Objective      To study the effect of hypoxia on human breast cancer mammospheres (MSs) culture and resistance to chemotherapy.       Methods      MDA-MB-231 cells were cultured in serum-free medium supplemented with growth factors under normoxia and hypoxia conditions

respecti

vely. The culture time and volume of the MSs were recorded. The survival rates (SR) of mammos-pheres-derived cells (MSDCs) and MDA-MB-231 cells incubated with various chemotherapy drugs with various concentrations were detected by MTT assay. The expression of hypoxia inducible factor 2α (HIF-2α) and ATP-binding cassette super family G2 (ABCG2) in the MSs were observed by immunofluorescence technique. The expression levels of CD44 and ABCG2 were detected by Western blotting. The expression levels of HIF-2α

ABCG2

glycoprotein P (P-gp) and multi-drug resistance gene 1 (MDR1) in the MSDCs were detected by Western blotting and real-time RT-PCR.       Results      The MSs of the hypoxia group had shorter culture time and greater volume than those of the normoxia group. The SR of the MSDCs incubated with various chemotherapy drugs with various concentrations in the hypoxia group were significantly higher than that of the MSDCs and MDA-MB-231 cells in the normoxia group (P0.05). Immunofluorescence showed that the fluorescence of HIF-2α and ABCG2 in the MSDCs of the hypoxia group was stronger than that of the normoxia group. The expression levels of CD44 and ALDH1 in the MSDCs of the hypoxia group were higher than those of the normoxia group. The protein expression levels of HIF-2α

ABCG2 and P-gp in the MSDCs of the hypoxia group were significantly higher than those in the MSDCs and MDA-MB-231 cells of the normoxia group (

P

0.05). The mRNA expression levels of HIF-2α

ABCG2 and MDR1 in the MSDCs of the hypoxia group were higher than those of the other groups.       Conclusion      Hypoxia can shorten the culture time for MDA-MB-231 cells forming MSs and increase the size of MSs. The pathway of HIF-2α

ABCG2

P-gp and MDR1 may be involved in the mechanism of the above phenomenon.