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

Abstract

We evaluated the effect of 3,3Ј-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 doseand time-dependent manner with effective concentrations ranging from 40 to 100 M. 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.

Blocking Epidermal Growth Factor Receptor Activation by 3,3′-Diindolylmethane Suppresses Ovarian Tumor Growth In Vitro and In Vivo

“…We have demonstrated previously that DIM inhibits the proliferation of ovarian cancer cells by inducing apoptosis (Kandala and Srivastava, 2010). We hypothesized that the growth suppressive effect of DIM in ovarian cancer cells was mediated by inhibiting EGFR activation.…”

“…It also is currently in clinical trials for prostate cancer (Heath et al, 2010). In our previous study, we showed that DIM exhibits antiproliferative properties in ovarian cancer cells by causing G 2 /M cell cycle arrest (Kandala and Srivastava, 2010). In the present study, we investigated the mechanism by which DIM inhibits the proliferation of ovarian cancer cells.…”

“…SKOV-3, OVCAR-3, and TOV-21G cells were exposed to varying concentrations of DIM alone or in combination with cisplatin. Cells were collected and lysed, and 20 to 80 g of protein was subjected to SDS gel electrophoresis followed by immunoblotting as described by us previously (Kandala and Srivastava, 2010).…”

“…The effect of DIM on cell cycle distribution in the presence and absence of EGF was assessed by flow cytometry after staining the cells with propidium iodide. Stained cells were analyzed using a flow cytometer (Accuri) as described by us previously (Kandala and Srivastava, 2010).…”

“…Accumulating epidemiological evidence indicates an inverse relationship between the intake of cruciferous vegetables and the risk of ovarian cancer (Zhang et al, 2002). Several studies, including those from our laboratory, have suggested that DIM possesses chemopreventive and therapeutic properties (Li et al, 2005;Ali et al, 2008;Kandala and Srivastava, 2010;Rajoria et al, 2011). Moreover, DIM was shown to be nontoxic to normal cells (Rahman and Sarkar, 2005).…”