β-榄香烯与吉非替尼逆转乳腺癌MCF-7细胞他莫西芬耐药的实验研究
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摘要
目的：内分泌治疗是激素受体阳性乳腺癌的重要治疗手段，但许多ER阳性的患者在内分泌治疗过程中出现ER转阴，导致获得性耐药，因此对内分泌治疗获得性耐药研究备受关注。对耐药机制的研究在ER水平和信号转导机制的研究则最被重视，内分泌耐药是ER和EGFR家族信号网络的双向作用共同调节的，ER阳性的细胞在MAPK信号表达上调后转为ER阴性。乳腺癌的综合治疗中，内分泌治疗多是序贯于放化疗之后，内分泌治疗过程中也可能伴有乳腺癌干细胞的富集，干细胞的富集是否构成内分泌耐药的原因，如何提高内分泌治疗的疗效，并延缓和逆转内分泌耐药是临床亟待解决的问题。中药β-榄香烯（β-Elemene，β-ELE）是从姜科植物温郁金中提取的抗癌有效成分，临床上应用于多种肿瘤的放疗增敏及化疗辅助，尚有逆转化疗耐药的研究，有报道与内分泌治疗具有协同作用。吉非替尼（Gefitinib，Iressa）是小分子酪氨酸激酶抑制剂，主要应用于肺癌的分子靶向治疗，近年来也应用于乳腺癌的内分泌联合治疗研究。据文献报道，化疗多药耐药逆转的研究中，具有逆转耐药的药物本身与化疗药物具备协同治疗的作用，与内分泌治疗有协同作用的药物是否具备逆转耐药的作用，对此我们进行一系列实验设计。首先，本研究模拟了人体乳腺癌治疗后复发的生物学特点，引入乳腺癌干细胞的概念，即在综合治疗后富集了少量的乳腺癌干细胞，在适当条件下进行增殖分化，导致乳腺癌复发的过程，通过体外干细胞培养条件下培养激素敏感的人乳腺癌MCF-7细胞，检测具备干细胞特性的悬浮球细胞雌激素受体（ER）的表达及对他莫西芬（Tamoxifen，TAM）的治疗敏感性，初步探讨内分泌耐药与乳腺癌干细胞的关系；并同时构建乳腺癌对TAM耐药的MCF-7/TAM细胞株（干细胞培养及传统低剂量诱导的方法），检测耐药细胞中ER在mRNA和蛋白表达水平改变，相关信号转导通路MAPK通路蛋白RAS、MEK1/2、p-ERK1/2水平变化，分析耐药MCF-7对TAM的耐药机制。其次，通过β-ELE、Gefitinib与TAM的联合应用，采用不同给药顺序干预乳腺癌MCF-7细胞的增殖，确定β-ELE和Gefitinib与TAM在内分泌治疗方面的联合应用价值，探索最佳给药顺序，确定其对MCF-7和MCF-7/TAM细胞的低剂量（细胞增殖抑制率<5%）。继而采用低剂量的β-ELE和Gefitinib作为逆转实验的干预剂量，处理对TAM耐药的MCF-7/TAM细胞，通过后者恢复对TAM敏感的现象，进一步检测治疗过程中ERα、ERβ在mRNA和蛋白表达水平的变化，以及MAPK通路蛋白RAS、MEK1/2、p-ERK1/2水平变化，分析β-ELE与Gefitinib在逆转乳腺癌内分泌耐药中的作用及机制。
方法：分别于常规培养及干细胞培养条件下（悬浮球培养）培养激素敏感的MCF-7细胞株，流式细胞仪检测分子表型CD44~+CD24~(–/low)与CD44~+CD24~+亚群细胞比例变化，免疫细胞化学法测定ERα和ERβ的表达变化，MTT法检测细胞对他莫西芬的敏感程度。培养乳腺癌MCF-7/TAM耐药细胞；分别应用TAM与β-ELE联合、TAM与Gefitinib联合作用于MCF-7细胞，设立序贯与混合联用方案，用MTT法检测不同用药顺序对MCF-7细胞的增殖抑制作用，计算二药联合作用指数(combine index，CI)，比较不同方案对细胞增殖抑制率的影响，并着重观察ELE和Gefitinib低剂量下TAM对MCF-7细胞的增殖抑制情况。流式细胞仪检测Gefitinib与ELE细胞毒性剂量对MCF-7细胞周期的影响。采用干细胞培养及传统低剂量诱导的方法构建出ERα阴性的乳腺癌TAM耐药细胞MCF-7/TAM，分别不同剂量和不同时间段β-ELE和Gefitinib处理MCF-7/TAM细胞，通过MTT法分析并确定β-ELE和Gefitinib对MCF-7/TAM和对MCF-7细胞的低剂量及最佳作用时间，据MTT结果用10ug/ml的β-ELE和Gefitinib处理MCF-7/TAM细胞48h，设立MCF-7组（M0），MCF-7/TAM组（M/T），MCF-7/TAM-ELE10ug/ml（E10），MCF-7/TAM-Gefitinib10ug/ml（G10）组进行后续试验，用MTT方法检测经处理后的E10与G10组细胞对TAM的敏感性，RT-PCR和免疫细胞化学观察ERα和ERβ在mRNA和蛋白水平的变化，并检测各组细胞MAPK通路RAS，MEK1/2、p-ERK1/2蛋白水平变化。实验结果应用SPSS16.0软件，计数资料ERα与ERβ的阳性表达采用卡方检验进行统计学分析，结果用百分率表示；凝胶电泳结果分析采用IMAGEJ与LabWorks4.6软件分析，计量资料确保符合正态分布，两组样本比较采用t检验，三组IC50值采用方差分析，结果以均数±标准差表示，P＜0.05为差异有统计学意义。
结果：干细胞培养条件下CD44~+CD24~(–/low)亚群细胞所占比例由常规培养的（0.27±0.08）%增至（1.60±0.08）%（p＜0.05），而CD44~+CD24~+亚群细胞比例由（5.59±0.88）%增至（30.63±4.40）%（p＜0.05）。干细胞培养条件培养下ERα和ERβ表达率较常规培养下调，分别由85.27%和90.53%降至69.43%和73.20%，差异有统计学意义（p均＜0.05），且对他莫西芬的敏感性降低，IC50值由(9.82±0.31)umol/L升至(16.46±0.50)umol/L，再次诱导分化后ER并未出现上调，对他莫西芬的敏感性仍旧降低（p＜0.05）。TAM与β-ELE序贯联用具有协同作用，序贯方法优于混合应用的叠加作用（p＜0.05）。TAM与Gefitinib联用不同顺序均具有协同作用，序贯联用方法优于混合应用（p＜0.05）。β-ELE5ug/ml及10ug/ml时ELE--TAM显示出优于TAM--ELE方案的趋势， Gefitinib5ug/ml及10ug/ml时Gefitinib--TAM显示出优于TAM--Gefitinib方案的趋势。流式细胞仪检测显示，β-ELE20ug/ml及40ug/ml作用于MCF-7细胞后，G0/G1期细胞比例由49.26%提高到64.04%和63.88%；Gefitinib10ug/ml及20ug/ml作用于MCF-7细胞后，G0/G1期细胞比例由49.26%提高到54.89%，68.35%。确定了β-ELE与Gefitinib与TAM的可联合性。后续试验发现，10μg/ml的β-ELE与Gefitinib作为干预剂量作用于M/T细胞48h后，细胞可重新对TAM敏感，细胞增殖受到抑制，实现耐药逆转。RT-PCR检测发现β-ELE治疗后可上调MCF-7/TAM细胞中ERα mRNA表达水平。与M0组相比，M/T组ERα mRNA与ERβ mRNA水平均见下调，以ERα mRNA下调最为显著(p＜0.05)。经β-ELE治疗后，E10组ERα mRNA明显上调(p＜0.05)，ERβ mRNA也在E10组出现上调(p＜0.05)。M0、M/T、E10各组ERα表达率分别为(95.04±1.81)%、(2.10±0.24)%、(82.34±3.21)%；各组ERβ表达率(96.13±1.07)%、(85.13±2.17)%、(74.33±3.07)%。ERα在M/T组细胞中的表达率显著下降p＜0.05)，在经过β-ELE治疗后表达率再次升高(p＜0.05)。而ERβ在M/T组细胞中的表达率仅有轻度下降(p＞0.05)，应用β-ELE治疗后表达率呈下降趋势。Western blot分析结果显示，M/T组MAPK通路蛋白Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M0组明显增强(p＜0.05)，经10μg/ml的β-ELE治疗48h后Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M/T组下调(p＜0.05)。在Gefitinib实验中，我们发现，Gefitinib上调MCF-7/TAM细胞中ERα mRNA和ERβ mRNA的水平，与M0组相比，M/T组ERα mRNA与ERβmRNA水平均见下调，以ERα mRNA下调最为显著(p＜0.05)。但是经Gefitinib治疗后，G10组ERα mRNA明显上调(p＜0.05)，ERβ mRNA也在G10组出现上调(p＜0.05)。免疫细胞化学显示，M0、M/T、G10各组ERα表达率分别为(95.04±1.81)%、(2.10±0.24)%、(75.04±2.88)%；各组ERβ表达率(96.13±1.07)%、(85.13±2.17)%、(90.25±2.15)%。ERα在M/T组细胞中的表达率显著下降(p＜0.05)，在经过Gefitinib治疗后表达率再次升高(p＜0.05)。而ERβ在M/T组细胞中的表达率仅有轻度下降(p＞0.05)，应用Gefitinib治疗后表达率呈上调趋势。Western blot分析结果显示，M/T组MAPK通路蛋白Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M0组明显增强(p＜0.05)，经10μg/ml的Gefitinib治疗48h后Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M/T组下调(p＜0.05)。
结论：
1.干细胞培养条件下可培养出具备干细胞特性的细胞亚群的悬浮球，且ER为阳性表达，但对TAM治疗敏感性减低，提示乳腺癌干细胞的富集可能是内分泌耐药的原因之一。
2.利用干细胞培养方法可以构建乳腺癌MCF-7细胞的TAM耐药细胞。耐药的主要原因可能与MAPK通路蛋白表达上调，导致ERα缺失有关。
3. β-ELE与Gefitinib均可阻滞MCF-7细胞于G0/G1期，增强TAM的抑制细胞增殖作用，与TAM药物间有协同作用。
4. β-ELE和Gefitinib通过上调ERα mRNA水平，使ERα受体再表达，恢复对TAM的治疗敏感性，逆转MCF-7细胞对TAM耐药。
5. MAPK通路是ERα受体再表达的重要途径，β-ELE和Gefitinib可以通过下调MAPK通道蛋白表达而实现ERα受体再表达。
Objective: Endocrine therapy is an important approach in the treatment ofestrogen receptor (ER)-positive breast cancer. However, the ER status of ER-positivepatients may be lost during endocrine therapy, leading to the acquired drug resistance,the loss of ERα and the up-regulation of growth factor receptor pathways are the mostwidely accepted causes of drug resistance. Therefore, great attention has been paid inresearches on the acquired resistance in endocrine therapy, and great emphasis has beenmade on ER level and detailed signal transduction in researches on the mechanism ofdrug resistance. Resistance to endocrine therapy is regulated by both ER and the EGFRfamily signalling network. Upregulation of the mitogen-activated protein kinase(MAPK) signal causes ER-positive cells to lose ER expression and become ER-negative.In the comprehensive treatment