Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against
tumorigenesis in multiple organs.
3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising
antitumor agent. Previous studies in our laboratory showed that
3,3'-diindolylmethane induced a G(1) cell cycle arrest in human
breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that
3,3'-diindolylmethane is a strong mitochondrial
H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L).
3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular
ATP level, and significantly stimulated mitochondrial
reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal
kinase. Using specific
kinase inhibitors (
SB203580 and
SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal
kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21
mRNA transcription. In addition,
antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that
3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which
3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human
breast cancer cells.