Chk1 and DNA-PK mediate TPEN-induced DNA damage in a ROS dependent manner in human colon cancer cells.
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 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. Treatment with antioxidants, however, failed to reverse cytotoxicity at high TPEN concentrations (10µM). 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. In addition, siRNA silencing of Chk1, DNA-PK and ATM abrogated the expression of γ-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.
a Department of Biology , American University of Beirut , Lebanon.
Omar Nasser
ON
Rahal
a Department of Biology , American University of Beirut , Lebanon.
Maamoun
M
Fatfat
a Department of Biology , American University of Beirut , Lebanon.
Carla
C
Hankache
a Department of Biology , American University of Beirut , Lebanon.
Bassam
B
Osman
b Department of Biology , Lebanese University , Lebanon.
Hala
H
Khalife
c Department of Physiology and Biophysics , Weill Cornell Medical College , Qatar.
Khaled
K
Machaca
a Department of Biology , American University of Beirut , Lebanon.
Hala-Gali
HG
Muhtasib
D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1 / Q000235:genetics* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000235:genetics* / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology*; D006801:Humans; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
D002614:Chelating Agents; D005029:Ethylenediamines; D017382:Reactive Oxygen Species; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051747:DNA-Activated Protein Kinase; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
Anticancer; DNA damage; antioxidant; copper; metal chelation; reactive oxygen species; redox cycling
Am J Physiol Gastrointest Liver Physiol. 2015 May 1;308(9):G757-66
25767260
Trends Mol Med. 2016 Apr;22(4):274-89
26970951
Drug Resist Updat. 2004 Apr;7(2):97-110
15158766
Cancer Res. 2008 Nov 1;68(21):8643-53
18974103
Cancer Biol Ther. 2003 May-Jun;2(3):233-5
12878854
Biometals. 2011 Feb;24(1):143-51
20957409
Cell Cycle. 2010 Sep 1;9(17):3460-9
20855954
Cell Death Dis. 2013 Jun 06;4:e660
23744360
Cancer Biol Ther. 2013 May;14(5):379-89
23380594
Nat Rev Cancer. 2005 Feb;5(2):102-12
15685195
Oxid Med Cell Longev. 2012;2012:313275
23320127
Chem Rev. 1999 Sep 8;99(9):2201-4
11749478
Cancer Lett. 2008 Jul 18;266(1):60-72
18374480
World J Gastroenterol. 2007 Jan 28;13(4):612-8
17278230
J Biol Chem. 1985 Mar 10;260(5):2719-27
3919006
Crit Rev Clin Lab Sci. 1985;22(2):97-184
3891229
Cancer Res. 2009 Feb 15;69(4):1259-62
19208831
BMC Cancer. 2014 Jul 21;14 :527
25047035
Cold Spring Harb Perspect Biol. 2013 Aug 01;5(8):a012724
23813586
Biometals. 2010 Dec;23 (6):997-1013
20524045
J Cell Biochem. 2008 May 1;104(1):202-12
17979179
Eur J Pharmacol. 2007 Feb 14;557(1):9-19
17169355
Cancer Biol Ther. 2008 Dec;7(12):1875-84
18981733
J Natl Cancer Inst. 1971 Jul;47(1):1-13
4328191
Nat Rev Drug Discov. 2009 Jul;8(7):579-91
19478820
Biol Trace Elem Res. 2014 May;158(2):259-67
24591003
Nature. 2009 Oct 22;461(7267):1071-8
19847258
Cancer Lett. 2008 Nov 28;271(2):251-9
18639975
Nature. 2001 May 17;411(6835):366-74
11357144