of breast cancer, endocrine therapy, usually positionedsequentially post to chemotherapy and radiotherapy, is potentially accompanied with theenriched stem cells of breast cancer, resulting in resistance to endocrine drugs.Therefore it is critical to improve the efficacy of endocrine therapy, to postpone theonset of the resistance and to reverse it. β-Elemene (β-ELE) is an effective anti-cancercomponent extracted from turmeric, a member of the Zingiberaceae family, and it hasbeen used clinically to enhance radiosensitisation and to assist chemotherapeutictreatment in treating multiple types of tumours. ELE has been studied as an agentcapable of reversing resistance to chemotherapy, Further, research on the application ofELE in endocrine therapy is sparse. Gefitinib, a small-molecular tyrosine kinaseinhibitor, has mainly been used in molecular targeted treatment(6-8),and in recent yearsalso been used in the study of breast cancer treatment combined with endocrine therapy.Studies on the reversal of multidrug resistance to chemotherapy have demonstrated thatdrugs that reverse drug resistance can synergistically inhibit cancer when used in combination with chemotherapy In this respect we have made serried of laboratorydesigns. Firstly, we introduced the concept of stem cell of breast cancer throughsimulating the biology of recurrent human breast cancer, i.e. recurrence induced byproliferation and differentiation of a little amount of stem cells of breast cancer enrichedafter the comprehensive treatment. We cultivated a MCF-7line sensitive to endocrinetherapy in vitro through stem cell cultivation, tested ER expressed by suspended spherecells with stem cells features and their response to tamoxifen, and explored thecorrelation between resistance to endocrine therapy and cancer stem cells. We alsoestablished a cell line CMF-7/TAM resistant to breast cancer through stem cellcultivation and low-dose induction) to detect the altered ER in levels of both mRNAand protein, the variation in proteins of RAS、MEK1/2、p-ERK1/2in the relatedpathway of MAPK, to discuss the mechanism of resistance to TAM in MCF-7.Secondly, we defined the value of β-ELE and Gefitinib in combination with TAM in theendocrine therapy through intervening the proliferation of MCF-7with β-ELE andGefitinib combined with TAM in different sequences, to explore the best dosagesequence, and define the non-toxic dosage to MCF-7and MCF-7/TAM. Then we usedthe non-toxic dosage of β-ELE and Gefitinib as the intervening dosage to treatMCF-7/TAM cell resistant to TAM, which later recovered the response to TAM.Furthermore, we detected the variation of ERα and ERβ in mRNA and protein duringthe intervention, and the altered protein levels of RAS, MEK1/2, p-ERK1/2involved inMAPK pathway to analyze the effect ofβ-ELE and Gefitinib in reversed resistance toendocrine therapy and the mechanism.
Methods: The MCF-7cells were cultured in normal culture or in stem cell culturein vitro (suspension sphere). The proportion of CD44~+CD24-/lowphenotype and CD44~+CD24~+phenotype cells were determined using flow cytometry (FCM). The ERa andERβ expression was detected using immunohistochemical method. The susceptibility totamoxifen was determined using MTT essay. The data are processed with t-test,Chi-square test and analysis of variance,respectively.Hormone-sensitive breast cancerMCF-7cell was treated with Tamoxifen added β-elemeneversus TAM added Gefitinib. To establish groups of combination and sequential medication.The MTT test was used to detect the repressional effection to MCF-7cells with different drug orders, focusing on observing the inhibition of cell proliferation under non-toxic dose of β-ELE versus Gefitinib and the effect of both drugs on cell cycle which wastested by flow cytometric. To calculate the combine index (CI) and compare cell prolife ration inhibition rate of two groups. For our subsequent experiments, we used the method of stem cell culture in vitro and inducing with low dose to create a tamoxifen (TAM)-resistant ERα-negative breast cancer cell line, MCF7/TAM cells. After treating MCF7/TAM cells with ELE versus Gefitinib in different doses and different times, the best non-cytotoxic doses and duration of action were determined using MTT essay.We treated cells with10μg/ml β-ELE and Gefitinib for48h,and divide into MCF-7group(M0)MCF-7/TAM group(M/T),MCF-7/TAM-ELE10ug/ml group(E10)and MCF-7/TAM-Gefitinib10ug/ml group（G10). To investigate the mechanism by which ELE reversed the drugresistance of MCF-7/TAM cells, we used RT-PCR to analyse the mRNA levels of ERαand ERβ. ERα and ERβ expression levels determined by immunohistochemistry.Expression levels of proteins in the MAPK (RAS and MEK1/2and p-ERK1/2)pathwayas analyzed by western blotting. Results from our experiments were statisticallyanalyzed using SPSS16.0software. Count data of ERα and ERβ expressed positivelywas analyzed with Chi-square test and expressed in percentage. IMAGEJ andLabWorks4.6were used to test results of gel electrophoresis. The quantitative data wasproved to be in the normal distribution. T-test was used for comparison between twogroups and variance analysis for analysis among three groups. All results wereexpressed in mean±standard deviation, with P<0.05considered as the statisticalsignificance.
