三氧化二砷对人乳腺癌雌激素受体表达的影响
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摘要
背景和目的：
近年来乳腺癌的发病率和死亡率增长速度较快,全世界每年患乳腺癌的女性约130万人,而死于乳腺癌的有50万人。虽然我国的乳腺癌属于低发,但近年来其发病率增长幅度较快,预测20年后将会成为我国发病率最高的恶性肿瘤。
雌激素受体(estrogen receptor ER)在乳腺癌的发生发展过程中,扮演着重要角色。在体内通过与雌激素的结合,激活含雌激素应答元件基因和含有其他转录因子结合元件基因的表达,进而导致乳腺癌的发生。通常情况下,以ER基因表达情况的不同可将乳腺癌分为ER阴性乳腺癌和ER阳性乳腺癌两种。其中ER阴性乳腺癌约占20%-35%,其恶性程度高、发展迅速、有较高的局部复发率,并易发生肺、肝、骨及脑等重要脏器的转移,预后较差。由于缺乏相应的受体不能进行内分泌和生物治疗,所以这类特殊乳腺癌的治疗仅限于化疗,但疗效欠佳。因此,作为一个分子标记物,ER在乳腺癌的内分泌治疗中进行效果预测、评估激素依赖性等方面具有重要的意义。
DNA甲基化和染色体重组是当前研究ER阴性乳腺癌中ER基因失活的两大主要学说。甲基化在肿瘤的发生、发展中有着重要作用,但又是一可逆过程。从理论上来说,如果使用药物对甲基化的抑癌基因和癌基因进行人为逆转,即可使因甲基化失活的基因重新获得表达。
三氧化二砷(As2O3)为中药砒霜的主要成分,近年来发现其对白血病、原发性肝癌等多种肿瘤治疗取得了较好的效果。主要通过诱导细胞凋亡来发挥其细胞毒性作用,如今多项研究证明其具有甲基化抑制作用。
本实验通过研究不同浓度的As2O3对人ER阴性乳腺癌细胞株MDA-MB-435进行药物诱导,使其基因启动子区CpG岛部分脱甲基而使其重新表达有功能的ERα,从而使得ER阴性乳腺癌细胞恢复对内分泌治疗药物的敏感性。
材料与方法：
(1)常规培养雌激素受体(ERα)阴性人乳腺癌细胞株MDA-MB-435,细胞浓度调整为5×105/ml后换含As2O3的培养液,至AS2O3终浓度分别为0.5μmol/L、1.0μmol/L、2.0μmol/L、4.0μmol/L,连续培养72h。以同期培养不加药的MDA-MB-435细胞作为阴性对照,ERα阳性人乳腺癌细胞株MCF-7作为阳性对照。
(2) RT-PCR方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERα的mRNA表达情况。
(3)甲基化特异性PCR (Methylation Specific PCR, MSP)方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERαDNA的5’-CpG岛甲基化的情况。
(4) Western blot方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERα的蛋白表达情况。
(5)免疫组化方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERα的蛋白表达情况。
(6)MTT方法检测经不同浓度As2O3处理后的MDA-MB-435细胞,再经三苯氧胺(TAM)处理,观察各组细胞的生长受抑情况。
(7)统计学方法：应用统计学处理软件SPSS 16.0,X2检验进行统计学分析,·差异显著性标准设为P<0.05。
结果：
(1) RT-PCR法检测阴性对照组无ERαmRNA基因；0.5μmol/L组未扩增出ERαmRNA基因；1.0μmol/L组、2.0μmol/L组、4.0μmol/L组均扩增出ERαmRNA基因。
(2)甲基化特异性PCR法检测阴性对照组仅甲基化引物扩增出特异PCR条带；0.5μmol/L组、1.0μmol/L组、2.0μmol/L组、4.0μmol/L组甲基化和去甲基化引物均扩增出特异PCR条带。
(3)经Western blot检测证实阴性对照组未显示蛋白条带；0.5μmol/L组也未显示蛋白条带,但1.0μmol/L组、2.0μmol/L和4.0μmol/L组显示出蛋白条带。
(4)经免疫组化法检测证实阴性对照组细胞阳性率为7.4%；/L组、/L组、/L组、/L组细胞阳性率分别为7.8%、43.8%、78.2%、41.5%。
(5)MTT法检测TAM作用于经As203处理的MDA-MB-435细胞后,细胞增殖明显受抑。阴性对照组受抑率为10.7%;0.5μmol/L组、/L组、2.0μmol/L组、/L组细胞受抑率分别是14.9%、30.7%、43.1%、56.6%。
结论：
适当浓度的As203通过对部分细胞的去甲基化作用,使ERa阴性人乳腺癌细胞株MDA-MB-435细胞ERa mRNA和蛋白重新表达,并可恢复对内分泌治疗药物(三苯氧胺)的敏感性,从而为ERa阴性乳腺癌患者实现内分泌治疗提供可靠的理论依据。
Background and objective:
In recent years, the morbidity and mortality of breast cancer are growing rapidly. About 130 million women suffer from breast cancer and 50 million people died of breast cancer each year in the world. Although breast cancer has a low morbidity in our country, the incidence of breast cancer is rising obviously. We predict that it will be the highest incidence of malignant tumor in 20 years.
