散发性乳腺癌中BCRP表达与ERα基因启动子甲基化相关性研究
|
摘要
散发性乳腺癌中BCRP表达与ERα基因启动子甲基化相关性研究
目的
探讨乳腺癌耐药相关蛋白(breast cancer resistance protein,BCRP)在散发性乳腺癌组织中的表达特征及其与雌激素受体α(Estrogen Receptorα,ERα)蛋白表达和ERα基因启动子甲基化的相关性,通过细胞实验观察DNA甲基转移酶抑制剂5'-氮杂-脱氧胞苷酸(5'-Aza-2'-deoxycytidine,5'-Aza-dC)及17-β雌二醇(17β-Estradiol,E_2)处理前后ERα阴性乳腺癌细胞株MDA-MB-435s ERα基因启动子甲基化及ERα、BCRP mRNA表达变化情况,探讨乳腺癌中BCRP表达与雌激素信号的相关性及乳腺癌内分泌治疗耐药与多药耐药间可能存在的内在联系,初步阐明去甲基化药物在乳腺癌内分泌治疗与化学治疗中的可能意义。
材料与方法
采用免疫组织化学(immunohistochemistry,IHC)染色法,检测146例散发性乳腺癌和22例乳腺纤维腺瘤组织中BCRP、ERα、PR、HER-2、P53蛋白的表达,分析BCRP表达与临床病理学参数之间的相关性;通过甲基化特异性PCR(Methylation specific PCR,MSP)法检测60例乳腺浸润性导管癌肿瘤及14例乳腺纤维腺瘤组织中ERα基因启动子区四个CpG岛密集区域甲基化情况,探讨乳腺浸润性导管癌组织中ERα基因启动子区甲基化特征及其与ERα和BCRP表达的相关性;通过MSP、逆转录PCR(Reverse transcript PCR,RT-PCR)等方法检测5'-Aza-dC和E_2处理前后ER阴性MDA-MB-435s乳腺癌细胞株ERα基因启动子甲基化及ERα、BCRP mRNA变化情况,探讨去甲基化药物对于BCRP表达的可能影响及其机制。
结果
1.散发性乳腺癌中BCRP表达与临床病理参数相关性:
(1) BCRP在乳腺癌组织中表达情况:乳腺癌中BCRP表达阳性率60.3%,纤维腺瘤中BCRP表达阳性率31.8%,两者相比有显著性差异(X~2=6.301,P=0.012);(2)BCRP表达与乳腺癌临床指标相关性:在乳腺癌组织中,BCRP表达水平与患者年龄、病理类型、临床分级及淋巴结转移均无关;(3)乳腺癌组织中BCRP与ERα、PR表达的相关性:结合ERα、PR表达与绝经情况分组,未绝经且ERα阳性患者乳腺肿瘤组织中BCRP表达阳性率(38.5%)显著低于未绝经ERα阴性(71.9%)和已绝经ERα阳性(60.3%)及已绝经ERα阴性患者(63.0%)(X~2=9.301,P=0.007);绝经与未绝经PR阳性或阴性患者之间BCRP表达均未见显著性差别;(4)乳腺癌组织中BCRP与HER-2、P53表达的相关性:HER-2阳性表达乳腺癌中BCRP表达阳性率显著高于HER-2阴性乳腺癌(81.0%vs.46.6%,X~2=17.321,P=0.001),P53阳性组与阴性组间BCRP表达未见显著差异。
2.浸润性导管癌中ERα基因启动子甲基化特征及其与ERα、BCRP表达相关性:
(1)乳腺癌与纤维腺瘤组织中的ERα表达与基因启动子甲基化:乳腺浸润性导管癌组织中ERα表达阳性率与纤维腺瘤比较未见显著性差异(48.3%vs.71.4%,X~2=2.429,P=0.119);乳腺癌组织甲基化发生率显著性高于纤维腺瘤组织(83.3%vs.28.6%,X~2=17.260,P=0.001);(2)乳腺癌中ERα基因启动子甲基化与ERα表达相关性:ERα阴性浸润性导管癌组织中ER2、ER3、ER4三个检测区域的甲基化发生率(63.3%,58.1%,83.9%)均显著高于ERα阳性组织(24.1%,17.2%,13.8%),以ERα表达水平与甲基化区域检出个数做Spearman相关性分析,二者呈显著负相关(r=-0.713,P=0.001);ERα阴性浸润性导管癌组织中ER4甲基化发生率显著高于其它三个检测区域(X~2=8.321,P=0.004);(3)乳腺癌中ERα基因启动子甲基化与BCRP蛋白表达的相关性:以BCRP表达水平与甲基化区域检出个数做Spearman相关性分析,二者呈较弱的正相关趋势(r=0.254,P=0.050);(4)乳腺癌中ERα基因启动子甲基化与临床病理参数的相关性:ERα基因启动子甲基化水平与乳腺癌患者年龄、临床分级、淋巴结转移及HER-2表达情况无关;孕激素受体阳性患者ERα基因启动子区甲基化水平显著低于孕激素受体阴性患者(X~2=9.598,P=0.002)。
3.5'-Aza-dC联合E_2对MDA-MB-435s细胞ERα基因启动子甲基化及ERα、BCRPmRNA表达的影响
(1)5'-Aza-dC对ERα基因启动子区甲基化的影响:采用10、50、100ng/ml终浓度的5'-Aza-dC处理MDA-MB-435s细胞,10ng/ml浓度处理后ER1、ER2、ER4三个检测区域发生了去甲基化,50、100ng/ml终浓度处理后MDA-MB-435s细胞ERα基因启动子四个甲基化检测区域均发生了去甲基化;(2)5'-Aza-dC对ERαmRNA表达的影响:溶剂及单纯3nmol/ml E_2处理组MDA-MB-435s细胞均未见明显PCR扩增阳性条带;单独采用100ng/ml终浓度的5'-Aza-dC处理MDA-MB-435s细胞96h后ERαmRNA相对表达量增加了16.3倍(0.814±0.031 vs.0.050±0.006);分别采用10、50、100ng/ml终浓度的5'-Aza-dC联合E_2处理MDA-MB-435s细胞96h后,10、50、100ng/ml浓度组ERαmRNA相对表达量分别增加了11.3(0.563±0.007 vs.0.050±0.006)、13.2(0.661±0.008 vs.0.050±0.006)、16.0(0.799±0.051 vs.0.050±0.006)倍;100ng/ml 5'-Aza-dC联合E_2组与单纯100ng/ml 5'-Aza-dC处理组间比较差别无统计学意义(t=1.358,P=0.232),10、50、100ng/ml 5'-Aza-dC分别联合3nmol/ml E_2处理组间ERαmRNA表达水平随5'-Aza-dC浓度增大而增加,差别具有显著性(F=85.182,P=0.001);(3)17-β-雌二醇对5'-Aza-dC处理前后BCRPmRNA表达的影响:MDA-MB-435s细胞单纯100ng/ml 5'-Aza-dC和3nmol/ml E_2处理组BCRP mRNA表达水平与溶剂组无显著性差异(F=3.256,P=0.425);10、50、100ng/ml 5'-Aza-dC联合3nmol/ml E_2处理MDA-MB-435s细胞96h后,三组BCRPmRNA相对表达量分别降低至溶剂对照组的81.5%(0.551±0.0177 vs.0.676±0.028)、51.7%(0.350±0.024 vs.0.676±0.028),42.8%(0.289±0.077 vs.0.676±0.028),各处理组间BCRP mRNA表达水平随5'-Aza-dC浓度增大而降低,差别具有显著性(F=67.232,P=0.001)。
结论
1.BCRP在乳腺癌组织中表达显著高于正常乳腺组织。BCRP在乳腺癌组织中表达水平患者与年龄、病理类型、临床分期、淋巴结转移等无关,在绝经前与ERα表达负相关、与HER-2表达正相关,与PR、p53表达未见相关性。
2.乳腺癌组织中ERα基因启动子甲基化水平显著高于乳腺良性病变。ERα阴性患者乳腺癌组织ERα基因启动子甲基化发生率显著高于ERα阳性患者,ERα蛋白表达水平与其基因启动子区甲基化位点个数呈高度负相关,乳腺癌组织中ERα失表达与其基因启动子甲基化有关,靠近编码区启动子序列甲基化更易引起ERα失表达。
3.5'Aza-dC可有效诱导MDA-MB-435s细胞ERα基因启动子去甲基化,并恢复其ERαmRNA表达;E_2对野生型MDA-MB-435s细胞BCRP表达无影响,但可显著下调ERα基因去甲基化MDA-MB-435s细胞的BCRP mRNA表达,表明ERα甲基化状态可能影响BCRP表达调控,提示ERα阴性乳腺癌易产生化疗耐药的机制可能与缺少雌激素信号对多药耐药蛋白表达调控有关。
Objective
To discuss the relationship of the expression of breast cancer resistance protein (BCRP) and Estrogen receptor alpha(ERα) with the methylation feature of promoter of ERαgene in breast cancer.Investigate the changes of methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after treated with methyltransferases inhibitor 5'-Aza-deoxycytidine(5'-Aza-dC).To investigate the correlation between the expression of BCRP and estrogen signaling and observe the possible effect of demethylation drugs on antihormone therapy to breast cancer,to elucidate the possible internal relation between endocrine therapy resistance and multidrug resistance as well as different pathological profiles in breast cancer.
