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Objective: Breast cancer is common female malignancy; the globalepidemiological studies have shown that in female malignant tumors, breast cancer ranks first in incidence and mortality. According to cancer stem cell hypothesis, cancer stem cells are the source of tumor recurrence and metastasis. Currently, more and more experiments confirmed that CD44+CD24-/low phenotype is a subset of breast cancer stem cells. Endocrine therapy is an essential hormone receptor-positive breast cancer treatment means. This study investigated the impact of the estradiol （ E2）, tamoxifen （TAM） and advanced breast cancer chemotherapy drugs commonly used in estrogen receptor positive to human breast cancer cell strainMCF-7 cell subsets in the CD44+CD24-/low, supply references in clinical ER+ breast cancer drug usage. Meanwhile, the thesis investigates the relativity between estrogen receptor-positive advanced breast cancer in peripheral blood CD45-CD44+CD24-/low cell subsets and clinical treatment topredict the efficacy of chemotherapy in advanced breast cancer patients.Methods:1 we cultured vitro ER+ human breast cancer cell strain MCF-7, selected the logarithmic growing cells to make test. Experiments were divided into control group, E2 group and E2+TAM group. The control group were added the same dose of drug solvent （ethanol） as experimental group, E2 group were joined the estrogen of 10-7mol/L, 10-8mol/L, 10-9mol/L of E2 respectively. Based on the three concentrations of E2 group, the E2+TAM group were added 10-6mol / L of TAM. After 10-days and 20-days culture, each was detected the proportion of cell subsets in the CD44+CD24-/low by flow cytometry.2 Selected the DMEM culture medium in estrogen with a final concentration of 10-8mol / L, after 10-days culture, the logarithmic growing cells MCF-7 were used for experiment. Advanced breast cancer chemotherapy common medicine: docetaxel （DTX）, paclitaxel （PTX）, doxorubicin （EPI）, fluorouracil （5-Fu） were the experimental drug. Applied tetrzolium-based colorimetric assay （MTT）, and determined inhibition ratio at different concentrations in every drug, chose concentration with inhibition ratio 50% （IC50） in every drug as the work concentration for experiment. Experiments were divided into control group, 24 hours group and 20 days group. In the control group, the same dose of the drug solvent was joined. The drugs with work concentration were added into 24 hours group and 20 days respectively. 24 hours group were detected the proportion of cell subsets in the CD44+CD24-/low by flow cytometry after 24 hours;20 day group stopped drug action after 24 hours, continued the culture for 19 days and treat the group as 24-hour group.3 Collected peripheral venous blood 5ml of estrogen receptor-positive recurrence and metastasis breast cancer patient in the day before the first cycle of chemotherapy and the day before the third cycle of chemotherapy. The proportion of cell subsets of CD45-CD44+CD24-/low in each group was deleted by flow cytometry to analyze the relativity with clinical treatment and predict the healing efficacy of chemotherapy.Results:1 The proportion of CD44+CD24-/low subsets in human breast cancer cell strain MCF-7 cells is （0.59±0.39） %. When E2 group with different concentration cultured for 10 days, the proportions of CD44+CD24-/low subsets in MCF-7 cells were （8.16±0.31） %, （9.61±0.66） %, and （7.10±0.21） %. Compared with the control group, the data were higher （P <0.05）. After 10 days culture, in E2 + TAM group the proportions of CD44+CD24-/low cell subsets were （5.73±0.59） %, （8.12±0.33） %, （6.11±0.44） %. Compared with the corresponding concentration of E2 group, data were decreased （P <0.05）. After 20 days culture, in E2 group with different concentration the proportions of CD44+CD24-/low subsets in the MCF-7 cells were （8.46±0.49） %, （9.66±0.50） %, （7.60±0.64） %, higher than the control Group （P <0.05）. After 20 days culture, in E2 + TAM group the proportions of CD44+CD24-/low cell subsets were（5.73±0.70）%, （7.73±0.64）%, （5.96±0.49）%, compared with the corresponding concentration of E2 group, the proportions of CD44+ CD24-/low cell subsets were all decreased （P <0.05）.2 MTT results: in a certain range of concentration, after 24 hours cell action with different concentrations of DTX, PTX, 5-Fu, EPI on MCF-7 cells, all drugs showed a dose- effect dependency relationship, among them, DTX, 5-Fu, EPI showed a linear relation, PTX showed a logarithmical relation. To ensure equal treatment factors on MCF-7 cells with all drugs in experiments, we selected the IC50 concentration of all drugs as the work concentration. Obtained related analysis to work out equations between drug concentration and inhibition ratio, and calculated data of each drug with IC50, DXT: 3.22umol/mL, PTX: 1.89umol/mL, 5-Fu: 55.46umol/mL, EPI: 0.827umol/mL. Made action with 10-8mol / L of E2 on MCF-7 cells for 10 days and 30 days, the proportions of CD44 + CD24-/low cell subsets worked as a comparison. After 24 hours, proportions of CD44+CD24-/low cell subsets in each drug groups （DTX, PTX ,5-Fu, EPI） were: （12.21±1.10）%, （23.42±2.76）%, （54.25±1.91）%, （41.37±1.57）%, were higher than control group （P <0.05）. The most obvious increase is the proportion of CD44+CD24-/low cell subsets in the MCF-7 caused by 5-FU, the minimum is the proportion of CD44+CD24-/low cell subsets in the MCF-7caused by DTX. After 20 days, the proportion of CD44+CD24-/low cell subsets respectively （DTX, PTX ,5-Fu, EPI）were: （8.68±0.90）% ,（8.08±0.82）%, （15.30±1.06） %, （14.39±1.45）%,compared with the control group, DOX, PTX decrease, but the difference was not significant statistically （P> 0.05）; 5-Fu, EPI increased compared with the control group （P <0.05）.3 The proportion of CD45-CD44+CD24-/low cell subset in the healthy volunteers: （1.80±0.10） %, 20 patients’proportion of CD45-CD44+CD24-/low cell subset of before chemotherapy: （6.49±1.77） %, higher than healthy volunteers obviously （P <0.05）. In 20 patients, 11 patients were stable disease （SD） and 7 patients were partial remission （PR） and 2 patients were disease progression （PD）. Proportion of CD45-CD44 + CD24-/low cell subsets in the peripheral blood of PR and SD patients decreased in different degree, proportion of CD45-CD44+CD24-/low cell subsets of two PD patients increased, definite conclusions could not work out as the few number of cases.Conclusions:1 Different concentrations of estrogen can make increase, in the action of same concentration of estrogen, adding tamoxifen can reduce the proportion of CD44+CD24-/low cell subsets in the MCF-7.2 The CD44+CD24-/low cell subsets in MCF-7 can resist docetaxel, paclitaxel, fluorouracil and adriamycin. The affect of docetaxel and paclitaxel on the the proportion of CD44+CD24-/low cell subsets in MCF-7 is not obvious, yet fluorouracil and adriamycin can increase the proportion CD44+CD24-/low cell subsets markedly, and as a result docetaxel and paclitaxel are the choice drugs in ER+ breast cancer chemotherapy.3 The proportion of CD45-CD44+CD24-/low cells in peripheral blood of estrogen receptor-positive advanced breast cancer is distinctly higher than healthy volunteers. The proportion of CD45-CD44+CD24-/low cells in peripheral blood of chemotherapy effective patients reduces, the proportion change of CD45-CD44+CD24-/low cells in peripheral blood of chemotherapy noneffective patients and clinical relativity relevance need further study.
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