乳腺癌他莫昔芬耐药机制的研究进展

他莫昔芬(Tamoxifen,TAM)对于治疗雌激素受体(ER)阳性的乳腺癌是一种重要的治疗策略.然而TAM耐药是内分泌治疗失败的一个主要原因.TAM耐药的潜在机制是多因素的,其中大部分仍是未知的.本文介绍了近年来有关乳腺癌TAM耐药机制的研究进展,为阐明TAM耐药机理及克服耐药性提供有价值的信息和思路.     

他莫昔芬对乳腺癌MCF-7细胞侵袭能力的影响及其作用机制

目的 探讨他莫昔芬(TAM)对乳腺癌MCF-7细胞侵袭能力的影响及其作用机制.方法 采用对数生长期的乳腺癌MCF-7细胞,以无血清无酚红高糖DMEM培养基对其进行24 h的培养,然后将其分别接种并分为对照组(无血清无酚红高糖DMEM培养基)、TAM组(含有1μmol/L TAM的无血清无酚红高糖DMEM培养基),采用Western blot法、RT-PCR法检测两组MCF-7细胞基质金属蛋白酶-9(MMP-9)、MMP-2、血管内皮细胞生长因子(VEGF)蛋白及mRNA表达水平,采用Transwell法检测MCF-7细胞的侵袭能力.结果 TAM组MCF-7细胞中VEGF、MMP-9、MMP-2蛋白表达水平均明显高于对照组,VEGF、MMP-9、MMP-2 mRNA相对表达水平均明显高于对照组,差异均有统计学意义(P﹤0.01);TAM组MCF-7细胞的侵袭能力相对数明显高于对照组,差异有统计学意义(P﹤0.01).结论 TAM可能发挥类雌激素功能,促进乳腺癌MCF-7细胞侵袭能力,这与其提高MCF-7细胞中VEGF、MMP-9、MMP-2表达水平有关.     

miR-206对三阴性乳腺癌细胞的增殖抑制作用及初步机制研究

目的 探讨miR-206对三阴性乳腺癌细胞MDA-MB-231增殖的影响及其作用机制。方法 转染miR-206 mimic和miR-NC至乳腺癌细胞株MDA-MB-231中,应用实时荧光定量PCR（qRT-PCR）技术检测miR-206相对表达水平;应用MTT法、克隆形成实验检测miR-206对MDA-MB-231细胞增殖能力的影响;流式细胞术检测miR-206对细胞周期的影响;Western blotting进一步验证miR-206对周期相关蛋白CyclinD2的影响。结果 qRT-PCR检测结果显示,miR-206 mimic转染至MDA-MB-231乳腺癌细胞48h后miR-206的相对表达水平为10.2±1.5。MTT法检测结果显示,miR-206 mimic转染至MDA-MB-231乳腺癌细胞6、24、48、72、96h后的增殖抑制率分别为（0±0.01）％、（0.12±0.03）％、（0.21±0.08）％、（0.28±0.11）％ 和（0.39±0.16）％;克隆形成实验结果显示,miR-206 mimic和miR-NC转染至MDA-MB-231乳腺癌细胞2周后的克隆数目分别为106±35和843±143,差异具有统计学意义（P＜0.01）。流式细胞术检测结果显示,miR-206 mimic转染至MDA-MB-231乳腺癌细胞48h后,明显地阻滞细胞周期于G1期;Western blotting检测显示miR-206表达下调了细胞周期蛋白CyclinD2的表达。结论 miR-206明显抑制了三阴性乳腺癌细胞株MDA-MB-231的增殖,其机制可能与下调细胞周期蛋白CyclinD2的表达有关,这将成为乳腺癌临床治疗一个新的靶点。     

