| Objective: Breast cancer is a heterogeneous disease that encompasses several distinct entities with remarkably different biological characteristics and clinical behavior, and threatened to female public health. The incidence of breast cancer is increasing year by year. Triple-negative breast cancer, namely TNBC, is characterized by estrogen receptor negative(ER), progesterone receptor negative(PR), and human epidermal growth factor receptor-2(HER-2) negative using histochemical staining. TNBC have the features of postoperative recur, easy to distant metastasis and poor prognosis, endocrine therapy and therapy targeted HER2 invalid. However, there is still absence of suitable treatment for this subtype tumor. In recent years, cancer stem cells(CSC) hypothesis is proposed which holds that those cells are the source of cancer recrudesce and metastasis. Therefore, the chemotherapeutic drugs on CSC of TNBC are critically important, and its may determines the success or failure of the TNBC treatment. Currently most studies show CD44+CD24-/low phenotypic cells are breast cancer stem-like cells. The aim of this study is to observe the effects of six chemotherapeutic drugs commonly used in clinical on the proliferation of human triple-negative breast cancer cell line MDA-MB-231 in vitro, and to know whether these drugs has the ability to arise the percentage of CD44+CD24-/low phenotype cells in MDA-MB-231. At the same time, we detect the CD44+CD24-/low cells content changes in the blood of 10 cases metastasis triple-negative breast cancer patients before and after chemotherapy, in order to provides the basis of the TNBC drug treatment. Methods:1 MDA-MB-231 cell line were maintained in vitro, using MTT assay to investigate the inhibitive effect rate among different chemotherapy drugs, as follows: paclitaxel(PTX), docetaxel(DOX), 5-fluorouracil(5-FU), epirubicin (EPI), vinorelbine(NVB), gemcitabine (GEM). 0.2,1 and 5 concentration of the drug peak plasma concentration (PPC), were stimulated 24h, 48h and 72h.2 The cells were randomly divided into control group (cultured under normal conditions) and six experimental groups of chemotherapy drugs, each group also set up 10 bottles of cells in culture. The working concentration of the six drugs are PPC acting on the MDA-MB-231,after 48 hours, morphological changes were observed and 5 bottles of each group were detected by flow cytometry. The other 5 bottles of cells remove the drugs after acting 48 hours and culture in normal conditions continued to recover which were observed the number of adherent cells seized about 80% of total bottom area, then detected the CD44+CD24-/low cells proportion by flow cytometry.3 Flow cytometry(FCM) was employed to investigate the CD44+CD24-/low cells content changes in circulating blood of 10 cases metastasis triple-negative breast cancer patients before and after chemotherapy. The other blood samples collected from 10 healthy volunteers as normal control group for comparison.Results:1 MTT results showed that: compare with the control group, PTX,DOX, EPI,5-FU,NVB,GEM at concentrations of 0.2,1 and 5 PPC were used to treat MDA-MB-231 cell line for 24h,48h and 72h,all significant inhibit the proliferation of MDA-MB-231 cells(P<0.05),and along with the increase of drug concentrations, inhibition rate also increases. At PPC 1 times 48 hours, the PTX inhibition is most obvious.2 Flow cytometry results showed that: the percentages of CD44+CD24-/low cells in control group was: 86.46%±4.72%. The role of drugs in 1 PPC treat 48h, the percentages of CD44+CD24-/low cells in each experimental group were: NVB(91.34%±4.17%), GEM(85.43%±2.76%), which no significant difference compared with the control group(P>0.05), DOX(74.78%±3.98%), PTX(71.67%±1.87%), EPI (66.38%±3.06%), 5-FU(42.41%±5.43%), which reduced significantly compared with the control group(P<0.05). The experimental group of 5-FU was the most obvious. Removed the drugs after acting 48 hours and cultured in normal conditions to recover, flow cytometry results showed that: the percentages of CD44+CD24-/low cells in experimental group were : NVB(86.46%±2.58%), GEM(85.97%±3.80%), which no significant difference compared with the control group(P>0.05), DOX(75.23%±2.91%), PTX(69.21%±5.55%), which reduced significantly compared with the control group(P<0.05). EPI and 5-FU group did not resume normal growth (repeat training 3 times).3 10 patients after second cycle effect assessment are stable disease (SD, Tumor Reduced in different degree). In the blood samples of 10 breast cancer patients before chemotherapy, the content of CD44+CD24-/low cells is between 54 and 793/100ul, the median is 160/100ul. In the 10 blood samples after chemotherapy, the content of CD44+CD24-/low cells is between 13 and 198/ul, the median is 48/100ul. In the blood samples of 10 volunteers, the content of CD44+CD24-/low cells is between 13 and 41/100ul, the median is 28/100ul.Statistics show that: between the volunteers team and breast cancer team,breast cancer before and after chemotherapy team,the difference of CD44+CD24-/low cells content have statistical significance (P<0.05).Conclusions:1 PTX,DOX,EPI,5-FU,NVB,GEM at concentrations of 0.2,1 and 5 PPC were used to treat MDA-MB-231 cell line for 24h,48h and 72h,all significant inhibit the proliferation of MDA-MB-231 cells,and along with the increase of drug concentrations, inhibition rate also increases. At PPC 1 times 48 hours, the group of PTX inhibition rate is most obvious.2 This study suggests that CD44+CD24-/low subpopulation as the breast cancer stem-like cells in the breast cancer MDA-MB-231 cell lines are not all generate resistance to all the chemotherapy drugs. The chemotherapeutic drugs of PTX, DOX, EPI and 5-FU all reduced its content, and the most obvious function is 5-FU.3 The content of CD44+CD24-/low breast cancer stem-like cells in volunteers is less than triple-negative breast cancer patients, the chemotherapy could reduce the content of CD44+CD24-/low breast cancer stem cells in the blood. There is a correlation between the proportion of breast cancer stem-like cells in the blood and the tumor load of the patients, it will possible become a valuable therapeutic reaction index in the future.
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