The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells

Background: To study whether hypoxia influences the stem-like properties of ovarian cancer cells and their biological behavior under hypoxia. Method: Ovarian cancer cell lines ES-2 and OVCAR-3 were cultivated in different oxygen tensions for proliferation, cell cycling and invasion analyses. The clonogenic potential of cells was examined by colony formation and sphere formation assays. Stem cell surface markers, SP and CD44(bright) and CD44(dim) cells were analyzed by flow cytometry. Protein expression of HIF-1 alpha, HIF-2 alpha, Ot3/4 and Sox2 were investigated by Western blotting. Results: Both cell lines cultivated at hypoxic condition grew relatively slowly with extended G0/G1 phase. However, if the cells were pre-treated under 1% O-2 for 48 hrs before brought back to normoxia, the cells showed significantly higher proliferation rate with higher infiltration capability, and significant more colonies and spheres, in comparison to the cells always cultivated under normoxia. CD44(bright) cells expressed significantly higher levels of Oct3/4 and Sox2 than the CD44(dim) cells and formed significantly more clones and spheres examined in vitro. Hypoxic treatment of the cells resulted in stronger CD44 expression in both cell lines, and stronger CD133 expression in the OVCAR-3 cell line. In parallel with these findings, significantly increased number of side population (SP) cells and up-regulated expression of Oct3/4 and Sox2 in both ES-2 and OVCAR-3 cell lines were observed. Conclusion: We conclude that ovarian cancer cells survive hypoxia by upgrading their stem-like properties through up-regulation of stemness-related factors and behave more aggressively when brought back to higher oxygen environment.