The involvement of Bax in zinc-induced mitochondrial apoptogenesis in malignant prostate cells

Abstract

Background: The development and progression of prostate cancer requires the transformation of normal zinc-accumulating epithelial cells to malignant cells that have lost the ability to accumulate zinc. This metabolic transformation is essential so that the tumor suppressive effects of zinc can be eliminated and the malignant process can proceed. One of the major effects of zinc is its prevention of prostate cell growth by its induction of apoptosis. The accumulation of cellular zinc has a direct effect on the mitochondria that results in the release of cytochrome c, which initiates the caspase cascade that leads to apoptosis. This effect is associated with the mitochondrial pore-forming process, but the mechanism by which zinc induces the release of cytochrome c and induces mitochondrial apoptogenesis has not been resolved. The present report provides for the first time information that implicates Bax in the zinc induction of mitochondrial apoptogenesis.

Results: The effects of zinc treatment on the Bax levels of PC-3 cells and on the mitochondria were determined. The exposure of isolated mitochondria to zinc results in an increase in membrane bound Bax, which is due to the mitochondrial insertion of endogenous resident Bax. The mitochondrial Bax/Bcl-2 ratio is increased by zinc treatment. Zinc treatment of PC-3 cells also increases the mitochondrial level of Bax. In addition, zinc treatment increases the cellular level of Bax and the cellular Bax/Bcl2 ratio. Down regulation of Bax in PC-3 cells eliminates the zinc induction of apoptosis. The increase in cellular Bax level appears to involve zinc induction of Bax gene expression.

Conclusion: This report extends and confirms that physiological levels of zinc induce apoptosis in prostate cells. The study provides evidence that zinc is directly involved in facilitating a Bax-associated pore formation process that initiates mitochondrial apoptogenesis. This is enhanced by an additional effect of zinc on increasing the cellular level of Bax. To avoid the anti-tumor apoptogenic effects of zinc, the malignant cells in prostate cancer posses genetic/metabolic adaptations that prevent the cellular accumulation of zinc.

Figure 1

The direct effect of zinc on the level of bound Bax in isolated mitochondria from PC-3 cells. The isolated mitochondria were exposed to medium supplemented with 15 μM zinc for 15 minutes or with unsupplemented medium (control).

Figure 2

The effects of zinc on Bax and Bcl-2 levels of mitochondria isolated from PC-3 cells that were exposed to medium supplemented with zinc. The cells were treated with medium supplemented with 15 μM zinc for 180 minutes versus no zinc treatment.

Figure 3

The effect of 15 μM zinc treatment of PC-3 cells on the total cellular levels of Bax and Bcl-2.

Figure 4

The effect of zinc on Bax knock-down PC-3 cells. PC-3 cells were transfected with Bax siRNA or with empty vector and cultured for 72 hours; followed by treatment of the cells with 15 μM zinc for 24 hours.

Figure 5

The effect of cyclohexamide treatment of PC-3 cells on the zinc-induced cellular increase in Bax. PC-3 cells were cultured in serum-free medium for 24 h and then treated with or without cycloheximide (CHX, 5 μg/ml) for 1 hour prior to zinc (15 μM) exposure for 6 hours.

Figure 6

A representation of the integrated apoptogenic effects of zinc. Zinc increases the Bax-associated mitochondrial pore forming process that results in the release of cytochrome c that initiates the caspase cascade leading to apoptosis. Zinc also increases the gene expression and cellular production of Bax and the Bax/Bcl-2 ratio, which facilitates the mitochondrial Bax-associated apoptogenic effect.