Methods
The protein expression of BCRP,ER,PR,HER-2 and P53 in 146 sporadic breast cancers and 22 fibroadenoma tissues were examined using immunohistochemistry.The correlations of BCRP with the clinical data as well as the pathological parameters were analyzed by correlate test.the methylation states of four CpG compact sites in promoter region of ERαgene in 60 infitrating ductal carcinomas(IDCs) and 14 fibro adenomas(FAs) were detected by methylation specific PCR(MSP),correlation analysis between methylation of promoter of ERαgene and its' expression as well as clinical parameter was performed.The methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after be treated with 5'-Aza-dC by MSP and RT-PCR.
Results
The positive immunostaining rate of BCRP in breast cancer was significantly higher than that in fibroadenoma tissues(60.3%vs.31.8%,X~2=6.301,P=0.012).The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state,but was significantly lower in ERαpositive pre menopause patients(38.5%,X~2=9.301,P=0.007).The expression of BCRP in HER-2 positive pateins were significantly higher than that in HER-2 negative pateins (81.0%vs.46.6%,X~2=17.321,P=0.001),but no correlation with P53 or PR.
The expression of ERαprotein in IDCs didn't show statistical difference compared with that in AFs;the methylation rate in IDCs were significantly higher than that in AFs(83.3%vs.28.6%,X~2=17.260,P=0.001);the methylation rates of checking site ER2,ER3,ER4 in ERαnegative IDCs(63.3%,58.1%,83.9%) were totally higher than that in ERαpositive IDCs(24.1%,17.2%,13.8%);The methylation frequency of the promoter region of ERαgene was dramatically negative correlated with expression of ERα(r=-0.713,P=0.001) but modest positive correlated with expression of BCRP (r=0.254,P=0.050) in IDCs;the methylation rate of ER4 was significantly higher than that of other three check sites in ERαnegative IDCs(X~2=8.321,P=0.004);the methylation frequency of the promoter region of ERαgene in progesterone receptor (PR)positive IDCs was significantly lower than that in PR negative IDCs(X~2=9.598, P=0.002),but not correlated with age,clinical stage,lymph node metastases or HER-2 state.
Three of four methylation detection site were demethylated in MDA-MB-435s cell line which after been treated with 10 ng/ml 5'-Aza-dC and all the four sites were demethylated in that treated with 50 or 100ng/ml 5'-Aza-dC for 96h.The expression of ERαmRNA was increased for 16.3 fold in MDA-MB-435s after been treated only with 100 ng/ml 5'-Aza-dC for 96h(0.814±0.031 vs.0.050±0.006);After been exposed to 10, 50 or 100ng/ml 5'-Aza-dC respectively in combination with 3nmol/l E_2 for 96h,the expressions of ERαmRNA in MDA-MB-435s were correspondingly increased for 11.3 fold(0.563±0.007 vs.0.050±0.006),13.2 fold(0.661±0.008 vs.0.050±0.006) and 16.0 fold(0.799±0.051 vs.0.050±0.006);the expressions of ERαmRNA increased followed the concentration of 5'-Aza-dC.Positive stain band of BCRP mRNA were present in every group,the expression of ERαmRNA didn't changed in MDA-MB-435s after been treated singly with 100 ng/ml 5'-Aza-dC or 3nmol/ml E_2 for 96h(F=3.256, P=0.425);After been exposed to 10,50 or 100ng/ml 5'-Aza-dC respectively in combination with 3nmol/l E_2 for 96h,the expressions of BCRP mRNA in MDA-MB-435s were correspondingly decreased to 81.5%(0.551±0.0177 vs.0.676±0.028),51.7%(0.350±0.024 vs.0.676±0.028),42.8%(0.289±0.077 vs.0.676±0.028) when compared with the control group,the expressions of BCRP mRNA decreased contrast with the concentration of 5'-Aza-dC.
Conclusion
1.the protein level of BCRP in sporadic breast cancer was higher than that in fibroadenoma tissues and negatively correlated with ERαstatues in pre-menopause pateints but positively correlated with HER-2 statues all the menopause state;The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state.
2.The aberrant methylation of the promoter region of ERαgene was detected in IDC and this aberrant methylation contribute to the loss of ERαexpression;the effect of the methylation on protein expression depends on the frequency of methylation or specific methylated CpG Island;PR expression was correlated with methylation of the promoter region of ERαgene.
