Relationship between antileukemic effects of high AA and HIF-1α expression.

<p>A) Quantitative real-time PCR analysis of <i>HIF-1α</i> mRNA expression in K562 and K562-HIF1α cells. Cells were treated with the vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. After high AA exposure, <i>HIF-1α</i> mRNA expression significantly reduced in K562 (*<i>P</i><0.01), but not in K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. High AA exposure significantly reduced the HIF-1α protein level in both types of cells. However, the HIF-1α protein level in K562-HIF1α cells was significantly higher than that in K562 cells after vehicle or high AA exposure. *<i>P</i><0.01, **<i>P</i><0.0001, ***<i>P</i><0.00001. The values represent the mean ± SD values of triplicate samples. C) Flow cytometric measurement of apoptosis of K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 18 h. There was a significant difference in the number of apoptotic (annexin V⁺ propidium iodide (PI)⁺) cells between high AA-treated K562 and K562-HIF1α cells (*<i>P</i><0. 001). The values represent the mean ± SD values of triplicate samples. D) Flow cytometric measurement of cleaved caspase-3 expressed by K562 and K562-HIF1α cells. Cells were treated with vehicle (gray lines) or high AA (black lines) for 1 h, washed, cultured, and analyzed after 24 h. Activation of caspase-3 by high AA was lower in K562-HIF1α than in K562 cells. E) Western blotting analysis of Mcl-1, Bcl-x_L, and Bcl-2 in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.05) and between the vehicle-treated and high AA-treated K562 cells (**<i>P</i><0.0001). There was no significant difference between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. F) Western blotting analysis of Sp1, Sp3, Sp4, and VEGF. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels of these molecules between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.01, ** <i>P</i><0.0001). There were significant differences in the expression levels of Sp1, Sp3, and Sp4 between the vehicle-treated and high AA-treated K562 or K562-HIF1α cells (^†<i>P</i><0.01, ^††<i>P</i><0.001, ^†††<i>P</i><0.0001). There was a significant difference in the expression level of VEGF between the vehicle-treated and high AA-treated K562 (^†††<i>P</i><0.0001), but not between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05).