HuR胞浆表达水平与接受他莫昔芬辅助治疗的雌激素受体阳性乳腺癌预后的相关性

目的 探讨接受他莫昔芬辅助内分泌治疗的乳腺癌患者人类抗原R (human antigen R,HuR)胞浆表达水平与生存的相关性。方法 选取2005年1月至2016年12月接受他莫昔芬辅助内分泌治疗的Ⅰ～Ⅲ期乳腺癌患者105例。免疫组化方法评估胞浆HuR表达水平。SPSS 21.0软件进行统计学分析。结果 HuR强表达与Ki-67增殖指数高显著相关。Kaplan-Meier生存分析显示,Ki-67指数高以及HuR表达水平强的患者DFS和OS显著更差。COX多因素预后分析显示,腋窝淋巴结转移状态(P=0.014,HR=6.845,95%CI:1.486～31.518)、Ki-67指数(P=0.038,HR=9.995,95%CI:1.141～87.577)、HuR表达水平(P=0.006,HR=6.820,95%CI:1.758～26.464)是独立的预后因素。结论 在接受他莫昔芬内分泌治疗的雌激素受体阳性乳腺癌患者中,HuR胞浆高表达者预后更差,HuR可能成为逆转他莫昔芬耐药的一个靶点。     

miR-342与乳腺癌ERα表达及他莫昔芬敏感性关系探讨

目的:探讨miR-342与乳腺癌临床病理的关系及是否参与调节ERα并影响他莫昔芬(TAM)的敏感性。方法:RT-PCR检测48例癌组织miR-342和ERαmRNA水平及其中24例癌旁组织miR-342的表达;免疫组化评价癌组织ER、PR、HER-2和VEGF的状态;RT-PCR检测乳腺癌细胞株MCF-7、SKBR-3及MB-231的miR-342、ERαmRNA水平;检测MCF-7细胞瞬时转染hsa-miR342(分mimic、inhibitor及各自阴性对照共4组)48h后miR-342和ERαmRNA的变化;1×10-8 mol/L 17-β雌二醇(E2)单独或联合2×10-5 mol/L TAM处理MCF-7细胞72h,CCK-8法测不同转染组细胞增殖;各转染组细胞1.5×10-5 mol/L TAM处理48h,流式细胞术分析细胞凋亡率。结果:miR-342及ERαmR-NA在ERα阳性乳腺癌组织及细胞中均明显升高,P<0.01;miR-342和ERαmRNA之间存在正相关,P=0.003。miR-342在HER-2阴性(P=0.001)及VEGF阴性(P=0.031)乳腺癌组织中上调;miR-342与PR、淋巴转移、病理分级等因素无明显关系,P>0.05;癌与癌旁miR-342表达差异无统计学意义,P=0.065。MCF-7细胞mimic组ERαmRNA表达为1.80±0.14,高于对照组的1.0±0.0,P=0.001;inhibitor组为0.747±0.087,较对照组低,P=0.037。TAM作用72h,mimic组细胞增殖率为(45.9±1.3)%,与对照组的(55.0±1.5)%相比明显抑制,P=0.001;inhibitor组增殖率为(72.9±1.9)%,较对照组升高,P=0.000。流式细胞术分析TAM处理后的细胞凋亡率,结果显示,mimic组凋亡率为(9.54±1.14)%,较对照组的(4.50±0.46)%增加,P=0.002;inhibitor组凋亡率为(3.06±0.42)%,较对照组的(4.95±0.59)%降低,P=0.011。结论:miR-342的表达可以一定程度预测ERα的表达水平,并作为分子标志预测乳腺癌细胞对TAM的敏感性;miR-342有望成为潜在靶点来参与乳腺癌内分泌治疗。     

他莫昔芬治疗乳腺癌的副作用

他莫昔芬(ｔａｍｏｘｉｆｅｎ,ＴＡＭ)为非甾体类抗雌激素药物。长期服用ＴＡＭ可使乳腺癌患者的生存期延长,乳腺癌复发减少,对侧乳腺癌的发生率降低。在多数大规模的临床试验中,参加者多能耐受ＴＡＭ的标准治疗剂量,但是有关ＴＡＭ在治疗乳腺癌过程对眼、肝脏、卵巢、骨?..     

