Figure 3.

EP depletes intracellular iron in leukemia cell lines. (A) HL60 cells were labeled with a 0.25μM concentration of the intracellular iron-chelating dye calcein-AM for 5 minutes. Cells were washed then treated with 0 μg/mL of EP, 5 μg/mL of EP, 10 μg/mL of EP, 25 ng/mL of TPO (− control), 100μM SIH (+ control), or 100μM DFO (+ control) for 1 hour (left panel) or 4 hours (right panel) at 37°C and cells were analyzed by FACS. Data represent the change in the MFI ± SD (n = 3) compared with untreated HL60 cells *P < .05; **P < .01; ***P < .001. (B) Five leukemia/lymphoma cell lines were labeled with calcein-AM and then treated with 5 μg/mL of EP for 4 hours. Cells were analyzed by FACS. Data represent the change in the MFI± SD (n = 3) compared with untreated cells. (C) The fold change variation of gene expression in HL60 cells treated with 3 μg/mL of EP or 100 ng/mL of TPO for 12 hours (left panel) or 36 hours (right panel) relative to untreated cells. EP up-regulated the transferrin receptor (TFRC) and down-regulated the ferritin light chain (FTL) and heavy chain (FTH1). (D) FACS analysis of CD71 expression in untreated HL60 cells (blue line) versus cells treated with 5 μg/mL of EP (orange line) for 24 hours (left panel). FACS analysis of CD71 expression in HL60 cells treated with 5 μg/mL of EP (orange line) versus cells preloaded with 500 μg/mL of ferric ammonium citrate (FAC) for 24 hours and then treated with 5 μg/mL of EP (blue line) for 24 hours (middle panel). FACS MFI ± SD (n = 3) of CD71 expression in HL60 cells and URE cells untreated, treated with 5 μg/mL of EP, or preloaded with 500 μg/mL of ferric ammonium citrate followed by treatment with 5 μg/mL of EP for 24 hours (right panel). P value (by t test) represents the difference in MFI between treated and untreated cells *P < .05; **P < .01; ***P < .001. CD71 is overexpressed in response to EP treatment and expression is decreased when cells are preloaded with iron.