Estrogen receptor (ER) is an important role in the development of breast cancer. In vivo, ER combine with estrogen and active the genes which contain estrogen response element and transcription factor response element, then lead to breast cancer. In general, we divide the breast cancer into ER negative breast cancer and ER positive breast cancer. Among about 20～35 percent of breast cancer patients, the estrogen receptor is negative. With the high malignant degree, rapid development, higher local recurrence, and metastases easily occurring in lung, liver, bone, brain and other important viscera, it has poor prognosis. Due to lack of corresponding receptor, the patients cannot apply endocrine and biological treatment. At present, the treatment of this kind of special type of breast cancer limits to chemotherapy, but it is far from perfect. Thus, as an important molecular marker, ER has vital significance to forecast the effect of endocrine therapy and evaluate the dependence of hormone in breast cancer.
The main theories of ER gene expression silencing are DNA methylation and chromosome restructuring in ER negative breast cancer. Methylation occurs early in the process of cancer, it plays an important role in the occurrence and development of cancer. DNA methylation is a reversible process. By theoretically, if we reverse methylated oncogenes and tumor-suppressor genes with drugs, it will be made the gene re-expressed.
Arsenic trioxide (AS2O3) is the main ingredient of arsenic's of Traditional Chinese medicine. In recent years, we find that it has better effects on many kinds of tumor cells, such as leukemia, liver cancer and so on. AS2O3 exert it's cytotoxicity by inducing cells apoptosis. Recent research suggests that it can demethylate.
This study is designed that AS2O3 effect on ERa negative breast cancer cell MDA-MB-435. As2O3 can demethylate gene promoter CpG island partially, and made ERa re-expressed. Consequently, ERa negative breast cancer patients can restore the sensitivity of endocrine drugs.
Materials and methods:
(1) ERa negative human breast cancer cell line MDA-MB-435 planted at 37℃and 5% CO2. Adjust cell density of 5×105/ml and treat with 1640 containing AS2O3 0.5μmol/L, 1.0μmol/L,2.0μmol/L,4.0μmol/L respectively. Then plant for 72 hours. MDA-MB-435 without being treated with containing As2O3 was taken as negative control. Human breast cancer cell line MCF-7 (ER positive) was taken as positive control.
(2) To detect the re-expression of the ERa mRNA by RT-PCR.
(3) To detect the status of 5'CpG island methylation of ERa gene by Methylation Specific PCR(MSP).
(4) To detect the re-expression of the ERa protein by Western blot.
(5) To detect the re-expression of the ERa protein by immunohismchemistry.
(6) MTT method tested whether Tamoxifen could induce the growth inhibition of MDA-MB-435 cell after treated with As2O3.
(7) Using SPSS 16.0 statistics processing software andχ2-test for statistical analysis. Significant of difference standard is P<0.05.