3.5'Aza-dC can not only efficaciously induced demethylation of the promoter of ERαgene but also restore the ERαexpression in a concentration dependent manner in ERαnegative breast cancer cell line.When used in combine with 5'Aza-dC,E_2 dramatically induced BCRP Expression down regulation in ERαnegative breast cancer cell line.These datas sugest that methylation of promoter of ERαgene might contrubite to MDR development in ERαnegative breast cancer due to Estrogen signaling deficiency caused BCRP regulation malfunction
目的
探讨乳腺癌耐药相关蛋白(breast cancer resistance protein,BCRP)在散发性乳腺癌组织中的表达特征及其与雌激素受体α(Estrogen Receptorα,ERα)蛋白表达和ERα基因启动子甲基化的相关性,通过细胞实验观察DNA甲基转移酶抑制剂5'-氮杂-脱氧胞苷酸(5'-Aza-2'-deoxycytidine,5'-Aza-dC)及17-β雌二醇(17β-Estradiol,E_2)处理前后ERα阴性乳腺癌细胞株MDA-MB-435s ERα基因启动子甲基化及ERα、BCRP mRNA表达变化情况,探讨乳腺癌中BCRP表达与雌激素信号的相关性及乳腺癌内分泌治疗耐药与多药耐药间可能存在的内在联系,初步阐明去甲基化药物在乳腺癌内分泌治疗与化学治疗中的可能意义。
材料与方法
采用免疫组织化学(immunohistochemistry,IHC)染色法,检测146例散发性乳腺癌和22例乳腺纤维腺瘤组织中BCRP、ERα、PR、HER-2、P53蛋白的表达,分析BCRP表达与临床病理学参数之间的相关性;通过甲基化特异性PCR(Methylation specific PCR,MSP)法检测60例乳腺浸润性导管癌肿瘤及14例乳腺纤维腺瘤组织中ERα基因启动子区四个CpG岛密集区域甲基化情况,探讨乳腺浸润性导管癌组织中ERα基因启动子区甲基化特征及其与ERα和BCRP表达的相关性;通过MSP、逆转录PCR(Reverse transcript PCR,RT-PCR)等方法检测5'-Aza-dC和E_2处理前后ER阴性MDA-MB-435s乳腺癌细胞株ERα基因启动子甲基化及ERα、BCRP mRNA变化情况,探讨去甲基化药物对于BCRP表达的可能影响及其机制。
结果
1.散发性乳腺癌中BCRP表达与临床病理参数相关性:
(1) BCRP在乳腺癌组织中表达情况:乳腺癌中BCRP表达阳性率60.3%,纤维腺瘤中BCRP表达阳性率31.8%,两者相比有显著性差异(X~2=6.301,P=0.012);(2)BCRP表达与乳腺癌临床指标相关性:在乳腺癌组织中,BCRP表达水平与患者年龄、病理类型、临床分级及淋巴结转移均无关;(3)乳腺癌组织中BCRP与ERα、PR表达的相关性:结合ERα、PR表达与绝经情况分组,未绝经且ERα阳性患者乳腺肿瘤组织中BCRP表达阳性率(38.5%)显著低于未绝经ERα阴性(71.9%)和已绝经ERα阳性(60.3%)及已绝经ERα阴性患者(63.0%)(X~2=9.301,P=0.007);绝经与未绝经PR阳性或阴性患者之间BCRP表达均未见显著性差别;(4)乳腺癌组织中BCRP与HER-2、P53表达的相关性:HER-2阳性表达乳腺癌中BCRP表达阳性率显著高于HER-2阴性乳腺癌(81.0%vs.46.6%,X~2=17.321,P=0.001),P53阳性组与阴性组间BCRP表达未见显著差异。
2.浸润性导管癌中ERα基因启动子甲基化特征及其与ERα、BCRP表达相关性:
(1)乳腺癌与纤维腺瘤组织中的ERα表达与基因启动子甲基化:乳腺浸润性导管癌组织中ERα表达阳性率与纤维腺瘤比较未见显著性差异(48.3%vs.71.4%,X~2=2.429,P=0.119);乳腺癌组织甲基化发生率显著性高于纤维腺瘤组织(83.3%vs.28.6%,X~2=17.260,P=0.001);(2)乳腺癌中ERα基因启动子甲基化与ERα表达相关性:ERα阴性浸润性导管癌组织中ER2、ER3、ER4三个检测区域的甲基化发生率(63.3%,58.1%,83.9%)均显著高于ERα阳性组织(24.1%,17.2%,13.8%),以ERα表达水平与甲基化区域检出个数做Spearman相关性分析,二者呈显著负相关(r=-0.713,P=0.001);ERα阴性浸润性导管癌组织中ER4甲基化发生率显著高于其它三个检测区域(X~2=8.321,P=0.004);(3)乳腺癌中ERα基因启动子甲基化与BCRP蛋白表达的相关性:以BCRP表达水平与甲基化区域检出个数做Spearman相关性分析,二者呈较弱的正相关趋势(r=0.254,P=0.050);(4)乳腺癌中ERα基因启动子甲基化与临床病理参数的相关性:ERα基因启动子甲基化水平与乳腺癌患者年龄、临床分级、淋巴结转移及HER-2表达情况无关;孕激素受体阳性患者ERα基因启动子区甲基化水平显著低于孕激素受体阴性患者(X~2=9.598,P=0.002)。
3.5'-Aza-dC联合E_2对MDA-MB-435s细胞ERα基因启动子甲基化及ERα、BCRPmRNA表达的影响
(1)5'-Aza-dC对ERα基因启动子区甲基化的影响:采用10、50、100ng/ml终浓度的5'-Aza-dC处理MDA-MB-435s细胞,10ng/ml浓度处理后ER1、ER2、ER4三个检测区域发生了去甲基化,50、100ng/ml终浓度处理后MDA-MB-435s细胞ERα基因启动子四个甲基化检测区域均发生了去甲基化;(2)5'-Aza-dC对ERαmRNA表达的影响:溶剂及单纯3nmol/ml E_2处理组MDA-MB-435s细胞均未见明显PCR扩增阳性条带;单独采用100ng/ml终浓度的5'-Aza-dC处理MDA-MB-435s细胞96h后ERαmRNA相对表达量增加了16.3倍(0.814±0.031 vs.0.050±0.006);分别采用10、50、100ng/ml终浓度的5'-Aza-dC联合E_2处理MDA-MB-435s细胞96h后,10、50、100ng/ml浓度组ERαmRNA相对表达量分别增加了11.3(0.563±0.007 vs.0.050±0.006)、13.2(0.661±0.008 vs.0.050±0.006)、16.0(0.799±0.051 vs.0.050±0.006)倍;100ng/ml 5'-Aza-dC联合E_2组与单纯100ng/ml 5'-Aza-dC处理组间比较差别无统计学意义(t=1.358,P=0.232),10、50、100ng/ml 5'-Aza-dC分别联合3nmol/ml E_2处理组间ERαmRNA表达水平随5'-Aza-dC浓度增大而增加,差别具有显著性(F=85.182,P=0.001);(3)17-β-雌二醇对5'-Aza-dC处理前后BCRPmRNA表达的影响:MDA-MB-435s细胞单纯100ng/ml 5'-Aza-dC和3nmol/ml E_2处理组BCRP mRNA表达水平与溶剂组无显著性差异(F=3.256,P=0.425);10、50、100ng/ml 5'-Aza-dC联合3nmol/ml E_2处理MDA-MB-435s细胞96h后,三组BCRPmRNA相对表达量分别降低至溶剂对照组的81.5%(0.551±0.0177 vs.0.676±0.028)、51.7%(0.350±0.024 vs.0.676±0.028),42.8%(0.289±0.077 vs.0.676±0.028),各处理组间BCRP mRNA表达水平随5'-Aza-dC浓度增大而降低,差别具有显著性(F=67.232,P=0.001)。
结论
1.BCRP在乳腺癌组织中表达显著高于正常乳腺组织。BCRP在乳腺癌组织中表达水平患者与年龄、病理类型、临床分期、淋巴结转移等无关,在绝经前与ERα表达负相关、与HER-2表达正相关,与PR、p53表达未见相关性。
2.乳腺癌组织中ERα基因启动子甲基化水平显著高于乳腺良性病变。ERα阴性患者乳腺癌组织ERα基因启动子甲基化发生率显著高于ERα阳性患者,ERα蛋白表达水平与其基因启动子区甲基化位点个数呈高度负相关,乳腺癌组织中ERα失表达与其基因启动子甲基化有关,靠近编码区启动子序列甲基化更易引起ERα失表达。
3.5'Aza-dC可有效诱导MDA-MB-435s细胞ERα基因启动子去甲基化,并恢复其ERαmRNA表达;E_2对野生型MDA-MB-435s细胞BCRP表达无影响,但可显著下调ERα基因去甲基化MDA-MB-435s细胞的BCRP mRNA表达,表明ERα甲基化状态可能影响BCRP表达调控,提示ERα阴性乳腺癌易产生化疗耐药的机制可能与缺少雌激素信号对多药耐药蛋白表达调控有关。
Objective
To discuss the relationship of the expression of breast cancer resistance protein (BCRP) and Estrogen receptor alpha(ERα) with the methylation feature of promoter of ERαgene in breast cancer.Investigate the changes of methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after treated with methyltransferases inhibitor 5'-Aza-deoxycytidine(5'-Aza-dC).To investigate the correlation between the expression of BCRP and estrogen signaling and observe the possible effect of demethylation drugs on antihormone therapy to breast cancer,to elucidate the possible internal relation between endocrine therapy resistance and multidrug resistance as well as different pathological profiles in breast cancer.
Methods
