Androgens induce oxidative stress and radiation resistance in prostate cancer cells though NADPH oxidase

Abstract

Androgen deprivation therapy (ADT) facilitates the response of prostate cancer (PC) to radiation. Androgens have been shown to induce elevated basal levels of reactive oxygen species (ROS) in PC, leading to adaptation to radiation-induced cytotoxic oxidative stress. Here, we show that androgens increase the expression of p22(phox) and gp91(phox) subunits of NADPH oxidase (NOX) and ROS production by NOX2 and NOX4 in PC. Pre-radiation treatment of 22Rv1 human PC cells with NOX inhibitors sensitize the cells to radiation similarly to ADT, suggesting that their future usage may spare the need for adjuvant ADT in PC patients undergoing radiation.

Figure 1

Effects of androgen on reactive oxygen species (ROS) production in 22Rv1 cells is abolished by inhibitors of NADPH oxidase (NOX), diphenyleneiodonium (DPI) and apocynin, as well as the general antioxidant N-acetylcysteine (NAC). (a) Nitroblue tetrazolium (NBT) reduction assay. (b) Dihydroethidium (DHE) staining top- fluorescent microscopy view ( × 400) and bottom- calculated relative fluorescent intensity. C= androgen-depleted medium comprising phenol-free medium and 10% charcoal-stripped fetal calf serum (CSFCS). R= same medium as C+10 nM R1881. R+B= same medium as C+10 nM R1881 and 10 μM bicalutamide.

Figure 2

Androgens increase the mRNA levels of NOX 2 and NOX4 enzymes compared with treatment with no androgens (C), treatment with R1881 (R) or the combination of R1881 and bicalutamide (R+B). (0)− mRNA control.

Figure 3

Androgens increase the protein expression of gp91^phox in human prostate cancer. (a) In-vitro (22Rv1 cells). C= androgen-depleted medium comprising phenol-free medium and 10% charcoal-stripped fetal calf serum (CSFCS). R= same medium as C+10 nM R1881. R+B= same medium as C+10 nM R1881 and 10 μM bicalutamide. (b) In-vivo. A+ testosterone supplemented, A− castrated SCID mice.

Figure 4

Androgens increase the protein expression of p22^phox in human prostate cancer. (a) In-vitro (22Rv1 cells). C= androgen-depleted medium comprising phenol-free medium and 10% charcoal-stripped fetal calf serum (CSFCS). R= same medium as C+10 nM R1881. R+B= same medium as C+10 nM R1881 and 10 μM bicalutamide. (b) In-vivo. A+ testosterone supplemented, A- castrated SCID mice.

Figure 5

The effect of apocynin on the in-vitro response of 22Rv1 cells to a single dose of 3 Gy radiation. C= androgen-depleted medium comprising phenol-free medium and 10% charcoal-stripped fetal calf serum (CSFCS). R= Same medium as C+10 nM R1881. R+B= same medium as C+10 nM R1881 and 10 μM bicalutamide.

Figure 6

Proposed model for the reversal of androgen-driven adaptation to radiation therapy in prostate cancer cells by apocynin or androgen deprivation therapy. Androgens increase the expression of NOX2, NOX4, p22^phox and gp91^phox resulting in increased basal production of reactive oxygen species (ROS). The cells in response, develop adaptive molecular machinery to protect from ROS accumulation, which leads to relative resistance to the toxic oxidative stress induced by radiation (as describe in details in reference 8). Inhibition of ROS production by NOX2 and NOX4 using apocynin or androgen deprivation therapy, reduces the basal ROS levels and prevents adaptive changes to oxidative stress, thus sensitizing the cells to the toxic oxidative effects of radiation.