PS341对他莫昔芬诱导乳腺癌MCF-7细胞凋亡影响及其机制的探讨

目的:研究蛋白酶体抑制剂PS341是否可增强他莫昔芬(TAM)诱导的乳腺癌细胞凋亡,并探讨可能的机制。方法:采用MTT方法测定细胞活力,采用流式细胞仪检测细胞凋亡,采用蛋白质印迹法检测Caspase-8的活化,PARP的裂解及死亡受体Fas蛋白的表达。结果:TAM以时间及浓度依赖的方式抑制乳腺癌MCF-7细胞增殖,24及48h的IC50分别为25和6μmol/L,25μmol/L的TAM处理24h可诱导21.9%的MCF-7细胞发生凋亡,10nmol/L的PS341预处理后给予25μmol/L的TAM处理24h细胞凋亡增至34.6%,PS341与TAM均可轻微上调Fas的表达水平,两药联合应用后可显著上调Fas的表达水平,并明显增强线粒体外凋亡通路的活性。结论:PS341与TAM联合应用可通过上调Fas的表达水平,增强TAM诱导的乳腺癌MCF-7细胞凋亡。     

人参多糖与他莫昔芬联合对人乳腺癌MCF-7细胞凋亡的影响及其机制研究

目的： 探讨人参多糖 (ginseng polysaccharide,GPS)与他莫昔芬 (tamoxifen,TAM)联合对人乳腺癌MCF-7细胞凋亡的影响及其机制。方法：以不同浓度 (0、20、40、80和160 μg/mL)GPS作用于人乳腺癌MCF-7细胞株48 h,以MTT法检测细胞增殖抑制情况,以GPS的最小有作用剂量与不同浓度 (0、0.5、1、2和4 μg/mL)TAM联合处理细胞48 h,以金氏公式筛选出协同抑制作用最明显的联合剂量,以该联合剂量处理MCF-7细胞48 h后,用DAPI染色法、流式细胞术检测细胞凋亡情况;用Giemsa染色检测细胞有丝分裂指数;用Western blot检测细胞中Fas、Caspase-9 以及Parp的表达情况。结果：MTT法检测提示GPS在48 h对MCF-7细胞的最小有作用剂量为40 μg/mL,金氏公式计算结果提示40 μg/mL GPS+1 μg/mL TAM联合用药协同抑制细胞增殖作用最强 (q=1.82);与GPS或TAM单独作用相比,DAPI染色发现联合用药组镜下可见大量凋亡小体;流式细胞仪检测发现,联合用药能够协同增加细胞凋亡率 ( q=2.19,P <0.05);Giemsa染色结果显示联合用药能够协同抑制细胞有丝分裂 (均P<0.05);Western blot检测发现,联合用药能增加细胞中Fas表达,促进Caspase-9以及Parp的活化。结论：GPS与TAM联合能够协同通过Fas信号通路促进人乳腺癌MCF-7细胞发生凋亡。     

他莫昔芬对转染ERβ1基因的MCF-7细胞生长特性的影响

目的研究在不同处理因子作用下,外源基因ERβ1的表达对MCF-7乳腺癌细胞系生长特性的影响。方法利用脂质体转染方法将ERβ1真核表达载体pcDNA3.1-EGFPERβ1导入MCF-7乳腺癌细胞系。采用Western blot方法检测转染细胞中ERβ1的蛋白表达水平,筛选阳性克隆。以亲本细胞MCF-7为对照,分别在雌激素和雌激素受体拮抗剂他莫昔芬作用下观察细胞的生长特点。结果在转染ERβ1基因的MCF-7细胞系中,Western blot检测证实ERβ1的蛋白表达水平显著增高。在无处理因子的情况下,外源基因ERβ1在MCF-7细胞系中的表达能抑制细胞生长。与亲本细胞MCF-7细胞相比,转染ERβ1的MCF-7细胞对雌激素的敏感性下降,但对他莫昔芬的敏感性无明显变化。结论外源性ERβ1基因在MCF-7乳腺癌细胞中的稳定表达不增加对他莫昔芬的耐药性,但使之对雌激素的敏感性下降。     

