Hypoxia influences stem cell-like properties in multidrug resistant K562 leukemic cells

Abstract

Objectives: The present study investigates the potential role of hypoxia in maintaining stem cell-like properties and therapeutic resistance in K562 leukemic cell.

Methods: Western blot, flow cytometry and cell viability assays were used to investigate the effects of hypoxia (1% O2) on cell proliferation, drug resistance and expression of the hypoxia inducible factor-2α (HIF-2α), the octamer-binding transcription factor 4 (Oct4), CD133, CD34 and the ATP-binding cassette sub-family G member 2 (ABCG2) as well as Smad2 phosphorylation in the drug resistant cell line K562/DOX and its parental cell line.

Results: Hypoxia induced growth inhibition and significantly upregulated HIF-2α, CD133, Oct4, CD34 and ABCG2 expression in the wild type K562 cells (p<0.05). The IC50 of doxorubicin was also enhanced about 2.5-fold in hypoxia. In contrast, the K562/DOX cells, which showed significantly higher ABCG2 expression and IC50 for various drugs, no significant difference in cell proliferation was observed between hypoxia and normoxia. The hypoxia-induced upregulation of HIF-2α, CD133, Oct4, CD34 and ABCG2 expression was significantly lower than in the wild type cells (p<0.05). Moreover, hypoxia induced the phosphorylation of Smad2 and additional treatment with SD-208, an inhibitor of the TGF-β receptor I kinase, resulted in a dose-dependent downregulation of CD133 and Oct4 in the K562/DOX cells.

Conclusions: Hypoxia plays an important role in enhancing the stem cell-like properties and to induce multidrug resistance of leukemia cells. The activation of the TGF-β/Smad2 signaling pathway may be involved in the regulation of this pathophysiological process.

Keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; ABC; AML; ATP-Binding Cassette; Acute myeloid leukemia; Ara-C; BM; DMSO; DOX; Drug concentrations inhibiting cell growth by 50%; Hypoxia; IC(50); Leukemia; MDR; MTT; MTX; Multi-drug resistance; PAGE; Stem cell-like properties; TBS-T; TGF-β; VCR; bone marrow; cytosine arabinoside; dimethyl sulfoxide; doxorubicin; methotrexate; multi-drug resistant; polyacrylamide gel electrophoresis; transforming growth factor-β; tris-buffered saline-Tween; vincristine.