摘要
目的研究三氧化二砷（As2O3）对雌激素受体α（ERα）阴性人乳腺癌细胞MDA-MB-435s的去甲基化作用及可能机制，观察As2O3联合他莫昔芬（TAM）对MDA—MB-435s细胞裸鼠移植瘤的疗效。方法采用四甲基偶氮唑蓝（MTT）法，检测不同浓度As2O3单用或与TAM联用对MDA—MB-435s细胞的增殖抑制作用。以不同浓度As2O3处理MDA—MB-435s细胞，同时建立MDA—MB-435s细胞裸鼠移植瘤模型，以不同剂量的As2O3单用或与TAM联用治疗裸鼠移植瘤。采用甲基化特异性聚合酶链反应（MSP），检测不同处理组MDA—MB-435s细胞和裸鼠移植瘤组织中ERa基因的甲基化状态。采用逆转录聚合酶链反应（RT-PCR），检测DNA甲基转移酶1（DNMT1）和ERαmRNA的表达变化。采用Westernblot法，检测DNMTl和ERa蛋白的表达变化。治疗过程中，每周测量裸鼠移植瘤的大小，绘制肿瘤生长曲线。结果不同浓度As203单用或与TAM联合处理组MDA—MB-435s细胞的增殖均受到明显抑制，其中4μmol／LAs2O3＋TAM组处理72h时的抑制率最高，达62．6％。1、2和4μmol／LAs2O3对MDA—MB-435s细胞有去甲基化作用，并可使DNMTlmRNA和蛋白的表达受到抑制，ERamRNA和蛋白恢复表达。不同剂量的As2O3单用或与TAM联合处理后，裸鼠移植瘤组织中ERa基因出现不同程度的去甲基化条带，DNMTlmRNA和蛋白的表达受到抑制，而ERamRNA和蛋白则逐渐恢复表达。不同剂量的As2O3单用或与TAM联用均能明显抑制裸鼠移植瘤的生长，以4mg／kgAs2O3＋5mg／kgTAM组的抑制作用最强，肿瘤重量抑制率和肿瘤体积抑制率分别为79．5％和76．4％。结论As2O3可以通过抑制DNMTl的活性使ERa阴性人乳腺癌MDA—MB-435s细胞的ERa基因去甲基化，并恢复其表达；恢复的ERμ可以使MDA—MB-435s细胞对内分泌治疗敏感。As2O3与TAM联用可能成为治疗ERa阴性乳腺癌患者的新途径。
Objective To study the demethylation effect of arsenic trioxide （As2O3 ） on ERa- negative human breast cancer MDA-MB-435s cells and its possible mechanisms, and to observe its treatment efficacy in combination with tamoxifen （TAM） after ERa re-expression. Methods MTT assay was used to examine the inhibitory effect of As2O3 treatment alone or in combination with TAM on cell proliferation. A nude mouse xenograft model was used to further examine the treatment efficacy in vivo. MSP was used to detect the methylation status of ERa gene after treated with As2O3 in MDA-MB-435s cells and the transplanted tumor tissues. RT-PCR was used to detect the mRNA expression of DNMT1 and Era. Western bolt was used to detect the DNMT1 and ERα protein expression. The diameter of xenograft tumors was measured weekly, and the tumor growth curve was drawn. Results The level of proliferation of the MDA- MB-435s cells was significantly suppressed after treatment with different concentration of As203 alone or As203 combined with TAM, and the 4 /~mol/L As203 ＋ TAM treatment for 72 h showed the highest inhibition rate （62.6%）. 1,2, 4/xmol/L As203 had demethylation effect on MDA-MB-435s cells, and the DNMT1 mRNA and protein expression was inhibited and accompanied by ERa mRNA and protein re- expression. The unmethylation specific bands of ERa A%O3 combined with TAM in the xenograft tumors. gene were enhanced after treated by As203 alone or The expression of DNMT1 mRNA and protein was inhibited, and accompanied by ERoL mRNA and protein re-expression. An significant decrease of volume and weight of the xenograft tumors in the AszO3 treated alone or combined with TAM groups was observed compared with those of the normal saline group or TAM alone group （ P 〈0.05 ） , and the 4 mg/kg As203 ＋ TAM group had the highest inhibition rate of tumor weight （79.5%） and volume （76.4%）. Conclusions ERa can be re-expressed in ERc^-negative breast cancer MDA-MB-435s cells after treated with As203 by inhibiting the DNMT1 activity. MDA-MB-435s cells are re-sensitized to endocrine therapy after ERa re- expression. As203 combined with TAM may provide a new therapeutic approach for patients with ERa- negative breast cancer in the clinic.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2012年第9期645-651,共7页
Chinese Journal of Oncology
基金
河南省科技攻关计划[（JB02）0524410092]