Results: In suspension culture as stem cells, the proportions of theCD44~+CD24~(–/low)CD44~+CD24~+phenotype cells were (1.60±0.08)%and(30.63±4.40)%respectively, significantly higher than (0.27±0.08)%and (5.59±0.88)%in normal medium (p<0.05). The positive expressions of ERα and ERβ were85.27%and90.53%in normal medium,69.43%and73.20%in suspension culture,respectively，and the differences were statistically significan（tp<0.05）. With regards totamoxifen sensitivity, IC50was （9.82±0.31） μmol/L in normal medium, and(16.46±0.50) μ mol/L in suspension culture. After the second induced differentiation,the positive expression of ER was not up-regulated and the susceptibility to tamoxifenremained low (p<0.05). TAM combined with β-ELE or Gefitinib in different orders allhave synergistic effect. Sequential application is better than superposition（p<0.05）.Under Non-toxic dose of β-ELE or Gefitinib（5ug/ml and10ug/ml）, priorityuse of β-ELE or Gefitinib is superior of TAM’s first use. Flow cytometric detected thatthe proportion of G0/G1phase in MCF-7cells,which were cultured with β-ELE in20ug/ml and40ug/ml dose density,increased from49.26%to64.04%and63.88%,and with gefitinib in10ug/ml and20ug/ml dose density,the proportion increased from49.26%to54.89%and68.35%. In the following research, response to TAM wasrecovered in cells with inhibited proliferation and reversed resistance, after treatment for48h with10μg/ml β-ELE and10μg/ml Gefitinib as the intervening dosage. Toinvestigate the mechanism by which ELE reversed the drug resistance of MCF-7/TAMcells, we used RT-PCR to analyse the mRNA levels of ERα and ERβ. Our resultsdemonstrated that the ERα and ERβ mRNA levels in the M/T group weredown-regulated compared with those of the M0group. The reduction in the level ofERα mRNA was significant (p<0.05). In addition, the ERα mRNA level in the E10group was significantly up-regulated after β-ELE treatment (p<0.05), and the ERβmRNA level was also increased after treatment in the E10group (p<0.05). The ERαexpression levels in the M0, M/T, and E10groups were95.04±1.81%,2.10±0.24%,and82.34±3.21%, respectively. The ERβ expression levels in each group were96.13±1.07%,85.13±2.17%, and74.33±3.07%, respectively. Our results demonstratedthat the ERα expression level was significantly reduced in cells in the M/T group(p<0.05) and was increased by ELE treatment (p<0.05). ERβ expression levels wereslightly reduced in cells of the M/T group (p＞0.05), a trend that was less significantafter ELE treatment. These results suggest that the TAM resistance of MCF-7cells waslargely associated with the loss of ERα expression, but the correlation of TAMresistance and ERβ expression needs to be further investigated. Our western blot resultsrevealed that the expression levels of proteins in MAPK pathway, including Ras,MEK1/2, and p-ERK1/2, were significantly increased in the M/T group compared withthose of the M0group (p<0.05). After the cells were treated with10μg/ml β-ELE for48h, the protein expression levels of Ras, MEK1/2, and p-ERK1/2were significantlydown-regulated compared with the levels of the M/T group (p<0.05), which indicatedthat TAM resistance in MCF-7cells is largely mediated by the MAPK pathway. Inaddition, these data indicate that β-ELE reverses the drug resistance of MCF-7/TAMcells by re-sensitising the drug-resistant cells to TAM through the modulation of theexpression of proteins involved in MAPK pathway.
To investigate the mechanism by which Gefitinib reversed the drug resistance ofMCF-7/TAM cells, we used RT-PCR to analyse the mRNA levels of ERα and ERβ. Ourresults demonstrated that the ERα and ERβ mRNA levels in the M/T group weredown-regulated compared with those of the M0group. The reduction in the level ofERα mRNA was significant (p<0.05). In addition, the ERα mRNA level in the E10 group was significantly up-regulated after Gefitinib treatment (p<0.05), and the ERβmRNA level was also increased after treatment in the E10group (p<0.05). The ERαexpression levels in the M0, M/T, and E10groups were95.04±1.81%,2.10±0.24%,and82.34±3.21%, respectively. The ERβ expression levels in each group were96.13±1.07%,85.13±2.17%, and74.33±3.07%, respectively. Our results demonstratedthat the ERα expression level was significantly reduced in cells in the M/T group(p<0.05) and was increased by Gefitinib treatment (p<0.05). ERβ expression levelswere slightly reduced in cells of the M/T group (p>0.05), a trend that was lesssignificant after Gefitinib treatment. These results suggest that the TAM resistance ofMCF-7cells was largely associated with the loss of ERα expression, but the correlationof TAM resistance and ERβ expression needs to be further investigated. Our westernblot results revealed that the expression levels of proteins in MAPK pathway, includingRas, MEK1/2, and p-ERK1/2, were significantly increased in the M/T group comparedwith those of the M0group (p<0.05). After the cells were treated with10μg/mlGefitinib for48h, the protein expression levels of Ras, MEK1/2, and p-ERK1/2weresignificantly down-regulated compared with the levels of the M/T group (p<0.05),which indicated that TAM resistance in MCF-7cells is largely mediated by the MAPKpathway. In addition, these data indicate that Gefitinib reverses the drug resistance ofMCF-7/TAM cells by re-sensitising the drug-resistant cells to TAM through themodulation of the expression of proteins involved in MAPK pathway.
Conclusion:1.Suspension sphere with cells can be incubated in stem cell cultureunder the cultivating condition of stem cells in vitro, with positive expression of ER anda lower sensitivity to tamoxifen, suggesting the enriched breast cancer cells may be oneof the reasons of endocrine resistance.
2.Breast cancer cell line MCF-7resistant to TAM can be established throughcultivation of stem cells. The resistance may be induced by the upregulated proteins inMAPK pathway resulting in loss of ERα.
3. either β-ELE or Gefitinib may block MCF-7cells in G0/G1,which enhances theeffect of TAM in inhibiting the proliferation and inducing apoptosis, showing asynergistic effect with TAM.
4.The ELE and Gefitinib-induced reversal of TAM resistance was mediated by theup-regulation of ERα mRNA and the re-expression of ERα through the MAPKpathway.
5. The MAPK pathway is critical for re-expression of ERα,which was achievedby changing the protain expression of the MAPK pathway.