The protein expression of BCRP,ER,PR,HER-2 and P53 in 146 sporadic breast cancers and 22 fibroadenoma tissues were examined using immunohistochemistry.The correlations of BCRP with the clinical data as well as the pathological parameters were analyzed by correlate test.the methylation states of four CpG compact sites in promoter region of ERαgene in 60 infitrating ductal carcinomas(IDCs) and 14 fibro adenomas(FAs) were detected by methylation specific PCR(MSP),correlation analysis between methylation of promoter of ERαgene and its' expression as well as clinical parameter was performed.The methylation state of promoter of ERαand the level of ERαand BCRP mRNA in ERαnegative MDA-MB-435s breast cancer cell line before or after be treated with 5'-Aza-dC by MSP and RT-PCR.
Results
The positive immunostaining rate of BCRP in breast cancer was significantly higher than that in fibroadenoma tissues(60.3%vs.31.8%,X~2=6.301,P=0.012).The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state,but was significantly lower in ERαpositive pre menopause patients(38.5%,X~2=9.301,P=0.007).The expression of BCRP in HER-2 positive pateins were significantly higher than that in HER-2 negative pateins (81.0%vs.46.6%,X~2=17.321,P=0.001),but no correlation with P53 or PR.
The expression of ERαprotein in IDCs didn't show statistical difference compared with that in AFs;the methylation rate in IDCs were significantly higher than that in AFs(83.3%vs.28.6%,X~2=17.260,P=0.001);the methylation rates of checking site ER2,ER3,ER4 in ERαnegative IDCs(63.3%,58.1%,83.9%) were totally higher than that in ERαpositive IDCs(24.1%,17.2%,13.8%);The methylation frequency of the promoter region of ERαgene was dramatically negative correlated with expression of ERα(r=-0.713,P=0.001) but modest positive correlated with expression of BCRP (r=0.254,P=0.050) in IDCs;the methylation rate of ER4 was significantly higher than that of other three check sites in ERαnegative IDCs(X~2=8.321,P=0.004);the methylation frequency of the promoter region of ERαgene in progesterone receptor (PR)positive IDCs was significantly lower than that in PR negative IDCs(X~2=9.598, P=0.002),but not correlated with age,clinical stage,lymph node metastases or HER-2 state.
Three of four methylation detection site were demethylated in MDA-MB-435s cell line which after been treated with 10 ng/ml 5'-Aza-dC and all the four sites were demethylated in that treated with 50 or 100ng/ml 5'-Aza-dC for 96h.The expression of ERαmRNA was increased for 16.3 fold in MDA-MB-435s after been treated only with 100 ng/ml 5'-Aza-dC for 96h(0.814±0.031 vs.0.050±0.006);After been exposed to 10, 50 or 100ng/ml 5'-Aza-dC respectively in combination with 3nmol/l E_2 for 96h,the expressions of ERαmRNA in MDA-MB-435s were correspondingly increased for 11.3 fold(0.563±0.007 vs.0.050±0.006),13.2 fold(0.661±0.008 vs.0.050±0.006) and 16.0 fold(0.799±0.051 vs.0.050±0.006);the expressions of ERαmRNA increased followed the concentration of 5'-Aza-dC.Positive stain band of BCRP mRNA were present in every group,the expression of ERαmRNA didn't changed in MDA-MB-435s after been treated singly with 100 ng/ml 5'-Aza-dC or 3nmol/ml E_2 for 96h(F=3.256, P=0.425);After been exposed to 10,50 or 100ng/ml 5'-Aza-dC respectively in combination with 3nmol/l E_2 for 96h,the expressions of BCRP mRNA in MDA-MB-435s were correspondingly decreased to 81.5%(0.551±0.0177 vs.0.676±0.028),51.7%(0.350±0.024 vs.0.676±0.028),42.8%(0.289±0.077 vs.0.676±0.028) when compared with the control group,the expressions of BCRP mRNA decreased contrast with the concentration of 5'-Aza-dC.
Conclusion
1.the protein level of BCRP in sporadic breast cancer was higher than that in fibroadenoma tissues and negatively correlated with ERαstatues in pre-menopause pateints but positively correlated with HER-2 statues all the menopause state;The expression of BCRP in breast cancer tissues was not associated with histological types, age,clinical stage or lymph node metastases state.
2.The aberrant methylation of the promoter region of ERαgene was detected in IDC and this aberrant methylation contribute to the loss of ERαexpression;the effect of the methylation on protein expression depends on the frequency of methylation or specific methylated CpG Island;PR expression was correlated with methylation of the promoter region of ERαgene.
3.5'Aza-dC can not only efficaciously induced demethylation of the promoter of ERαgene but also restore the ERαexpression in a concentration dependent manner in ERαnegative breast cancer cell line.When used in combine with 5'Aza-dC,E_2 dramatically induced BCRP Expression down regulation in ERαnegative breast cancer cell line.These datas sugest that methylation of promoter of ERαgene might contrubite to MDR development in ERαnegative breast cancer due to Estrogen signaling deficiency caused BCRP regulation malfunction
引文
1 Faneyte IF, Kristel PM, Maliepaard M, et al.Expression of the breast cancer resistance protein in breast cancer.Clin Cancer Res. 2002 ; 8(4):1068-1074.
