Article Figures & Data
Figures
- Figure 1.
Drp1 levels are positively correlated with AR status in prostate cancer cells. A, total cell lysates (20 μg) from the indicated prostate cancer cells were analyzed by Western blot analyses using specific antibodies against Drp1, AR, PSA, COX IV, and GAPDH. B, the above blots were scanned and the densitometric values for Drp1 and AR were normalized to GAPDH and plotted as a percentage of the values. The values for the cell line CWR-R1, which express the highest levels of Drp1 and AR were set at 100% and a correlation coefficient was calculated on the basis of these values using GraphPad Prism software. C, total RNA from prostate cancer cells was isolated, cDNA was prepared, and Drp1 mRNA levels were analyzed by qRT-PCR. Drp1 mRNA levels were normalized to GAPDH and the data expressed relative to mRNA levels of normal prostate epithelial cells, P69. D, Drp1 expression in prostate cancer (n = 7) and hormone-refractory metastatic prostate cancer (n = 6) were analyzed by Western blotting. Expression of PSA served as an indicator of androgen responsiveness, whereas GAPDH was used as loading control. CaP, prostate cancer.
- Figure 2.
Androgens upregulate Drp1 mRNA and protein expression. A, left, cells were grown in medium containing 9% FBS or CSS; right, cells were treated with R1881 (10 nmol/L) for 24 hours. Total cell lysates were analyzed by Western blotting for the levels of Drp1, PSA, and AR. Hsp90α was used as a loading control. B, total RNA was isolated, and Drp1 (left) and PSA (right) mRNA levels were measured by qRT-PCR (**, P ≤ 0.01; ***, P ≤ 0.001, n = 3). C, cells were treated with increasing concentrations of R1881 for 24 hours and total cell lysates were analyzed for the levels of Drp1 protein. D, cells were treated with R1881 for different periods and Drp1 mRNA expression was analyzed by qRT-PCR (P values were compared with the CSS controls, n = 3).
- Figure 3.
Decreasing AR function using the antiandrogen, bicalutamide, or siRNA resulted in decreased Drp1 expression. A and B, LNCaP cells were treated with varying concentrations of bicalutamide (BIC) in the presence of R1881 (10 nmol/L), and total cell lysates were analyzed by Western blotting (A) or total RNA was analyzed by qRT-PCR for Drp1 mRNA levels (B). For qRT-PCR, results represent the means ± SEM of 3 separate experiments with **, P < 0.01; ***, P < 0.001 relative to R1881 treatment in the absence of bicalutamide. C, LNCaP cells were transfected either with siRNA against AR or scrambled siRNA. Total cell lysates were analyzed for the expression of Drp1, AR, and β-actin (loading control). D, cells were treated with R1881 (10 nmol/L) for 8 or 12 hours, either in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX; 50 μg/mL, 1 hour before and during R1881 treatment). Drp1 mRNA levels were analyzed by qRT-PCR (left). Total cell lysates were analyzed for the expression of Drp1 (right). The results represent the means ± SEM of 3 separate experiments.
- Figure 4.
ChIP-seq and Illumina BeadArray expression analyses confirmed the presence of an AR-binding site in the Drp1 gene. A, AR ChIP-seq data analysis showing .wig data of an AR-binding site located in the Drp1 gene. As denoted by “KLK2” in the figure, the sequence in red denotes only AR-binding sites that are s enriched or more enriched than the binding site in kallikrein 2 (KLK2), a prostate-associated protease that activates PSA. The data predict an AR-binding site that is downstream of the RNA Pol II site at the start of the Drp1 (also known as dynamin-like protein 1 or DNM1L) gene, located at chr12: 32,724,000–32,725,500. B, LNCaP cells were treated with R1881 for different time periods and isolated mRNA was subjected to Illumina BeadArray analysis. The 2 left plots represent results obtained with 2 separate probes for Drp1. TMPRSS2 was analyzed as a positive control for an androgen-responsive gene. Open circles represent cells treated with androgen and blue circles with vehicle (EtOH). The analysis approach generates the autocorrelation factor (acf) for each gene as described previously (28).
