Bio－Rad半干转移仪进行转膜，120 a转移 30 min
左右。转移完毕后，NC膜用封闭缓冲液（20 mM Tis·HCI，150 mM NaCI，0．l％
2
Tween20，5％脱脂奶粉，pH 7．5）室温封闭 1小时。加入用封闭液稀释的兔抗
Cx43多抗（Zymed co＊ Catalog No．71－0700，l：2 000稀释度）室温摇动温育 1
小时，TBST洗膜后加入辣根过氧化物酶（HRP）标记的二抗记ymed Laboratories
IncJ Catalog No．sl＊ 120，l：2 000稀释度），室温摇动温育 1’J’ 时，洗膜。将膜
暴露于电化学发光（ECL）检测试剂中作用3～5 min（A液＊液一100：l），弃去
ECL，膜吸干水份后用保鲜膜包好，暗室中在X光胶片上曝光3—30 min不等，
显影液中显影 5 min，在清水中漂洗后放入定影液中，至胶片透明。
【结果IQC在0．1～mpM浓度对GnC无明显影响，反而能桔抗T助引起的GnC
抑制，但在 100 pM时能抑制 GJIC功能。其它受试的 4种植物性雌激素均能不
同程度地抑制细胞 GJIC的功能，ENL在 0二叫 pM范围内都有显著性抑制，但
缺乏明确的剂量－依赖关系；ZEL和 GEN在 0．lpM对 GJIC没有影响，但在 l叫
pM都能显著性地抑制GJIC，并呈明确的剂量－依赖关系；COM的抑制作用不是
简单的线性关系，在0．lpM就有显著性抑制，但lpM的抑制作用没有统计学
显著性，而 10 pM以上能显著性抑制
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 GJIC
HaCaT cells were cultured in DMEM containing 10% fetal bovine serum, 2 mM
glutamine, 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=0
2. Western blotting analysis
NIH3T3 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 transferred
to 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
引文
1. Brzezinski A, Debi A. Phytoestrogens: the "natural" selective estrogen receptor modulators? Eur J Obstet Gynecol Reprod Biol 1999; 85(1): 47-51.
2. Ganry O. Phytoestrogen and breast cancer prevention. Eur J Cancer Prev 2002;11(6): 519-522.
3. Adlercreutz H. Phytoestrogens and breast cancer. J Steroid Biochem Mol Biol 2002; 83(1-5): 113-118.
4. Castle EP, Thrasher JB. The role of soy phytoestrogens in prostate cancer. Urol Clin North Am 2002; 29(1): 71-81.
5. Morrissey C, Watson RW. Phytoestrogens and prostate cancer. Curr Drug Targets 2003; 4(3): 231-241.
6. Gallo D, Giacomelli S, Cantelmo F, Zannoni GF, Ferrandina G, Fruscella E, et al. Chemoprevention of DMBA-induced mammary cancer in rats by dietary soy. Breast Cancer Res Treat 2001; 69(2): 153-164.
7. Mentor-Marcel R, Lamartiniere CA, Eltoum IE, Greenberg NM, Elgavish A. Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP). Cancer Res 2001; 61(18): 6777-6782.
8. Dabrosin C, Chen J, Wang L, Thompson LU. Flaxseed inhibits metastasis and
decreases extracellular vascular endothelial growth factor in human breast cancer xenografts. Cancer Lett 2002; 185(1): 31-37.
9. Sarkar FH, Li Y. Mechanisms of cancer chemoprevention by soy isoflavone genistein. Cancer Metastasis Rev 2002; 21 (3-4): 265-280.
10. Peeters PH, Keinan-Boker L, van der Schouw YT, Grobbee DE. Phytoestrogens and breast cancer risk: review of the epidemiological evidence. IARC Sci Publ 2002; 156: 331-336.
11. Mishra SI, Dickerson V, Najm W. Phytoestrogens and breast cancer prevention: What is the evidence? Am J Obstet Gynecol 2003; 188(5): S66-70.
12. Benassayag C, Perrot-Applanat M, Ferre F. Phytoestrogens as modulators of steroid action in target cells. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 777(1-2): 233-248.
13. Cotroneo MS, Wang J, Fritz WA, Eltoum IE, Lamartiniere CA. Genistein action in the prepubertal mammary gland in a chemoprevention model. Carcinogenesis 2002; 23(9): 1467-1474.
14. Newbold RR, Banks EP, Bullock B, Jefferson WN. Uterine adenocarcinoma in mice treated neonatally with genistein. Cancer Res 2001; 61(11): 4325-4328.
15. Chen S, Zhou D, Okubo T, Kao YC, Eng ET, Grebe B, et al. Prevention and treatment of breast cancer by suppressing aromatase activity and expression. Ann N Y Acad Sci 2002; 963: 229-238.
