We recently reported that novel ring-substituted analogs of
3,3'-diindolylmethane (ring-DIMs) induce apoptosis and
necrosis in
androgen-dependent and -independent
prostate cancer cells. In this paper, we have focused on the mechanism(s) associated with ring-DIM-mediated cell death, and on identifying the specific intracellular target(s) of these compounds. The 4,4'- and 7,7'-dichloroDIMs and 4,4'- and 7,7'-dibromoDIMs induced the death of LNCaP, C42B and DU145
prostate cancer cells, but not that of immortalized normal human prostate epithelial (RWPE-1) cells. Ring-DIMs caused the early loss of mitochondrial membrane potential (
MMP) and decreased mitochondrial
ATP generation in
prostate cancer cells.
Cyclosporin A, an inhibitor of the
mitochondrial permeability transition pore, inhibited ring-DIM-mediated cell death, and
salubrinal, an inhibitor of ER stress, inhibited cell death mediated only by 4,4'-dihaloDIMs. We found that although
salubrinal did not inhibit the onset of ER stress, it prevented 4,4'-dibromoDIM mediated loss of
MMP.
Salubrinal potentiated cell death in response to 7,7'-dihaloDIMs and DIM, and this effect concurred with increased loss of
MMP. Using in silico 3-D docking affinity analysis, we identified Ca2+/
calmodulin-dependent
kinase II (
CaMKII) as a potential direct target for the most toxic ring-DIM, 4,4'-dibromoDIM. An inhibitor of
CaMKII,
KN93, but not its inactive analog KN92, abrogated cell death mediated by 4,4'-dibromoDIM. The ring-DIMs induced ER stress and autophagy, but these processes were not necessary for ring-DIM-mediated cell death. Inhibition of autophagy with
bafilomycin A1,
3-methyladenine or by LC3B gene silencing sensitized LNCaP and C42B, but not ATG5-deficient DU145 cells to ring-DIM- and DIM-mediated cell death. We propose that autophagy induced by the ring-DIMs and DIM has a cytoprotective function in
prostate cancer cells.