Growth inhibition of androgen-responsive prostate cancer cells with brefeldin A targeting cell cycle and androgen receptor

Abstract

Background: Androgen ablation is one of the viable therapeutic options for patients with primary hormone (androgen)-dependent prostate cancer. However, an antibiotic brefeldin A (BFA) has been shown to exhibit the growth inhibitory effect on human cancer cells. We thus investigated if BFA might inhibit proliferation of androgen-responsive prostate cancer LNCaP cells and also explored how it would be carried out, focusing on cell cycle and androgen receptor (AR).

Methods: Androgen-mediated cellular events in LNCaP cells were induced using 5alpha-dihydrotestosterone (DHT) as an androgenic mediator. Effects of BFA on non-DHT-stimulated or DHT-stimulated cell growth were assessed. Its growth inhibitory mechanism(s) was further explored; performing cell cycle analysis on a flow cytometer, assessing AR activity by AR binding assay, and analyzing AR protein expression using Western blot analysis.

Results: DHT (1 nM) was capable of stimulating LNCaP cell growth by ~40% greater than non-stimulated controls, whereas BFA (30 ng/ml) completely inhibited such DHT-stimulated proliferation. Cell cycle analysis showed that this BFA-induced growth inhibition was associated with a ~75% reduction in the cell number in the S phase and a concomitant increase in the G1 cell number, indicating a G1 cell cycle arrest. This was further confirmed by the modulations of specific cell cycle regulators (CDK2, CDK4, cyclin D1, and p21WAF1), revealed by Western blots. In addition, the growth inhibition induced by BFA was accompanied by a profound (~90%) loss in AR activity, which would be presumably attributed to the significantly reduced cellular AR protein level.

Conclusions: This study demonstrates that BFA has a potent growth inhibitory activity, capable of completely inhibiting DHT (androgen)-stimulated LNCaP proliferation. Such inhibitory action of BFA appears to target cell cycle and AR: BFA led to a G1 cell cycle arrest and the down-regulation of AR activity/expression, possibly accounting for its primary growth inhibitory mechanism. Thus, it is conceivable that BFA may provide a more effective therapeutic modality for patients with hormone-dependent prostate cancer.

Figure 1

Effects of BFA on LNCaP cell growth. (A) Dose-dependent effects of BFA on LNCaP cell growth were assessed: after cells were cultured with 0-50 ng/ml of BFA for 72 h, those viable cell numbers were determined and expressed by the percent (%) relative to cell number in control (100%). (B) Effects of BFA on DHT-stimulated cell growth were examined: cells were grown with DHT (1 nM), BFA (30 ng/ml), or their combination for 72 h, and cell growth was assessed by the % of controls. All data are mean ± SD (standard deviation) from three separate experiments (*p < 0.05 compared with controls).

Figure 2

Effects of BFA on cell cycle. (A) LNCaP cells were treated with DHT (1 nM), BFA (30 ng/ml), or BFA/DHT combination for 72 h and cell cycle analysis was performed as described in Methods. Cell cycle phase distributions or the number (%) of cells present at the G₁, S, or G₂/M phases in each experimental condition was determined and plotted. The data were mean ± SD from three separate experiments and subjected to statistical analysis; however, only those mean values (without SD) were used for plotting the graph for a clear illustration. (B) After cells were treated with or without BFA (30 ng/ml) for 72 h, the expressions of specific cell cycle regulators such as CDK2, CDK4, cyclin D₁, and p21^WAF1 were analyzed on Western blots and autoradiographs of those regulators (on the X-ray film) are shown.

Figure 3

Effects of BFA on androgen receptor (AR) binding activity. Cells were cultured with BFA (30 ng/ml) for 24, 48, or 72 h, and AR binding assays were performed as described in Methods. Specific AR activity was calculated and expressed by cpm incorporated/10⁶ cells. The data are mean ± SD from three independent experiments (*p < 0.03 compared with controls at 0 h).

Figure 4

Effects of BFA on AR expression. (A) After cells were cultured with BFA (30 ng/ml), DHT (1 nM), or BFA/DHT combination for 72 h, cell lysates (10 μg) were prepared and analyzed for AR expression using Western blots and autoradiograph of those AR protein bands (110 kDa) is shown. Beta (β)-actin is also shown as a protein loading control. (B) Following the exposure of cells to BFA (30 ng/ml) for 0, 6, 12, or 24 h, the time-dependent reduction in AR expression was analyzed on Western blots. Intensities of AR expressions detected on autoradiograph were quantified using a scan densitometer and expressed by the arbitrary values (*p < 0.05) relative to controls (at 0 h) normalized to 100. The data are mean ± SD from three independent experiments.

Figure 5

Effect of BFA on PSA secretion regulated by DHT. Cells were cultured with BFA (30 ng/ml), DHT (1 nM), or BFA/DHT combination for 24, 48, and 72 h. Spent media were collected at each time point and assayed for secreted PSA. The amount of PSA secreted was expressed by ng/ml and all data are mean ± SD from three independent experiments (*p < 0.05; **p < 0.005 compared with controls).