16. Choi YH, Lee WH, Park KY, Zhang L. p53-independent induction of p21 (WAF1/CIP1), reduction of cyclin B1 and G2/M arrest by the isoflavone genistein in human prostate carcinoma cells. Jpn J Cancer Res 2000; 91(2): 164-173.
17. Yamasaki H, Naus CC. Role of connexin genes in growth control. Carcinogenesis, 1996; 17(6): 1199-1213.
18. Yamasaki H, Krutovskikh V, Mesnil M, et al. Role of connexin (gap junction) genes in cell growth control and carcinogenesis. C R Acad Sci Ⅲ, 1999; 322(2-3): 151-159.
19. Zimmer DB, Green CR, Evans WH, et al. Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single
membrane structures. J Biol Chem. 1987; 262(16): 7751-7763.
20. Cooper CD, Solan JL, Dolejsi MK. et al. Analysis of connexin phosphorylation sites. Methods, 2000(2): 196-204.
21. Warn-Cramer BJ, Lampe PD, Kurata WE, et al. Characterization of the mitogen-activated protein kinase phosphorylation sites on the connexin-43 gap junction protein. J Biol Chem 1996; 271(7): 3779-3786.
22. Haque SJ, Flati V, Deb A, et al. Roles of protein-tyrosine phosphatases in Statl alpha-mediated cell signaling. J Biol Chem 1995; 270(43): 25709-25714.
23. Lampe PD, TenBroek EM, Burt JM, et al. Phosphorylation of Connexin43 on serine 368 by protein kinase C regulates gap junctional communication. J Cell Biol 2000; 149(7): 1503-1512.
24. Ren P, Mehta PP, Ruch RJ. Inhibition of gap junctional intercellular communication by tumor promoters in connexin 43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C. Carcinogenesis 1998; 19(1): 169-175.
25. Wade MH, Trosko JE, Schindler M. A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 1986; 232 (4749): 525-528.
26. Schindler M, Trosko JE, Wade MH. Fluorescence photobleaching assay of tumor promoter 12-O-tetradecanoylphorbol 13-acetate inhibition of cell-cell communication. Methods Enzymol 1987; 141:439-447.
27．李楠，尹岭，苏振伦，主编．激光扫描共聚焦显微术．北京：人民军医出版社，1997．108-115．
28. Wang Y, Rose B. An inhibition of gap junctional communication by cadherins. J Cell Sci 1997; 110(Pt3): 301-309.
29．Sambrook J, Fritsch EF, Maniatis T. 金冬雁，黎孟枫，译．分子克隆实验指南[M]．第2版．北京：科学出版社，1995．873～891．
30．罗玲，吴凯南．槲皮素的癌化学预防作用研究进展．中草药 2001；32(4)：378-379．
31．施明．类黄酮抗肿瘤作用的研究进展．国外医学卫生学分册 2001；28(2)：96-99．
32. Tsutsui T, Tamura Y, Yagi E, Someya H, Hori I, Metzler M, Barrett JC. Cell-transforming activity and mutagenicity of 5 phytoestrogens in cultured mammalian cells. Int J Cancer 2003; 105(3): 312-320.
33. Kwak BR, Jongsma HJ. Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating condictions. Mol Cell Biochem 1996; 157: 93-99.
34. Musil LS, Goodenough DA. Biochemical analysis of connexin43 intercellular transport, phosphorylation and assembly into gap junctional plaques. J Cell Biol 1991; 115: 1357-1374.
35. Goldberg GS, Lau AF. Dynamics of connexin43 phosphorylation in pp60~(v-src)-transformed cells. Biochem J 1993; 295(Pt3): 735-742.
36. Musil LS, Cunningham BA, Edelman GM, et al. Differential of the gap junction protein connexin43 in junctional communication-competent and-deficient cell lines. J Cell Biol 1990; 111(5 Pt1): 2077-2088.
37. Rishi RK. Phytoestrogens in health and illness. Indian J Pharmacol 2002; 34: 311-320.
38. Burns CJ, Howell SL, Persaud SJ, et al. Effects of tyrosine kinase inhibitors on beta-cell proliferation. Biochem Soc Trans 1997; 25(1): 117S.
39. Ale-Agha N, Stahl W, Sies H. Epicatechin effects in rat liver epithelial cells: stimulation of gap junctional communication and counteraction of its loss due to the tumor promoter 12-o-tetradecanoylphorbol-13-acetate. Biochem Pharmacol 2002; 63(12): 2145-2149.
40. Schultze-Mosgau MH, Dale IL, Gant TW, et al. Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells. Eur J Cancer 1998; 34(9): 1425-1431.
41. Rivedal E, Yamasaki H, Sanner T. Inhibition of gap junctional intercellular communication in Syrian hamster embryo cells by TPA, retinoic acid and DDT. Carconogenesis 1994; 15(4): 689-694.