方法：分别于常规培养及干细胞培养条件下（悬浮球培养）培养激素敏感的MCF-7细胞株，流式细胞仪检测分子表型CD44~+CD24~(–/low)与CD44~+CD24~+亚群细胞比例变化，免疫细胞化学法测定ERα和ERβ的表达变化，MTT法检测细胞对他莫西芬的敏感程度。培养乳腺癌MCF-7/TAM耐药细胞；分别应用TAM与β-ELE联合、TAM与Gefitinib联合作用于MCF-7细胞，设立序贯与混合联用方案，用MTT法检测不同用药顺序对MCF-7细胞的增殖抑制作用，计算二药联合作用指数(combine index，CI)，比较不同方案对细胞增殖抑制率的影响，并着重观察ELE和Gefitinib低剂量下TAM对MCF-7细胞的增殖抑制情况。流式细胞仪检测Gefitinib与ELE细胞毒性剂量对MCF-7细胞周期的影响。采用干细胞培养及传统低剂量诱导的方法构建出ERα阴性的乳腺癌TAM耐药细胞MCF-7/TAM，分别不同剂量和不同时间段β-ELE和Gefitinib处理MCF-7/TAM细胞，通过MTT法分析并确定β-ELE和Gefitinib对MCF-7/TAM和对MCF-7细胞的低剂量及最佳作用时间，据MTT结果用10ug/ml的β-ELE和Gefitinib处理MCF-7/TAM细胞48h，设立MCF-7组（M0），MCF-7/TAM组（M/T），MCF-7/TAM-ELE10ug/ml（E10），MCF-7/TAM-Gefitinib10ug/ml（G10）组进行后续试验，用MTT方法检测经处理后的E10与G10组细胞对TAM的敏感性，RT-PCR和免疫细胞化学观察ERα和ERβ在mRNA和蛋白水平的变化，并检测各组细胞MAPK通路RAS，MEK1/2、p-ERK1/2蛋白水平变化。实验结果应用SPSS16.0软件，计数资料ERα与ERβ的阳性表达采用卡方检验进行统计学分析，结果用百分率表示；凝胶电泳结果分析采用IMAGEJ与LabWorks4.6软件分析，计量资料确保符合正态分布，两组样本比较采用t检验，三组IC50值采用方差分析，结果以均数±标准差表示，P＜0.05为差异有统计学意义。
结果：干细胞培养条件下CD44~+CD24~(–/low)亚群细胞所占比例由常规培养的（0.27±0.08）%增至（1.60±0.08）%（p＜0.05），而CD44~+CD24~+亚群细胞比例由（5.59±0.88）%增至（30.63±4.40）%（p＜0.05）。干细胞培养条件培养下ERα和ERβ表达率较常规培养下调，分别由85.27%和90.53%降至69.43%和73.20%，差异有统计学意义（p均＜0.05），且对他莫西芬的敏感性降低，IC50值由(9.82±0.31)umol/L升至(16.46±0.50)umol/L，再次诱导分化后ER并未出现上调，对他莫西芬的敏感性仍旧降低（p＜0.05）。TAM与β-ELE序贯联用具有协同作用，序贯方法优于混合应用的叠加作用（p＜0.05）。TAM与Gefitinib联用不同顺序均具有协同作用，序贯联用方法优于混合应用（p＜0.05）。β-ELE5ug/ml及10ug/ml时ELE--TAM显示出优于TAM--ELE方案的趋势， Gefitinib5ug/ml及10ug/ml时Gefitinib--TAM显示出优于TAM--Gefitinib方案的趋势。流式细胞仪检测显示，β-ELE20ug/ml及40ug/ml作用于MCF-7细胞后，G0/G1期细胞比例由49.26%提高到64.04%和63.88%；Gefitinib10ug/ml及20ug/ml作用于MCF-7细胞后，G0/G1期细胞比例由49.26%提高到54.89%，68.35%。确定了β-ELE与Gefitinib与TAM的可联合性。后续试验发现，10μg/ml的β-ELE与Gefitinib作为干预剂量作用于M/T细胞48h后，细胞可重新对TAM敏感，细胞增殖受到抑制，实现耐药逆转。RT-PCR检测发现β-ELE治疗后可上调MCF-7/TAM细胞中ERα mRNA表达水平。与M0组相比，M/T组ERα mRNA与ERβ mRNA水平均见下调，以ERα mRNA下调最为显著(p＜0.05)。经β-ELE治疗后，E10组ERα mRNA明显上调(p＜0.05)，ERβ mRNA也在E10组出现上调(p＜0.05)。M0、M/T、E10各组ERα表达率分别为(95.04±1.81)%、(2.10±0.24)%、(82.34±3.21)%；各组ERβ表达率(96.13±1.07)%、(85.13±2.17)%、(74.33±3.07)%。ERα在M/T组细胞中的表达率显著下降p＜0.05)，在经过β-ELE治疗后表达率再次升高(p＜0.05)。而ERβ在M/T组细胞中的表达率仅有轻度下降(p＞0.05)，应用β-ELE治疗后表达率呈下降趋势。Western blot分析结果显示，M/T组MAPK通路蛋白Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M0组明显增强(p＜0.05)，经10μg/ml的β-ELE治疗48h后Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M/T组下调(p＜0.05)。在Gefitinib实验中，我们发现，Gefitinib上调MCF-7/TAM细胞中ERα mRNA和ERβ mRNA的水平，与M0组相比，M/T组ERα mRNA与ERβmRNA水平均见下调，以ERα mRNA下调最为显著(p＜0.05)。但是经Gefitinib治疗后，G10组ERα mRNA明显上调(p＜0.05)，ERβ mRNA也在G10组出现上调(p＜0.05)。免疫细胞化学显示，M0、M/T、G10各组ERα表达率分别为(95.04±1.81)%、(2.10±0.24)%、(75.04±2.88)%；各组ERβ表达率(96.13±1.07)%、(85.13±2.17)%、(90.25±2.15)%。ERα在M/T组细胞中的表达率显著下降(p＜0.05)，在经过Gefitinib治疗后表达率再次升高(p＜0.05)。而ERβ在M/T组细胞中的表达率仅有轻度下降(p＞0.05)，应用Gefitinib治疗后表达率呈上调趋势。Western blot分析结果显示，M/T组MAPK通路蛋白Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M0组明显增强(p＜0.05)，经10μg/ml的Gefitinib治疗48h后Ras、MEK1/2、p-ERK1/2蛋白表达水平均较M/T组下调(p＜0.05)。
结论：
1.干细胞培养条件下可培养出具备干细胞特性的细胞亚群的悬浮球，且ER为阳性表达，但对TAM治疗敏感性减低，提示乳腺癌干细胞的富集可能是内分泌耐药的原因之一。
2.利用干细胞培养方法可以构建乳腺癌MCF-7细胞的TAM耐药细胞。耐药的主要原因可能与MAPK通路蛋白表达上调，导致ERα缺失有关。
3. β-ELE与Gefitinib均可阻滞MCF-7细胞于G0/G1期，增强TAM的抑制细胞增殖作用，与TAM药物间有协同作用。
4. β-ELE和Gefitinib通过上调ERα mRNA水平，使ERα受体再表达，恢复对TAM的治疗敏感性，逆转MCF-7细胞对TAM耐药。
5. MAPK通路是ERα受体再表达的重要途径，β-ELE和Gefitinib可以通过下调MAPK通道蛋白表达而实现ERα受体再表达。
Objective: Endocrine therapy is an important approach in the treatment ofestrogen receptor (ER)-positive breast cancer. However, the ER status of ER-positivepatients may be lost during endocrine therapy, leading to the acquired drug resistance,the loss of ERα and the up-regulation of growth factor receptor pathways are the mostwidely accepted causes of drug resistance. Therefore, great attention has been paid inresearches on the acquired resistance in endocrine therapy, and great emphasis has beenmade on ER level and detailed signal transduction in researches on the mechanism ofdrug resistance. Resistance to endocrine therapy is regulated by both ER and the EGFRfamily signalling network. Upregulation of the mitogen-activated protein kinase(MAPK) signal causes ER-positive cells to lose ER expression and become ER-negative.In the comprehensive treatment of breast cancer, endocrine therapy, usually positionedsequentially post to chemotherapy and radiotherapy, is potentially accompanied with theenriched stem cells of breast cancer, resulting in resistance to endocrine drugs.Therefore it is critical to improve the efficacy of endocrine therapy, to postpone theonset of the resistance and to reverse it. β-Elemene (β-ELE) is an effective anti-cancercomponent extracted from turmeric, a member of the Zingiberaceae family, and it hasbeen used clinically to enhance radiosensitisation and to assist chemotherapeutictreatment in treating multiple types of tumours. ELE has been studied as an agentcapable of reversing resistance to chemotherapy, Further, research on the application ofELE in endocrine therapy is sparse. Gefitinib, a small-molecular tyrosine kinaseinhibitor, has mainly been used in molecular targeted treatment(6-8),and in recent yearsalso been used in the study of breast cancer treatment combined with endocrine therapy.Studies on the reversal of multidrug resistance to chemotherapy have demonstrated thatdrugs that reverse drug resistance can synergistically inhibit cancer when used in combination with chemotherapy In this respect we have made serried of laboratorydesigns. Firstly, we introduced the concept of stem cell of breast cancer throughsimulating the biology of recurrent human breast cancer, i.e. recurrence induced byproliferation and differentiation of a little amount of stem cells of breast cancer enrichedafter the comprehensive treatment. We cultivated a MCF-7line sensitive to endocrinetherapy in vitro through stem cell cultivation, tested ER expressed by suspended spherecells with stem cells features and their response to tamoxifen, and explored thecorrelation between resistance to endocrine therapy and cancer stem cells. We alsoestablished a cell line CMF-7/TAM resistant to breast cancer through stem cellcultivation and low-dose induction) to detect the altered ER in levels of both mRNAand protein, the variation in proteins of RAS、MEK1/2、p-ERK1/2in the relatedpathway of MAPK, to discuss the mechanism of resistance to TAM in MCF-7.Secondly, we defined the value of β-ELE and Gefitinib in combination with TAM in theendocrine therapy through intervening the proliferation of MCF-7with β-ELE andGefitinib combined with TAM in different sequences, to explore the best dosagesequence, and define the non-toxic dosage to MCF-7and MCF-7/TAM. Then we usedthe non-toxic dosage of β-ELE and Gefitinib as the intervening dosage to treatMCF-7/TAM cell resistant to TAM, which later recovered the response to TAM.Furthermore, we detected the variation of ERα and ERβ in mRNA and protein duringthe intervention, and the altered protein levels of RAS, MEK1/2, p-ERK1/2involved inMAPK pathway to analyze the effect ofβ-ELE and Gefitinib in reversed resistance toendocrine therapy and the mechanism.
