EP inhibits cell cycling and leads to a block in the G1 phase. (A) HL60 and URE cells were treated with increasing concentrations of EP. Cell viability was measured by cell counts with trypan blue exclusion (top panel), and MTS assays (bottom panel) were performed every 24 hours for 72 hours. Data represent the means ± SD of viable cells performed in triplicate and MTS proliferative index. *P < .05; **P < .01. (B) HL60 cells (i) and URE cells (ii) were incubated in 10μM Cell Tracker Orange for 30 minutes, washed, and analyzed by FACS (red line = hour 0). Cells were treated without EP (blue line) or with 5 μg/mL of EP (orange line) and FACS analysis was performed every 24 hours for 3 days. Lower mean fluorescence indicates increased cell division. The fold change of HL60 cells (iii) and URE cells (iv) of FACS mean fluorescence intensity (MFI) ± SD (n = 3) of Cell Tracker Orange–labeled HL60 cells treated with 5 μg/mL of EP relative to untreated cells is shown. *P < .05; **P < .01; ***P < .001. EP slows cell division in HL60 and URE cells because higher MFI represents slower cell division. (C) Cell-cycle analysis of HL60 cells (i) and URE cells (ii) with or without 5 or 10 μg/mL of EP for 48 hours. EP induces a cell-cycle block in G1 phase with a subsequent decrease in the S phase. (D) The fold change variation of gene expression by microarray in HL60 cells treated with 3 μg/mL of EP for 36 hours relative to untreated cells. EP down-regulated genes necessary for the transition from the G1 to the S phase.
