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3,3'-Diindolylmethane (DIM) and its ring-substituted halogenated analogs (ring-DIMs) induce differential mechanisms of survival and death in androgen-dependent and -independent prostate cancer cells.

Goldberg AA, Draz H, Montes-Grajales D, Olivero-Verbél J, Safe SH, Sanderson JT - Genes Cancer (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: INRS-Institut Armand-Frappier, Laval, Québec, Canada ; Critical Care Division and Meakins-Christie Laboratories, Faculty of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Ring-DIM mediated cell death is dependent on the mitochondrial permeability transition pore (mPTP)Percentage of intact LNCaP (A) and C42B (B) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). TMRE fluorescence of LNCaP (C) or C42B (D) cells after a 4 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cell extracts after a 24 hour exposure to 4,4′-Br2DIM (E), 7,7′-Cl2DIM (F) or DIM (G) with or without a 4 hour pre-treatment with CsA.
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Figure 3: Ring-DIM mediated cell death is dependent on the mitochondrial permeability transition pore (mPTP)Percentage of intact LNCaP (A) and C42B (B) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). TMRE fluorescence of LNCaP (C) or C42B (D) cells after a 4 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cell extracts after a 24 hour exposure to 4,4′-Br2DIM (E), 7,7′-Cl2DIM (F) or DIM (G) with or without a 4 hour pre-treatment with CsA.

Mentions: We next assessed whether CsA, an inhibitor of the mitochondrial permeability transition pore, could abrogate the toxicity of cells exposed to the ring-DIMs or DIM. We found that pre-treatment with 5 μM of CsA prevented ring-DIM-mediated loss of cell viability, but did not affect DIM-induced death of LNCaP or C42B cells (Fig. 3A, B). In LNCaP and C42B cells, CsA prevented the loss of MMP caused by the 4,4′-dihaloDIMs and, to a lesser extent, DIM, but had no effect on the loss of MMP mediated by the 7,7′-dihaloDIMs (Fig. 3C, D). We confirmed that CsA also prevented 4,4′-Br2DIM-mediated cell death and loss of MMP in AI DU145 cells (Supplementary Fig. S1E and B). However, CsA had no effect on the phosphorylation status of eIF2α or the expression levels of CHOP and ATF4 in LNCaP or C42B cells treated with either of the three compounds (Fig. 3E-G); this was confirmed using only 4,4′- Br2DIM in DU145 cells (Supplementary Fig. S1F). Treatment with CsA alone did not affect cell viability or eIF2α phosphorylation in LNCaP, C42B or DU145 cells (Supplementary Fig. S2A, B); nor did it significantly affect MMP (Supplementary Fig. S2C).


3,3'-Diindolylmethane (DIM) and its ring-substituted halogenated analogs (ring-DIMs) induce differential mechanisms of survival and death in androgen-dependent and -independent prostate cancer cells.

Goldberg AA, Draz H, Montes-Grajales D, Olivero-Verbél J, Safe SH, Sanderson JT - Genes Cancer (2015)

Ring-DIM mediated cell death is dependent on the mitochondrial permeability transition pore (mPTP)Percentage of intact LNCaP (A) and C42B (B) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). TMRE fluorescence of LNCaP (C) or C42B (D) cells after a 4 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cell extracts after a 24 hour exposure to 4,4′-Br2DIM (E), 7,7′-Cl2DIM (F) or DIM (G) with or without a 4 hour pre-treatment with CsA.
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Figure 3: Ring-DIM mediated cell death is dependent on the mitochondrial permeability transition pore (mPTP)Percentage of intact LNCaP (A) and C42B (B) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). TMRE fluorescence of LNCaP (C) or C42B (D) cells after a 4 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM, or DIM with or without a 4 h pre-treatment with cyclosporin A (CsA). Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cell extracts after a 24 hour exposure to 4,4′-Br2DIM (E), 7,7′-Cl2DIM (F) or DIM (G) with or without a 4 hour pre-treatment with CsA.
Mentions: We next assessed whether CsA, an inhibitor of the mitochondrial permeability transition pore, could abrogate the toxicity of cells exposed to the ring-DIMs or DIM. We found that pre-treatment with 5 μM of CsA prevented ring-DIM-mediated loss of cell viability, but did not affect DIM-induced death of LNCaP or C42B cells (Fig. 3A, B). In LNCaP and C42B cells, CsA prevented the loss of MMP caused by the 4,4′-dihaloDIMs and, to a lesser extent, DIM, but had no effect on the loss of MMP mediated by the 7,7′-dihaloDIMs (Fig. 3C, D). We confirmed that CsA also prevented 4,4′-Br2DIM-mediated cell death and loss of MMP in AI DU145 cells (Supplementary Fig. S1E and B). However, CsA had no effect on the phosphorylation status of eIF2α or the expression levels of CHOP and ATF4 in LNCaP or C42B cells treated with either of the three compounds (Fig. 3E-G); this was confirmed using only 4,4′- Br2DIM in DU145 cells (Supplementary Fig. S1F). Treatment with CsA alone did not affect cell viability or eIF2α phosphorylation in LNCaP, C42B or DU145 cells (Supplementary Fig. S2A, B); nor did it significantly affect MMP (Supplementary Fig. S2C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: INRS-Institut Armand-Frappier, Laval, Québec, Canada ; Critical Care Division and Meakins-Christie Laboratories, Faculty of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus