| Background:Breast cancer is one of the most common malignancies in women worldwhile, and its incidence rate is still rising. In developped countries such as America, the incidence rate of breast cancer occupies the first place of the malignancies in women, accounting for 30-32 percent. The same situation happens in some developped cities in China. In that case, breast cancer has been a serious disease threatening the physical and mental health of women all over the world.Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in many chronic inflammatory diseases. Recent studies have shown that IL-6 plays a primary role in the pathophysiology of cancer. In breast cancer, tumor tissue exhibits high expression levels of IL-6 compared with matched normal breast tissue samples, which also correlate with more advanced tumor grades. Furthermore, elevated serum IL-6 levels correlate with advanced breast tumor stage, increased number of metastatic sites, and poor survival in patients with breast cancer.Cancer stem-like cells (CSCs) are a highly tumorigenic cell type. CSCs exist as a small subset within tumors and are hypothesized to be critical initiators of cancers, as well as sustaining tumor growth and producing metastases. They also mediate resistance to conventional anti-tumor therapies. Breast cancer is the first human tumor for which a putative CSC subpopulation has been isolated as CD44+CD24-/low cells. These breast CSCs (BrCSCs) exhibit high tumorigenicity when injected into immunocompromized mice, and possess characteristics that are associated with normal stem cells; specifically, they have the ability to give rise to all cell types found in a particular cancer sample.In view of the important role of IL-6 in the malignant features of cancer, we were interested to explore the relationship between IL-6 and BrCSCs. Recent studies have indicated that IL-6 is capable of inducing an epithelial-mesenchymal transition (EMT) phenotype in human breast cancer cells. The induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the generation of cells with stem cell properties. We hypothesized that IL-6 promotes the generation of cancer cells with stem-like properties by induction of an EMT.Purpose;1) To explore the role of IL-6 in the induction of epithelial-mesenchymal-transition of breast cancer MCF-7 cells.2) To explore the potential contribution of EMT to the generation of breast CD44+CD24-/low cells.3) To evaluate the proliferation ability, doxorubicin resistance and radiation radioresistance of the cells generated by IL6-induced epithelial-mesenchymal-transition.Methods:1) Cell cultureHuman breast cancer cell line MCF7 cells was cultured in EMEM medium containing 10% FBS. IL-6 (Peprotech, Rocky Hill, NJ, USA) was added to all cultures at final concentrations of 50 ng/ml in EMEM containing 10% FBS.2) Flow cytometric analysisCells were trypsinized, suspended into single-cell mixtures, washed with phosphate buffered saline (PBS), and incubated on ice for 30 min with monoclonal antibodies specific for human cell surface markers CD44-FITC (eBioscience, San Diego, CA, USA) or CD24-PE (eBioscience). In negative control experiments, cells were incubated with fluorescence-labeled isotype-matched pre-immune IgG instead. Cells were washed and analyzed using a flow cytometer (BD FACS Aria, San Jose, CA, USA).3) Real time RT-PCRCells were harvested, and RNA was extracted using Trizol (Invitrogen) following the manufacturer’s protocol. One microgram of total RNA was reverse transcribed into cDNA using the Superscript First-Strand Synthesis System (Invitrogen). Real-time polymerase chain reactions (PCRs) using the SYBR Green PCR Master Mix were performed using an ABI PRISM 7500 Sequence Detection System (Perkin-Elmer/Applied Biosystems, Rotkreuz, Switzerland). Data are shown after normalization to 18S expression.4) Western blotPrimary antibodies included mouse anti-E-cadherin (1:5,000; BD Biosciences), mouse anti-vimentin (1:500; Clone V9, Dako, Glostrup, Denmark), and rabbit anti-CD44 (1:5,000; GeneTex Inc., Irvine, CA, USA). Secondary antibodies included rabbit anti-mouse IgG-HRP (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-rabbit IgG-HRP (1:1,000; GE Healthcare, Chalfont St Giles, Uk). HRP-conjugated monoclonal mouse anti-GAPDH (kangchen, Shanghai, China) was used as an internal parameter. All antibodies were diluted with 5% milk in PBS containing 0.1% Tween-20 (PBS-T) and incubated for either 1 h at room temperature or overnight at 4℃. All Western blots were visualized with ECL Western blotting substrate (Pierce, Rockford, IL, USA).5) Assessment of proliferation and doxorubicin resistanceCell proliferation potential and the relative resistance to doxorubicin were evaluated by cell proliferation assays using a Cell Counting Kit-8 (CCK8, Tiyuan Biotechnologies, Guangzhou, China). Cells were plated at a concentration of 1×103 cells per well (for growth advantage assays) or 1×104 cells per well (for doxorubicin resistance) into 96-well culture plates. For the cell proliferation potential assay,10 μl CCK-8 solution was added on days 1,2,3,4, and 5. For the doxorubicin resistance assay,10 μl of CCK-8 solution was added to each well of the plate 4 h, and 1,2,3,4, and 5 days after treatment with doxorubicin at a final concentration of 10 μg/mL. After the addition of CCK-8 solution, plates were incubated for 4 h, and absorbance was then measured at 450 nm using a microplate reader (SpectraMax M5, Sunnyvale, CA, USA).6) Irradiation and clonogenic assayCells were dissociated by trypsinization and mechanical agitation with a Pasteur pipette into single cell suspensions in RPMI 1640 medium supplemented with 10% FBS. The cells were seeded in 6-well plates at the indicated densities, and then incubated overnight before the irradiation treatment. Cells were irradiated from a vertical direction at a dose rate of 400 cGy/min with 6-MV X-rays produced by a Varian 2100C linear accelerator at the Southern Medical University. Cells were irradiated for the time required to receive a total dose of 0,2,4,6, or 8 Gy. Negative control cells were sham-irradiated. Following the irradiation, the cells were incubated for 15 days at 37℃ in a 5% CO2 environment to allow the formation of colonies. The resulting colonies were fixed with 100% ethanol and stained with 1% crystal violet. Colonies containing>50 cells were counted as clonogenic survivors. Two independent experiments were performed, each in triplicate. Plating efficiency (PE)= colonies observed/number of cells plated, Surviving fraction (SF)= colonies counted/[cells seeded x (PE/100). Using GraphPad Prism 5 software (GraphPad, La Jolla, CA, USA), the data were fitted into the following single-hit multitarget formula: S=1(1-e-D/D0)N.7) Statistical AnalysisIn all experiments, differences among groups were analyzed by variance (ANOVA) method or Student’s t test using SPSS (13.0). Bonferroni method was used for multiple comparisons. A probability level of 0.05 was chosen for statistical significance.Results:1) IL-6 induced epithelial-mesenchymal transition of breast cancer MCF7 cellsMorphological changes from a cobblestone to a spindle-like morphology, a classical marker of EMT induction, were seen 5 days after IL-6 exposure. Quantitative real-time PCR analysis showed a gene expression pattern that was consistent with EMT, including E-cadherin repression and the concomitant induction of vimentin and N-cadherin. Western blot analyses also showed the down-regulation of E-cadherin expression and the induction of vimentin.2) IL-6 culture increased the percentage of CD44+CD24-/low cells in breast cancer MCF7 cellsHuman breast cancer MCF7 cells were cultured in EMEM medium containing 10% FBS. MCF7 cells contain a very low proportion of CD44+CD24-/low cells. After 5 days of exposure to 50 ng/mL IL-6, the number of CD44+CD24-/low cells had significantly increased (P=0.000,t=-14.69), as shown by the FACS analysis.3) IL-6 enhanced radioresistance of MCF7 cellsThe MCF7 cells treated by IL-6 exhibited increased resistance to radiation compared with untreated-control cells (control:SF2Gy=0.63+0.014; IL-6: SF2Gy=0.80+0.009, P=0.009, t=-10.30). Furthermore, the CD44+CD24-/low cells isolated from MCF7 cells induced by IL-6 also exhibited increased resistance to radiation compared with untreated-control cells (P=0.004, t=-16.77).Conclusions:Interleukin-6 promoted the proliferation ability, doxorubicin resistance and radioresistance of breast cancer MCF7 cells by induction of CD44+CD24-/low cancer stem cells. |
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