Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells

Abstract

Recently, we showed that the metal chelator TPEN targets colon cancer cells through redox cycling of copper. Here, we studied the DNA damage potential of TPEN and deciphered the role of Chk1, ATM and DNA-PK in TPEN-induced toxicity in 3 human colon cancer cell lines, HCT116, SW480 and HT29. We also investigated the role of reactive oxygen species (ROS) in TPEN-induced DNA damage. TPEN reduced cell viability in a dose-and time-dependent manner. Cytotoxicity was associated with significant DNA damage and higher expression of g-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 g-H2AX levels in the presence of antioxidants. Treatment with antioxidants, however, failed to reverse cytotoxicity at high TPEN concentrations (10mM). TPEN-induced cell death was also dependent on the redox cycling of copper since the copper chelator neocuproine inhibited DNA damage and reduced pChk1, g-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. In addition, siRNA silencing of Chk1, DNA-PK and ATM abrogated the expression of g-H2AX and reversed cell death, suggesting that Chk1 and DNA-PK mediate TPEN-induced cytotoxicity in colon cancer cells. This study shows for the first time the involvement of Chk1, DNA-PK and ATM in TPEN-induced DNA damage and confirms our previous findings that ROS generation and the redox cycling of copper in response to TPEN are the main mechanisms by which this compound induces cell death in human colon cancer cells. Inhibition of ATM or DNA-PK did not reverse cytotoxicity at high TPEN concentrations that cause excessive levels of ROS and irreversible cellular damage.Abbreviations: ROS, reactive oxygen species; XIAP, X-linked inhibitor of apoptosis; DNA-PK, DNA-dependent protein kinase; ATM, ataxia telangiectasia mutated; ATR, serine/threonine protein kinase ataxia telangiectasia; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DCFH, 2 0 ,7 0 -Dichlorofluorescin diacetate; NAC, N-acetyL-cysteine; CAT, catalase; DSB, double strand break; SSB, single strand break; Neo, neocuproine; PI, propidium iodide; DDR, DNA damage response

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