Biological characteristics of the side population of human ovarian epithelial cancer cell line

Abstract

Objectives Existence of side population (SP) in human ovarian cancer cell line OVCAR3 and characteristics of both stem cells and cancer cells were detected. Relation between SP phenotype and ABCG2 protein( ATP-binding cassette superfamily G member 2/breast cancer resistance protein, BCRP) was examined,and the potential to use OVCAR3 as tools for investigating the characteristics of cancer stem cells (CSCs)was estimated as well. Methods Hoechst 33342 efflux SP cells and dye-uptaking non-SP cells were separated by flow cytometry from human serous ovarian adenocarcinoma cell line OVCAR3. Cell colonyforming efficiency (CFE) of both subgroups ware evaluated. Soft agar assay was performed to compare the growth capacity of SP cells and non-SP cells. ABCG2 expression was detected by Western blotting and immunofluoreacence using an anti-ABCG2 monoclonal antibody. Results Flow cytometric analysis confirmed the presence of small subpopulations of cells excluding Hoechst dye in OVCAP,3 cells (2. 0% ) in a verapamil-sensitive manner. Hoechst dye uptake in OVCAR3 cells was enhanced by addition of verapamil, consistent with activity of ABCG2 protein. CFE of both SP and non-SP was 46.17%±23.27% and 6.17%±3.06%, respectively(P<0. 05). The Hoechst dye excluding SP cells exhibited immunoreactivity to stem cell markers ABCG2, demonstrated by Western blotting. They could grow in soft agar plate and formed cell sphere. Conclusions Human epithelial ovarian carcinoma cell line OVCAR3 contains a small subpoptdation of cells that exhibits a cancer stem cell-like phenotype. Special significance is the expression of ABCG2 in human tumor cells, a calcium-sensitive cell surface protein that acts to exclude Hoechst dye. These cells play a principal role in maintaining cancer cell line. It can be seen that cancer cell lines are potentially useful tools for investigating the characteristics of CSCs under controlled experimental conditions. Key words: Ovarian neoplasms;  Tumor stem cells;  ATP-binding cassette superfamily G member 2; Side-population cells;  Hoechst 33342