他莫昔芬对乳腺癌患者卵巢的影响

他莫昔芬(tamoxifen,TAM)作为一种非类固醇类抗雌激素药物,自70年代初开始应用于治疗乳腺癌以来,已作为乳腺癌术后辅助治疗和预防复发的主要用药[1]。但长期服用TAM的乳腺癌患者常潜在妇科并发症,我们对我院2000年1月至2004年12月102例服TAM治疗的绝经前后乳腺癌妇女卵巢囊肿发生情况进行分析并探讨对策。1材料和方法1.1研究对象观察组:乳腺癌患者102例,年龄26~75岁,平均年龄59岁。绝经前31例,绝经后71例。对照组:乳腺癌患者46例,年龄32~73岁,平均年龄57岁,绝经前26例,绝经后20例。1.2TAM治疗观察组:口服TAM10mg,一日2次。TAM治疗时…     

维生素E琥珀酸酯对MCF-7乳腺癌细胞三苯氧胺敏感性的影响

目的检测维生素E琥珀酸酯(VES)对MCF-7乳腺癌细胞三苯氧胺敏感性的影响。方法人乳腺癌细胞MCF-7以VES刺激24h,VES浓度为5μg/ml和10μg/ml。随后以1μM,2.5μM,5μM和10μM的三苯氧胺作用24h,以MTT法测定对细胞增殖的抑制作用,RT-PCR法检测VES对HER-2mRNA表达的影响,以免疫组化法检测VES作用前后HER-2产物c-erbB-2表达的变化。结果TAM对MCF-7乳腺癌细胞具有增殖抑制作用,VES处理后,乳腺癌细胞对TAM的敏感性升高。RT-PCR显示VES作用后乳腺癌细胞HER-2mRNA转录水平有一定程度的下降,乳腺癌细胞c-erbB-2的表达降低。结论VES对MCF-7乳腺癌细胞三苯氧胺的敏感性具有增强作用,可能与降低HER-2基因表达产物有关。     

雌激素受体阳性可手术乳腺癌三苯氧胺辅助治疗的远期疗效

目的：探讨三苯氧胺（TAM）辅助治疗雌激素受体阳性可手术乳腺癌的远期疗效。方法：414例雌激素受体阳性可手术乳腺癌患者按服用TAM与否分成TAM组和非TAM组，并随访观察至少10年，用Kaplan-Meier法计算两组生存率和无瘤生存率，Log-rank统计分析行显著性检验，Cox模型进行多因素分析，结果：TAM组生存率和无瘤生存率均高于非TAM组，服药时间与生存率和无瘤生存率均呈正相关（P＜0．05），进一步分层比较，腋淋巴结阴性患者两指标无差异（P＞0．05），腋淋巴结阳性患者两指标差异有显著性（P＜0．05），结论：TAM用于雌激素受体阳性可手术乳腺癌患者术后辅助治疗可提高远期生存率和无瘤生存率。     

三苯氧胺与乳腺癌患者子宫内膜病理改变的非随机性临床观察

目的：随访口服三苯氧胺的乳腺癌患者，观察在随访期内发生的子宫内膜病理改变类型。方法：80例乳腺癌患者口服三苯氧胺20mg/日，每隔6个月作双合诊检查及子宫内膜活体组织检查。结果：可评价患者共67例，共行200次子宫内膜活检，60例仍为正常子宫内膜表现，TAM应用中位持续时间30个月，2例绝经前患者子宫内膜活检表现为单纯性增生过长，1例绝经后患者表现为萎缩性子宫内膜与复合性增生过长并存，1例绝经后患者切除子宫，术后内膜病是表现为增生性息肉、单纯必囊性增生。7例患者发生子宫内膜息肉，结论：长期口服三苯氧胺在绝经前后患者中均能发生子宫内膜单纯增生过长，复合性增生过长及息肉改变，未见有癌的发生。     

