【作者】 刘铁成；

【导师】 李德春；

【作者基本信息】 苏州大学 ， 普通外科学， 2011， 博士

【摘要】 乳腺癌是一种全身性疾病,极易早期发生血道转移,严重威胁着妇女的健康。乳腺癌的治疗原则相应地发生了巨大变革,由以往单纯局部治疗转入局部与全身相结合的综合治疗时代。内分泌治疗作为乳腺癌全身治疗的主要方法之一,因其疗效确切、副作用少和给药方便而倍受青睐。据统计,约45%乳腺癌患者因雌激素(ERα)受体阴性而失去了内分泌治疗这一有效的治疗模式,所以性激素受体是决定内分泌治疗疗效的关键因素。DNA的甲基化以及组蛋白的去乙酰化是表观遗传调控中一种重要的共价修饰,可能调控着激素受体基因的表达。目前关于乳腺癌中性激素受体基因表达调控的分子机制尚不十分清楚,为了进一步了解乳腺癌中性激素受体表达的转录调控和翻译后调节,我们进行了如下的研究。研究目的联合应用DNA甲基转移酶抑制剂(DNA methyltransferase inhibitor, DNMTi) 5-氮基-2-脱氧胞苷(5-aza-2’deoxycytidine,AZA)和组蛋白去乙酰化酶抑制剂(Histone deacetylase inhibitor,HDACi)曲古菌素A(trichostatin A,TSA)作用于乳腺癌细胞株SKBR-3,研究能否通过这两种药物诱导性激素受体原本阴性乳腺癌细胞株SKBR-3重新表达功能性的雌雄激素受体(ERα和AR),并探讨经过药物诱导重新表达激素受体的SKBR-3细胞增殖能力的变化以及对于内分泌治疗的敏感性的改变。另外,研究单药TSA能否引起乳腺癌细胞株SKBR-3和MCF-7中激素受体的改变,并探讨经过药物处理后其增殖能力的变化。材料与方法1.联合应用AZA和TSA或单药TSA作用于乳腺癌细胞系SKBR-3后,应用反转录聚合酶链式反应(Reverse transcription-Polymerase chain reaction ,RT-PCR)方法检测处理后SKBR-3细胞株ERα、PR、AR、pS2以及PSA的mRNA表达水平。2. AZA与TSA联合处理乳腺癌细胞SKBR-3细胞爬片,单药TSA分别处理乳腺癌细胞SKBR-3或MCF-7细胞爬片。根据药物处理共分六组:①(Vehicle) SKBR-3组;②(AZA+TSA) SKBR-3组;③(Vehicle) MCF-7组;④(TSA) MCF-7组;⑤(Vehicle) SKBR-3组;⑥(TSA) SKBR-3组。通过细胞免疫化学(immunocytochemistry ,ICC)SP法来检测药物处理前后乳腺癌细胞中ERα、PR、AR、pS2及PSA的蛋白表达水平的改变。3.通过水溶性四唑盐(water-soluble tetrazolium salt,WST)方法来检测乳腺癌细胞的增殖能力变化。(1)AZA+TSA联合诱导SKBR-3细胞株后,以WST法测定不同浓度梯度(①vehicle②lnmol/L③10nmo1/L④l00nmol/L⑤1μmo1/L⑥l0μmo1/L)MA作用下肿瘤细胞的增殖能力,并确定疗效最佳的MA浓度。(2)取疗效最佳的MA浓度,根据药物处理将AZA+TSA诱导前后乳腺癌细胞SKBR-3共分六组:①vehilce对照组;②AZA+TSA组;③AZA+TSA+E2组;④AZA+TSA+E2+TAM组;⑤AZA+TSA+E2+MA组;⑥AZA+TSA+E2+TAM+MA组,以WST法来测定各组肿瘤细胞增殖能力的变化。(3)根据药物处理将单药TSA处理的乳腺癌细胞SKBR-3和MCF-7共分四个组:①(Vehicle) SKBR-3组;②(TSA)SKBR-3组;③(Vehicle)MCF-7组;④(TSA) MCF-7组。以WST法测定各组肿瘤细胞在12、24、48、72、96小时的增殖能力。研究结果1. AZA+TSA或TSA都能诱导激素受体阴性的乳腺癌细胞株SKBR-3重新表达ERα、AR、PR、pS2以及PSA的mRNA。单药组的mRNA水平低于双药组(p<0.05),二组的mRNA水平显著低于MCF-7对照组(p<0.01)。2.药物处理后乳腺癌细胞株ERα、PR、AR、pS2以及PSA表达的蛋白水平的改变。(1)经AZA+TSA诱导后SKBR-3细胞爬片中部分细胞重新表达ERα、AR、PR、pS2和PSA的蛋白,但表达程度远不及MCF-7细胞(p<0.01),且爬片中单位高倍视野内(ICC,×400)细胞数目较处理前减少,但未发现细胞爬片中细胞溶解现象。(2)单药TSA处理后MCF-7细胞爬片中ERα、AR、PR、pS2和PSA的蛋白表达量较处理前明显减少(p<0.05),且爬片中单位高倍视野内(ICC,×400)细胞数目较处理前明显减少,部分细胞出现溶解现象。(3)单药TSA处理后SKBR-3细胞爬片中不同程度的恢复ERα、AR、PR、PS2和PSA的蛋白表达,但表达程度也远不及TSA处理后MCF-7细胞爬片中的残留量(p<0.05),且爬片中单位高倍视野内(ICC,×400)细胞数目较处理前显著减少,绝大部分细胞出现溶解现象。3.药物处理前后乳腺癌细胞的增殖能力。(1)在不同浓度MA作用下,诱导后SKBR-3细胞株表现出不同的增殖能力,呈浓度效应关系,MA浓度越大肿瘤细胞增殖能力越弱,在10μmol/L时肿瘤细胞增殖能力最弱。(2)药物AZA 2.5μmol/L+TSA 100ng/ml诱导后细胞株SKBR-3增殖能力下降(p<0.05);加入1nmol/L 17β-estrodial后,AZA+TSA+E2组细胞增殖能力略有上升,但无显著性差异(p>0.05);进一步加入TAM或MA ,与AZA+TSA+E2组相比,AZA+TSA+E2+TAM组和AZA+TSA+E2+MA组细胞增殖能力呈相似程度的下降(p<0.05);最后联合应用TAM和MA,与单药组相比, AZA+TSA+E2+TAM+MA组细胞增殖能力进一步下降(p<0.05)。(3)经TSA处理后的SKBR-3细胞株的增殖能力于24h后开始逐渐较处理前降低,且呈时间效应关系,时间越长两组间增殖能力差异越显著;经TSA处理后的MCF-7细胞株的增殖能力于48h以后才开始逐渐较处理前减低,时间越长两组间增殖能力差异越显著;96h时TSA处理前后MCF-7细胞增殖能力降低的幅度也明显低于SKBR-3细胞株降低的幅度(OD值0.6387 vs 1.1437)(p<0.05)。