2 Burger H, Foekens JA, Look MP, et al. RNA expression of breast cancer resistance protein, lung resistance-related protein, multidrug resistance-associated proteins 1 and 2, and multidrug resistance gene 1 in breast cancer: correlation with chemotherapeutic response. Clin Cancer Res. 2003 ; 9(2):827-836.
3 曾爱华,何蕴韶,韩非．BCRP基因在乳腺癌组织中的表达及意义．山东医药．2005； 45(1)：14—16．
4 Tan B, Piwnica-Worms D, Ratner L. Multidrug resistance transporters and modulation. Curr Opin Oncol. 2000; 12(5):450-458.
5 Sugimoto Y, Tsukahara S, Ishikawa E, et al. Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/ pharmacodynamics. Cancer Sci. 2005; 96(8):457-465.
6 Anderson E, Clarke RB. Steroid receptors and cell cycle in normal mammary epithelium. J Mammary Gland Biol Neoplasia, 2004, 99(3):3-13.
7 Casalini P, Iorio MV, Galmozzi E, et al. Role of HER receptors family in development and differentiation. J Cell Physiol. 2004; 200(3):343-350.
8 Mass RD.The HER receptor family: a rich target for therapeutic developmentInt. J Radiat Oncol Biol Phys. 2004; 58(3):932-940.
9 Masood S. Prognostic/predictive factors in breast cancer. Clin Lab Med. 2005; 25(4):809-825.
10 Allred DC, Harvey JM, Berardo M, et al. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998; 11(2):155-168.
11 Kalofonos HP, Grivas PD. Monoclonal antibodies in the management of solid tumors. Curr Top Med Chem. 2006;6(16):1687-1705
12 Wang J, Hu Z, Ye Z. Effect of trastuzumab in combination with IFN alpha-2b on HER2 and MRP1 of ACHN. J Huazhong Univ Sci Technolog Med Sci. 2005; 25(6):326-328.
13 Gradishar WJ, Cella D. Selective estrogen receptor modulators and prevention of invasive breast cancer. JAMA, 2006, 23(8):2784-2786.
14 Normanno N, Di Maio M, De Maio E, et al. Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer. 2005; 4(16):721-747.
15 Kuukasjarvi T, Kononen J, Helin K, et al. Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy. J Clin Oncol. 1996; 14(5): 2584-2589.
16 Ottaviano YL, Issa JP, Parl FF, et al. Methylation of the estrogen receptor gene CpG Island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res. 1994; 54(10): 2552-2555.
17 Lapidus RG, Nass SJ, Butash KA, et al. Mapping of ER gene CpG island methylation-specific polymerase chain reaction. Cancer Res. 1998; 58(12): 2515-2519.
18 Cordera F, Jordan VC. Steroid receptors and their role in the biology and control of breast cancer growth. Semin Oncol. 2006; 33(6):631-641.
19 Ariazi EA, Ariazi JL, Cordera F, et al. Estrogen receptors as therapeutic targets in breast cancer Curr Top Med Chem. 2006;6(3): 181-202.
20 Cunha GR, Cooke PS, Kurita T. Role of stromal-epithelial interactions inhormonal responses. Arch Histol Cytol. 2004; 67(5):417-434.
21 Streuli CH, Haslam SZ. Control of mammary gland development and neoplasia by stromal-epithelial interactions and extracellular matrix. J Mammary Gland Biol Neoplasia 1998;3:107-108.
22 Ellis MJ, Rigden CE. Initial versus sequential adjuvant aromatase inhibitor therapy: a review of the current data. Curr Med Res Opin. 2006; 22(12): 2479-2487.
23 Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007; 128(4): 683-692.
24 Widschwendter M, Siegmund KD, Muller HM, et al. Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen. Cancer Res. 2004; 64(11): 3807-3813.
25 Bibikova M, Lin Z, Zhou L, et al. High-throughput DNA methylation profiling using universal bead arrays. Genome Res. 2006; 3: 383-393.
26 Ee PL, Kamalakaran S, Tonetti D, et al. Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene. Cancer Res, 2004, 64(4): 1247-51.
27 Callinan PA, Feinberg AP. The emerging science of epigenomicsHum Mol Genet. 2006; 15 (1):R95-101.
28 Hsi LC,Xi X,Wu Y,et al.The methyltransferase inhibitor 5-aza-2-deoxycytidine induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells.Mol Cancer Ther.2005;4(11):1740-1746.
29 Pulukuri SM,Rao JS.Activation of p53/p21Waf1/Cip1 pathway by 5-aza-2'-deoxycytidine inhibits cell proliferation,induces pro-apoptotic genes and mitogen-activated protein kinases in human prostate cancer cells.Int J Oncol.2005;26(4):863-871.
30 Bheemanaik S,Reddy YV,Rao DN.Structure,function and mechanism of exocyclic DNA methyltransferases.Biochem J.2006;399(2):177-190.
31 Gowher H,Jeltsch A.Mechanism of inhibition of DNA methyltransferases by cytidine analogs in cancer therapy Cancer Biol Ther.2004;3(11):1062-8.
32 Yang X,Phillips DL,Ferguson AT,et al.Synergistic activation of functional estrogen receptor(ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells.Cancer Res.2001;61(19):7025-7029
33 Imai Y,Ishikawa E,Asada S,et al.Estrogen-mediated post transcriptional down-regulation of breast cancer resistance protein/ABCG2.Cancer Res.2005;65(2):596-604.
34 Wang H,Zhou L,Gupta A,et al.Regulation of BCRP/ABCG2 expression by progesterone and 17beta-estradiol in human placental BeWo ceils.Am J Physiol Endocrinol Metab.2006;290(5):E798-807.
35 范江,陆劲松,殷文瑾,等.药物诱导MDA-MB-435细胞表达ERα及其对内分泌治疗的敏感性.中华肿瘤杂志.2006;28(12):886-889,
1. Banrett - Connor E , Grady D. Hormone replacement therapy , heart disease , and other considerations . Annu Rev Public Health, 1998 ,19(1) :55 - 72.
2. Pace P , Taylor J , Suntharalingam S , et al. Human estrogen receptor beta binds DNA in a manner similar to and dimerizes with estrogen receptor alpha. J Biol Chem , 1997 , 272 (41) :25832-25838.
3. Mosselman S , Polman J , Dijkema R. ERβ: identification and characterization of a novel human estrogen receptor. FEBS Lett, 2005 , 392(1): 49 - 53.
4. Mcinerney EM, Katzenellenbogen BS. Different regions in ac2 tivation function - 1 of the human estrogen receptor required for antiestrogen - and estradiol - dependent transcription acti2 vation. J Biol Chem ,1996 , 271 (39): 24172 - 24178.