Result:
(1) It is confirmed by the test of RT-PCR that expression of ERa mRNA were not found in group of negative control, neither in group of 0.5μmol/L. But ERa mRNA were detectable in groups of 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
(2) By MSP technique, methylation of ERa gene was found in group of negative control, methylation of ERa gene and demethylation of ERa gene were both found in groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
(3) It is confirmed by the test of Western blot that expression of ERa protein were not found in group of negative control and in group of 0.5μmol/L. But ERa protein were detectable in groups of 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
(4) By immunohistochemistry technique, we found that, in group of negative control, the positive rate was 7.4%. In groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L, the positive rate is respectively 7.8%,43.8%,78.2% and 41.5%.
(5) By the test of MTT, we found that tamoxifen can effect on the growth of MDA-MB-435 cells treated with AS2O3. We found that, in group of negative control, the inhibition rate was 10.7%. Inhibition rate was respectively 14.9%,30.7%,43.1% and 56.6% in groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
Conclusion:
A proper concentration of AS2O3 could effect on a part cells through demethylation and induce ERa gene re-expression in human breast cancer cell line MDA-MB-435. And it can restore to sensitivity of endocrine therapy drug(tamoxifen). Consequently, this study provides the reliable theory base for treating ERa negative breast cancer patients with endocrine therapy.
近年来乳腺癌的发病率和死亡率增长速度较快,全世界每年患乳腺癌的女性约130万人,而死于乳腺癌的有50万人。虽然我国的乳腺癌属于低发,但近年来其发病率增长幅度较快,预测20年后将会成为我国发病率最高的恶性肿瘤。
雌激素受体(estrogen receptor ER)在乳腺癌的发生发展过程中,扮演着重要角色。在体内通过与雌激素的结合,激活含雌激素应答元件基因和含有其他转录因子结合元件基因的表达,进而导致乳腺癌的发生。通常情况下,以ER基因表达情况的不同可将乳腺癌分为ER阴性乳腺癌和ER阳性乳腺癌两种。其中ER阴性乳腺癌约占20%-35%,其恶性程度高、发展迅速、有较高的局部复发率,并易发生肺、肝、骨及脑等重要脏器的转移,预后较差。由于缺乏相应的受体不能进行内分泌和生物治疗,所以这类特殊乳腺癌的治疗仅限于化疗,但疗效欠佳。因此,作为一个分子标记物,ER在乳腺癌的内分泌治疗中进行效果预测、评估激素依赖性等方面具有重要的意义。
DNA甲基化和染色体重组是当前研究ER阴性乳腺癌中ER基因失活的两大主要学说。甲基化在肿瘤的发生、发展中有着重要作用,但又是一可逆过程。从理论上来说,如果使用药物对甲基化的抑癌基因和癌基因进行人为逆转,即可使因甲基化失活的基因重新获得表达。
三氧化二砷(As2O3)为中药砒霜的主要成分,近年来发现其对白血病、原发性肝癌等多种肿瘤治疗取得了较好的效果。主要通过诱导细胞凋亡来发挥其细胞毒性作用,如今多项研究证明其具有甲基化抑制作用。
本实验通过研究不同浓度的As2O3对人ER阴性乳腺癌细胞株MDA-MB-435进行药物诱导,使其基因启动子区CpG岛部分脱甲基而使其重新表达有功能的ERα,从而使得ER阴性乳腺癌细胞恢复对内分泌治疗药物的敏感性。
材料与方法：
(1)常规培养雌激素受体(ERα)阴性人乳腺癌细胞株MDA-MB-435,细胞浓度调整为5×105/ml后换含As2O3的培养液,至AS2O3终浓度分别为0.5μmol/L、1.0μmol/L、2.0μmol/L、4.0μmol/L,连续培养72h。以同期培养不加药的MDA-MB-435细胞作为阴性对照,ERα阳性人乳腺癌细胞株MCF-7作为阳性对照。
(2) RT-PCR方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERα的mRNA表达情况。
(3)甲基化特异性PCR (Methylation Specific PCR, MSP)方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERαDNA的5’-CpG岛甲基化的情况。
(4) Western blot方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERα的蛋白表达情况。
(5)免疫组化方法检测经不同浓度As2O3处理后的MDA-MB-435细胞ERα的蛋白表达情况。