结论1. AZA+TSA联合应用能诱导性激素受体全阴性的乳腺癌细胞SKBR-3重新表达功能性的ERα和AR;诱导后重新表达ERα和AR的SKBR-3表现出增殖能力下降,两药联合诱导对于肿瘤细胞有一定抑制作用,并恢复肿瘤细胞对雌激素的生长依赖和内分泌治疗的敏感性。2.诱导后重新表达ERα和AR的SKBR-3恢复对孕激素治疗的敏感性,且呈浓度效应关系,即MA浓度越大肿瘤细胞增殖能力越弱。小剂量MA可以取得与TAM相同的疗效,二者联合应用疗效优于单药,说明两者联合应用具有协同作用,为以后乳腺癌患者接受他莫西芬治疗后妇科并发症的防治提供了实验依据。3. TSA即可以通过诱导激素受体的表达提高受体阴性的乳腺癌患者对内分泌治疗的敏感性,也可以通过降解激素受体的途径治疗对内分泌治疗耐药的激素受体阳性患者。更重要的是提示,TSA可能既通过增强组蛋白的乙酰化水平,松解染色质的构象,重新激活抑癌基因的表达,又通过增强非组蛋白的乙酰化水平,抑制HSP90的分子伴侣功能,降解癌基因蛋白,介导肿瘤细胞的分化及凋亡,全方位、多环节地发挥抗肿瘤作用。这为HDAC抑制剂作为乳腺癌新型抗肿瘤药物提供了理论依据。更多还原

【Abstract】 BackgroudBreast cancer was known as a systemic disease because it often caused earlier hematogeneous metastasis,which heavily threatened women’s health. Accordingly,great change happened in the therapy strategy of breast cancer ,from the past local treatments alone to local and systemic treatment in combination. Endocrine therapy as one of systemic treatments was well welcomed because of its factual effects ,less adverse-effects and convenience. There were about 45% breast cancer patients would have lost the chance of endocrine therapy because estrogen receptor alpha ( ERa ) was negative.So we could drow that human sex hormone reccpters (SHR) were the main prognostic indicators of endocrine therapy. DNA methylation and histone acetylation are important covalent chromatin modification in epigenetic modification, which might regulate the expression of SHR gene. However,very little is known about the molecular mechanisms of the regulation of SHR gene expression itself.To better understand transcriptional and translational regulation of SHR gene expression,we carded out this study and the major results are summarized below.ObjectiveTo investigate whether DNMT inhibitor 5-AZA-CdR (AZA) and HDAC inhibitor tri- chostatin A(TSA) in combination can synergistically induce ERa and AR reexpression in initially Era- and AR- negative breast cancer cells SKBR-3 and restore its response to estrogen and endocrine therapy.Moreover,to investigate whether TSA can induce the chan- ge of ERa and AR expression level in SKBR-3 or MCF-7 cells and change their prolife- ration after chemical treatment.Methods1. The mRNA expression of ERa、PR、AR、pS2 and PSA in SKBR-3 cells treated with TSA and AZA in combination or TSA alone was detected by RT-PCR.2. Human breast cancer cells SKBR-3 were treated with TSA and AZA in combina- tion,breast cancer cells SKBR-3 or MCF-7 were treated with TSA alone,and then there are six treatment groups established:①Group (Vehicle) SKBR-3,②Group (AZA+TSA) SKBR-3,③Group (Vehicle) MCF-7,④Group (TSA) MCF-7,⑤Group (Vehicle) SKBR-3,⑥Group (TSA) SKBR-3. The protein expression of ERa、PR、AR、pS2 and PSA in the treated SKBR-3 and MCF-7 cells was detected by immunocytochemistry(ICC) method.3. The water-soluble tetrazolium salt (WST) method was used to analyze the cells proliferation.