Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells

<div><p>Androgen receptor (AR) signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT) treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS), TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP) in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2) were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1) were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins, such that androgen signaling sensitizes mitochondria to apoptotic signaling, thus rendering HPr-1AR more vulnerable to cell death signals. Our study offers insight into AR-mediated regulation of prostate epithelial cell death signaling.</p></div

Androgen-sensitized apoptosis of HPr-1AR cells involves caspase activation.

<p>(A) HPr-1AR cells were treated with 10 nM DHT or vehicle control for 18 hours and co-treated with 1 μM STS for 0 to 10 hours. Immunoblot analysis was performed using antibodies that detect the cleaved and active forms of caspase-9 (35 kDa) and caspase-3 (19 and 17 kDa). HPr-1AR cells pretreated with DHT show rapid activation of caspase-9 and caspase-3 upon STS co-treatment, whereas DHT or STS treatment alone show little or no caspase activation. (B) Cells were treated with 1 nM DHT or vehicle control in the absence or presence of 5–10 μM ENZ for 18 hours and then co-treated with 1 μM STS or vehicle control for 6 hours. The DHT-induced cleavage of caspase-9 and caspase-3 in STS-treated HPr-1AR cells is completely suppressed by AR antagonist, ENZ. (C) Cells were treated with 1–10 nM DHT or vehicle control for 18 hours and then co-treated with TNFated or vehicle control for 10 hours. Immunoblot analysis was performed using an additional antibody to detect the cleaved and active form of caspase-8 (18 kDa), an initiator caspase that is activated in response to extrinsic apoptotic stimuli, such as TNFe. DHT and TNF and synergistically enhance cleavage of caspase-9 and caspase-3, whereas DHT or TNF or treatment alone shows no significant activation of caspase-9 or caspase-3. The arrows and corresponding molecular weights indicate the different caspase forms. (D) HPr-1AR cells were treated with 1 nM DHT or vehicle control for 24 hours and co-treated with 200 μM H₂O₂ for 0 to 10 hours. HPr-1AR cells pretreated with DHT show rapid activation of caspase-3 upon H₂O₂ co-treatment, whereas DHT or H₂O₂ alone show little or no caspase-3 activation. (E) RWPE-AR cells were treated with 1 nM DHT or vehicle control for 38 hours and then co-treated with 1 μM STS or vehicle control for 5 to 10 hours. RWPE-AR cells pretreated with DHT show rapid and robust activation of caspase-3 upon STS co-treatment (11-fold at 5 hours and 23-fold at 10 hours) compared to RWPE-AR cells pretreated with vehicle as a control. Immunoblot results were quantified and represented as the mean ± SEM (n ± S. Comparisons between different treatments were performed using two-way ANOVA followed by Tukey's honest significant difference test.</p

Androgen and staurosporine synergize to induce mitochondrial depolarization in HPr-1AR.

<p>(A-E) HPr-1AR cells were treated with 10 nM DHT or vehicle control for 20 hours and then co-treated with 2 μM STS or vehicle control for 4.5 hours. Cells treated with vehicle control were used as the negative control. In addition, 2 μM CCCP was added to an aliquot of the negative control cells to depolarize their mitochondria and generate the positive control. Cells were stained with JC-1 dye and analyzed using flow cytometry. For each treatment, the ratios of red to green fluorescent intensities are displayed in histograms. Blue brackets at the left of each panel specify the population of cells with depolarized mitochondria. Normal cells (A) have a wide and bell-shaped distribution of JC-1 stained mitochondria, whereas cells with depolarized mitochondria have a sharp and left-shifted distribution (B). (F) Quantification of the depolarized cell proportion for each treatment revealed that DHT or STS alone do not significantly increase mitochondrial depolarization. However, co-treatment with DHT and STS significantly increases the depolarized population by 4-fold or more. Data represent the mean ± SEM (n = 4). Comparisons between different treatments were performed using two-way ANOVA followed by Tukey’s honest significant difference test.</p

Anchoring visual search in scenes : assessing the role of anchor objects on eye movements during visual search

The arrangement of the contents of real-world scenes follows certain spatial rules that allow for extremely efficient visual exploration. What remains underexplored is the role different types of objects hold in a scene. In the current work, we seek to unveil an important building block of scenes—anchor objects. Anchors hold specific spatial predictions regarding the likely position of other objects in an environment. In a series of three eye tracking experiments we tested what role anchor objects occupy during visual search. In all of the experiments, participants searched through scenes for an object that was cued in the beginning of each trial. Critically, in half of the scenes a target relevant anchor was swapped for an irrelevant, albeit semantically consistent, object. We found that relevant anchor objects can guide visual search leading to faster reaction times, less scene coverage, and less time between fixating the anchor and the target. The choice of anchor objects was confirmed through an independent large image database, which allowed us to identify key attributes of anchors. Anchor objects seem to play a unique role in the spatial layout of scenes and need to be considered for understanding the efficiency of visual search in realistic stimuli

Androgen and staurosporine synergize to decrease the relative ATP concentration in HPr-1AR cells.

