Fig 1.
Androgen and staurosporine synergize to decrease the relative ATP concentration in HPr-1AR cells.
(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 24 hours and then co-treated with 0.5 μM STS for 0, 3, 6, or 9 hours (h). In comparison to control-treated HPr1AR 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, * P < 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 μM PD0332991, a selective inhibitor of 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 ≥ 4).
Fig 2.
Androgen sensitizes HPr-1AR and RWPE-AR to apoptotic cell death.
(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, TNFα+CHX, 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 TNFα+CHX. (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 ≥ 3). Comparisons between multiple treatment groups were performed using three- or two-way ANOVA followed by Tukey's honest significant difference test (S2 Table).
Fig 3.
Androgen-sensitized apoptosis of HPr-1AR and RWPE-AR cells involves caspase activation.
(A) HPr-1AR and RWPE-AR 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 DHTinduced 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 cotreated with TNFα+CHX 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 TNFα. DHT and TNFα+CHX synergistically enhance cleavage of caspase-9 and caspase-3, whereas DHT or TNFα+CHX 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 ≥ 3). Comparisons between different treatments were performed using two-way ANOVA followed by Tukey's honest significant difference test.
Fig 5.
Transcription and protein synthesis are necessary for androgen-sensitized apoptosis of HPr-1AR.
Cells were treated with 1 nM DHT or vehicle control in the absence or presence of transcription inhibitor, 20 μg/mL 5,6-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 HPr1AR. Data represent the mean (n = 3). Comparisons between multiple treatment groups were performed using threeway ANOVA followed by Tukey's honest significant difference test (S2 Table). (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).