5. Denger S , Reid G, Brand H , et al. Tissue - specific expres2 sion of human ER - alpha and ER - beta in male. Mol Cell Endocrinol, 2003 , 178(1 - 2): 155 -160.
6. Pakdel F , Metivier R , Flouriot G, et al. Two estrogen recep2 tor (ER) isoforms with different estrogen dependencies are gen2 erated from the trout ER gene. Endocrinology ,2000 , 141 (2): 571 - 580.
7. Petersen DN , Tkalcevic GT, Koza - Taylor PH , et al. Identi2 fication of estrogen receptor ERβ2 , a functional variant estro2 gen receptorpexpressed in normal rat tissues. Endocrinology, 1998 ,139(3): 1082 -1092.
8. Hodges YK, Tung L , Yan XD , et al. Estrogen receptoraand β:revalence of estrogen receptorPmRNA in human vascular smooth muscle and transcriptional effects. Circulation , 2000 ,101(15):1792 -1798.
9. Cunha GR, Cooke PS, Kurita T. Role of stromal-epithelial interactions inhormonal responses. Arch Histol Cytol 2004; 67:417-434.
10. Streuli CH, Haslam SZ. Control of mammary gland development and neoplasia by stromal-epithelial interactions and extracellular matrix. J Mammary Gland Biol Neoplasia 1998;3:107-108.
11. Anderson E, Clarke RB. Steroid receptors and cell cycle in normal mammary epithelium. J Mammary Gland Biol Neoplasia 2004; 9:3-13.
12. Wang W, Dong L, Saville B, Safe S. Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-YSp1/estrogen receptor interactions. Mol Endocrinol 1999; 13:1373-1387.
13. Nemere I, Pietras RJ, Blackmore PF. Membrane receptors for steroid hormones: signal transduction and physiological significance. J Cell Biochem 2003; 88:438-445.
14. Cheskis BJ. Regulation of cell signalling cascades by steroid hormones. J Cell Biochem 2004; 93:20-27.
15. Levin ER. Integration of the extra-nuclear and nuclear actions of estrogen. Mol Endocrinol 2005;19:1951-1959.
16. Shupnik MA. Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation. Oncogene 2004; 23:7979-7989.
17. Guo X, Razandi M, Pedram A, et al. Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta. J Biol Chem 2005; 280:19704-19710.
18. Chambliss KL, Simon L, Yuhanna IS, et al. Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERa domains with known nuclear functions. Mol Endocrinol 2005; 19:277-289.
19. Watters JJ, Chun TY, Kim YN, et al. Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells. Mol Endocrinol 2000; 14:1872-1881.
20. Song RX, McPherson RA, Adam L, et al. Linkage of rapid estrogen action to MAPK activation by ERa-Shc association and Shc pathway activation. Mol Endocrinol 2002; 16:116-127.
21. McEwen B, Akama K, Alves S, et al. Tracking the estrogen receptor in neurons: implications for estrogen-induced synapse formation. PNAS 2001; 98:7093-7100. 14. Warner M, Gustafsson JA. Nongenomic effects of estrogen: why all the uncertainty? Steroids 2006; 71:91-95.
22. Castoria G, Barone MV, Di Domenico M, et al. Nontranscriptional action of oestradiol and progestin triggers DNA synthesis. EMBO J 1999; 18:2500-2510.
23. Levin ER. Cellular functions of plasma membrane estrogen receptors. Steroids 2002; 67:471-475.
24. O'Malley BW. A life-long search for the molecular pathways of steroid hormone action. Mol Endocrinol 2005; 19:1402-1411.
25. Edwards DP, Wardell SE, Boonyaratanakornkit V. Progesterone receptor interacting coregulatory proteins and cross talk with cell signaling pathways. J Steroid Biochem Mol Biol 2002; 83:173-186.
26. Migliaccio A, Castoria G, Di Domenico M, et al. Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model. Nat Rev Drug Discov 2004; 3:27-41.
27. Song RX, Zhang Z, Santen RJ. Estrogen rapid action via protein complex formation involving ERa and Src. Trends Endocrinol Metab 2005; 16: 347-353.
28. Razandi M, Pedram A, Greene GL, et al. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERa and ERb expressed in Chinese hamster ovary cells. Mol Endocrinol 1999; 13:307-319.
29. Li L, Haynes MP, Bender JR. Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. Proc Natl Acad Sci U S A 2003; 100:4807-4812.
30. Toran-Allerand CD, Guan X, MacLusky NJ, et al. ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci 2002; 22:8391-8401.
31. Hammes A, Andreassen TK, Spoelgen R, et al. Role of endocytosis in cellular uptake of sex steroids. Cell 2005; 122:751-762.
32. Catalano MG, Comba A, Fazzari A, et al. Sex steroid binding protein receptor (SBP-R) is related to a reduced proliferation rate in human breast cancer. Breast Cancer Res Treat 1997; 42:227-234.
33. Thomas P, Pang Y, Filardo EJ, et al. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 2005; 146:624-632.
34. Nethrapalli IS, Tinnikov AA, Krishnan V, et al. Estrogen activates mitogen-activated protein kinase in native, nontransfected CHO-K1, COS-7, and RAT2 fibroblast cell lines. Endocrinology 2005; 146:56-63.
35. Razandi M, Pedram A, Merchenthaler I, et al. Plasma membrane estrogen receptors exist and functions as dimers. Mol Endocrinol 2004; 18:2854-2865.
36. Song RX, Barnes CJ, Zhang Z, et al. The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane. Proc Natl Acad Sci U S A 2004; 101:2076-2081.
37. Zhang Z, Maier B, Santen RJ, et al. Membrane association of estrogen receptor alpha mediates estrogen effect on MAPK activation. Biochem Biophys Res Commun 2002; 294:926-933.
38. Pappas, Kim SH, Funk CC, et al. Estrogen dendrimer conjugates that preferentially activate extranuclear, non-genomic versus genomic pathways of estrogen action. Mol Endocrinol 1995; 20:491-502.
39. Razandi, Shaul PW. Estrogen modulation of endothelial nitric oxide synthase. Endocr Rev 2002; 23:665-686.
40. Filardo EJ, Quinn JA, Bland KI, et al. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000; 14:1649-1660.
41. Thomas P, Castoria G, Di Domenico M, et al. Sex steroid hormones act as growth factors. J Steroid Biochem Mol Biol 2002; 83:31-35.
42. Pedram A, Alton G, Pedram A, et al. Identification of a structural determinant necessary for the localization and function of estrogen receptor alpha at the plasma membrane. Mol Cell Biol 2003; 23:1633-1646.
43. Robert X, Pallas DC, Surks HK, et al. Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha. Proc Natl Acad Sci U S A 2004; 101:17126-17131.
44. Chen JQ, Yager JD. Estrogen's effects on mitochondrial gene expression: mechanisms and potential contributions to estrogen carcinogenesis. Ann N Y Acad Sci 2004; 1028:258-272.
45. Razandi M, Oh P, Pedram A, et al. ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Mol Endocrinol 2002; 16:100-115.