(6)MTT方法检测经不同浓度As2O3处理后的MDA-MB-435细胞,再经三苯氧胺(TAM)处理,观察各组细胞的生长受抑情况。
(7)统计学方法：应用统计学处理软件SPSS 16.0,X2检验进行统计学分析,·差异显著性标准设为P<0.05。
结果：
(1) RT-PCR法检测阴性对照组无ERαmRNA基因；0.5μmol/L组未扩增出ERαmRNA基因；1.0μmol/L组、2.0μmol/L组、4.0μmol/L组均扩增出ERαmRNA基因。
(2)甲基化特异性PCR法检测阴性对照组仅甲基化引物扩增出特异PCR条带；0.5μmol/L组、1.0μmol/L组、2.0μmol/L组、4.0μmol/L组甲基化和去甲基化引物均扩增出特异PCR条带。
(3)经Western blot检测证实阴性对照组未显示蛋白条带；0.5μmol/L组也未显示蛋白条带,但1.0μmol/L组、2.0μmol/L和4.0μmol/L组显示出蛋白条带。
(4)经免疫组化法检测证实阴性对照组细胞阳性率为7.4%；/L组、/L组、/L组、/L组细胞阳性率分别为7.8%、43.8%、78.2%、41.5%。
(5)MTT法检测TAM作用于经As203处理的MDA-MB-435细胞后,细胞增殖明显受抑。阴性对照组受抑率为10.7%;0.5μmol/L组、/L组、2.0μmol/L组、/L组细胞受抑率分别是14.9%、30.7%、43.1%、56.6%。
结论：
适当浓度的As203通过对部分细胞的去甲基化作用,使ERa阴性人乳腺癌细胞株MDA-MB-435细胞ERa mRNA和蛋白重新表达,并可恢复对内分泌治疗药物(三苯氧胺)的敏感性,从而为ERa阴性乳腺癌患者实现内分泌治疗提供可靠的理论依据。
Background and objective:
In recent years, the morbidity and mortality of breast cancer are growing rapidly. About 130 million women suffer from breast cancer and 50 million people died of breast cancer each year in the world. Although breast cancer has a low morbidity in our country, the incidence of breast cancer is rising obviously. We predict that it will be the highest incidence of malignant tumor in 20 years.
Estrogen receptor (ER) is an important role in the development of breast cancer. In vivo, ER combine with estrogen and active the genes which contain estrogen response element and transcription factor response element, then lead to breast cancer. In general, we divide the breast cancer into ER negative breast cancer and ER positive breast cancer. Among about 20～35 percent of breast cancer patients, the estrogen receptor is negative. With the high malignant degree, rapid development, higher local recurrence, and metastases easily occurring in lung, liver, bone, brain and other important viscera, it has poor prognosis. Due to lack of corresponding receptor, the patients cannot apply endocrine and biological treatment. At present, the treatment of this kind of special type of breast cancer limits to chemotherapy, but it is far from perfect. Thus, as an important molecular marker, ER has vital significance to forecast the effect of endocrine therapy and evaluate the dependence of hormone in breast cancer.
The main theories of ER gene expression silencing are DNA methylation and chromosome restructuring in ER negative breast cancer. Methylation occurs early in the process of cancer, it plays an important role in the occurrence and development of cancer. DNA methylation is a reversible process. By theoretically, if we reverse methylated oncogenes and tumor-suppressor genes with drugs, it will be made the gene re-expressed.
Arsenic trioxide (AS2O3) is the main ingredient of arsenic's of Traditional Chinese medicine. In recent years, we find that it has better effects on many kinds of tumor cells, such as leukemia, liver cancer and so on. AS2O3 exert it's cytotoxicity by inducing cells apoptosis. Recent research suggests that it can demethylate.
This study is designed that AS2O3 effect on ERa negative breast cancer cell MDA-MB-435. As2O3 can demethylate gene promoter CpG island partially, and made ERa re-expressed. Consequently, ERa negative breast cancer patients can restore the sensitivity of endocrine drugs.
Materials and methods:
(1) ERa negative human breast cancer cell line MDA-MB-435 planted at 37℃and 5% CO2. Adjust cell density of 5×105/ml and treat with 1640 containing AS2O3 0.5μmol/L, 1.0μmol/L,2.0μmol/L,4.0μmol/L respectively. Then plant for 72 hours. MDA-MB-435 without being treated with containing As2O3 was taken as negative control. Human breast cancer cell line MCF-7 (ER positive) was taken as positive control.
(2) To detect the re-expression of the ERa mRNA by RT-PCR.
(3) To detect the status of 5'CpG island methylation of ERa gene by Methylation Specific PCR(MSP).
(4) To detect the re-expression of the ERa protein by Western blot.
(5) To detect the re-expression of the ERa protein by immunohismchemistry.
(6) MTT method tested whether Tamoxifen could induce the growth inhibition of MDA-MB-435 cell after treated with As2O3.
(7) Using SPSS 16.0 statistics processing software andχ2-test for statistical analysis. Significant of difference standard is P<0.05.
Result:
(1) It is confirmed by the test of RT-PCR that expression of ERa mRNA were not found in group of negative control, neither in group of 0.5μmol/L. But ERa mRNA were detectable in groups of 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
(2) By MSP technique, methylation of ERa gene was found in group of negative control, methylation of ERa gene and demethylation of ERa gene were both found in groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
(3) It is confirmed by the test of Western blot that expression of ERa protein were not found in group of negative control and in group of 0.5μmol/L. But ERa protein were detectable in groups of 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
(4) By immunohistochemistry technique, we found that, in group of negative control, the positive rate was 7.4%. In groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L, the positive rate is respectively 7.8%,43.8%,78.2% and 41.5%.
(5) By the test of MTT, we found that tamoxifen can effect on the growth of MDA-MB-435 cells treated with AS2O3. We found that, in group of negative control, the inhibition rate was 10.7%. Inhibition rate was respectively 14.9%,30.7%,43.1% and 56.6% in groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L.
Conclusion:
A proper concentration of AS2O3 could effect on a part cells through demethylation and induce ERa gene re-expression in human breast cancer cell line MDA-MB-435. And it can restore to sensitivity of endocrine therapy drug(tamoxifen). Consequently, this study provides the reliable theory base for treating ERa negative breast cancer patients with endocrine therapy.
引文
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[30].傅海英,沈建箴,沈松菲等.巢式MSP检测砷剂诱导人多发性骨髓瘤U266细胞系p16 基因去甲基化及转录[J].中国实验血液学杂志,2007,(15)：79-85
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[23].苏世振,布立敬,李红智,等.乳腺癌雌激素受体α基因启动子甲基化与其蛋白表达的关系[J].温州医学院学报,2009,39(2)：133-136
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[26].Billam M, Sobolewski MD, Davidson NE. Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells [J]. Breast Cancer Res Treat,2010,120: 581～592
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[30].傅海英,沈建箴,沈松菲等.巢式MSP检测砷剂诱导人多发性骨髓瘤U266细胞系p16 基因去甲基化及转录[J].中国实验血液学杂志,2007,(15)：79-85