42. Hii CS, Ferrante A, Schmidt S, et al. Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated. Carconogenesis 1995; 16(7): 1505-1511.
43. Hossain MZ, Jagdale AB, Ao P, et al. Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate. J Cell Physiol 1999; 179(1): 87-96.
44. Cohen GB, Ren R, Baltimore D. Modular binding domains in signal transduction proteins. Cell 1995; 80: 237-248.
45. Yamasaki H, Omori Y, Zaidan-Dagli ML, et al. Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis. Cancer Detect Prev 1999; 23(4): 273-279.
46. Wilson MR, Close TW, Trosko JE. Cell population dynamics apoptosis, mitosis and cell-cell communication during disruption of homeostatsis, Exper Cell Res 2000; 254(2): 257-268.
47. Goodenough DA, Goliger JA, Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem 1996; 65: 475-502.
48. Sharov VS, Briviba K, Sies H. Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation. IUBMB Life 1999; 48(4): 379-384.
2. Ganry O. Phytoestrogen and breast cancer prevention. Eur J Cancer Prev 2002;11(6): 519-522.
3. Adlercreutz H. Phytoestrogens and breast cancer. J Steroid Biochem Mol Biol 2002; 83(1-5): 113-118.
4. Castle EP, Thrasher JB. The role of soy phytoestrogens in prostate cancer. Urol Clin North Am 2002; 29(1): 71-81.
5. Morrissey C, Watson RW. Phytoestrogens and prostate cancer. Curr Drug Targets 2003; 4(3): 231-241.
6. Gallo D, Giacomelli S, Cantelmo F, Zannoni GF, Ferrandina G, Fruscella E, et al. Chemoprevention of DMBA-induced mammary cancer in rats by dietary soy. Breast Cancer Res Treat 2001; 69(2): 153-164.
7. Mentor-Marcel R, Lamartiniere CA, Eltoum IE, Greenberg NM, Elgavish A. Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP). Cancer Res 2001; 61(18): 6777-6782.
8. Dabrosin C, Chen J, Wang L, Thompson LU. Flaxseed inhibits metastasis and
decreases extracellular vascular endothelial growth factor in human breast cancer xenografts. Cancer Lett 2002; 185(1): 31-37.
9. Sarkar FH, Li Y. Mechanisms of cancer chemoprevention by soy isoflavone genistein. Cancer Metastasis Rev 2002; 21 (3-4): 265-280.
10. Peeters PH, Keinan-Boker L, van der Schouw YT, Grobbee DE. Phytoestrogens and breast cancer risk: review of the epidemiological evidence. IARC Sci Publ 2002; 156: 331-336.
11. Mishra SI, Dickerson V, Najm W. Phytoestrogens and breast cancer prevention: What is the evidence? Am J Obstet Gynecol 2003; 188(5): S66-70.
12. Benassayag C, Perrot-Applanat M, Ferre F. Phytoestrogens as modulators of steroid action in target cells. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 777(1-2): 233-248.
13. Cotroneo MS, Wang J, Fritz WA, Eltoum IE, Lamartiniere CA. Genistein action in the prepubertal mammary gland in a chemoprevention model. Carcinogenesis 2002; 23(9): 1467-1474.
14. Newbold RR, Banks EP, Bullock B, Jefferson WN. Uterine adenocarcinoma in mice treated neonatally with genistein. Cancer Res 2001; 61(11): 4325-4328.
15. Chen S, Zhou D, Okubo T, Kao YC, Eng ET, Grebe B, et al. Prevention and treatment of breast cancer by suppressing aromatase activity and expression. Ann N Y Acad Sci 2002; 963: 229-238.
16. Choi YH, Lee WH, Park KY, Zhang L. p53-independent induction of p21 (WAF1/CIP1), reduction of cyclin B1 and G2/M arrest by the isoflavone genistein in human prostate carcinoma cells. Jpn J Cancer Res 2000; 91(2): 164-173.
17. Yamasaki H, Naus CC. Role of connexin genes in growth control. Carcinogenesis, 1996; 17(6): 1199-1213.
18. Yamasaki H, Krutovskikh V, Mesnil M, et al. Role of connexin (gap junction) genes in cell growth control and carcinogenesis. C R Acad Sci Ⅲ, 1999; 322(2-3): 151-159.
19. Zimmer DB, Green CR, Evans WH, et al. Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single
membrane structures. J Biol Chem. 1987; 262(16): 7751-7763.
20. Cooper CD, Solan JL, Dolejsi MK. et al. Analysis of connexin phosphorylation sites. Methods, 2000(2): 196-204.
21. Warn-Cramer BJ, Lampe PD, Kurata WE, et al. Characterization of the mitogen-activated protein kinase phosphorylation sites on the connexin-43 gap junction protein. J Biol Chem 1996; 271(7): 3779-3786.
