Induction of apoptosis in human prostate cancer cell line, PC3, by 3,3'-diindolylmethane through the mitochondrial pathway

Abstract

Prostate cancer is the most common malignancy and the second leading cause of male death in Western countries. Prostate cancer mortality results from metastases to the bones and lymph nodes and progression from androgen-dependent to androgen-independent disease. Although androgen ablation was found to be effective in treating androgen-dependent prostate cancer, no effective life-prolonging therapy is available for androgen-independent cancer. Epidemiological studies have shown a strong correlation between consumption of cruciferous vegetables and a lower risk of prostate cancer. These vegetables contain glucosinolates, which during metabolism give rise to several breakdown products, mainly indole-3-carbinol (I3C), which may be condensed to polymeric products, especially 3,3'-diindolylmethane (DIM). It was previously shown that these indole derivatives have significant inhibitory effects in several human cancer cell lines, which are exerted through induction of apoptosis. We have previously reported that I3C and DIM induce apoptosis in prostate cancer cell lines through p53-, bax-, bcl-2- and fasL-independent pathways. The objective of this study was examination of the apoptotic pathways that may be involved in the effect of DIM in the androgen-independent prostate cancer cell line, PC3, in vitro. Our results suggest that DIM induces apoptosis in PC3 cells, through the mitochondrial pathway, which involves the translocation of cytochrome c from the mitochondria to the cytosol and the activation of initiator caspase, 9, and effector caspases, 3 and 6, leading to poly ADP-ribose polymerase (PARP) cleavage and induction of apoptosis. Our findings may lead to the development of new therapeutic strategies for the treatment of androgen-independent prostate cancer.

Figure 1

Pro-caspase 8 and 9 levels following treatment of PC3 cells with 75 μM DIM for 8–72 h. Cells were treated with DIM and their total protein fraction was extracted, separated on SDS–PAGE and exposed to specific antibodies using Western blotting, as described in ‘Materials and methods’. (A) Western blotting results. C^* denotes a representative control group. The figures shown are representatives of three independent experiments. (B) Densitometer results. Data presented are averages of three independent experiments (±s.e.) and are expressed as percentages of the respective controls. ^**P<0.01; ^***P<0.001.

Figure 2

Pro-caspase 3 and 6 levels following treatment of PC3 cells with 75 μM DIM for 8–72 h. The experimental details and the designations of letters (A–C) were as described for Figure 1. ^*P<0.05; ^***P<0.001.

Figure 3

Effect of DIM on caspase activation in PC3 cells. Determination of enzyme activity was applied by a colorimetric assay using specific caspase substrates, as described in ‘Materials and methods’. Data presented are averages of three experiments, each conducted in duplicates (±s.e.). ^**P<0.01; ^***P<0.001.

Figure 4

Release of cytochrome c to the cytosol in PC3 cells treated with 75 μM DIM for 8–72 h. Cells were treated with DIM and their cytosol protein fraction was extracted, separated on SDS–PAGE and exposed to specific antibody using Western blotting, as described in ‘Materials and methods’. (A) Western blotting results. C^* denotes a representative control group. The figure shown is a representative of three independent experiments. (B) Densitometer results. Data presented are averages of three independent experiments (±s.e.) and are expressed as percentages of the respective controls. ^*P<0.05; ^***P<0.001.

Figure 5

Schematic description of the apoptotic pathway in PC3 cells treated with DIM. Exposure of PC3 cells to DIM triggers the release of cytochrome c from the mitochondria. As a result, initiator caspase 9 is cleaved and activated, and cleaves and activates the effector caspases 3 and 6. These caspases cleave vital cell proteins such as PARP and cause the typical morphological and biochemical characteristics of apoptosis in these cells.

Figure 6

Actin levels following treatment of PC3 cells with 75 μM DIM for 8–72 h. The experimental details and the designations of letters (A–C) were as described for Figure 1.