Isolation of Breast Cancer Stem Cell from MDA-MB231 Cell Line Using Vincristine

Abstract

© 2016, Universidad de la Frontera. All rights reserved. SUMMARY: Cancer has been considered as a stem cell disease. Suspension culture combined with anti-cancer drugs has recently been proposed for isolation of cancer stem cells (CSCs). In the current study, Vincristine as an anti-cancer drug combined with suspension culture was used for isolation and purification of CSCs from human breast cancer cell line (MDA-MB231). The cells were treated with different concentrations of vincristine (0, 2, 4, 6 and 8 ng/ml). Stem cells were identified with the expression of OCT4, nanog, SOX2 and nucleostemin genes by RT-PCR. Mammosphere forming unit was measured upon suspension culture containing EGF, bFGF, LIF, B27, insulin and BSA. The isolated mammospheres were investigated for CD44 expression. Results showed that 4 ng/ml of vincristine for 72 hours could be utilized as the best and most reliable dose which eliminates around 80 % of non-cancer stem cells with no destructive effect on CSCs’ viability (P > 0.05). RT-PCR demonstrated that drug treated cells expressed OCT4, nanog, SOX2 and nucleostemin. Mammosphere formation unit of cells pretreated with vincristine was significantly higher than unpretreated ones (P > 0.05). Immunofluorescence staining for CD44 depicted high expression of CSC marker among the isolated mammospheres. Vincristine combined with suspension culture can be considered as an appropriate method to isolate CSC.