22. Haque SJ, Flati V, Deb A, et al. Roles of protein-tyrosine phosphatases in Statl alpha-mediated cell signaling. J Biol Chem 1995; 270(43): 25709-25714.
23. Lampe PD, TenBroek EM, Burt JM, et al. Phosphorylation of Connexin43 on serine 368 by protein kinase C regulates gap junctional communication. J Cell Biol 2000; 149(7): 1503-1512.
24. Ren P, Mehta PP, Ruch RJ. Inhibition of gap junctional intercellular communication by tumor promoters in connexin 43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C. Carcinogenesis 1998; 19(1): 169-175.
25. Wade MH, Trosko JE, Schindler M. A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 1986; 232 (4749): 525-528.
26. Schindler M, Trosko JE, Wade MH. Fluorescence photobleaching assay of tumor promoter 12-O-tetradecanoylphorbol 13-acetate inhibition of cell-cell communication. Methods Enzymol 1987; 141:439-447.
27．李楠，尹岭，苏振伦，主编．激光扫描共聚焦显微术．北京：人民军医出版社，1997．108-115．
28. Wang Y, Rose B. An inhibition of gap junctional communication by cadherins. J Cell Sci 1997; 110(Pt3): 301-309.
29．Sambrook J, Fritsch EF, Maniatis T. 金冬雁，黎孟枫，译．分子克隆实验指南[M]．第2版．北京：科学出版社，1995．873～891．
30．罗玲，吴凯南．槲皮素的癌化学预防作用研究进展．中草药 2001；32(4)：378-379．
31．施明．类黄酮抗肿瘤作用的研究进展．国外医学卫生学分册 2001；28(2)：96-99．
32. Tsutsui T, Tamura Y, Yagi E, Someya H, Hori I, Metzler M, Barrett JC. Cell-transforming activity and mutagenicity of 5 phytoestrogens in cultured mammalian cells. Int J Cancer 2003; 105(3): 312-320.
33. Kwak BR, Jongsma HJ. Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating condictions. Mol Cell Biochem 1996; 157: 93-99.
34. Musil LS, Goodenough DA. Biochemical analysis of connexin43 intercellular transport, phosphorylation and assembly into gap junctional plaques. J Cell Biol 1991; 115: 1357-1374.
35. Goldberg GS, Lau AF. Dynamics of connexin43 phosphorylation in pp60~(v-src)-transformed cells. Biochem J 1993; 295(Pt3): 735-742.
36. Musil LS, Cunningham BA, Edelman GM, et al. Differential of the gap junction protein connexin43 in junctional communication-competent and-deficient cell lines. J Cell Biol 1990; 111(5 Pt1): 2077-2088.
37. Rishi RK. Phytoestrogens in health and illness. Indian J Pharmacol 2002; 34: 311-320.
38. Burns CJ, Howell SL, Persaud SJ, et al. Effects of tyrosine kinase inhibitors on beta-cell proliferation. Biochem Soc Trans 1997; 25(1): 117S.
39. Ale-Agha N, Stahl W, Sies H. Epicatechin effects in rat liver epithelial cells: stimulation of gap junctional communication and counteraction of its loss due to the tumor promoter 12-o-tetradecanoylphorbol-13-acetate. Biochem Pharmacol 2002; 63(12): 2145-2149.
40. Schultze-Mosgau MH, Dale IL, Gant TW, et al. Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells. Eur J Cancer 1998; 34(9): 1425-1431.
41. Rivedal E, Yamasaki H, Sanner T. Inhibition of gap junctional intercellular communication in Syrian hamster embryo cells by TPA, retinoic acid and DDT. Carconogenesis 1994; 15(4): 689-694.
42. Hii CS, Ferrante A, Schmidt S, et al. Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated. Carconogenesis 1995; 16(7): 1505-1511.
43. Hossain MZ, Jagdale AB, Ao P, et al. Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate. J Cell Physiol 1999; 179(1): 87-96.
44. Cohen GB, Ren R, Baltimore D. Modular binding domains in signal transduction proteins. Cell 1995; 80: 237-248.
45. Yamasaki H, Omori Y, Zaidan-Dagli ML, et al. Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis. Cancer Detect Prev 1999; 23(4): 273-279.
46. Wilson MR, Close TW, Trosko JE. Cell population dynamics apoptosis, mitosis and cell-cell communication during disruption of homeostatsis, Exper Cell Res 2000; 254(2): 257-268.
47. Goodenough DA, Goliger JA, Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem 1996; 65: 475-502.
48. Sharov VS, Briviba K, Sies H. Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation. IUBMB Life 1999; 48(4): 379-384.