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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

KN93 abrogates 4,4′-Br2DIM mediated cell deathPercentage of intact LNCaP (A) and C42B (B) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with KN92 or KN93. (C) TMRE fluorescence of LNCaP and C42B cells after a 4 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (D) Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cells after a 24 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (E) Protein levels of Ca2+/calmodulin-dependent protein kinase (CaMKII) beta and gamma subunits in LNCaP, C42B and DU145 cells.
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Figure 7: KN93 abrogates 4,4′-Br2DIM mediated cell deathPercentage of intact LNCaP (A) and C42B (B) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with KN92 or KN93. (C) TMRE fluorescence of LNCaP and C42B cells after a 4 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (D) Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cells after a 24 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (E) Protein levels of Ca2+/calmodulin-dependent protein kinase (CaMKII) beta and gamma subunits in LNCaP, C42B and DU145 cells.

Mentions: The involvement of the various CaMKII subunits in DIM or ring-DIM-mediated toxicity in prostate cancer cells was assessed using a selective CaMKII inhibitor, KN93. In LNCaP, C42B and DU145 cells, a 4 hour pre-treatment with KN93 resulted in a marked reduction of cell death caused by 4,4′-Br2DIM, but not 7,7′-Cl2DIM or DIM (Fig. 7A-B, Supplementary Fig. S1E). Additionally, KN92, an inactive derivative of KN93, did not abrogate cell death induced by 4,4′-Br2DIM (Fig. 7A-B, Supplementary Fig. S1E). Pre-treatment with KN93 restored the loss of MMP caused by 4,4′-Br2DIM in all three prostate cancer cell lines (Fig. 7C, Supplementary Fig. S1B). Pre-treatment of LNCaP and C42B cells with KN93 reduced 4,4′-Br2DIM-mediated eIF2α phosphorylation, but it did not affect 4,4′-Br2DIM-mediated increases in CHOP and ATF4 levels (Fig. 7D). In DU-145 cells, pre-treatment with KN93 did not alter the increases in eIF2α phosphorylation or levels of ATF4 and CHOP caused by 4,4′-Br2DIM (Supplementary Fig. S1H). In addition, the expression of the beta and gamma subunits of CaMKII in LNCaP C42B, and DU145 cells was confirmed (Fig. 7E). The alpha and delta subunits of CaMKII were not found (data not shown).


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)

KN93 abrogates 4,4′-Br2DIM mediated cell deathPercentage of intact LNCaP (A) and C42B (B) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with KN92 or KN93. (C) TMRE fluorescence of LNCaP and C42B cells after a 4 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (D) Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cells after a 24 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (E) Protein levels of Ca2+/calmodulin-dependent protein kinase (CaMKII) beta and gamma subunits in LNCaP, C42B and DU145 cells.
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Figure 7: KN93 abrogates 4,4′-Br2DIM mediated cell deathPercentage of intact LNCaP (A) and C42B (B) cells after a 24 hour exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with KN92 or KN93. (C) TMRE fluorescence of LNCaP and C42B cells after a 4 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (D) Phosphorylation of eIF2α, and levels of ER stress proteins in LNCaP and C42B cells after a 24 hour exposure to 4,4′-Br2DIM with or without a 4 hour pre-treatment with KN93. (E) Protein levels of Ca2+/calmodulin-dependent protein kinase (CaMKII) beta and gamma subunits in LNCaP, C42B and DU145 cells.
Mentions: The involvement of the various CaMKII subunits in DIM or ring-DIM-mediated toxicity in prostate cancer cells was assessed using a selective CaMKII inhibitor, KN93. In LNCaP, C42B and DU145 cells, a 4 hour pre-treatment with KN93 resulted in a marked reduction of cell death caused by 4,4′-Br2DIM, but not 7,7′-Cl2DIM or DIM (Fig. 7A-B, Supplementary Fig. S1E). Additionally, KN92, an inactive derivative of KN93, did not abrogate cell death induced by 4,4′-Br2DIM (Fig. 7A-B, Supplementary Fig. S1E). Pre-treatment with KN93 restored the loss of MMP caused by 4,4′-Br2DIM in all three prostate cancer cell lines (Fig. 7C, Supplementary Fig. S1B). Pre-treatment of LNCaP and C42B cells with KN93 reduced 4,4′-Br2DIM-mediated eIF2α phosphorylation, but it did not affect 4,4′-Br2DIM-mediated increases in CHOP and ATF4 levels (Fig. 7D). In DU-145 cells, pre-treatment with KN93 did not alter the increases in eIF2α phosphorylation or levels of ATF4 and CHOP caused by 4,4′-Br2DIM (Supplementary Fig. S1H). In addition, the expression of the beta and gamma subunits of CaMKII in LNCaP C42B, and DU145 cells was confirmed (Fig. 7E). The alpha and delta subunits of CaMKII were not found (data not shown).

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