- Figure 5.
Androgens regulate Drp1 in androgen-sensitive and androgen-independent (VCaP and C4-2) but not in androgen-refractory, AR-negative (DU145 and PC3) prostate cancer cells. A, androgen-sensitive VCaP cells were treated with R1881 and bicalutamide, and total cell lysates were analyzed by Western blotting for Drp1 expression. B, ADI C4-2 cells were treated and analyzed (as in A). C and D, androgen-refractory, AR-negative DU145 (C) and PC3 cells (D) were treated with R1881 (10 nmol/L) for 24 hours. All cells were grown in steroid-depleted medium for 48 hours before R1881 treatment and analyzed (as in A).
- Figure 6.
Androgen facilitated mitochondrial fission induced by CGP. Stably transfected mito-green LNCaP cells were treated with R1881 (1 nmol/L) for 24 hours with or without CGP (50 μmol/L) for the last 1 hour, the cells were washed with HBSS and live cells were observed in a confocal fluorescent microscope. A, representative photographs of cells in each treatment group is shown, illustrating the shape of the mitochondria. These pictures are from confocal imaging (Zen software) using “Z-stack” option and signals from all the planes were considered using the “Maximum Intensity Projection” option. B, cells exhibiting more than 80% punctate (fragmented) mitochondria were counted and are presented as a percentage of the total number of cells counted. At least 200 cells were examined in each dish and values represent the means ± SEM from at least 3 separate experiments with **, P ≤ 0.01; ***, P ≤ 0.001.
- Figure 7.
Androgen enhanced CGP-induced apoptosis: mediation by Drp1. A, androgen increased cell proliferation. LNCaP cells were treated with R1881 (1 nmol/L), bicalutamide (50 μmol/L), or CGP (50 μmol/L) as described above. Cell proliferation was measured using a Quick Cell Proliferation assay kit (BioVision). Data are expressed as means ± SEM, with the parentheses indicating the P values (n = 5; left). Right, cells were treated with R1881 (1 nmol/L) for 48 hours, fixed overnight with 70% ethanol, treated with RNase, and then stained with propidium iodide and analyzed using flow cytometry to determine the S-phase of the cell cycle (***, P ≤ 0.0002; n = 3). B, androgens increased phosphorylation of Drp1-Ser-616. LNCaP cells were treated with R1881 (1 nmol/L) and CGP (50 μmol/L for 18 hours) as described earlier. Cell lysates were subjected to Western blot analysis and Drp1 phosphorylation at Ser-616 and total Drp1 determined. Bottom, the densitometric values for phospho-Ser-616 Drp1 levels were normalized to the levels of total Drp1 in the above blot. C, androgens increased CGP-induced apoptosis. LNCaP cells were treated with R1881 (1 nmol/L) for 24 hours. Cells were treated with CGP (50 μmol/L) for an additional 4 or 18 hours in the presence or absence of R1881. Cell lysates were used for an apoptosis assay using a M30-Apoptosense analyses kit. Results are presented as the percentage of apoptosis compared with the CSS control (*, P ≤ 0.05; **, P ≤ 0.01; n = 3). D, DN-Drp1 reduced androgen-mediated CGP-induced apoptosis. LNCaP cells were transfected with DN-Drp1 and treated with R1881 (1 nmol/L) and CGP (50 μmol/L for 18 hours) as described earlier. Cell lysates were analyzed for apoptosis using an M30-Apoptosense analysis kit. Results are presented as the percentage of the respective controls (**, P ≤ 0.01; n = 3). E, CGP induced a decrease in Mfn1. LNCaP cells were treated (as in B), and total cell lysates were analyzed for Mfn1 and Mfn2 levels.
Volume 9, Issue 8
