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Molecular Pharmacology

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Research ArticleArticle

Activation of Checkpoint Kinase 2 by 3,3′-Diindolylmethane Is Required for Causing G2/M Cell Cycle Arrest in Human Ovarian Cancer Cells

Prabodh K. Kandala and Sanjay K. Srivastava
Molecular Pharmacology August 2010, 78 (2) 297-309; DOI: https://doi.org/10.1124/mol.110.063750
Prabodh K. Kandala
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Sanjay K. Srivastava
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Abstract

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 μM. Growth-inhibitory effects of DIM were mediated by cell cycle arrest in G2/M phase in all the three cell lines. G2/M arrest was associated with DNA damage as indicated by phosphorylation of H2A.X at Ser139 and activation of checkpoint kinase 2 (Chk2) in all the three cell lines. Other G2/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 G2/M arrest and apoptosis mediated by DIM. To further establish the involvement of Chk2 in DIM-mediated G2/M arrest, cells were transfected with dominant-negative Chk2 (DN-Chk2). Blocking Chk2 activation by DN-Chk2 completely protected cells from DIM-mediated G2/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 G2/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 G2/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.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported in part by the National Institutes of Health National Cancer Institute [Grants R01-CA106953, R01-CA129038] and the Texas Tech University Health Sciences Center, School of Pharmacy.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.063750.

  • ABBREVIATIONS:

    Chk2
    checkpoint kinase 2
    DIM
    3,3′-diindolylmethane
    DN-Chk2
    dominant-negative checkpoint kinase 2
    ROS
    reactive oxygen species
    NAC
    N-acetyl cysteine
    FBS
    fetal bovine serum
    DCFDA
    2′-7′-dichlorofluorescin diacetate
    TUNEL
    terminal deoxynucleotidyl transferase dUTP nick-end labeling
    dbs
    double-strand breaks
    ATCC
    American Type Culture Collection
    CHX
    cycloheximide
    KO
    knock out
    Cdc25C
    cell division cycle 25C
    CDK
    cyclin-dependent kinase
    ATM
    ataxia telangiectasia mutated
    MG132
    N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal.

  • Received January 25, 2010.
  • Accepted May 5, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (2)
Molecular Pharmacology
Vol. 78, Issue 2
1 Aug 2010
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Research ArticleArticle

Activation of Checkpoint Kinase 2 by 3,3′-Diindolylmethane Is Required for Causing G2/M Cell Cycle Arrest in Human Ovarian Cancer Cells

Prabodh K. Kandala and Sanjay K. Srivastava
Molecular Pharmacology August 1, 2010, 78 (2) 297-309; DOI: https://doi.org/10.1124/mol.110.063750

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Research ArticleArticle

Activation of Checkpoint Kinase 2 by 3,3′-Diindolylmethane Is Required for Causing G2/M Cell Cycle Arrest in Human Ovarian Cancer Cells

Prabodh K. Kandala and Sanjay K. Srivastava
Molecular Pharmacology August 1, 2010, 78 (2) 297-309; DOI: https://doi.org/10.1124/mol.110.063750
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