Based on the important role of ODC in ESCC progression, ESCC cells were treated with DFMO, a potent substrate analog and specific irreversible inhibitor of ODC.23 The data showed that treatment with DFMO dose-dependently decreased ODC activity in ESCC cells (Fig. 5a). Inhibition of ODC activity was associated with suppression of proliferation and colony formation of ESCC cells (Fig. 5b, c), indicating that DFMO attenuates ESCC progression by targeting ODC.Fig. 5DFMO inhibits ESCC cells in vitro. a The effect of DFMO on ODC activity in KYSE450 and KYSE510 cells was measured as the release of CO2 from L-[1-C14] ornithine and results are shown as percentage of control group (set at 100%). b Anchorage-dependent cell growth was measured by MTS assay. c For measuring anchorage-independent growth, cells were cultured for 3 weeks with different concentrations of DFMO in soft agar and then colonies were counted using a microscope and the Image-Pro Plus software (v.6.0) program. After treatment with DFMO for 72 h, d total apoptosis and e cell cycle were analyzed by flow cytometry. The expression of markers associated with cell cycle and apoptosis was analyzed by Western blot. All data are shown as means ± S.D. of triplicate values from 3 independent experiments