<p>(A) HPr-1AR cells were treated with a range of DHT concentrations or vehicle control for 18 hours and then co-treated with 1 μM STS or vehicle control for 6 hours. Relative ATP concentrations available for biochemical processes in metabolically active cells were quantified using a luciferase-based bioassay for relative ATP levels in cultured cells. In comparison to vehicle control, STS and to a lesser extent DHT significantly decrease the relative ATP concentration of HPr-1AR cells at 24 hours. In addition, ANOVA revealed significant interaction between 1 μM STS and 0.1–10 nM DHT, which is visually evident from the unparallel trends of the white bars and black bars in the plot. Estimates of the interaction effect and corresponding p-values are indicated. Negative interaction terms indicate synergy whereas positive values indicate antagonism between DHT and STS. (B) Cells were treated with 10 nM DHT or vehicle control for 10 hours and then co-treated with 0.5 μM STS for 0, 3, 6, or 9 hours (h). In comparison to control-treated HPr-1AR cells (circles), DHT-treated HPr-1AR cells (squares) had 40%, 72%, and 76% reductions in ATP levels after 3, 6, and 9 hours of STS co-treatment, respectively. For time course analysis, significance differences between androgen treatment and vehicle control were determined at each time point using Student’s t-test and adjusted using the Bonferroni method, * <i>P</i> < 0.05. (C) Cells were treated with 1 nM DHT or vehicle control in the absence or presence of 5 μM enzalutamide (ENZ) for 18 hours and then co-treated with 1 μM STS or vehicle control for 6 hours. AR antagonist, ENZ significantly suppresses the synergistic interaction between DHT and STS. (D) Cells were treated with 5 STS. (suppresses the es between andof CDK4/6 kinase activity, for 18 hours to mimic the inhibitory effect of DHT on HPr-1AR cell cycle progression and growth, and then these cells were co-treated with 1 μM STS or vehicle control for 6 hours. The positive interaction term indicates that the synergy between DHT and STS on ATP depletion is not dependent on growth suppression and suggests an antagonistic effect between STS and PD0332991. Data represent the mean ± SEM (n ta4).</p

AR-mediated transcriptional regulation of apoptotic genes in HPr-1AR.

<p>(A) Cells were treated with 10 nM DHT or vehicle control for 24 hours. Total RNA was isolated, cDNA was synthesized by reverse transcription, and the relative mRNA levels of apoptosis-related genes were quantified by QPCR analysis. The colorimetric representation shows genes, indicated by HUGO Gene Nomenclature Committee gene symbols, whose transcripts were induced (red), unresponsive (black), and repressed (green) by androgen. The color intensity reflects the relative fold change (brightest red = 4-fold increase and brightest green = 4-fold decrease) in transcript level for DHT- versus vehicle control (CTL)-treated cells (n = 3). The 2-fold cutoff boundaries (orange lines) and 1.5-fold cutoff boundaries (gray lines) were determined from the mean change in expression level for DHT- versus CTL-treated cells. (B) Cells were treated with 1 nM DHT or vehicle control in the absence or presence of 5 μM ENZ for 24 hours. QPCR analysis demonstrates that the pro-apoptotic genes (BCL2L11 and AIFM2) are DHT-induced, whereas the pro-survival genes (BCL2L1 and MCL1) are DHT-repressed. Further, the AR antagonist, ENZ, completely suppressed the androgen-responsive mRNA changes. Data represent the mean ± SEM (n = 3) in log₂ scale. Comparisons between different treatments were performed using two-way ANOVA followed by Tukey’s honest significant difference test. (C) HPr-1AR cells were treated with 10 nM DHT for 24 hours. Immunoblot analysis of BCL2 family gene products demonstrates that the expression of pro-apoptotic BCL2L11/BIM is androgen-induced, whereas pro-survival proteins, BCL2L1/BCL-XL and MCL1, are androgen-repressed. Representative immunoblots (left panel) and the quantified results (right panels) are shown. Data represent the mean ± SEM (n = 4). Comparisons between DHT- versus vehicle-treated cells were made using Student’s t-test, * <i>P</i> < 0.05.</p

Transcription and protein synthesis are necessary for androgen-sensitized apoptosis of HPr-1AR.

