Abstract #2966: Activation of check point kinase 2 by 3,3\#8217;-Diindolylmethane is responsible for causing G2/M cell cycle arrest in human ovarian cancer cells.

Ovarian cancer ranks second among gynecologic cancers and causes more deaths than any other cancers of female reproductive system. Hence there is a need for novel strategies to control and treat this malignancy. Epidemiological studies suggest that consumption of Brassica vegetables reduces the risk of ovarian cancer. 3,3\#8217;-Diindolylmethane (DIM) is an active metabolite of Indole-3-carbinol, which is present in Brassica vegetables such as cabbage, broccoli, kale etc. In our studies, we used BR-DIM, a formulated version of DIM that is currently being evaluated in clinical trials against cervical cancer. Treatment of SKOV-3 or OVCAR-3 human ovarian cancer cells with varying concentration of BR-DIM for 24h resulted in the significantly reduced survival of both the cell lines. Our results reveal that exposure of SKOV-3 or OVCAR-3 cells to BR-DIM for 24h caused about 2.4 fold retention of cells in G2/M phase. Arrest of cells in G2/M phase was as early as 12h following BR-DIM treatment in both the cell lines. Our western blot analysis, demonstrate that BR-DIM treatment cause DNA damage as evidenced by phosphorylation of H2A.X at Ser-139, which is an indicator of the presence of DNA double strand breaks. Further, our results demonstrate significant activating phosphorylation of Chk2 at Thr-168. Chk2 is a checkpoint kinase that gets activated in response to DNA damage. We also observed reduced expression of cyclin dependent kinase1 (Cdk1) and induction of p21 expression in response to BR-DIM treatment. p21 is a cdk inhibitor and is known to negatively regulate G2/M transition. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor and then with BR-DIM resulted in the abrogation of Chk2 activation as well as G2/M arrest. To further confirm the involvement of Chk2 in G2/M arrest, cells were transfected with dominant negative Chk2 (Chk2-DN) prior to BR-DIM treatment. Our results demonstrate that Chk2-DN completely blocked not only the activation of Chk2 but also G2/M arrest. Taken together, our results for the first time demonstrate Chk2 as a novel molecular target of BR-DIM in mediating G2/M cell cycle arrest in human ovarian cancer cells. [Supported in part by RO1 grant CA 106953 (to S.K.S) awarded by the National Cancer Institute].

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2966.