46. Cabodi S, Moro L, Baj G, Smeriglio M, Di Stefano P, Gippone S, Surico N, Silengo L, Turco E, Tarone G, Defilippi P. p130Cas Interacts with estrogen receptor a and modulates non-genomic estrogen Endocr Rev 2005; 29:621-632.
2 Burger H, Foekens JA, Look MP, et al. RNA expression of breast cancer resistance protein, lung resistance-related protein, multidrug resistance-associated proteins 1 and 2, and multidrug resistance gene 1 in breast cancer: correlation with chemotherapeutic response. Clin Cancer Res. 2003 ; 9(2):827-836.
3 曾爱华,何蕴韶,韩非．BCRP基因在乳腺癌组织中的表达及意义．山东医药．2005； 45(1)：14—16．
4 Tan B, Piwnica-Worms D, Ratner L. Multidrug resistance transporters and modulation. Curr Opin Oncol. 2000; 12(5):450-458.
5 Sugimoto Y, Tsukahara S, Ishikawa E, et al. Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/ pharmacodynamics. Cancer Sci. 2005; 96(8):457-465.
6 Anderson E, Clarke RB. Steroid receptors and cell cycle in normal mammary epithelium. J Mammary Gland Biol Neoplasia, 2004, 99(3):3-13.
7 Casalini P, Iorio MV, Galmozzi E, et al. Role of HER receptors family in development and differentiation. J Cell Physiol. 2004; 200(3):343-350.
8 Mass RD.The HER receptor family: a rich target for therapeutic developmentInt. J Radiat Oncol Biol Phys. 2004; 58(3):932-940.
9 Masood S. Prognostic/predictive factors in breast cancer. Clin Lab Med. 2005; 25(4):809-825.
10 Allred DC, Harvey JM, Berardo M, et al. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998; 11(2):155-168.
11 Kalofonos HP, Grivas PD. Monoclonal antibodies in the management of solid tumors. Curr Top Med Chem. 2006;6(16):1687-1705
12 Wang J, Hu Z, Ye Z. Effect of trastuzumab in combination with IFN alpha-2b on HER2 and MRP1 of ACHN. J Huazhong Univ Sci Technolog Med Sci. 2005; 25(6):326-328.
13 Gradishar WJ, Cella D. Selective estrogen receptor modulators and prevention of invasive breast cancer. JAMA, 2006, 23(8):2784-2786.
14 Normanno N, Di Maio M, De Maio E, et al. Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer. 2005; 4(16):721-747.
15 Kuukasjarvi T, Kononen J, Helin K, et al. Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy. J Clin Oncol. 1996; 14(5): 2584-2589.
16 Ottaviano YL, Issa JP, Parl FF, et al. Methylation of the estrogen receptor gene CpG Island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res. 1994; 54(10): 2552-2555.
17 Lapidus RG, Nass SJ, Butash KA, et al. Mapping of ER gene CpG island methylation-specific polymerase chain reaction. Cancer Res. 1998; 58(12): 2515-2519.
18 Cordera F, Jordan VC. Steroid receptors and their role in the biology and control of breast cancer growth. Semin Oncol. 2006; 33(6):631-641.
19 Ariazi EA, Ariazi JL, Cordera F, et al. Estrogen receptors as therapeutic targets in breast cancer Curr Top Med Chem. 2006;6(3): 181-202.
20 Cunha GR, Cooke PS, Kurita T. Role of stromal-epithelial interactions inhormonal responses. Arch Histol Cytol. 2004; 67(5):417-434.
21 Streuli CH, Haslam SZ. Control of mammary gland development and neoplasia by stromal-epithelial interactions and extracellular matrix. J Mammary Gland Biol Neoplasia 1998;3:107-108.
22 Ellis MJ, Rigden CE. Initial versus sequential adjuvant aromatase inhibitor therapy: a review of the current data. Curr Med Res Opin. 2006; 22(12): 2479-2487.
23 Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007; 128(4): 683-692.
24 Widschwendter M, Siegmund KD, Muller HM, et al. Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen. Cancer Res. 2004; 64(11): 3807-3813.
25 Bibikova M, Lin Z, Zhou L, et al. High-throughput DNA methylation profiling using universal bead arrays. Genome Res. 2006; 3: 383-393.
26 Ee PL, Kamalakaran S, Tonetti D, et al. Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene. Cancer Res, 2004, 64(4): 1247-51.
27 Callinan PA, Feinberg AP. The emerging science of epigenomicsHum Mol Genet. 2006; 15 (1):R95-101.
28 Hsi LC,Xi X,Wu Y,et al.The methyltransferase inhibitor 5-aza-2-deoxycytidine induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells.Mol Cancer Ther.2005;4(11):1740-1746.
29 Pulukuri SM,Rao JS.Activation of p53/p21Waf1/Cip1 pathway by 5-aza-2'-deoxycytidine inhibits cell proliferation,induces pro-apoptotic genes and mitogen-activated protein kinases in human prostate cancer cells.Int J Oncol.2005;26(4):863-871.
30 Bheemanaik S,Reddy YV,Rao DN.Structure,function and mechanism of exocyclic DNA methyltransferases.Biochem J.2006;399(2):177-190.
31 Gowher H,Jeltsch A.Mechanism of inhibition of DNA methyltransferases by cytidine analogs in cancer therapy Cancer Biol Ther.2004;3(11):1062-8.
32 Yang X,Phillips DL,Ferguson AT,et al.Synergistic activation of functional estrogen receptor(ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells.Cancer Res.2001;61(19):7025-7029
33 Imai Y,Ishikawa E,Asada S,et al.Estrogen-mediated post transcriptional down-regulation of breast cancer resistance protein/ABCG2.Cancer Res.2005;65(2):596-604.
34 Wang H,Zhou L,Gupta A,et al.Regulation of BCRP/ABCG2 expression by progesterone and 17beta-estradiol in human placental BeWo ceils.Am J Physiol Endocrinol Metab.2006;290(5):E798-807.
35 范江,陆劲松,殷文瑾,等.药物诱导MDA-MB-435细胞表达ERα及其对内分泌治疗的敏感性.中华肿瘤杂志.2006;28(12):886-889,
1. Banrett - Connor E , Grady D. Hormone replacement therapy , heart disease , and other considerations . Annu Rev Public Health, 1998 ,19(1) :55 - 72.
2. Pace P , Taylor J , Suntharalingam S , et al. Human estrogen receptor beta binds DNA in a manner similar to and dimerizes with estrogen receptor alpha. J Biol Chem , 1997 , 272 (41) :25832-25838.
3. Mosselman S , Polman J , Dijkema R. ERβ: identification and characterization of a novel human estrogen receptor. FEBS Lett, 2005 , 392(1): 49 - 53.
4. Mcinerney EM, Katzenellenbogen BS. Different regions in ac2 tivation function - 1 of the human estrogen receptor required for antiestrogen - and estradiol - dependent transcription acti2 vation. J Biol Chem ,1996 , 271 (39): 24172 - 24178.
5. Denger S , Reid G, Brand H , et al. Tissue - specific expres2 sion of human ER - alpha and ER - beta in male. Mol Cell Endocrinol, 2003 , 178(1 - 2): 155 -160.
