CAJ | 학술논문

目的研究三氧化二砷(As2 O3)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As2 O3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As2 O3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As2 O3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ERα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Western blot法,检测DNMT1和ERα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As2O3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4 μmol/LAs2O3+ TAM组处理72 h时的抑制率最高,达62.6％.1、2和4 μmol/L As2O3对MDA-MB-435s细胞有去甲基化作用,并可使DNMT1 mRNA和蛋白的表达受到抑制,ERαmRNA和蛋白恢复表达.不同剂量的As2 O3单用或与TAM联合处理后,裸鼠移植瘤组织中ERα基因出现不同程度的去甲基化条带,DNMT1 mRNA和蛋白的表达受到抑制,而ERα mRNA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4 mg/kg As2O3 +5 mg/kg TAM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DNMT1的活性使ERα阴性人乳腺癌MDA-MB-435s细胞的ERα基因去甲基化,并恢复其表达；恢复的ERα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ERα阴性乳腺癌患者的新途径.
목적 연구삼양화이신(As2 O3)대자격소수체α(ERα)음성인유선암세포MDA-MB-435s적거갑기화작용급가능궤제,관찰As2 O3연합타막석분(TAM)대MDA-MB-435s세포라서이식류적료효.방법 채용사갑기우담서람(MTT)법,검측불동농도As2 O3단용혹여TAM련용대MDA-MB-435s세포적증식억제작용.이불동농도As2 O3처리MDA-MB-435s세포,동시건립MDA-MB-435s세포라서이식류모형,이불동제량적As2O3단용혹여TAM련용치료라서이식류.채용갑기화특이성취합매련반응(MSP),검측불동처리조MDA-MB-435s세포화라서이식류조직중ERα기인적갑기화상태.채용역전록취합매련반응(RT-PCR),검측DNA갑기전이매1(DNMT1)화ERαmRNA적표체변화.채용Western blot법,검측DNMT1화ERα단백적표체변화.치료과정중,매주측량라서이식류적대소,회제종류생장곡선.결과 불동농도As2O3단용혹여TAM연합처리조MDA-MB-435s세포적증식균수도명현억제,기중4 μmol/LAs2O3+ TAM조처리72 h시적억제솔최고,체62.6％.1、2화4 μmol/L As2O3대MDA-MB-435s세포유거갑기화작용,병가사DNMT1 mRNA화단백적표체수도억제,ERαmRNA화단백회복표체.불동제량적As2 O3단용혹여TAM연합처리후,라서이식류조직중ERα기인출현불동정도적거갑기화조대,DNMT1 mRNA화단백적표체수도억제,이ERα mRNA화단백칙축점회복표체.불동제량적As2O3단용혹여TAM련용균능명현억제라서이식류적생장,이4 mg/kg As2O3 +5 mg/kg TAM조적억제작용최강,종류중량억제솔화종류체적억제솔분별위79.5％화76.4％.결론 As2O3가이통과억제DNMT1적활성사ERα음성인유선암MDA-MB-435s세포적ERα기인거갑기화,병회복기표체；회복적ERα가이사MDA-MB-435s세포대내분비치료민감.As2O3여TAM련용가능성위치료ERα음성유선암환자적신도경.
Objective To study the demethylation effect of arsenic trioxide (As2O3) on ERα-negative human breast cancer MDA-MB-435s cells and its possible mechanisms,and to observe its treatment efficacy in combination with tamoxifen (TAM) after ERα 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 ERα 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 Erα.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 As2O3 alone or As2O3 combined with TAM,and the 4 μmol/L As2O3 + TAM treatment for 72 h showed the highest inhibition rate (62.6％).1,2,4 μmol/L As2O3 had demethylation effect on MDA-MB-435s cells,and the DNMT1 mRNA and protein expression was inhibited and accompanied by ERα mRNA and protein re-expression.The unmethylation specific bands of ERα gene were enhanced after treated by As2O3 alone or As2O3 combined with TAM in the xenograft tumors.The expression of DNMT1 mRNA and protein was inhibited,and accompanied by ERα mRNA and protein re-expression.An significant decrease of volume and weight of the xenograft tumors in the As2O3 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 As2O3 +TAM group had the highest inhibition rate of tumor weight (79.5％) and volume (76.4％).Conclusions ERα can be re-expressed in ERα-negative breast cancer MDA-MB-435s cells after treated with As2O3 by inhibiting the DNMT1 activity.MDA-MB-435s cells are re-sensitized to endocrine therapy after ERα re-expression.As2O3 combined with TAM may provide a new therapeutic approach for patients with ERα-negative breast cancer in the clinic.