<p>(A) Cells were treated with 1 nM DHT or vehicle control in the absence or presence of transcription inhibitor, 20 in the5,6-dichlororibofuranosylbenzimidazole (DRB), for 16 hours, and then these cells were co-treated with 1 μM STS or vehicle control for 4 hours to induce apoptosis. Cells were harvested, stained with annexin V and PI, and the intensities of annexin V and PI stained cells were quantified by flow cytometry. DRB treatment significantly suppressed the androgen-sensitized apoptosis of HPr-1AR. (B) HPr-1AR cells were treated with 1 nM DHT or vehicle control in the absence or presence of protein synthesis inhibitor, 25 μg/mL CHX, for 16 hours, and then these cells were co-treated with 1 μM STS or vehicle control for 4 hours to induce apoptosis. Cells were harvested, stained with annexin V and PI, and analyzed by flow cytometry. CHX co-treatment completely suppressed the androgen-sensitized apoptosis of HPr-1AR. Data represent the mean (n = 3). Comparisons between multiple treatment groups were performed using three-way ANOVA followed by Tukey's honest significant difference test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156145#pone.0156145.s005" target="_blank">S2 Table</a>). (C) Immunoblot analysis of cell lysates reveals that DHT-induced caspase-3 cleavage in STS-treated HPr-1AR cells is significantly suppressed by the inhibition of transcription (DRB) and protein synthesis (CHX).</p

Androgen sensitizes HPr-1AR and RWPE-AR to apoptotic cell death.

<p>(A) HPr-1AR cells were treated with 1 nM DHT or vehicle control in the absence or presence of 5 μM ENZ for 19 hours and then co-treated with 1 μM STS or vehicle control for 5 hours. Cells were harvested, stained with annexin V and PI, and the fluorescence intensities of annexin V and PI stained cells were quantified by flow cytometry. Viable live cells (annexin V-negative and PI-negative, gray dots), early apoptotic cells (annexin V-positive and PI-negative, blue dots), and late apoptotic cells (annexin V-positive and PI-positive, orange dots) are indicated. (B) Quantification of the fraction of viable live (gray bar with black number), early apoptotic (blue bar with white number), and late apoptotic cells (orange bar with gray number) is shown from the dot plots in Fig 2A. DHT treatment alone does not trigger cell death in HPr-1AR. However, DHT sensitizes HPr-1AR to STS-induced apoptosis. In addition, AR antagonist, ENZ, suppresses the synergistic interaction between DHT and STS, which significantly increases the live cell proportion. (C) HPr-1AR cells were treated with 1 nM DHT or vehicle control for 12 hours and then co-treated with apoptosis inducer, TNFoptos, or vehicle control for 11 hours. The fluorescence intensities of annexin V and PI stained cells were then quantified by flow cytometry. DHT sensitizes HPr-1AR cells to apoptotic death induced by TNF sens. (D) HPr-1AR cells were treated with 1 nM DHT or vehicle control for 20 hours and then co-treated with apoptosis inducer, H₂O₂, or vehicle control for 24 hours. The fluorescence intensities of annexin V and PI stained cells were then quantified by flow cytometry. DHT sensitizes HPr-1AR cells to apoptotic death induced by H₂O₂. (E) RWPE-AR cells were treated with 1 nM DHT or vehicle control in the absence or presence of 5 μM ENZ for 30 hours and then co-treated with 1 μM STS or vehicle control for 10 hours. The fluorescence intensities of annexin V and PI stained cells were then quantified by flow cytometry. DHT treatment alone does not induce cell death in RWPE-AR. However, DHT sensitizes RWPE-AR to STS-induced apoptosis. Further, ENZ co-treatment completely suppresses the synergistic interaction between DHT and STS, fully rescuing the live cell proportion of RWPE-AR. Data represent the mean (n yn3). Comparisons between multiple treatment groups were performed using three- or two-way ANOVA followed by Tukey's honest significant difference test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156145#pone.0156145.s005" target="_blank">S2 Table</a>).</p