6. Pakdel F , Metivier R , Flouriot G, et al. Two estrogen recep2 tor (ER) isoforms with different estrogen dependencies are gen2 erated from the trout ER gene. Endocrinology ,2000 , 141 (2): 571 - 580.
7. Petersen DN , Tkalcevic GT, Koza - Taylor PH , et al. Identi2 fication of estrogen receptor ERβ2 , a functional variant estro2 gen receptorpexpressed in normal rat tissues. Endocrinology, 1998 ,139(3): 1082 -1092.
8. Hodges YK, Tung L , Yan XD , et al. Estrogen receptoraand β:revalence of estrogen receptorPmRNA in human vascular smooth muscle and transcriptional effects. Circulation , 2000 ,101(15):1792 -1798.
9. Cunha GR, Cooke PS, Kurita T. Role of stromal-epithelial interactions inhormonal responses. Arch Histol Cytol 2004; 67:417-434.
10. Streuli CH, Haslam SZ. Control of mammary gland development and neoplasia by stromal-epithelial interactions and extracellular matrix. J Mammary Gland Biol Neoplasia 1998;3:107-108.
11. Anderson E, Clarke RB. Steroid receptors and cell cycle in normal mammary epithelium. J Mammary Gland Biol Neoplasia 2004; 9:3-13.
12. Wang W, Dong L, Saville B, Safe S. Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-YSp1/estrogen receptor interactions. Mol Endocrinol 1999; 13:1373-1387.
13. Nemere I, Pietras RJ, Blackmore PF. Membrane receptors for steroid hormones: signal transduction and physiological significance. J Cell Biochem 2003; 88:438-445.
14. Cheskis BJ. Regulation of cell signalling cascades by steroid hormones. J Cell Biochem 2004; 93:20-27.
15. Levin ER. Integration of the extra-nuclear and nuclear actions of estrogen. Mol Endocrinol 2005;19:1951-1959.
16. Shupnik MA. Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation. Oncogene 2004; 23:7979-7989.
17. Guo X, Razandi M, Pedram A, et al. Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta. J Biol Chem 2005; 280:19704-19710.
18. Chambliss KL, Simon L, Yuhanna IS, et al. Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERa domains with known nuclear functions. Mol Endocrinol 2005; 19:277-289.
19. Watters JJ, Chun TY, Kim YN, et al. Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells. Mol Endocrinol 2000; 14:1872-1881.
20. Song RX, McPherson RA, Adam L, et al. Linkage of rapid estrogen action to MAPK activation by ERa-Shc association and Shc pathway activation. Mol Endocrinol 2002; 16:116-127.
21. McEwen B, Akama K, Alves S, et al. Tracking the estrogen receptor in neurons: implications for estrogen-induced synapse formation. PNAS 2001; 98:7093-7100. 14. Warner M, Gustafsson JA. Nongenomic effects of estrogen: why all the uncertainty? Steroids 2006; 71:91-95.
22. Castoria G, Barone MV, Di Domenico M, et al. Nontranscriptional action of oestradiol and progestin triggers DNA synthesis. EMBO J 1999; 18:2500-2510.
23. Levin ER. Cellular functions of plasma membrane estrogen receptors. Steroids 2002; 67:471-475.
24. O'Malley BW. A life-long search for the molecular pathways of steroid hormone action. Mol Endocrinol 2005; 19:1402-1411.
25. Edwards DP, Wardell SE, Boonyaratanakornkit V. Progesterone receptor interacting coregulatory proteins and cross talk with cell signaling pathways. J Steroid Biochem Mol Biol 2002; 83:173-186.
26. Migliaccio A, Castoria G, Di Domenico M, et al. Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model. Nat Rev Drug Discov 2004; 3:27-41.
27. Song RX, Zhang Z, Santen RJ. Estrogen rapid action via protein complex formation involving ERa and Src. Trends Endocrinol Metab 2005; 16: 347-353.
28. Razandi M, Pedram A, Greene GL, et al. Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERa and ERb expressed in Chinese hamster ovary cells. Mol Endocrinol 1999; 13:307-319.
29. Li L, Haynes MP, Bender JR. Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. Proc Natl Acad Sci U S A 2003; 100:4807-4812.
30. Toran-Allerand CD, Guan X, MacLusky NJ, et al. ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci 2002; 22:8391-8401.
31. Hammes A, Andreassen TK, Spoelgen R, et al. Role of endocytosis in cellular uptake of sex steroids. Cell 2005; 122:751-762.
32. Catalano MG, Comba A, Fazzari A, et al. Sex steroid binding protein receptor (SBP-R) is related to a reduced proliferation rate in human breast cancer. Breast Cancer Res Treat 1997; 42:227-234.
33. Thomas P, Pang Y, Filardo EJ, et al. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 2005; 146:624-632.
34. Nethrapalli IS, Tinnikov AA, Krishnan V, et al. Estrogen activates mitogen-activated protein kinase in native, nontransfected CHO-K1, COS-7, and RAT2 fibroblast cell lines. Endocrinology 2005; 146:56-63.
35. Razandi M, Pedram A, Merchenthaler I, et al. Plasma membrane estrogen receptors exist and functions as dimers. Mol Endocrinol 2004; 18:2854-2865.
36. Song RX, Barnes CJ, Zhang Z, et al. The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane. Proc Natl Acad Sci U S A 2004; 101:2076-2081.
37. Zhang Z, Maier B, Santen RJ, et al. Membrane association of estrogen receptor alpha mediates estrogen effect on MAPK activation. Biochem Biophys Res Commun 2002; 294:926-933.
38. Pappas, Kim SH, Funk CC, et al. Estrogen dendrimer conjugates that preferentially activate extranuclear, non-genomic versus genomic pathways of estrogen action. Mol Endocrinol 1995; 20:491-502.
39. Razandi, Shaul PW. Estrogen modulation of endothelial nitric oxide synthase. Endocr Rev 2002; 23:665-686.
40. Filardo EJ, Quinn JA, Bland KI, et al. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000; 14:1649-1660.
41. Thomas P, Castoria G, Di Domenico M, et al. Sex steroid hormones act as growth factors. J Steroid Biochem Mol Biol 2002; 83:31-35.
42. Pedram A, Alton G, Pedram A, et al. Identification of a structural determinant necessary for the localization and function of estrogen receptor alpha at the plasma membrane. Mol Cell Biol 2003; 23:1633-1646.
43. Robert X, Pallas DC, Surks HK, et al. Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha. Proc Natl Acad Sci U S A 2004; 101:17126-17131.
44. Chen JQ, Yager JD. Estrogen's effects on mitochondrial gene expression: mechanisms and potential contributions to estrogen carcinogenesis. Ann N Y Acad Sci 2004; 1028:258-272.
45. Razandi M, Oh P, Pedram A, et al. ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Mol Endocrinol 2002; 16:100-115.
46. Cabodi S, Moro L, Baj G, Smeriglio M, Di Stefano P, Gippone S, Surico N, Silengo L, Turco E, Tarone G, Defilippi P. p130Cas Interacts with estrogen receptor a and modulates non-genomic estrogen Endocr Rev 2005; 29:621-632.