Figure 1.
AhR mRNA and protein expression in prostate cancer cell lines.
A. Total cellular proteins were isolated from LNCaP, DU145, PC3 and PC3M prostate cancer cell lines. Proteins were separated by SDS polyacrylamine gel electrophoresis and blotted using an anti-AhR antibody. Anti-β-actin was used as a loading control. Image J was used to obtain desitometric measures from 3 independent membranes. Each bar represents mean±SEM (n = 3) and were analyzed by student t-test. Statistically significant differences (*p<0.05) compared to LNCaP control. B. Total RNAs were isolated and quantitative RT-PCR was performed to determine the mRNA expression of AhR in the prostate cancer cell lines. mRNA levels were normalized using L-19 which serves as an internal control. Each bar represents mean±SEM (n = 3) and were analyzed by student t-test. (*) denotes statistically significant differences between groups.
Figure 2.
Subcellular localization of AhR in prostate cancer cells.
A. Nuclear and cytoplasmic fractionation: Cell lines were grown on 100 mm dishes until ∼75% confluent, washed with cold PBS and cellular fractions were isolated by per manufactures instructions using a NE-PER Extraction kit. The nuclear and cytoplasmic fractions were analyzed by western blotting for AhR protein expression. The relative level of cytoplasmic AhR was normalized with β-tubulin expression and the relative level of nuclear AhR was normalized with topoisomerase expression. Bars represent mean±SEM of the corrected values from three independent experiments and (*) denotes constitutive nuclear levels of AhR that are significantly different between cell lines. B. Subcellular localization of AhR in prostate cancer cell lines by immunocytochemical staining: Cells were grown on coverslips and fixed with methanol:acetone. AhR was visualized by staining with rabbit anti-AhR polyclonal antibodies followed by FITC-conjugated goat anti-rabbit antibody. The nuclei were counter-stained with DAPI fluorescence dye. Images from FITC and DAPI-fluorescence channels were merged. Images were captured on an Olympus wide fluorescence microscope (400x magnification).
Figure 3.
Constitutive AhR transcriptional activity in advanced prostate cancer cell lines.
A. Each prostate cancer cell line was transfected with an XRE reporter plasmid, as well as with positive and negative control reporter plasmids using attractene. Following transfection, a dual luciferase assay was performed. Promoter activity values are expressed as arbitrary florescence units (AFU). Each bar represents mean±SEM (n = 3) and were analyzed by student t-test. (*) denotes statistically significant differences (*P<0.05). B. qRT-PCR analysis of CYP1B1 mRNA expression in prostate cancer cells. Cells were treated with 50 µM of AhR inhibitor (CH223191) or vehicle control (DMSO) for 24 h and total RNAs were isolated and quantitative RT-PCR was performed to determine the mRNA expression of CYP1B1 in each prostate cancer cell lines. mRNA levels were normalized using L-19 which serves as an internal control. Each bar represents mean±SEM (n = 3) and were analyzed by student t-test. (*) denotes statistically significant differences (*P<0.05) compared to LNCaP prostate cancer cell line.
Figure 4.
Inhibition of AhR signaling decreases growth of prostate cancer cells.
A. Cells were grown in a 96 well plate at 5.0×103 cells per well. The cells were treated with DMSO or 1 µM, 5 µM, 10 µM, 25 µM or 50 µM of CH223191. Cell growth was measured using Promega CellTiter 96 Cell Proliferation Assay per manufactures instructions. B. Confirmation of AhR protein expression in DU145 cells with scrambled vector control (SCR) and shRNA lentivirus against AhR (-AhR) by western blotting. 20 µg of protein isolated from was analyzed by western blotting. β-action is used as a loading control. Proliferation of DU145(SCR) and DU145(-AhR) cells were analyzed using the CellTiter 96 Cell Proliferation Assay in the presence and absence of 50 µM of CH223191. C. The cells were grown in charcoaled stripped media and treated with DMSO (CON), 20 µM casodex (CDX), 50 µM CH223191 or a combination of CDX and CH223191 for 72 hours. Cell growth was measured using Promega CellTiter 96 Cell Proliferation Assay per manufactures instructions. Each bar represents mean±SEM (n = 3), *p<.05. D. Cells were serum starved for 24hrs and grown in charcoal stripped (CSS) media. Cells where then treated with DMSO (Con) or 50 µM CH223191 for an additional 24, 48 or 72 hours. At each endpoint, cells were analyzed for DNA content using the cell proliferation assay as described in the materials and methods. Each data point represents mean±SEM (n = 3). (*) denotes a significant difference compared to respective DMSO control.
Figure 5.
AhR expression in prostate cancer tissues.
A. Representative Grade 1, Grade 2 and Grade 3 prostate cancer tissues: Upper panel stained with anti-AhR polyclonal antibody; lower panel stained with hematoxylin and eosin. The arrows depict positive anti-AhR reactivity in the nucleus of grade 2 and grade 3 tumors. B. Total anti-AhR reactivity was scored in grade 1, grade 2 and grade 3 prostate cancer tissues based upon intensity level in both cytoplasm and nucleus (0–1). Total AhR score was determined by combining individual cytoplasmic and nuclear scores.
Table 1.
Cytoplasmic and nuclear expression of AhR in prostate cancer tissues.
Table 2.
AhR reactivity by Gleason score: Prostate cancer tissues were catorized based upon Gleason score 2–6 (well differentiated), 7 (moderately differentiated) or 8–10 (poorly differentiated).