Interrelationship between Estradiol and Tamoxifen Responses for Clinical Breast Carcinoma Cells Cultured on Contact-sensitive Plates

An in vitro assay system for predicting the estradiol (E₂) sensitivity of clinical cancer cells was applied to 54 patients with breast carcinoma to compare the responses to E₂ and tamoxifen (TAM) with the estrogen receptor (ER) status. We found that 18 of the 35 cases in the ER-positive group and 6 of the 19 cases in the ER-negative group were stimulated by E₂. It is suggested that ER status alone can not predict the response of cultured cells to E₂ in clinical breast cancer. Cell growth of 11/35 (31%) of the ER-positive cases and that of 8/19 (42%) of the ER-negative cases was inhibited by E₂. Since the cases inhibited by E₂ could not be distinguished by ER status alone, an assay system based on a quantitative proliferative response was considered necessary. There were 20 (83%) cases of inhibition by TAM among the 24 stimulated by E₂. Only 18/35 (51%) of the ER-positive group exhibited growth inhibition by TAM. In our (CSP) assay, 20 (83%) of the 24 cases stimulated by E₂ were inhibited by TAM, 10 (91%) of the 11 E₂-insensitive cases were insensitive to TAM and 13 (68%) of the 19 cases inhibited by E₂ were stimulated by TAM. In short, TAM response and E₂ response tended to be inversely related (43/54=80%, P <0.01). Furthermore, the E₂-response rate showed a good correlation with the TAM-response rate (R₂= 0.825). These results indicate the feasibility of predicting individual tumor responses to either E₂ or TAM by using CSPs.     

In vitro Sensitivity Test of Breast Cancer Cells to Hormonal Agents in a Radionucleotide-incorporation Assay

Breast cancer cell lines (MCF-7, T47D, BT-20 and STT-11) and fresh cells from malignant effusions of eight breast cancer patients were examined for their in vitro sensitivity to 17β-estradiol (E₂), tamoxifen and toremifene in a miniaturized, improved nucleic acid precursor incorporation assay (MINI assay). Seven of the eight patients received either tamoxifen or toremifene following a MINI assay and the correlation was examined between in vitro sensitivity and clinical responses to the hormonal agents. In cell lines, E₂ stimulated thymidine incorporation by estrogen receptor (ER)-rich cells, MCF-7 and T47D, but not by ER-poor cells, BT-20 and STT-11. Tamoxifen induced both ER-mediated and -unmediated effects in ER-rich cells. The latter effect was also observed in ER-poor cells. Toremifene had less ER-unmediated effect in all of the cells tested than tamoxifen did. The ER-mediated effect of toremifene was weaker than that of tamoxifen in cell lines but was equipotent to tamoxifen in fresh cells. E₂ affected thymidine incorporation by cells withdrawn from patients who showed a partial response to the anti-estrogens. No clear correlation was demonstrated between in vitro sensitivity to anti-estrogens of fresh cells and clinical response to these agents. The present results suggest that 1) the MINI assay is a useful system to investigate hormonal effects on breast cancer cell lines; 2) clinical responses to anti-estrogens are not predicted by in vitro response to the agents but might be predicted by the in vitro response to E₂; and 3) toremifene has a smaller non-specific effect on breast cancer cells than tamoxifen and is equipotent to tamoxifen in the ER-mediated effect in vitro .     

