In vitro activity: TPEN (also known as TPED) is a specific cell-permeable heavy metal chelator. TPEN targets colon cancer cells through redox cycling of copper. TPEN reduced cell viability in a dose- and time-dependent manner. Cytotoxicity was associated with significant DNA damage and higher expression of γ-H2AX protein and activation of ATM/ATR signaling pathway. Cell death by TPEN was dependent on ROS generation as evidenced by the reversal of cell viability, and DNA damage and the abrogation of γ-H2AX levels in the presence of antioxidants. TPEN-induced cell death was also dependent on the redox cycling of copper since the copper chelator neocuproine inhibited DNA damage and reduced pChk1, γ-H2AX, and ATM protein expression. Cell death by low TPEN concentrations, involved ATM/ATR signaling in all 3 cell lines, since pre-incubation with specific inhibitors of ATM and DNA-PK led to the recovery of cells from TPEN-induced DNA damage.

Kinase Assay: Cells were seeded in 6 well culture plates. After 24 hrs, growth medium was replaced with low serum (2% FBS). In this experiment, cells were exposed to TPEN for 10 min in the presence or absence of NAC or CAT, after which they were incubated with 10 μM of the CM-H2DCFDA dye (Sigma-Aldrich, D6883) for 20 min. Cells were washed, harvested by centrifugation, and the pellet was re-suspended in 500 μl PBS (Lonza, BE17-517Q) followed by flow cytometry.

Cell Assay: Cells were plated in 96-well plates and treated with different concentrations of TPEN, Neo or copper sulfate. The inhibition of cell growth by TPEN was measured by the Cell Titer 96 non-radioactive cell proliferation kit (Promega Corp, G4000). The cell growth assay is an MTT-based method that measures the ability of metabolically active cells to convert tetrazolium salt into a blue formazan product the absorbance of which is recorded at 570 nm.