Methods: The MCF-7cells were cultured in normal culture or in stem cell culturein vitro (suspension sphere). The proportion of CD44~+CD24-/lowphenotype and CD44~+CD24~+phenotype cells were determined using flow cytometry (FCM). The ERa andERβ expression was detected using immunohistochemical method. The susceptibility totamoxifen was determined using MTT essay. The data are processed with t-test,Chi-square test and analysis of variance,respectively.Hormone-sensitive breast cancerMCF-7cell was treated with Tamoxifen added β-elemeneversus TAM added Gefitinib. To establish groups of combination and sequential medication.The MTT test was used to detect the repressional effection to MCF-7cells with different drug orders, focusing on observing the inhibition of cell proliferation under non-toxic dose of β-ELE versus Gefitinib and the effect of both drugs on cell cycle which wastested by flow cytometric. To calculate the combine index (CI) and compare cell prolife ration inhibition rate of two groups. For our subsequent experiments, we used the method of stem cell culture in vitro and inducing with low dose to create a tamoxifen (TAM)-resistant ERα-negative breast cancer cell line, MCF7/TAM cells. After treating MCF7/TAM cells with ELE versus Gefitinib in different doses and different times, the best non-cytotoxic doses and duration of action were determined using MTT essay.We treated cells with10μg/ml β-ELE and Gefitinib for48h,and divide into MCF-7group(M0)MCF-7/TAM group(M/T),MCF-7/TAM-ELE10ug/ml group(E10)and MCF-7/TAM-Gefitinib10ug/ml group（G10). To investigate the mechanism by which ELE reversed the drugresistance of MCF-7/TAM cells, we used RT-PCR to analyse the mRNA levels of ERαand ERβ. ERα and ERβ expression levels determined by immunohistochemistry.Expression levels of proteins in the MAPK (RAS and MEK1/2and p-ERK1/2)pathwayas analyzed by western blotting. Results from our experiments were statisticallyanalyzed using SPSS16.0software. Count data of ERα and ERβ expressed positivelywas analyzed with Chi-square test and expressed in percentage. IMAGEJ andLabWorks4.6were used to test results of gel electrophoresis. The quantitative data wasproved to be in the normal distribution. T-test was used for comparison between twogroups and variance analysis for analysis among three groups. All results wereexpressed in mean±standard deviation, with P<0.05considered as the statisticalsignificance.
Results: In suspension culture as stem cells, the proportions of theCD44~+CD24~(–/low)CD44~+CD24~+phenotype cells were (1.60±0.08)%and(30.63±4.40)%respectively, significantly higher than (0.27±0.08)%and (5.59±0.88)%in normal medium (p<0.05). The positive expressions of ERα and ERβ were85.27%and90.53%in normal medium,69.43%and73.20%in suspension culture,respectively，and the differences were statistically significan（tp<0.05）. With regards totamoxifen sensitivity, IC50was （9.82±0.31） μmol/L in normal medium, and(16.46±0.50) μ mol/L in suspension culture. After the second induced differentiation,the positive expression of ER was not up-regulated and the susceptibility to tamoxifenremained low (p<0.05). TAM combined with β-ELE or Gefitinib in different orders allhave synergistic effect. Sequential application is better than superposition（p<0.05）.Under Non-toxic dose of β-ELE or Gefitinib（5ug/ml and10ug/ml）, priorityuse of β-ELE or Gefitinib is superior of TAM’s first use. Flow cytometric detected thatthe proportion of G0/G1phase in MCF-7cells,which were cultured with β-ELE in20ug/ml and40ug/ml dose density,increased from49.26%to64.04%and63.88%,and with gefitinib in10ug/ml and20ug/ml dose density,the proportion increased from49.26%to54.89%and68.35%. In the following research, response to TAM wasrecovered in cells with inhibited proliferation and reversed resistance, after treatment for48h with10μg/ml β-ELE and10μg/ml Gefitinib as the intervening dosage. Toinvestigate the mechanism by which ELE reversed the drug resistance of MCF-7/TAMcells, we used RT-PCR to analyse the mRNA levels of ERα and ERβ. Our resultsdemonstrated that the ERα and ERβ mRNA levels in the M/T group weredown-regulated compared with those of the M0group. The reduction in the level ofERα mRNA was significant (p<0.05). In addition, the ERα mRNA level in the E10group was significantly up-regulated after β-ELE treatment (p<0.05), and the ERβmRNA level was also increased after treatment in the E10group (p<0.05). The ERαexpression levels in the M0, M/T, and E10groups were95.04±1.81%,2.10±0.24%,and82.34±3.21%, respectively. The ERβ expression levels in each group were96.13±1.07%,85.13±2.17%, and74.33±3.07%, respectively. Our results demonstratedthat the ERα expression level was significantly reduced in cells in the M/T group(p<0.05) and was increased by ELE treatment (p<0.05). ERβ expression levels wereslightly reduced in cells of the M/T group (p＞0.05), a trend that was less significantafter ELE treatment. These results suggest that the TAM resistance of MCF-7cells waslargely associated with the loss of ERα expression, but the correlation of TAMresistance and ERβ expression needs to be further investigated. Our western blot resultsrevealed that the expression levels of proteins in MAPK pathway, including Ras,MEK1/2, and p-ERK1/2, were significantly increased in the M/T group compared withthose of the M0group (p<0.05). After the cells were treated with10μg/ml β-ELE for48h, the protein expression levels of Ras, MEK1/2, and p-ERK1/2were significantlydown-regulated compared with the levels of the M/T group (p<0.05), which indicatedthat TAM resistance in MCF-7cells is largely mediated by the MAPK pathway. Inaddition, these data indicate that β-ELE reverses the drug resistance of MCF-7/TAMcells by re-sensitising the drug-resistant cells to TAM through the modulation of theexpression of proteins involved in MAPK pathway.
To investigate the mechanism by which Gefitinib reversed the drug resistance ofMCF-7/TAM cells, we used RT-PCR to analyse the mRNA levels of ERα and ERβ. Ourresults demonstrated that the ERα and ERβ mRNA levels in the M/T group weredown-regulated compared with those of the M0group. The reduction in the level ofERα mRNA was significant (p<0.05). In addition, the ERα mRNA level in the E10 group was significantly up-regulated after Gefitinib treatment (p<0.05), and the ERβmRNA level was also increased after treatment in the E10group (p<0.05). The ERαexpression levels in the M0, M/T, and E10groups were95.04±1.81%,2.10±0.24%,and82.34±3.21%, respectively. The ERβ expression levels in each group were96.13±1.07%,85.13±2.17%, and74.33±3.07%, respectively. Our results demonstratedthat the ERα expression level was significantly reduced in cells in the M/T group(p<0.05) and was increased by Gefitinib treatment (p<0.05). ERβ expression levelswere slightly reduced in cells of the M/T group (p>0.05), a trend that was lesssignificant after Gefitinib treatment. These results suggest that the TAM resistance ofMCF-7cells was largely associated with the loss of ERα expression, but the correlationof TAM resistance and ERβ expression needs to be further investigated. Our westernblot results revealed that the expression levels of proteins in MAPK pathway, includingRas, MEK1/2, and p-ERK1/2, were significantly increased in the M/T group comparedwith those of the M0group (p<0.05). After the cells were treated with10μg/mlGefitinib for48h, the protein expression levels of Ras, MEK1/2, and p-ERK1/2weresignificantly down-regulated compared with the levels of the M/T group (p<0.05),which indicated that TAM resistance in MCF-7cells is largely mediated by the MAPKpathway. In addition, these data indicate that Gefitinib reverses the drug resistance ofMCF-7/TAM cells by re-sensitising the drug-resistant cells to TAM through themodulation of the expression of proteins involved in MAPK pathway.
