An Investigation On Resistance To Tamoxifen Reversed By Elemene And Gefitinib In MCF-7Cells

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^–/lowCD44⁺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.