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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

Mitochondrial dysfunction and ER stress are early events in ring-DIM- and DIM-mediated prostate cancer cell deathTetramethylrhodamine ethyl ester (TMRE) fluorescence of LNCaP (A) and C42B (B) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Relative mitochondrial ATP levels of LNCaP (C) and C42B (D) cells treated with 5 mM 2-deoxy-D-glucose after a 1 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Phosphorylation of eIF2α, and levels of ER stress proteins were assayed by immunoblot of LNCaP (E) and C42B (F) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM.
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Figure 2: Mitochondrial dysfunction and ER stress are early events in ring-DIM- and DIM-mediated prostate cancer cell deathTetramethylrhodamine ethyl ester (TMRE) fluorescence of LNCaP (A) and C42B (B) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Relative mitochondrial ATP levels of LNCaP (C) and C42B (D) cells treated with 5 mM 2-deoxy-D-glucose after a 1 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Phosphorylation of eIF2α, and levels of ER stress proteins were assayed by immunoblot of LNCaP (E) and C42B (F) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM.

Mentions: To further investigate the mechanism of ring-DIM-induced toxicity, we looked at MMP, mitochondrial ATP generation and ER stress in response to ring-DIM exposure. After only 1 hour of exposure to the 4,4′-dihaloDIMs, MMP decreased by 36-40% in LNCaP and 54-60% in C42B cells, whereas MMP decreased by only 30% in LNCaP and 36 % in C42B cells after a 1 hour treatment with DIM. The 7,7′-dihaloDIMs (Fig. 2A, B) decreased MMP by only 15-18% in LNCaP and 16-24% in C42B cells. The observed decreases in MMP were sustained for at least 8 hours. DIM and all ring-DIMs, except 7,7′-Br2DIM, significantly decreased mitochondrial ATP generation in both cell lines by up to 80% (Fig. 2C, D). In DU145 cells treated with 4,4-Br2DIM, we observed decreases in MMP by 63% and ATP generation by 45% compared to controls (Supplementary Fig. S1B, C).


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)

Mitochondrial dysfunction and ER stress are early events in ring-DIM- and DIM-mediated prostate cancer cell deathTetramethylrhodamine ethyl ester (TMRE) fluorescence of LNCaP (A) and C42B (B) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Relative mitochondrial ATP levels of LNCaP (C) and C42B (D) cells treated with 5 mM 2-deoxy-D-glucose after a 1 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Phosphorylation of eIF2α, and levels of ER stress proteins were assayed by immunoblot of LNCaP (E) and C42B (F) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM.
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Related In: Results  -  Collection

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Figure 2: Mitochondrial dysfunction and ER stress are early events in ring-DIM- and DIM-mediated prostate cancer cell deathTetramethylrhodamine ethyl ester (TMRE) fluorescence of LNCaP (A) and C42B (B) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Relative mitochondrial ATP levels of LNCaP (C) and C42B (D) cells treated with 5 mM 2-deoxy-D-glucose after a 1 h exposure to 4,4′-Br2DIM, 7,7′-Br2DIM, 4,4′-Cl2DIM, 7,7′-Cl2DIM or DIM. Phosphorylation of eIF2α, and levels of ER stress proteins were assayed by immunoblot of LNCaP (E) and C42B (F) cells after 0, 1, 4, and 8 hrs of exposure to 4,4′-Br2DIM, 7,7′-Cl2DIM or DIM.
Mentions: To further investigate the mechanism of ring-DIM-induced toxicity, we looked at MMP, mitochondrial ATP generation and ER stress in response to ring-DIM exposure. After only 1 hour of exposure to the 4,4′-dihaloDIMs, MMP decreased by 36-40% in LNCaP and 54-60% in C42B cells, whereas MMP decreased by only 30% in LNCaP and 36 % in C42B cells after a 1 hour treatment with DIM. The 7,7′-dihaloDIMs (Fig. 2A, B) decreased MMP by only 15-18% in LNCaP and 16-24% in C42B cells. The observed decreases in MMP were sustained for at least 8 hours. DIM and all ring-DIMs, except 7,7′-Br2DIM, significantly decreased mitochondrial ATP generation in both cell lines by up to 80% (Fig. 2C, D). In DU145 cells treated with 4,4-Br2DIM, we observed decreases in MMP by 63% and ATP generation by 45% compared to controls (Supplementary Fig. S1B, C).

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