Semenogelin I promotes prostate cancer cell growth via functioning as an androgen receptor coactivator and protecting against zinc cytotoxicity

Abstract

A seminal plasma protein, semenogelin I (SgI), contributes to sperm clotting, upon binding to Zn(2+), and can be proteolyzed by prostate-specific antigen (PSA), resulting in release of the trapped spermatozoa after ejaculation. In contrast, the role of SgI in the development and progression of any types of malignancies remains largely unknown. We previously demonstrated that SgI was overexpressed in prostate cancer tissues and its expression was enhanced by zinc treatment in LNCaP cells. In the current study, using cell lines stably expressing SgI, we investigated its biological functions, in conjunction with zinc, androgen, and androgen receptor (AR), in prostate cancer. Zinc, without SgI, inhibited cell growth of both AR-positive and AR-negative lines. Co-expression of SgI prevented zinc inhibiting dihydrotestosterone-mediated proliferation of AR-positive cells, whereas SgI and/or dihydrotestosterone showed marginal effects in AR-negative cells. Similar effects of SgI overexpression in LNCaP on dihydrotestosterone-induced cell invasion, such as its significant enhancement with zinc, were seen. Overexpression of SgI in LNCaP and CWR22Rv1 cells also augmented dihydrotestosterone-mediated PSA expression (mRNA, protein) in the presence of zinc. However, culture in the conditioned medium containing secreted forms of SgI failed to significantly increase cell viability with or without zinc. In luciferase reporter gene assays, SgI showed even slight inhibitory effects (8% and 15% decreases in PC3 and CWR22Rv1, respectively) at 0 μM zinc and significant stimulatory effects (2.1- and 3.2-fold) at 100 μM zinc on dihydrotestosterone-enhanced AR transactivation. Co-immunoprecipitation then demonstrated dihydrotestosterone-induced physical interactions between AR and SgI. These results suggest that intracellular SgI, together with zinc, functions as an AR coactivator and thereby promotes androgen-mediated prostate cancer progression.

Keywords: Androgen receptor; prostate cancer; prostate-specific antigen; semenogelin; zinc.

Figure 1

SgI protein expression and secretion in prostate cancer lines stably expressing SgI. Cell extracts (A) or acetone-precipitated proteins in conditioned media (serum-free, no additional zinc) (B) from LNCaP-V/SgI, DU145-V/SgI, PC3-V/SgI, and CWR22Rv1-V/SgI were analyzed on western blots, using an antibody to SgI (52 kDa) or β-actin (42 kDa). Fresh human seminal vesicle (SV) tissue was used as a positive control.

Figure 2

Cell viability of prostate cancer lines stably expressing SgI. CWR22Rv1-V/SgI (A), LNCaP-V/SgI (B), PC3-V/SgI (C), and DU145-V/SgI (D) were cultured in phenol red-free medium supplemented with 5% charcoal-stripped FBS in the presence or absence of 100 μM zinc and 1 nM DHT for 96 hours. CWR22Rv1 (E) and DU145 (F) were cultured in conditioned medium (containing 10% normal FBS) derived from CWR22Rv1-V/SgI culture in the presence or absence of 100 μM zinc for 96 hours. Proliferation was assayed with MTT, and growth rates are presented relative to cell number in respective lines with mock treatment [lanes 1 (A-F) and 5 (A-D); set as 100%]. Each value represents the mean + SD of at least three determinations.

Figure 3

Progression of prostate cancer lines stably expressing SgI. (A) LNCaP-V/SgI cells cultured in the Matrigel-coated transwell chamber for 36 hours in the presence or absence of 300 μM zinc and 1 nM DHT were used for transwell assay. The number of invaded cells in five random fields was counted under a light microscope, using a 40x objective. Invasion ability is presented relative to that in each cell line with mock treatment (lane 1 or 5; set as 1-fold). Each value represents the mean + SD of at least three independent experiments. (B) LNCaP-V/SgI cells cultured in phenol red-free medium supplemented with 5% charcoal-stripped FBS in the presence or absence of 300 μM zinc and 1 nM DHT for 48 hours were subjected to a quantitative RT-PCR. Expression of PSA gene was normalized to that of GAPDH. Transcription amount is presented relative to that of mock treatment in each cell line (lane 1 or 5; set as 1-fold). Each value represents the mean + SD from at least three independent experiments. CWR22Rv1 cells (C) transiently transfected with pSG5 or pSG5-SgI were cultured in phenol red-free medium supplemented with 5% charcoal-stripped FBS in the presence or absence of 100 μM zinc and 1 nM DHT for 48 hours, and LNCaP-V/SgI cells (D) were similarly cultured with 300 μM zinc ± 1 nM DHT for 48 hours, as indicated. Cell extracts were then analyzed on western blots, using an antibody to PSA (33 kDa) or β-actin.

Figure 4

The effects of SgI on AR transcriptional activity in prostate cancer cells. PC3 cells were co-transfected with pSG5-AR, MMTV-Luc, pRL-TK, and either pSG5 or pSG5-SgI (AR:SgI = 1:5), and cultured in phenol red-free medium supplemented with 5% charcoal-stripped FBS along with mock (ethanol), zinc [(A) 0 μM; (B) 15 μM; (C) 100 μM], and/or 1 nM DHT for 24 hours. Similarly, CWR22Rv1 cells were co-transfected with MMTV-Luc, pRL-TK, and either pSG5 or pSG5-SgI, and treated with mock (ethanol) or 1 nM DHT in the absence (D) or presence (E) of 100 μM zinc for 24 hours. The luciferase activity is presented relative to that of mock treatment (first lanes; set as 1-fold). Each value represents the mean + SD of at least three determinations.

Figure 5

Co-precipitation of AR and SgI. Cell lysates from 293T transfected with pSG5-AR and pSG5-SgI (A) or LNCaP (B) treated with mock (ethanol) or 1 nM DHT were incubated with an anti-AR polyclonal antibody or normal rabbit IgG and then with A/G-agarose beads. The complex was resolved on a 10% SDS-polyacrylamide gel and blotted with an anti-AR or anti-SgI antibody.