Androgen induces adaptation to oxidative stress in prostate cancer: implications for treatment with radiation therapy

Abstract

Radiation therapy is a standard treatment for prostate cancer (PC). The postulated mechanism of action for radiation therapy is the generation of reactive oxygen species (ROS). Adjuvant androgen deprivation (AD) therapy has been shown to confer a survival advantage over radiation alone in high-risk localized PC. However, the mechanism of this interaction is unclear. We hypothesize that androgens modify the radioresponsiveness of PC through the regulation of cellular oxidative homeostasis. Using androgen receptor (AR)(+) 22rv1 and AR(-) PC3 human PC cell lines, we demonstrated that testosterone increased basal reactive oxygen species (bROS) levels, resulting in dose-dependent activation of phospho-p38 and pAKT, and increased expression of clusterin, catalase, and manganese superoxide dismutase. Similar data were obtained in three human PC xenografts; WISH-PC14, WISH-PC23, and CWR22, growing in testosterone-supplemented or castrated SCID mice. These effects were reversible through AD or through incubation with a reducing agent. Moreover, testosterone increased the activity of catalase, superoxide dismutases, and glutathione reductase. Consequently, AD significantly facilitated the response of AR(+) cells to oxidative stress challenge. Thus, testosterone induces a preset cellular adaptation to radiation through the generation of elevated bROS, which is modified by AD. These findings provide a rational for combined hormonal and radiation therapy for localized PC.

Figure 1

Androgens induce relative resistance to oxidative stress challenge in 22rv1 human PC cells that can be reversed by AD. (A) Survival of 22rv1 cells in response to 24 hours of incubation with increasing doses of hydrogen peroxide, as determined by neutral red viability assay. LD = lethal dose. (B) Survival of 22rv1 cells in response to γ-radiation, as determined by colony formation assay. CT = control cells growing in a culture medium with CSFCS and without phenol red; T = cells growing in the same medium but with the addition of 10 nM R1881; T + Bic = cells growing in the same medium but with the addition of both 10 nM R1881 and 10 µM bicalutamide. Results are derived from at least three experiments and are expressed as mean ± SD.

Figure 2

Androgens increase bROS levels in an AR-dependent manner. (A) Dose-dependent effect of R1881 on ROS levels in 22rv1 cells, as measured by NBT reduction assay. Addition of 10 µM bicalutamide antagonizes this effect. (B) The androgen-induced increase in bROS, as measured by NBT reduction assay, is evident only in PC cells expressing the AR (22rv1), and not in PC3 cells not expressing the AR. (C) Upper figure: DHE fluorescence measurements (representative microscopic view of the DHE fluorescence of 22rv1 cells in the presence and in the absence of R1881 and/or bicalutamide). Lower figure: Quantification of bROS levels (as measured by confocal fluorescent microscopy) following normalization to cells' metabolic activity (measured using WST-1 assay) under different hormonal manipulations. CT = control cells growing in a culture medium with CSFCS and without phenol red; T = cells growing in the same medium but with the addition of 10 nM R1881; T + Bic = cells growing in the same medium but with the addition of both 10 nM R1881 and 10 µM bicalutamide. Results are derived from at least three experiments and are expressed as mean ± SD.

Figure 3

Androgens increase bROS levels in an AR-dependent manner. In vivo effect on human PC xenografts. (A) In vivo expression of oxidative stress-induced lipid peroxidation as detected by the presence of 4-HNE protein adducts, judging by immunoblotting with anti-4-HNE antibodies. WISH-PC23, WISH-PC14, and CWR22 xenografts were grown as subcutaneous tumors in castrated (androgen^-) or testosterone-supplemented (androgen⁺) SCID mice. (B) In vivo expression of 8-OHdG in WISH-PC14, WISH-PC23, and LuCAP-35 human PC xenografts grown as subcutaneous tumors in castrated (A^-) or testosterone-supplemented (A⁺) SCID mice (original magnification, x400).

