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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-DIMs and DIM induce protective autophagy(A) Levels of LC3B I and II protein in LNCaP, C42B and DU145 cells after a 24 hr exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. Percentage of intact LNCaP (B, E),C42B (C, F) and DU145 (D, G) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with bafilomycin A1 (BafA1) or 3-methyladenine (3-MA). Percentage of intact LNCaP (H) and C42B (I) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 24 hour pre-treatment with LC3B siRNA. (J) Protein levels of LC3B I and II in LNCaP and C42B cells after treatment with or without LC3B siRNA.
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Figure 6: Ring-DIMs and DIM induce protective autophagy(A) Levels of LC3B I and II protein in LNCaP, C42B and DU145 cells after a 24 hr exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. Percentage of intact LNCaP (B, E),C42B (C, F) and DU145 (D, G) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with bafilomycin A1 (BafA1) or 3-methyladenine (3-MA). Percentage of intact LNCaP (H) and C42B (I) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 24 hour pre-treatment with LC3B siRNA. (J) Protein levels of LC3B I and II in LNCaP and C42B cells after treatment with or without LC3B siRNA.

Mentions: We investigated the potential of DIM and the ring-DIMs to induce autophagy in prostate cancer cells. A dose-dependent increase in LC3B conversion was observed in LNCaP and C42B cells, but not in autophagy-deficient DU145 cells, exposed to sub-toxic concentrations of 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM for 24 hours (Fig. 6A). Pre-treatment of cells with autophagy inhibitors bafilomycin A1 (Fig. 6B-D) or 3-MA (Fig. 6E-G) sensitized LNCaP and C42B cells, but not DU145 cells, to sub-toxic concentrations of 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. We next silenced the LC3B gene in LNCaP and C42B cells using siRNA and then exposed them to sub-toxic concentrations of 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. We observed significant decreases in viability of cells treated with the LC3B-selective siRNA, but not control siRNA, with each of the three compounds (Fig. 6H, I). We confirmed the effectiveness of the LC3B-selective siRNA in decreasing the expression of LC3B in LNCaP and C42B cells to almost undetectable levels relative to control siRNA (Fig. 6J).


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-DIMs and DIM induce protective autophagy(A) Levels of LC3B I and II protein in LNCaP, C42B and DU145 cells after a 24 hr exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. Percentage of intact LNCaP (B, E),C42B (C, F) and DU145 (D, G) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with bafilomycin A1 (BafA1) or 3-methyladenine (3-MA). Percentage of intact LNCaP (H) and C42B (I) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 24 hour pre-treatment with LC3B siRNA. (J) Protein levels of LC3B I and II in LNCaP and C42B cells after treatment with or without LC3B siRNA.
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Figure 6: Ring-DIMs and DIM induce protective autophagy(A) Levels of LC3B I and II protein in LNCaP, C42B and DU145 cells after a 24 hr exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. Percentage of intact LNCaP (B, E),C42B (C, F) and DU145 (D, G) cells after a 24 h exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with bafilomycin A1 (BafA1) or 3-methyladenine (3-MA). Percentage of intact LNCaP (H) and C42B (I) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 24 hour pre-treatment with LC3B siRNA. (J) Protein levels of LC3B I and II in LNCaP and C42B cells after treatment with or without LC3B siRNA.
Mentions: We investigated the potential of DIM and the ring-DIMs to induce autophagy in prostate cancer cells. A dose-dependent increase in LC3B conversion was observed in LNCaP and C42B cells, but not in autophagy-deficient DU145 cells, exposed to sub-toxic concentrations of 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM for 24 hours (Fig. 6A). Pre-treatment of cells with autophagy inhibitors bafilomycin A1 (Fig. 6B-D) or 3-MA (Fig. 6E-G) sensitized LNCaP and C42B cells, but not DU145 cells, to sub-toxic concentrations of 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. We next silenced the LC3B gene in LNCaP and C42B cells using siRNA and then exposed them to sub-toxic concentrations of 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM. We observed significant decreases in viability of cells treated with the LC3B-selective siRNA, but not control siRNA, with each of the three compounds (Fig. 6H, I). We confirmed the effectiveness of the LC3B-selective siRNA in decreasing the expression of LC3B in LNCaP and C42B cells to almost undetectable levels relative to control siRNA (Fig. 6J).

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