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[36].Nahta R, Yuan L X, Du Y, et al. Lapatinib induces apoptosis in trastuzumab resistant breast cancer cells:effects on insulinlike growth factor signaling [J]. Mol cancer Ther,2007,6 (2): 667～674
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[41].王瑞,李林蔚,王瑞林,等.人乳腺癌细胞雌激素受体基因的去甲基化及其表达[J].中华肿瘤杂志,2006,28(12)：894-897
[1]. Reik Wj Dean W, Walter J. Epigenetic reprogramming in mammalian velopment.Science, 2001,93(5532):1089-1093
[2].丁波泥.天花粉蛋白抑制乳腺癌生长及逆转ERa基因甲基化的研究[D].[博士学位论文].长沙：中南大学,2008
[3]. Etoh T,Kanai Y,Ushijima S,et al. Increased DNA methyltransferase I (DNMTI) protein ex-pression correlates significantly with poorer tumor differentiation and frequent DNA hyper-methylation of multiple CpG islands in gastric cancers.Am J Pathol,2004,164 (2):689～699
[4]. Lama G, Angelucci C,Bruzzese N,et al. Sensitivity of human melanoma cells to oestrogens,tamoxifen and quercetin:is there any relationship with type Ⅰ and Ⅱ estrogens binding site expression[J]. Melanoma Res,1998,8(4):313
[5].柯跃斌,夏菠,等.DNA去甲基化与基因激活过程[J].癌变.畸形.突变,2010,22(2)：149-154
[6], Hsieh CL.Evidence that protein binding specifies sites of DNA demethylation [J]. Mol Cell Biol,1999,19(1):46～56
[7].李培坤,耿小平.DNA甲基化抑制剂研究进展[J].国际药学研究杂志,2010,37(3)：198-203
[8]. Momparler RL. Pharmacology of 5-aza-2-deoxycytidine (decitabine) [J]. Sem in Hematol, 2005,42(1):9～16
[9]. Issa JP, Kantarjian HM, Kirkpatrick P. Azacitidine [J]. Nat Rev Drug Discover,2005,4 (4): 275-276
[10].Kaminskas E, FarrellA, Abraham S, et al. Approval summary:azacitidine for treatment of myelodys plastic syndrome subtypes [J]. Clin Cancer Res,2005,11 (10):3604～3608
[11].Issa JP, Gharibyan V, Cortes J, et al. Phase Ⅱ study of kwdose decitabine in patients with chronicmyelogenous leukemia resistant to imatinib mesylate [J].Clin Oncol,2005,23 (17):3948～-3956
[12].Samlowski WE, Leachman SA, WadeM, et al. Evaluation of 72 day continuous intravenous infusion of decitabine:inhibition of promoter specific and global genomic DNA methylation [J]. Clin Oncol,2005,23 (17):3897～3905
[13].徐昕,戴秋新,徐茂忠,等.地西他滨对淋巴瘤细胞株Raji中P15INK4B基因的去甲基化作用[J].山东大学学报(医学版),2010,48(7)：62-68
[14].Atceq B, Unterberger A, szyf M, et al. 1‰ nacological inhibition of DNA methylation induces proinvasive and prometastatic genes in duo and in vivo[J]. Neoplasia,2008,10(3): 266-278
[15].Kurkjian C, Kummar S, Murgo AJ, et al. DNA methylation:its role in cancer development and therapy [J]. Curr Probl Cancer,2008,32 (5):187～-235
[16].Griffiths EA, Gore SD. DNA methyltransferase and histone deacetylase inhibitors in the treatment of myelodysp lastic syndromes [J]. Sem in Hem atol,2008,45 (1):23～30
[17].Kaminskas E, Farrell AT, Wang YC, et al. FDA drug approval summary:azacitidine (5— azacytidine, Vidaza) for injectable suspension [J]. Oncologist,2005,10 (3):176～182
[18].Cheng JC, Yoo C B, Weisenberger D J, et al. Preferential response of cancer cells to zebμlarine [J]. Cancer Cel,2004,6 (2):151 ～158
[19].Hurd P J, Whitmarsh A J, Baldwin G S, et al. Mechanism-based inhibition of 5-cytosine DNA methyltransferases by Hpyrimidinone [J]. Mol Biol,1999,286 (2):389～401
[20].Marquez V E, Barchij JR, Kelley JA, et al. Zebμlarine:a unique molecule for an epigenetically based strategy in cancer chemotherapy [J]. Nucleo tides nucleic Acids, 2005,24(5-7):305～318
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