Background: Breast cancer is the second most common cancer worldwide. N, N, N’, N’-Tetrakis
(2-pyridylmethyl)-ethylenediamine (TPEN) is a lipid-soluble zinc metal chelator that induces apoptosis in
cancer cells through oxidative stress (OS). However, the effectiveness and the mechanisms involved in TPENinduced
cell death in mammary adenocarcinoma cells in vitro and in vivo are still unclear.
Objective: This study aimed to evaluate the cytotoxic effect of TPEN in mouse embryonic fibroblasts (MEFs, as
normal control cells) and mammary adenocarcinoma cancer cells (TS/A cells) in vitro and in a mammary tumor
model in vivo.
Methods: Cells were treated with TPEN (0-3 µM), and changes in nuclear chromatin and DNA, mitochondrial
membrane potential (ΔΨm), and intracellular reactive oxygen species (ROS) levels were determined by both
fluorescence microscopy and flow cytometry. Cell proliferation and the cell cycle were also analyzed. Cellular
markers of apoptosis were evaluated by Western blot. Finally, the effect of TPEN in a mammary adenocarcinoma
tumor model in vivo was determined by immunohistological analyses.
Results: TPEN induced apoptosis in TS/A cells in a dose-dependent manner, increasing nuclear chromatin condensation,
DNA fragmentation, cell cycle arrest and ΔΨm loss. Additionally, TPEN increased dichlorofluorescein
fluorescence (DCF+) intensity, indicative of ROS production; increased DJ-1-Cys106-sulfonate expression,
a marker of intracellular H2O2 stress; induced p53 and PUMA upregulation; and activated caspase-3.
Moreover, TPEN induced mammary cancer cell elimination and tumor size reduction in vivo 48 h after treatment
through an OS-induced apoptotic mechanism.
Conclusion: TPEN selectively induces apoptosis in TS/A cells through an H2O2-mediated signaling pathway.
Our findings support the use of TPEN as a potential treatment for breast cancer.