5-氮杂脱氧胞苷对乳腺癌细胞MDA-MB-231ER_α、E-cad表达和增殖侵袭能力的影响
摘要
目的:探讨在乳腺癌细胞中,去甲基化药物5-氮杂脱氧胞苷对雌激素受体α(estrogen receptor alpha, ERα)基因,上皮性钙粘连素(E-cadherin, E-cad)基因表达的影响,及其对细胞增殖和侵袭能力的影响,为乳腺癌的基因治疗提供一定的指导意义。
方法:(1)使用去甲基化药物5-氮杂脱氧胞苷处理MDA-MB-231细胞,利用细胞生长曲线、MTT法及流式细胞仪检测5-氮杂脱氧胞苷对MDA-MB-231细胞增殖和周期的影响;(2)通过RT-PCR和免疫组化检测5-氮杂脱氧胞苷对ERα阴性乳腺癌细胞MDA-MB-231 ERα、E-cad及基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达的影响;(3)通过Boyden小室侵袭实验检测5-氮杂脱氧胞苷对乳腺癌细胞浸润能力的影响;(4)用限制性酶PCR检测5-氮杂脱氧胞苷作用前后的乳腺癌细胞中ERα基因启动子及第一外显子区域的甲基化情况。
结果:(1)5-氮杂脱氧胞苷能使MDA-MB-231细胞的生长速度明显减慢,细胞周期被阻滞在G0/G1期;(2)5-氮杂脱氧胞苷部分恢复ERα阴性乳腺癌MDA-MB-231细胞ERα及E-cad基因的表达;(3)5-氮杂脱氧胞苷使MDA-MB-231细胞中MMP-2基因表达下降,及该细胞的侵袭能力降低;(4)MDA-MB-231细胞ERα基因中检测到HpaⅡ甲基化位点,5-氮杂脱氧胞苷作用后未检测到甲基化位点。
结论:(1)5-氮杂脱氧胞苷可有效抑制乳腺癌细胞生长并导致细胞周期的阻滞,以及明显降低乳腺癌细胞的侵袭能力;(2)5-氮杂脱氧胞苷可以部分的去甲基化和恢复ERα的表达;(3)利用5-氮杂脱氧胞苷可部分恢复ERα和E-cad的表达及降低MMP-2的表达,这可能为其引起乳腺癌MDA-MB-231细胞生物学行为改变的机制之一。
Objectiv:To explore the effect of 5-aza-2'-deoxycytidine(5-Aza-CdR) on ERαexpression, E-cad expression, proliferation and invasion of human breast cancer cell line MDA-MB-231, and it could provide instructive value for the gene therapy of human breast carcinoma.
Method:1. The breast cancer cell line MDA-MB-231 was treated with 5-Aza-CdR. Cell growth was evaluated by cell counting and MTT assay, and cell cycle was analyzed by flow cytometry. 2. Immunohistochemistry and RT-PCR were used to measure the changes of the ERαexpression, E-cad expression and MMP-2 expression in the MDA-MB-231 cells after 5-Aza-CdR treatment. 3. The invasion ability of MDA-MB-231 cells was tested by Boyden chamber assay. 4. PCR using the extracted DNA and methylation–sensitive restriction enzymes, was performed to detect the methylation of a CpG island in the ERαgene.
Results: 1. Compared with the control, the cell growth was significantly reduced and the cell cycle was stuck at G0/G1 phase after treatment. 2. The expression of ERαgene and E-cad gene was partly restored in ERαnegative human breast cancer cell line (MDA-MB-231) after treatment. 3. The expression of MMP-2 in the cell line MDA-MB-231 is downregulated and the invasion ability of MDA-MB-231 cell was decreased after treatment. 4. MDA-MB-231 cell was found methylation-positive in the exon 1 of ERαgene. No methylation was found in 5-Aza-CdR treated cells.
Conclusion: 1. The 5-Aza-CdR treatment in MDA-MB-231 cells leads to cell proliferation inhibition, cell cycle arrest and cell invasiveness decline. 2. Methylation was partly reversed and the ERαexpression was partly restored in MDA-MB-231 cells by 5-Aza-CdR. 3. Our findings suggest that the partial restoration of ERαexpression and E-cad expression, and the decrease of MMP-2 expression with 5-Aza-CdR may be a mechanism of biological behaviour change in MDA-MB-231 cells.
方法:(1)使用去甲基化药物5-氮杂脱氧胞苷处理MDA-MB-231细胞,利用细胞生长曲线、MTT法及流式细胞仪检测5-氮杂脱氧胞苷对MDA-MB-231细胞增殖和周期的影响;(2)通过RT-PCR和免疫组化检测5-氮杂脱氧胞苷对ERα阴性乳腺癌细胞MDA-MB-231 ERα、E-cad及基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达的影响;(3)通过Boyden小室侵袭实验检测5-氮杂脱氧胞苷对乳腺癌细胞浸润能力的影响;(4)用限制性酶PCR检测5-氮杂脱氧胞苷作用前后的乳腺癌细胞中ERα基因启动子及第一外显子区域的甲基化情况。
结果:(1)5-氮杂脱氧胞苷能使MDA-MB-231细胞的生长速度明显减慢,细胞周期被阻滞在G0/G1期;(2)5-氮杂脱氧胞苷部分恢复ERα阴性乳腺癌MDA-MB-231细胞ERα及E-cad基因的表达;(3)5-氮杂脱氧胞苷使MDA-MB-231细胞中MMP-2基因表达下降,及该细胞的侵袭能力降低;(4)MDA-MB-231细胞ERα基因中检测到HpaⅡ甲基化位点,5-氮杂脱氧胞苷作用后未检测到甲基化位点。
结论:(1)5-氮杂脱氧胞苷可有效抑制乳腺癌细胞生长并导致细胞周期的阻滞,以及明显降低乳腺癌细胞的侵袭能力;(2)5-氮杂脱氧胞苷可以部分的去甲基化和恢复ERα的表达;(3)利用5-氮杂脱氧胞苷可部分恢复ERα和E-cad的表达及降低MMP-2的表达,这可能为其引起乳腺癌MDA-MB-231细胞生物学行为改变的机制之一。
Objectiv:To explore the effect of 5-aza-2'-deoxycytidine(5-Aza-CdR) on ERαexpression, E-cad expression, proliferation and invasion of human breast cancer cell line MDA-MB-231, and it could provide instructive value for the gene therapy of human breast carcinoma.
