Abstract 2564: The anticancer molecule TPEN induces DNA damage in human colon cancer cells

Abstract

Abstract The maintenance of optimal metal levels is an essential aspect of cell homoeostasis. However, in many types of cancer these metal levels especially iron, zinc and copper diverge from normal levels. We have recently shown that the zinc chelator TPEN increases the generation of reactive oxygen species (ROS) which selectively kills colon cancer cells. We have also provided evidence that the redox cycling of copper is responsible for TPEN anticancer effects. In this study, we aimed to further decipher the mechanism of TPEN-induced cell death in colon cancer cells through studying its effect on DNA damage. HCT116 p53+/+ human colon cancer cells were seeded were treated with 5μM TPEN at 50% confluence The inhibition of cell growth was measured by MTT, while ROS production was measured by the DCFH Assay using flow cytometry. siRNAs against DNApk and Chk2 was used to investigate the involvement of these DNA damage sensors in TPEN activity. DNA damage was assessed by the comet assay. Phosphorylation of ATM, ATR, Chk1, Chk2 and H2AX by TPEN were detected immunocytochemically by multiparameter cytometry. Expression levels of Chk1/2, ATR and DNApK were determined by western blotting. We show that cell death by TPEN is associated with significant DNA damage, an effect that was dependent on ROS generation and on the redox cycling of copper, as evidenced by reversal of DNA damage in the presence of antioxidants (NAC, CAT) or the copper chelator neocuproine (Neo). DNA damage was associated with increased expression of p-H2AX and a significant activation of ATM/ATR signaling molecules, specifically p-ATM, p-ATR and p-Chk1. Interestingly, silencing DNApk and Chk2 reversed DNA damage caused by TPEN, suggesting the involvement of DNApk and ATM/ATR pathways in TPEN-mediated effects. This study shows for the first time the involvement of DNApk and Chk2 in TPEN-induced DNA damage and confirms our previous findings that the redox cycling of copper is the main mechanism by which TPEN induces cell death in human colon cancer cells. Citation Format: Hala Gali-Muhtasib, Omar Rahal, Maamoun Fatfat, Carla Hankache, Bassam Osman, Hala Khalife, Khaled Machaca. The anticancer molecule TPEN induces DNA damage in human colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2564. doi:10.1158/1538-7445.AM2015-2564