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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

Cyclosporin A (CsA) abrogates salubrinal-mediated sensitization of prostate cancer cells to mitochondrial dysfunction using sub-toxic concentrations of 7,7′-dihaloDIMs or DIMThe percentage of intact LNCaP (A) and C42B (B) cells pre-treated for 4 hours with salubrinal, salubrinal and CsA, or 0.3% DMSO, followed by a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM. TMRE fluorescence of LNCaP (C) and C42B (D) cells pre-treated for 4 hours with salubrinal and cyclosporin A (CsA), or 0.3% DMSO followed by a 4 hour exposure to 7,7′-Br2DIM or 7,7′-Cl2DIM. (E) Phosphorylation of eIF2α in C42B cell extracts after a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with salubrinal, salubrinal and CsA, or 0.3 % DMSO.
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Figure 5: Cyclosporin A (CsA) abrogates salubrinal-mediated sensitization of prostate cancer cells to mitochondrial dysfunction using sub-toxic concentrations of 7,7′-dihaloDIMs or DIMThe percentage of intact LNCaP (A) and C42B (B) cells pre-treated for 4 hours with salubrinal, salubrinal and CsA, or 0.3% DMSO, followed by a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM. TMRE fluorescence of LNCaP (C) and C42B (D) cells pre-treated for 4 hours with salubrinal and cyclosporin A (CsA), or 0.3% DMSO followed by a 4 hour exposure to 7,7′-Br2DIM or 7,7′-Cl2DIM. (E) Phosphorylation of eIF2α in C42B cell extracts after a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with salubrinal, salubrinal and CsA, or 0.3 % DMSO.

Mentions: Next, we asked if salubrinal could sensitize prostate cancer cells to cell death induced by the 7,7′-dihaloDIMs and DIM. Pre-treatment of LNCaP and C42B cells with salubrinal enhanced the loss of cell viability caused by concentrations of the 7,7′-dihaloDIMs or DIM that are otherwise sub-toxic (defined as all cells being intact and not visibly Hoechst- or PI-stained) in cells that are exposed to the 7,7′-dihaloDIMs alone. Loss of viability mediated by co-treatment of LNCaP or C42B cells with salubrinal and sub-toxic concentrations of 7,7′-dihaloDIMs was attenuated by pre-treatment with CsA (Fig. 5A, B). Consistent with the lack of abrogation of the cytotoxicity of DIM by CsA alone (Fig 3A, B) CsA was also incapable of abrogating the cytotoxicity of DIM that was potentiated by pre-treatment with salubrinal (Fig. 5A, B)


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)

Cyclosporin A (CsA) abrogates salubrinal-mediated sensitization of prostate cancer cells to mitochondrial dysfunction using sub-toxic concentrations of 7,7′-dihaloDIMs or DIMThe percentage of intact LNCaP (A) and C42B (B) cells pre-treated for 4 hours with salubrinal, salubrinal and CsA, or 0.3% DMSO, followed by a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM. TMRE fluorescence of LNCaP (C) and C42B (D) cells pre-treated for 4 hours with salubrinal and cyclosporin A (CsA), or 0.3% DMSO followed by a 4 hour exposure to 7,7′-Br2DIM or 7,7′-Cl2DIM. (E) Phosphorylation of eIF2α in C42B cell extracts after a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with salubrinal, salubrinal and CsA, or 0.3 % DMSO.
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Figure 5: Cyclosporin A (CsA) abrogates salubrinal-mediated sensitization of prostate cancer cells to mitochondrial dysfunction using sub-toxic concentrations of 7,7′-dihaloDIMs or DIMThe percentage of intact LNCaP (A) and C42B (B) cells pre-treated for 4 hours with salubrinal, salubrinal and CsA, or 0.3% DMSO, followed by a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM. TMRE fluorescence of LNCaP (C) and C42B (D) cells pre-treated for 4 hours with salubrinal and cyclosporin A (CsA), or 0.3% DMSO followed by a 4 hour exposure to 7,7′-Br2DIM or 7,7′-Cl2DIM. (E) Phosphorylation of eIF2α in C42B cell extracts after a 24 hour exposure to mildly toxic concentrations of 7,7′-Br2DIM, 7,7′-Cl2DIM or DIM with or without a 4 hour pre-treatment with salubrinal, salubrinal and CsA, or 0.3 % DMSO.
Mentions: Next, we asked if salubrinal could sensitize prostate cancer cells to cell death induced by the 7,7′-dihaloDIMs and DIM. Pre-treatment of LNCaP and C42B cells with salubrinal enhanced the loss of cell viability caused by concentrations of the 7,7′-dihaloDIMs or DIM that are otherwise sub-toxic (defined as all cells being intact and not visibly Hoechst- or PI-stained) in cells that are exposed to the 7,7′-dihaloDIMs alone. Loss of viability mediated by co-treatment of LNCaP or C42B cells with salubrinal and sub-toxic concentrations of 7,7′-dihaloDIMs was attenuated by pre-treatment with CsA (Fig. 5A, B). Consistent with the lack of abrogation of the cytotoxicity of DIM by CsA alone (Fig 3A, B) CsA was also incapable of abrogating the cytotoxicity of DIM that was potentiated by pre-treatment with salubrinal (Fig. 5A, B)

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