Identification Of Stem Cell-like Properties For Side Population (SP) Cells Of Neuroblastoma And Effects Of Estrogen And Bisphenol A On The SP Cells

BackgroundsNeuroblastoma (NB) is a common malignant pediatric tumor and accounts for 8-10% of childhood cancers. Long-term survival in high-risk cases is still less than 50% in current treatment strategies, thus research on the genesis and progress of NB is urgently needed to improve treatment. Our results of research on the genesis and progress of NB showed that estrogen (E) and bisphenol A (BPA) which is the most common environmental estrogen not only promoted the growth but also improved the invasion and metastasis for human NB SK-N-SH cells, i.e. E and BPA promote the progress of NB. On the basis of our research results for NB, we presume that E and BPA maybe contributes to the genesis of NB. It is well known that cancer stem cells (CSC) are the origin cells of tumors, thus we investigate the effects of E and BPA on the CSCs of SK-N-SH cells in order to identify if E and BPA have a tumorigenic ability for NB.PurposeThe aim of this study was to sort the side population (SP) cells from SK-N-SH cell line and identify if the sorted SP cells have the cancer stem cell-like properties. At last, the effects of E and BPA on the SP cells were investigated to understand if E and BPA promote the genesis of NB.Materials and Methods1. The detection of SP cells in SK-N-SH cell line and optimization of the detecting procedure:The comparison of detecting effect for SP cells by different incubating time, different concentrations of verapamil was performed to select the suitable incubating time and verapamil concentration. Morphology observation and cell counting kit-8 (CCK-8) assay for re-cultured cells following different concentrations of Hoechst exposure were carried out to confirm the toxicity of Hoechst to SK-N-SH cells. Flow cytometry analysis for cell cycle, Annexin V-FITC and propidium iodide staining flow cytometry, and detection of the SP cells for re-cultured cells following different concentrations of Hoechst exposure were used to investigate the toxicity mechanism of Hoechst to SK-N-SH cells. The effective and relatively nontoxic concentration of Hoechst for SP analysis in SK-N-SH cells was selected by comparing the SP cells detecting effect and cytotoxicity of different concentrations of Hoechst.2. The sorting of SK-N-SH SP cells and identification for their cancer stem cell-like characteristics:SP and non-SP (NSP) cells were separated from the SK-N-SH cell line by flow cytometry and cultured in normal growth medium or serum-free medium containing some growth factors, and then their characteristics were analyzed by light and electron microscopy for morphology; we also used western blotting to analyze marker proteins, CCK-8 assay for proliferative ability, series differentiation assays for differentiation properties; single cell clone experiment, Matrigel invasion assays and tumorigenicity assays were performed to analyze their malignant potential.3. The research of the effects of E and BPA on the SK-N-SH SP cells: Following by E2 or BPA exposure, the detection of SP cells was performed by flow cytometry, and the expression of stem cell marker proteins c-kit and ABCG2 was analyzed by western bloting. The growth-promoting effect of E2 or BPA on SK-N-SH parent cells was confirmed by the CCK-8 assay and flow cytometry cell cycle analysis. The SK-N-SH SP and NSP sub-types were treated by E2 or BPA, respectively, and then the number of viable cells for these two sub-types was evaluated by CCK-8 assay.Results1. The detection of SP cells in SK-N-SH cells and optimization of the detecting procedure:The incubation time of 60 minute, the verapamil concentration of 50μmol/L are suitable to detect the SP cells in SK-N-SH cell line. SK-N-SH cells had strong toxic reaction to Hoechst even at lower concentrations. The mechanism of the strong Hoechst sensitivity of SK-N-SH cells relates to the perturbation of cell cycle, the acute cell death and apoptosis induced by Hoechst. We ultimately selected the Hoechst concentration of 5μM to carry out the SP analysis for SK-N-SH cell line after we compared the SP cells detection effect and cytotoxicity of different Hoechst concentrations.2. The sorting of SK-N-SH SP cells and identification for their cancer stem cell-like characteristics:We successfully sorted the SP and NSP cells from SK-N-SH cell line. SP cells are small, have neurites, grow as clumps of cells with weakly substrate-adherent, and have few cytoplasmic organelles and high nuclear-cytoplasmic ratio; SP cells express high levels of the stem cell marker protein c-kit, have high proliferative and malignant ability, and have a capacity for fast differentiation. NSP cells are large and flat, do not have neurites, grow as a contact-inhibited monolayer with tightly substrate-adherent, and have many cytoplasmic organelles and low nuclear-cytoplasmic ratio; NSP cells express the differentiated cell marker proteins peripherin and S100 at high levels, have slow differentiate ability and have low proliferative and malignant ability.3. The research of the effects of E and BPA on the SK-N-SH SP cells:Both E2 and BPA gradually decreases the percentage of SP cells in SK-N-SH cell line to the level that can not be detected by flow cytometry and depresses the expression of the stem cell marker proteins c-kit and ABCG2. Both E2 and BPA significantly promotes growth not only for SK-N-SH parent eells but also for its SP and NSP sub-types, but the growth-promoting effect of E2 and BPA for NSP cells is significant higher than for SP cells.Conclusions1. It is essential to minimize or, at the very least, be aware of Hoechst toxicity in SP analysis. The detecting procedure of SP cells needs to be carefully optimized for each new cell line studied.2. SK-N-SH SP cells have cancer stem cell-like properties.3. E2 and BPA have lower effects on the SP cells which are enriched for the origin cells of tumor, thus E2 and BPA maybe don't have the capacity to promote the genesis of NB.