乳腺癌患者术后服用三苯氧胺后子宫内膜病变的临床病理分析

目的分析乳腺癌患者术后服用三苯氧胺(tamoxifen,TAM)后子宫内膜的病理变化。方法研究组为服用TAM出现阴道不规则出血或B型超声提示子宫内膜异常患者69例;对照组为同时期非乳腺癌因绝经后出血就诊的患者60例。TAM组又分为绝经前、后两组(20例、49例)及根据服用TAM时间(T)长短,分为T≤1年组、1年＜T＜2年组、T≥2年组,进行宫腔镜检查及子宫内膜活检。结果TAM组子宫内膜息肉及子宫内膜增生明显高于对照组(P<0.001,P<0.05)。绝经后TAM组较绝经前TAM组更易发生子宫内膜病变(P<0.01),且子宫内膜重度不典型增生和子宫内膜癌均发生在绝经后组。随着用药时间的延长,子宫内膜病变的发生增加(P<0.01),且子宫内膜癌均发生在服药2年后。结论乳腺癌患者使用TAM治疗发生子宫内膜病变的风险增加,尤其对于绝经后使用且用药2年以上的妇女。     

Survival Benefit of Tamoxifen in Estrogen Receptor-Negative and Progesterone Receptor-Positive Low Grade Breast Cancer Patients

Purpose

This study aimed to analyze the efficacy and prognostic significance of adjuvant tamoxifen in breast cancer patients with various hormone receptor statuses.

Methods

Typically, 1,260 female breast cancer patients were recruited in this study. The correlation between estrogen receptor (ER)/progesterone receptor (PR) phenotypes and clinical characteristics was investigated, and the survival rate was assessed after 5-year follow-up.

Results

The 5-year overall survival (85%) was better in women under the age of 50 years. Patients with ER+/PR+ tumors had a better 5-year survival rate (94%); those with ER-/PR- tumors experienced the worst outcome (74% survival rate); whereas single-positive cases were in between. In 97 out of 128 patients with ER-/PR+ tumors, tamoxifen was given as adjuvant hormonal therapy, and it increased the survival benefit in the lower grade group in terms of overall survival and disease-free survival (p=0.01 and p=0.03, respectively).

Conclusion

For high-grade tumors with ER-/PR+, adjuvant tamoxifen therapy may have no survival benefit, whereas for the patients with low-grade ER-/PR+ tumors, adjuvant tamoxifen therapy is highly suggestive.     

Soy Foods Might Weaken the Sensitivity of Tamoxifen in Premenopausal Patients With Lumina A Subtype of Breast Cancer

Background

Based on estrogen active substances, many women consume soy foods in the belief that it could prevent breast cancer (BC). Women with different molecular subtypes would be likely to have diverse reactions to soy foods, especially those with the estrogen-receptor-positive (ER⁺) subtype. The aim of the current study is to identify the differentially expressed genes (DEGs) on soy foods in premenopausal patients with Lumina A subtype of BC (LABC) after soy food treatment, and to further investigate the critical molecule change.

Association of miR-1266 with Recurrence/Metastasis Potential in Estrogen Receptor Positive Breast Cancer Patients

The Homeobox B13 (HOXB13):Interleukin 17 Receptor B (IL17BR) index of estrogen receptor (ER)-positivebreast cancer (ER (+) BC) patients may be a potential biomarker of recurrence/ metastasis. However, effects ofmicroRNA (miRNA) binding to the 3’ untranslated region (3´UTR) of HOXB13 and IL17BR and its function onrecurrence/metastasis in ER (+) BC remains elusive. The aims of this study were to determine the expressionof miRNAs that bind to 3´UTR of HOXB13 and IL17BR in ER (+) BC patients and asess the effects of thesemiRNAs on recurrence/metastasis. The expression profiles of HOXB13 and IL17BR were evaluated using RTPCRin tumors and normal tissue samples from 40 ER (+) BC patients. The expression level of 4 miRNAs, whichwere predicted to bind the 3´UTR of HOXB13 and IL17BR using TargetScan, microRNA.org and miRDB onlinedatabases, were further evaluated with RT-PCR. Our findings demonstrated that high miR-1266 levels might besignificant prognostic factor for recurrence/metastasis occurrence (3.05 fold p=0.004) and tamoxifen response(3.90 fold; p=0.2514) in ER (+) BC cases. Although we suggest that modulation of miR-1266 expression may bean important mechanism underlying the chemoresistance of ER (+) BC, advanced studies and validation arerequired.