(1) SKBR-3 cells proliferation treated with TSA and AZA in combination was ana- lyzed in the different concentration of megestrol acetate (MA):①vehicle,②lnmol/L,③10nmo1/L④l00nmol/L⑤1μmo1/L⑥l0μmo1/L.(2) In the study of the combination treatment by AZA and TSA, there were six treat- ment groups in breast cancer cells SKBR-3 after the optimal concentration of megestrol acetate (MA) was established:①vehicle,②AZA+TSA,③AZA+TSA+E2,④AZA+TSA +E2+TAM,⑤AZA+TSA+E2+MA,⑥AZA+TSA+E2+TAM+MA.(3)After the treatment by TSA alone ,there were four treatment groups established in breast cancer cells SKBR-3 and MCF-7:①(Vehicle)SKBR-3,②(TSA)SKBR-3,③(Vehicle)MCF-7,④(TSA)MCF-7.The proliferation rate of every group was analyzed at different time :12h, 24h, 48h , 72h, 96h.Results1. After treatment by AZA and TSA in combination or TSA alone, ERa、AR、PR、pS2 and PSA mRNA reexpressed in the initially ERa- and AR-negative breast cancer cells SKBR-3, whereas the mRNA expression level in the one chemical treatment was less than that in the combination treatment(P<0.05),and both of them was much less than that in MCF-7 cells(P<0.01).2. In the protein expression analysis:(1) After the combination treatment, ERa、PR、AR、pS2 and PSA protein re-expre- ssed in the initially ER- and AR-negotive SKBR-3 cells .The protein expression level indu- ced by AZA and TSA was much less than that in MCF-7 cells(P<0.01). In per field of high multiple vision(×400)in the glass plate where human breast cancer line SKBR-3 was se- eded,the number of cells present of AZA and TSA was less than that absent of the two che- micals.No cell death was found.(2) After treatment by the one chemical, ERa、PR、AR、pS2 and PSA protein re-ex- pressed in the initially ERa- and AR- negative breast cancer cells SKBR-3 whereas they were suppressed in the initially ERa-and AR- positive breast cancer cells MCF-7.However, the protein expression level in SKBR-3 cells treated with TSA was less than the remainder in MCF-7 cells treated with TSA(P<0.05).More cells death was found in the glass plate where human breast cancer line SKBR-3 was seeded than that of breast cancer cells MCF -7.3. In the proliferation rate analysis:(1) Breast cancer cells SKBR-3 induced by AZA and TSA was treated for 96 hours by Megestrol acetate (MA) at different concentrations (1nmol/L–10μmol/L).The dose-resp- onse study demonstrated lower proliferation rate at the higher concentration of megestrol acetate (MA),the optimal concentration of MA was 10μmol/L.