Conclusion:1.Suspension sphere with cells can be incubated in stem cell cultureunder the cultivating condition of stem cells in vitro, with positive expression of ER anda lower sensitivity to tamoxifen, suggesting the enriched breast cancer cells may be oneof the reasons of endocrine resistance.
2.Breast cancer cell line MCF-7resistant to TAM can be established throughcultivation of stem cells. The resistance may be induced by the upregulated proteins inMAPK pathway resulting in loss of ERα.
3. either β-ELE or Gefitinib may block MCF-7cells in G0/G1,which enhances theeffect of TAM in inhibiting the proliferation and inducing apoptosis, showing asynergistic effect with TAM.
4.The ELE and Gefitinib-induced reversal of TAM resistance was mediated by theup-regulation of ERα mRNA and the re-expression of ERα through the MAPKpathway.
5. The MAPK pathway is critical for re-expression of ERα,which was achievedby changing the protain expression of the MAPK pathway.
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7. Fountzilas G, Pectasides D, Kalogera-Fountzila A, Skarlos D, Kalofonos HP, Papadimitriou C,Bafaloukos D, Lambropoulos S, Papadopoulos S, Kourea H, Markopoulos C, Linardou H,Mavroudis D, Briasoulis E, Pavlidis N, Razis E, Kosmidis P, Gogas H. Paclitaxel andcarboplatin as firstline chemotherapy combined with gefitinib (IRESSA) in patients withadvanced breast cancer: a phase I/II study conducted by the Hellenic Cooperative OncologyGroup, Breast cancer Res Treat2005Jul;92(1):1-9
8. Shou J,Massarweh S,Osborne CK, Wakeling AE, Ali S, Weiss H, Schiff R. Mechanisms oftamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positivebreast cancer, Natl Cancer Inst2004Jun;96(12):926-35
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10.李国权,谢冰冰,陈英海,李小龙,邹丽娟. β-榄香烯对人肺腺癌裸鼠移植瘤放疗增敏作用与HIF-1αCAⅨ的相关性研究,中国肿瘤临床2011;38(6):312-5
11.胡军,金伟,杨佩满. β-榄香烯逆转人乳腺癌MCF-7/ADM细胞对阿霉素耐药性的研究,中华肿瘤杂志2004;26(5):268-70
12.周小娟,郑棋,赵小丽,马海琳.榄香烯联合三苯氧胺对乳腺癌MCF-7细胞株的抑制效应,西安交通大学学报2007;28(1):74-7
13.马海琳,周小娟,刘娟. β-榄香烯联合三苯氧胺抑制乳腺癌MCF-7细胞生长的实验研究,现代肿瘤医学2008;16(4):510-4
14. Salami S,Karami-Tehrani F. Biochemical studies of apoptosis induced by tamoxifen in estrogenreceptor positive and negative breast cancer cell lines, Clin Biochem2003Jun;36(4):247-53
15. Christine Pratt MA,Niu M,White D. Differential regulation of protein expression，growth andapoptosis by natural and synthetic retinoids, Cell Biochem2003Nov;90(4):692-708.
16. Nakayama Y,Sakamoto H,Satoh K, Yamamoto T..Tamoxifen and gonadal steroids inhibit coloncancer growth in association with inhibition of thymidylate synthase，survivin and telomeraseexpression through estro-gen receptor beta mediated system, Cancer Lett2000Dec;161(1):63-71
17.唐发清,顾焕华,胡智,唐敏,翁新宪,易薇,曹亚. EB病毒潜伏膜蛋白1通过Survivin介导细胞增殖和抑制细胞凋亡,中国生物化学与分子生物学报2003;19(5):646-52
1. Johnston SR. Acquired tamoxifen resistance in human breast cancer-potential mechanisms andclinical implications, Anticancer Drugs1997Nov;8(10):911-30
2. Harvey JM, Clark GM, Osborne CK, AllredDC. Estrogen receptor status byimmunohistochemistry is superior to the ligand-binding assay for predicting response toadjuvant endocrine therapy in breast cancer, J Clin Oncol1999May;17(5):1474-81
3. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance,Nat Rev Cancer2002Fed;2(2):101-12
4. Gee JM, Harper ME, Hutcheson IR, Madden TA, Barrow D, Knowlden JM, McClelland RA,Jordan N, Wakeling AE, Nicholson RI. The antiepidermal growth factor receptor agent gefitinib(ZD1839/Iressa) improves antihormone response and prevents development of resistance inbreast cancer in vitro，Endo crino logy Nov;144(11):5105-17. Epub2003Aug7
5. Johnston SR. Enhancing the efficacy of hormonal agents with selected targeted agents, ClinBreast Cancer2009Jun;9Suppl1:S28-36
6. Zhang F, Xu L, Qu X, Zhao M, Jin B, Kang J, Liu Y, Hu X. Synergistic antitumor effect ofβ-elemene and etoposide is mediated via induction of cell apoptosis and cell cycle arrest innon-small cell lung carcinoma cells, Mol Med Report2011Nov-Dec;4(6):1189-93
7. Wang G, Li X, Huang F, Zhao J, Ding H, Cunningham C, Coad JE, Flynn DC, Reed E, Li QQ.Antitumor effect of beta-elemene in non-small-cell lung cancer cells is mediatedvia inductionof cell cyle arrest and apoptotic cell death, Cell MolLife Sci2005Apr;62(7-8):881-93.
8. Li G, Xie B, Li X, Chen Y, Wang Q, Xu Y, Xu-Welliver M, Zou L. Down-Regulation ofSurvivin and Hypoxia-Inducible Factor-1α by β-elemene Enhances the Radiosensitivity of LungAdenocarcinoma Xenograft, Cancer Biother Radiopharm2012Feb;27(1):56-64
9. Hu J, Jin W, Yang PM. Reversal of resistance to adriamycin in human breast cancer cell lineMCF-7/ADM by beta-elemene, Zhonghua Zhong Liu Za Zhi2004May;26(5):268-70
10. Ma HL, Zhou XJ,Liu J.Experimental study on β-elemene combined with tamoxifen in hibitingthe apoptosis of breast cancer cell strain MCF-7, Modern Oncology2008;16(4):510-4
11. Creighton CJ, Hilger AM, Murthy S, Rae JM, Chinnaiyan AM, El-Ashry D. Activation ofmitogen-activated protein kinase in estrogen receptor alpha-positive breast cancer cells in vitroinduces an in vivo molecular phenotype of estrogen receptor alpha-negative human breasttumors, Cancer Res2006Apr;66(7):3903-11
12. Oh AS, Lorant LA, Holloway JN, Miller DL, Kern FG, El-Ashry D. Hyperactivation of MAPKinduces loss of ERalpha expression in breast cancer cells, Mol Endocrinol2001Aug;15(8):1344-59
13. Bayliss J, Hilger A, Vishnu P, Diehl K, El-Ashry D. Reversal of the estrogen receptor negativephenotype in breast cancer and restoration of an tiestrogen response,2007Dec;13(23):7029-36
14. Santen RJ, Song RX, Zhang Z, Yue W, Kumar R. Adaptive hypersensitivity to estrogen:mechanism for sequential responses to hormonal therapy in breast cancer, Clin Cancer Res2004Jan;10(1Pt2):337S-45S
15. Pan GD, Yang JQ, Yan LN, Chu GP, Liu Q, Xiao Y, Yuan L.Reversal of multi-drug resistanceby pSUPER-shRNA-mdr1in vivo and in vitro, World J Gastroenterol2009Jan;15(4):431-440
16. Chen FP, Hsu T, Hu CH, Wang WD, Wang KC, Teng LF. Expression of estrogen receptors alfaand beta mRNA and alkaline phosphatase in the differentiation of osteoblasts from elderlypostmenopausal women: comparison with osteoblasts from osteosarcoma cell lines, Taiwan JObstet Gynecol2006Dec;45(4):307-12
17. Fu Z Y, Han J X, Zhang H Y. Effect s of emodin on gene expression profile in small cell lungcancer NCI-H446cell. Chin Med J2007;120(19):1710-5
18. Clark AS,West K,Streicher S, Dennis PA. Constitutive and inducible Akt activity promotesresistance to chemotherapy，trastuzumab，or tamoxifen in breast cancer cells, Mol Cancer Ther2002Jul;1(9):707-17
19. Song RX, McPherson RA, Adam L, Bao Y, Shupnik M, Kumar R, Santen RJ. Linkage of rap idestrogen action of MAPK activation by ERα-Shc association and Shc path-way activation, MolEndocrinol2002Jan;16(1):116-27
20. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F.Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex inMCF-7cells, EMBO J1996Mar;15(6):1292-300
21. Krueger JS, Keshamouni VG, Atanaskova N, Reddy KB. Temporal and quantitative regulationof mitogen-activated protein kinase (MAPK) modulates cell motility and invasion, Oncogene2001;20:4209-18.
