Activation of checkpoint kinase 2 by 3,3'-diindolylmethane is required for causing G2/M cell cycle arrest in human ovarian cancer cells.
We evaluated the effect of 3,3'-diindolylmethane (DIM) in ovarian cancer cells. DIM treatment inhibited the growth of SKOV-3, TOV-21G, and OVCAR-3 ovarian cancer cells in both a dose- and time-dependent manner with effective concentrations ranging from 40 to 100 muM. Growth-inhibitory effects of DIM were mediated by cell cycle arrest in G(2)/M phase in all the three cell lines. G(2)/M arrest was associated with DNA damage as indicated by phosphorylation of H(2)A.X at Ser139 and activation of checkpoint kinase 2 (Chk2) in all the three cell lines. Other G(2)/M regulatory molecules such as Cdc25C, Cdk1, cyclin B1 were down-regulated by DIM. Cycloheximide or Chk2 inhibitor pretreatment abrogated not only activation of Chk2 but also G(2)/M arrest and apoptosis mediated by DIM. To further establish the involvement of Chk2 in DIM-mediated G(2)/M arrest, cells were transfected with dominant-negative Chk2 (DN-Chk2). Blocking Chk2 activation by DN-Chk2 completely protected cells from DIM-mediated G(2)/M arrest. These results were further confirmed in Chk2 knockout DT40 lymphoma cells, in which DIM failed to cause cell cycle arrest. These results clearly indicate the requirement of Chk2 activation to cause G(2)/M arrest by DIM in ovarian cancer cells. Moreover, blocking Chk2 activation also abrogates the apoptosis-inducing effects of DIM. Furthermore, our results show that DIM treatment cause ROS generation. Blocking ROS generation by N-acetyl cysteine protects the cells from DIM-mediated G(2)/M arrest and apoptosis. Our results establish Chk2 as a potent molecular target of DIM in ovarian cancer cells and provide the rationale for further clinical investigation of DIM.
Kandala
Prabodh K
PK
Department of Biomedical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
Srivastava
Sanjay K
SK
D017209:Apoptosis; D002455:Cell Division; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005260:Female; D005434:Flow Cytometry; D016195:G2 Phase; D006801:Humans; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D007211:Indoles / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000473:pathology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D007211:Indoles; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C016392:3,3'-diindolylmethane
Cancer Prev Res (Phila). 2009 Jun;2(6):581-9
19470789
Gynecol Oncol. 1999 Feb;72(2):215-9
10021304
Free Radic Biol Med. 2008 May 15;44(10):1833-45
18342637
Cancer Res. 2001 Jul 1;61(13):4990-3
11431331
Carcinogenesis. 2005 Apr;26(4):771-8
15661811
J Biol Chem. 2004 Jun 11;279(24):25813-22
15073169
Mol Cell Biol. 2002 Sep;22(18):6521-32
12192050
J Nutr. 2006 Nov;136(11):2728-34
17056792
Apoptosis. 2008 Dec;13(12):1465-78
19002586
Mol Cancer Ther. 2002 Jun;1(8):639-49
12479224
Am J Pathol. 2004 May;164(5):1511-8
15111296
Chem Res Toxicol. 1992 Mar-Apr;5(2):188-93
1643248
Carcinogenesis. 2002 Aug;23(8):1297-305
12151347
Toxicol Appl Pharmacol. 2007 Jan 15;218(2):174-85
17174997
Oncogene. 2009 Mar 12;28(10):1366-78
19151762
Mol Biol Cell. 2004 Sep;15(9):3965-76
15181148
Br J Cancer. 2009 May 5;100(9):1425-33
19401701
Genes Dev. 2004 May 15;18(10):1144-53
15131084
Biochem Pharmacol. 2007 Feb 15;73(4):469-80
17118344
J Natl Cancer Inst. 2009 Feb 4;101(3):176-93
19176463
Oncogene. 2008 Feb 7;27(7):896-906
17684483
Nature. 1997 Apr 24;386(6627):761, 763
9126728
Gynecol Oncol. 2002 Jul;86(1):85-90
12079305
Science. 1998 Nov 20;282(5393):1497-501
9822382
Mol Cancer Ther. 2008 Jun;7(6):1708-19
18566242
EMBO J. 1999 Mar 1;18(5):1223-34
10064589
Cancer Lett. 2005 Dec 18;230(2):260-70
16297712
PLoS Genet. 2010 Feb 26;6(2):e1000863
20195506
Cancer Res. 2007 Apr 1;67(7):3310-9
17409440
Br J Cancer. 2002 Mar 4;86(5):712-7
11875731
Cell Prolif. 2000 Oct;33(5):261-74
11063129
Nature. 2000 Nov 23;408(6811):433-9
11100718
Cancer Res. 2006 May 1;66(9):4880-7
16651444
Cancer Res. 2006 May 1;66(9):4952-60
16651453
CA Cancer J Clin. 2008 Mar-Apr;58(2):71-96
18287387
Biochem Pharmacol. 2002 Mar 15;63(6):1085-97
11931841
Int J Cancer. 2001 Sep;93(6):911-5
11519057
Am J Pathol. 2006 Nov;169(5):1833-42
17071604
Oncogene. 2004 Apr 12;23(16):2797-808
15077143
EMBO J. 2004 Jul 7;23(13):2554-63
15192702
DNA Repair (Amst). 2004 Aug-Sep;3(8-9):889-900
15279774
Cancer Epidemiol Biomarkers Prev. 2004 Sep;13(9):1521-7
15342455
EMBO J. 2002 Oct 1;21(19):5195-205
12356735
J Biol Chem. 2008 Jul 11;283(28):19322-8
18480045
Carcinogenesis. 2009 Oct;30(10):1744-53
19549704