Figure 4

Androgens induce the activation and expression of stress proteins in 22rv1 cells that can be reverted by AD. (A) pAKT(ser473) expression. (B) pp38 expression. (C) pp38 activity as determined by the phosphorylation of the ATF-2 client protein. (D) Clusterin expression. (A–D) Lower panels show relative expression in graphs. Results are derived from a minimum of three experiments and are expressed as mean ± SD. (E) The expression of HSP70, HSP27, and HSP90 in 22rv1 cells is not differentially affected by androgen supplementation or deprivation, but is increased in response to oxidative stress (overnight incubation with 50 µM H₂O₂). CT = control cells growing in a culture medium with CSFCS and without phenol red; Bic = cells growing in the same medium but with the addition of 10 µM bicalutamide; T = cells growing in the same medium but with the addition of 10 nM R1881; T + Bic = cells growing in the same medium but with the addition of both 10 nM R1881 and 10 µM bicalutamide. Results are derived from at least three experiments and are expressed as mean ± SD.

Figure 5

In vivo effects of androgen supplementation and deprivation on the expression of stress molecules and antioxidative enzymes. Androgens induce in vivo activation of the stress molecules pAKT and p38, and the expression of clusterin, the antioxidative enzyme MnSOD, and catalase, as documented in three human PC xenografts: WISH-PC23, WISH-PC14, and CWR22. These tumors were grown in castrated (androgen^-) or testosterone-supplemented (androgen⁺) SCID mice. Note that the global expression of pAKT and p38 is unchanged by androgen supplementation or deprivation, as opposed to their activated (phosphorylated) forms.

Figure 6

Androgens increase the antioxidative capacity of 22rv1 cells. (A) Dose-dependent effect of R1881 on the antioxidative capacity of the cells. (B) AD, using 10 µM bicalutamide, can reduce the antioxidative capacity of 22rv1 cells that are supplemented with 10 nM R1881. (C) The expression and activity of catalase and MnSOD (D and E) in 22rv1 cells are significantly increased by androgen supplementation and are reduced by AD. (F) The activity of GR in 22rv1 cells is significantly increased by androgen supplementation and is reduced by AD. Results are derived from at least three experiments and are expressed as mean ± SD. CT = control cells growing in a culture medium with CSFCS and without phenol red; Bic = cells growing in the same medium but with the addition of 10 µM bicalutamide; T = cells growing in the same medium but with the addition of 10 nM R1881; T + Bic = cells growing in the same medium but with the addition of both 10 nM R1881 and 10 µM bicalutamide.

Figure 7

The androgen-induced activation and expression of stress molecules and antioxidative enzymes stem from their induction by elevated bROS. (A) Incubation with 5 mM NAC reduced bROS under all hormonal conditions. Upper figure: DHE fluorescent test (representative microscopic view of the DHE fluorescence of 22rv1 cells in the presence and in the absence of R1881 and/or bicalutamide). Lower figure: Quantification of bROS levels (as measured with confocal fluorescent microscopy) following normalization to the cells' metabolic activity under different hormonal manipulations. Combined preincubation of 22rv1 cells with both R1881 and the reducing agent NAC erases the differential effects of different hormonal treatments on the expression of clusterin (B), pAKT(ser473) (C), and pp38 (D). (E) Combined preincubation of 22rv1 cells with both R1881 and the reducing agent NAC abolishes relative resistance to radiation, which is conferred to cells by androgens. Results show the survival of 22rv1 cells in a colony formation assay following a single treatment with 3-Gy radiation. CT = control cells growing in a culture medium with CSFCS and without phenol red; Bic = cells growing in the same medium but with the addition of 10 µM bicalutamide; T = cells growing in the same medium but with the addition of 10 nM R1881; T + Bic = cells growing in the same medium but with the addition of both 10 nM R1881 and 10 µM bicalutamide. Results are derived from at least three experiments and are expressed as mean ± SD.

Figure 8

Proposed model of androgen-driven adaptation for radiation therapy in PC. Androgens induce higher bROS in PC cells, resulting in both the increased expression and activity of stress molecules (pAKT and pp38 protein kinases) and clusterin, and the increased activity of antioxidative enzymes stimulating cellular antioxidant capacity. AD therapy reduces bROS levels, thus overturning the increased activity of stress molecules and antioxidative enzymes and facilitating oxidative stress-based therapies such as radiation.