22. Jelovac D, Sabnis G, Long BJ, Macedo L, Goloubeva OG, Brodie AM. Activation ofmitogen-activated protein kinase in xenografts and cells during prolonged treatment witharomatase inhibitor letrozole. Cancer Res2001Jul;20(31):4209-18
23. Martin LA, Farmer I, Johnston SR, Ali S, Dowsett M. Elevated ERK1/ERK2/estrogen receptorcross-talk enhances estrogen-mediated signaling during long-term estrogen deprivation, EndocrRelat Cancer2005Jul;12Suppl1:S75-84
24. Allred DC, M ohs in SK, Fuqua SA. Histological and biological evolution of humanpremalignant breast disease, Endocr Relat Cancer2001Mar;8(1):47-61
25. Yao YQ, Xu YH, Lu J, Zhou HY, Wang YZ. Effect of p38MAPK on elemene-induced cellcycle arrest in C6glioblastoma cells, Chinese Medical Journal2008Jan;88(1):56-8
26. Li L, Xu L, Qu X, Zhao M, Yu P, Kang J, Liu Y, Hu X.Cbl-regulated Akt and ERK signals areinvolved in β-elemene-induced cell apoptosis in lung cancer cells, Mol Med Report2011Nov-Dec;4(6):1243-6
27. Gruvberger-Saal SK, Bendahl PO, Saal LH, Laakso M, Hegardt C, Edén P, Peterson C,Malmstr m P, Isola J, Borg A, Fern M.Estrogen Receptor beta Expression Is Associated withTamoxifen Response in ERalpha-Negative Breast Carcinoma, Clin Cancer Res2007Apr;13(7):1987-94
28. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS. Differentialligand activation of estrogen receptors ERalpha and ERbeta at AP1sites, Science1997Sep;277(5331):1508-10
29. Hodges LC, Cook JD, Lobenhofer EK, Li L, Bennett L, Bushel PR, Aldaz CM, Afshari CA,Walker CL.Tamoxifen functions as a molecularagonist inducing cell cycle-associated genes inbreast cancer cells, Mol Cancer Res2003Feb;1(4):300-11
30. Str m A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen receptor βinhibits17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D, Proc NatlAcad Sci2004Feb10;101(6):1566-71
31. Borgquist S, Holm C, Stendahl M, Anagnostaki L, Landberg G, Jirstr m K.Oestrogen receptorsalpha and beta show different associations to clinicopathological parameters and theirco-expression might predict a better response to endocrine treatment in breast cancer, J ClinPathol2008Feb;61(2):197-203
32. Hopp TA, Weiss HL, Parra IS, Cui Y, Osborne CK, Fuqua SA.Low levels of estrogen receptorbeta protein predict resistance to tamoxifen therapy in breast cancer, Clin Cancer Res2004Nov15;10(22):7490-9
33. Iwao K, Miyoshi Y, Egawa C, Ikeda N, Tsukamoto F, Noguchi S. Quantitative analysis ofestrogen receptor-alpha and-beta messenger RNA expression in breast carcinoma by real-timepolymerase chain reaction, Cancer Cancer2000Oct;89(8):1732-8
34. Lapidus RG, Nass SJ, Butash KA, Parl FF, Weitzman SA, Graff JG, Herman JG, Davidson NE.Mapping of ER gene CpG island methylation-specific polymerase chain reaction, Cancer Res1998Jun;58(12):2515-9
35. Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB, Davidson NE. Methylation of theestrogen receptor gene CpG island marks loss of estrogen receptor expression in human breastcancer cells, Cancer Res1994May;54(10):2552-5
36. Zhao L, Wang L, Jin F, Ma W, Ren J, Wen X, He M, Sun M, Tang H, Wei M. Silencing ofestrogen receptor alpha(ERalpha) gene by promoter hypermethylation is a frequent event inChinese women with sporadic breast cancer, Breast Cancer Res Treat2009Sep;117(2):253-9.Epub2008Sep24
37. Kawai H, Li H, Avraham S, Jiang S, Avraham HK. Overexpression of histone deacetylaseHDAC1modulates breast cancer progression by negative regulation of estrogen receptor alpha,Int J Cancer2003Nov;107(3):353-8
38. Fan J, Yin WJ, Lu JS, Wang L, Wu J, Wu FY, Di GH, Shen ZZ, Shao ZM.ER alpha negativebreast cancer cells restore response to endocrine therapy by combination treatment with bothHDAC inhibitor and DNMT inhibitor, J Cancer Res Clin Oncol2008Aug;134(8):883-90
39. El-Osta A, Wolffe AP. DNA methylation and histone deacetylation in the control of geneexpression: basic biochemistry to human development and disease, Gene Expr2000;9(1-2):63-75
40. Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T. The methyl-CpG-binding proteinMeCP2links DNA methylation to histone methylation, J Biol Chem2003Feb7;278(6):4035-40. Epub2002Nov9
1. Johnston SR. Acquired tamoxifen resistance in human breast cancer-potential mechanisms andclinical implications, Anticancer Drugs1997Nov;8(10):911-30
2. Harvey JM, Clark GM, Osborne CK, AllredDC. Estrogen receptor status byimmunohistochemistry is superior to the ligand-binding assay for predicting response toadjuvant endocrine therapy in breast cancer, J Clin Oncol1999May;17(5):1474-81
3. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance,Nat Rev Cancer2002Fed;2(2):101-12
4. Gee JM, Harper ME, Hutcheson IR, Madden TA, Barrow D, Knowlden JM, McClelland RA,Jordan N, Wakeling AE, Nicholson RI. The antiepidermal growth factor receptor agent gefitinib(ZD1839/Iressa) improves antihormone response and prevents development of resistance inbreast cancer in vitro，Endo crino logy Nov;144(11):5105-17. Epub2003Aug7
5. Johnston SR. Enhancing the efficacy of hormonal agents with selected targeted agents, ClinBreast Cancer2009Jun;9Suppl1:S28-36
6. Janmaat ML, Rodriguez JA, Gallegos-Ruiz M, Kruyt FA, Giaccone G. Enhanced cytotoxicityinduced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3kinasepathways in non-small cell lung cancer cells, Int J Cancer2006Jan1;118(1):209-14
7. Huang YS, Huang B, Wu YL. Manifestation of leukoencephalopathy in a patient with advancednon-small cell lung cancer following treatment with gefitinib, Chin Med J (Engl)2011Nov;124(22):3834-7
8. Park HS, Lee HJ, Im JG, Goo JM, Lee CH, Park CM, Chun EJ. Gefitinib-induced pneumonitisin non-small cell lung cancer: radiological and clinical findings in five patients, Clin Imaging2007Sep-Oct;31(5):306-12.
9. Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H, Schiff R. Mechanisms oftamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positivebreast cancer, Natl Cancer Inst2004Jun16;96(12):926-35.