(2) The growth of SKBR-3 cells treated with AZA and TSA was suppressed signifi- cantly compared with the vehicle control(P<0.05);and then 1nmol/L 17β-estrodial added, the proliferation rate of the induced cells was upregulated again but not significantly(p> 0.05); TAM or MA added respectively again ,the growth of cells treated with AZA +TSA +E2+ MA as well as AZA +TSA +E2+TAM was suppressed compared with AZA +TSA +E2 group (P < 0.05); At last, the growth of the induced cells treated with TAM and MA in combination was further suppressed than that of cells treated with TAM or MA alone (P<0.05).(3)The growth of both SKBR-3 and MCF-7 cells was significantly suppressed by TSA alone compared with the vehicle group (P<0.05). In addition, the suppression of SKBR-3 cells proliferation rate by TSA happened much earlier and was much more than that of MCF-7 cells (24h vs 48h, OD 1.1437 vs 0.6387).Conclusion1.After treatment with AZA and TSA,ERa- and AR-negative SKBR-3 cells can ex- press functional ERa and AR and become responsive to estrogen and endocrine therapy , suggesting that the two chemicals may play important roles in restoring sensitivity of the SHR-negotive breast cancer to endocrinetherapy.Moreover,AZA and TSA could suppress SKBR-3 cells proliferation , suggesting that they could inhibits the growth of breast can- cer by changing its biology.2.Breast cancer cells SKBR-3 chemically induced ERa and AR re-expression res- tored dose-response to MA, which demonstrated lower proliferation rate in higher concen- tration of megestrol acetate (MA). In addition, the combination treatment with TAM and MA could synergistically inhibit the growth of induced breast cancer cells via functional ER and AR ,respectively, suggesting that small dose of MA(8mg/d)often used in hormone replacement therapy (HRT) maybe a potent plus for postmenopausal patients treated with long-term TAM to prevent the most unpleasant adverse effect of TAM,raised risk of endo- metrial cancer. However, this hypothesis is just from an experiment in vitro and must be further certified by clinical trials.3.TSA can not only improve sensitivity of the initially ERa and AR negative breast cancer cells to endocrine therapy by chemical induction of ERa and AR re-expression but also inhibit ERa and AR positive breast cancer cells growth by down-regulation of ERa and AR expression. It was more important to infer that TSA could not only induce the ex- pression of tumor suppressor genes but also down-regulate the expression of oncogenic protein to inhibit breast cancer cells proliferation by induction of their diffrention and apo- ptosis.更多还原