TPEN selectively kills colon cancer cells. A) Morphology of HCT116, NCM460, and FHS74Int cell lines with and without treatment with 5 μM TPEN for 24 h. B)Cell viability measured using the MTT assay as a function of TPEN concentrations in NCM 460, FHS74Int, HCT 116, SW480, LoVo and HT-29 cells (mean ± SD, n = 3). C) The mean percentage of cells in each cell cycle phase after DNA staining with PI and flow cyotmetry. D) TUNEL and Annexin assays in control cells and cells treated with TPEN 3 and 5 μM (mean ± SD, n = 3, ** p < 0.01, significant difference with respect to control). E) Rhodamine assay measuring disruption of mitochondrial membrane potential after TPEN treatment at 3, 5 and 10 μM (mean ± SD, n = 3). F) Time-dependent release of cytochrome c from mitochondria into the cytoplasm after TPEN treatment. G-I) TPEN alters the expression of several proteins involved in the apoptotic pathway in a time dependent manner. PARP, caspase-9 and caspase-3 were cleaved in response to TPEN. Bax levels were increased at 12 and 24 h of treatment.