10. Osborne CK, Neven P, Dirix LY, Mackey JR, Robert J, Underhill C, Schiff R, Gutierrez C,Migliaccio I, Anagnostou VK, Rimm DL, Magill P, Sellers M. Gefitinib or placebo incombination with tamoxifen in patients with hormone receptor-positive metastatic breast cancer:a randomized phase II study,2011Mar1;17(5):1147-59. Epub2011Jan10
11. Creighton CJ, Hilger AM, Murthy S, Rae JM, Chinnaiyan AM, El-Ashry D. Activation ofmitogen-activated protein kinase in estrogen receptor alpha-positive breast cancer cells in vitroinduces an in vivo molecular phenotype of estrogen receptor alpha-negative human breasttumors, Cancer Res2006Apr;66(7):3903-11
12. Oh AS, Lorant LA, Holloway JN, Miller DL, Kern FG, El-Ashry D. Hyperactivation of MAPKinduces loss of ERalpha expression in breast cancer cells, Mol Endocrinol2001Aug;15(8):1344-59
13. Bayliss J, Hilger A, Vishnu P, Diehl K, El-Ashry D. Reversal of the estrogen receptor negativephenotype in breast cancer and restoration of an tiestrogen response,2007Dec;13(23):7029-36
14. Santen RJ, Song RX, Zhang Z, Yue W, Kumar R. Adaptive hypersensitivity to estrogen:mechanism for sequential responses to hormonal therapy in breast cancer, Clin Cancer Res2004Jan;10(1Pt2):337S-45S
15. Pan GD, Yang JQ, Yan LN, Chu GP, Liu Q, Xiao Y, Yuan L.Reversal of multi-drug resistanceby pSUPER-shRNA-mdr1in vivo and in vitro, World J Gastroenterol2009Jan;15(4):431-440
16. Chen FP, Hsu T, Hu CH, Wang WD, Wang KC, Teng LF. Expression of estrogen receptors alfaand beta mRNA and alkaline phosphatase in the differentiation of osteoblasts from elderlypostmenopausal women: comparison with osteoblasts from osteosarcoma cell lines, Taiwan JObstet Gynecol2006Dec;45(4):307-12
17. Fu Z Y, Han J X, Zhang H Y. Effect s of emodin on gene expression profile in small cell lungcancer NCI-H446cell. Chin Med J2007;120(19):1710-5
18. Clark AS,West K,Streicher S, Dennis PA. Constitutive and inducible Akt activity promotesresistance to chemotherapy，trastuzumab，or tamoxifen in breast cancer cells, Mol Cancer Ther2002Jul;1(9):707-17
19. Song RX, McPherson RA, Adam L, Bao Y, Shupnik M, Kumar R, Santen RJ. Linkage of rap idestrogen action of MAPK activation by ERα-Shc association and Shc path-way activation, MolEndocrinol2002Jan;16(1):116-27
20. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F.Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex inMCF-7cells, EMBO J1996Mar;15(6):1292-300
21. Krueger JS, Keshamouni VG, Atanaskova N, Reddy KB. Temporal and quantitative regulationof mitogen-activated protein kinase (MAPK) modulates cell motility and invasion, Oncogene2001;20:4209-18.
22. Jelovac D, Sabnis G, Long BJ, Macedo L, Goloubeva OG, Brodie AM. Activation ofmitogen-activated protein kinase in xenografts and cells during prolonged treatment witharomatase inhibitor letrozole. Cancer Res2001Jul;20(31):4209-18
23. Martin LA, Farmer I, Johnston SR, Ali S, Dowsett M. Elevated ERK1/ERK2/estrogen receptorcross-talk enhances estrogen-mediated signaling during long-term estrogen deprivation, EndocrRelat Cancer2005Jul;12Suppl1:S75-84
24. Allred DC, M ohs in SK, Fuqua SA. Histological and biological evolution of humanpremalignant breast disease, Endocr Relat Cancer2001Mar;8(1):47-61
25. Normanno N, De Luca A, Maiello MR, Campiglio M, Napolitano M, Mancino M, CarotenutoA, Viglietto G, Menard S. The MEK/MAPK pathway is involved in the resistance of breastcancer cells to the EGFR tyrosine kinase inhibitor gefitinib,2006May;207(2):420-7
26. Matsuo M, Sakurai H, Ueno Y, Ohtani O, Saiki I. Activation of MEK/ERK and PI3K/Aktpathways by fibronectin requires integrin alphav-mediated ADAM activity in hepatocellularcarcinoma: a novel functional target for gefitinib, Cancer Sci,2006Feb;97(2):155-62
27. Gruvberger-Saal SK, Bendahl PO, Saal LH, Laakso M, Hegardt C, Edén P, Peterson C,Malmstr m P, Isola J, Borg A, Fern M.Estrogen Receptor beta Expression Is Associated withTamoxifen Response in ERalpha-Negative Breast Carcinoma, Clin Cancer Res2007Apr;13(7):1987-94
28. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS. Differentialligand activation of estrogen receptors ERalpha and ERbeta at AP1sites, Science1997Sep;277(5331):1508-10
29. Hodges LC, Cook JD, Lobenhofer EK, Li L, Bennett L, Bushel PR, Aldaz CM, Afshari CA,Walker CL.Tamoxifen functions as a molecularagonist inducing cell cycle-associated genes inbreast cancer cells, Mol Cancer Res2003Feb;1(4):300-11
30. Str m A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen receptor βinhibits17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D, Proc NatlAcad Sci2004Feb10;101(6):1566-71
31. Borgquist S, Holm C, Stendahl M, Anagnostaki L, Landberg G, Jirstr m K.Oestrogen receptorsalpha and beta show different associations to clinicopathological parameters and theirco-expression might predict a better response to endocrine treatment in breast cancer, J ClinPathol2008Feb;61(2):197-203
32. Hopp TA, Weiss HL, Parra IS, Cui Y, Osborne CK, Fuqua SA.Low levels of estrogen receptorbeta protein predict resistance to tamoxifen therapy in breast cancer, Clin Cancer Res2004Nov15;10(22):7490-9
33. Iwao K, Miyoshi Y, Egawa C, Ikeda N, Tsukamoto F, Noguchi S. Quantitative analysis ofestrogen receptor-alpha and-beta messenger RNA expression in breast carcinoma by real-timepolymerase chain reaction, Cancer Cancer2000Oct;89(8):1732-8
34. Lapidus RG, Nass SJ, Butash KA, Parl FF, Weitzman SA, Graff JG, Herman JG, Davidson NE.Mapping of ER gene CpG island methylation-specific polymerase chain reaction, Cancer Res1998Jun;58(12):2515-9
35. Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB, Davidson NE. Methylation of theestrogen receptor gene CpG island marks loss of estrogen receptor expression in human breastcancer cells, Cancer Res1994May;54(10):2552-5
36. Zhao L, Wang L, Jin F, Ma W, Ren J, Wen X, He M, Sun M, Tang H, Wei M. Silencing ofestrogen receptor alpha(ERalpha) gene by promoter hypermethylation is a frequent event inChinese women with sporadic breast cancer, Breast Cancer Res Treat2009Sep;117(2):253-9.Epub2008Sep24
37. Kawai H, Li H, Avraham S, Jiang S, Avraham HK. Overexpression of histone deacetylaseHDAC1modulates breast cancer progression by negative regulation of estrogen receptor alpha,Int J Cancer2003Nov;107(3):353-8
38. Fan J, Yin WJ, Lu JS, Wang L, Wu J, Wu FY, Di GH, Shen ZZ, Shao ZM.ER alpha negativebreast cancer cells restore response to endocrine therapy by combination treatment with bothHDAC inhibitor and DNMT inhibitor, J Cancer Res Clin Oncol2008Aug;134(8):883-90
39. El-Osta A, Wolffe AP. DNA methylation and histone deacetylation in the control of geneexpression: basic biochemistry to human development and disease, Gene Expr2000;9(1-2):63-75
40. Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T. The methyl-CpG-binding proteinMeCP2links DNA methylation to histone methylation, J Biol Chem2003Feb7;278(6):4035-40. Epub2002Nov9
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