Method:1. The breast cancer cell line MDA-MB-231 was treated with 5-Aza-CdR. Cell growth was evaluated by cell counting and MTT assay, and cell cycle was analyzed by flow cytometry. 2. Immunohistochemistry and RT-PCR were used to measure the changes of the ERαexpression, E-cad expression and MMP-2 expression in the MDA-MB-231 cells after 5-Aza-CdR treatment. 3. The invasion ability of MDA-MB-231 cells was tested by Boyden chamber assay. 4. PCR using the extracted DNA and methylation–sensitive restriction enzymes, was performed to detect the methylation of a CpG island in the ERαgene.
Results: 1. Compared with the control, the cell growth was significantly reduced and the cell cycle was stuck at G0/G1 phase after treatment. 2. The expression of ERαgene and E-cad gene was partly restored in ERαnegative human breast cancer cell line (MDA-MB-231) after treatment. 3. The expression of MMP-2 in the cell line MDA-MB-231 is downregulated and the invasion ability of MDA-MB-231 cell was decreased after treatment. 4. MDA-MB-231 cell was found methylation-positive in the exon 1 of ERαgene. No methylation was found in 5-Aza-CdR treated cells.
Conclusion: 1. The 5-Aza-CdR treatment in MDA-MB-231 cells leads to cell proliferation inhibition, cell cycle arrest and cell invasiveness decline. 2. Methylation was partly reversed and the ERαexpression was partly restored in MDA-MB-231 cells by 5-Aza-CdR. 3. Our findings suggest that the partial restoration of ERαexpression and E-cad expression, and the decrease of MMP-2 expression with 5-Aza-CdR may be a mechanism of biological behaviour change in MDA-MB-231 cells.
引文
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[4] Halbersztadt A, Halon A, Pajak J, et al. The role of matrix metalloproteinases in tumor invasion and metastasis [J]. Ginekol Pol. 2006, 77(1):63~71.
[5] ‘’Stetler-Stevenson WG.Aznavoorian S,Liotta LA,Tumor cell interactions with the extracellular matrix during invasion and metastasis [J].Annu Rev Cell Biol. 1993, 9:541-573.
[6] Kawai H,Li H,Avraham S,et a1.Overexpression of histone deacetylase HDAC1 modulates breast cancer progression by negative regulation of estrogen receptor alpha [J].Int J Cancer. 2003, 107(3):353-358.
[7] Glacinti L ,Claudio PP, Lopez M, et al. Epigenetic Information and Estrogen Receptor Alpha Expression in Breast Cancer [J].The Oncologist. 2006,11(1):1-8.
[8] Zhang S, Wang YJ, Wang YJ, et al.Inhibitory effect of 5-Aza-2'-deoxycytidine on human nasopharyngeal carcinoma xenograft in nude mice[J].Aizhen. 2005, 24(10):1201-1205.
[9] Hui R, Macmillan RD, Kenny FS, et al. INK4a gene expression and methylation in primary breast cancer: overexpression of p16INK4a messenger RNA is a marker of poor prognosis [J]. Clin Cancer Res. 2000, 6(7):2777-2787.
[10] Lliopoulos D, Fabbri M, Druck T, et al.Inhibition of breast cancer cell growth in vitro and in vivo :effect of restoration of Wwox expression[J].Clin Cancer res. 2007, 13(1):268-274.
[11] Pinzone JJ, Stevenson H, Strobl JS, et al. Molecular and cellular determinants ofestrogen receptor alpha expression[J]. Mol Cell Biol. 2004, 24(11):4605-4612.
[12] Razandi M,Pedram A, Park ST, et al. Proximal Events in Signaling by Plasma Membrane Estrogen Receptors[J] J Biol Chem. 2003, 278(4):2701-2712.
[13] Talvensaari-Mattila A,Paakko P,Hoyhtya M,et a1.Matrix metallo proteinase-2 immunoreactive protein : a marker of aggressiveness in breast carcinoma [J].Cancer(Phila). l998, 83(6):1153-1162.
[14] 刘真真, 崔树德,魏冰.。MMP-2 与 TIMP-2 在浸润性乳腺癌组织表达的临床意义及相关性研究肿瘤防治杂志[J]。2004, 11(9):909-912.
[15] Jinga DC, Blidaru A , Condrea I , et al. MMP-9 and MMP-2 gelatinases and TIMP-1 and TIMP-2 inhibitors in breast cancer: correlations with prognostic factors[J]. J Cell Mol Med. 2006, 10(2):499-510.
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