Article Figures & Data
Figures
- Figure 1.
BCO2 expression is downregulated in human prostate cancer tissues and cell lines. A, BCO2 protein expression in normal human prostate, BPH, and prostate cancer tissues. The positive control lane (+) contains protein extracted from HEK-293 cells engineered to overexpress the bco2 gene. Western blot analysis was conducted using anti-rabbit antibody against BCO2 protein N-terminal peptide. A representative tissue blot is shown. B, BCO2 expression in normal PrEC and prostate cancer cell lines LNCaP, PC3, C4-2, and DU145. Cell lysates were isolated and 30 μg of protein was subjected to NuPAGE. Equal loading of protein was confirmed by reprobing with a β-actin monoclonal antibody. C, real-time RT-PCR of bco2 gene expression in normal PrEC and the prostate cancer cell lines LNCaP, PC3, C4-2 and DU145. Total RNA was isolated from indicated cell lines, and real-time RT-PCR was performed as described in “Materials and Methods.” Relative fold bco2 expression is presented as 2−ΔΔCt relative to the ΔΔCt of DU145 cells. Data, mean ± SD (n = 3). *, P < 0.05.
- Figure 2.
Demethylation induces bco2 expression in prostate cancer cells. Normal prostatic epithelial (PrEC) and prostate cancer (LNCaP, PC3, C4-2, DU145) cell lines were treated with 10 μmol/L of the methyltransferase inhibitor 5-aza-2dC or vehicle (DMSO) for 96 hours. Total RNA was isolated and real-time RT-PCR was performed as described in “Materials and Methods.” Relative fold bco2 expression is presented as 2−ΔΔCt relative to controls. Data, mean ± SD (n = 3). *, P < 0.05 compared with controls (DMSO).
- Figure 3.
Lycopene is differentially accumulated in prostatic epithelial cells and upregulates bco2 gene expression in specific prostate cancer cells. A, normal PrEC and prostate cancer cell lines were treated either with 1 or 3 μmol/L lycopene for 24 hours. Cellular lycopene was extracted and analyzed by HPLC in individual cell lines as described in “Materials and Methods.” Lycopene concentrations in PrEC and prostate cancer cell lines are presented as pmol per million (m) cells. B, prostate cancer cell lines (LNCaP and DU145) were treated with 1 μmol/L lycopene or vehicle (DMSO) for 24 hours. Total RNA was isolated and real-time RT-PCR was performed as described in “Materials and Methods.” Relative fold expression is presented as 2−ΔΔCt compared with DMSO control. Data, mean ± SD (n = 3). *, P < 0.05, lycopene concentration versus control.
- Figure 4.
Lycopene and a lycopene metabolite inhibit cell growth in specific prostate cancer cells. In these representative experiments, LNCaP cells (8 × 104) were seeded onto 6-well plates, then treated with 1 μmol/L lycopene or apo-10-lycopenal, or vehicle (DMSO) for up to 3 days. Every 24 hours, duplicate samples were trypsinized and counted. Trypan blue exclusion showed no differences in cell death among different conditions (data not shown). Data, mean ± SD (n = 3). *, P < 0.05, lycopene concentration versus control, or apo-10-lycopenal versus control. A, LNCaP cells. B, DU145 cells.
- Figure 5.
Reexpression of BCO2 inhibits prostate cancer cell proliferation and colony formation. LNCaP (A) or DU145 (B) cells were transiently transfected with an empty vector (pcDNA3), or BCO1 or BCO2 expression constructs for 24 hours, then treated with 1 μmol/L lycopene or DMSO control for 24 hours. Cell number was estimated by the MTT method as described in “Materials and Methods.” Data, mean ± SD (n = 3). *, P < 0.05, BCO2 expression versus vector control. C, Western blot analysis of BCO2 protein expression in pcDNA3, BCO2, or BCO2-mt transfected DU145 cells at 24 hours posttransfection (β-actin detection as loading control). D, colony formation assay for transfected cell lines described in C. Quantification of colonies per well from nine 3-cm dishes for transfected cell lines as above. Data, mean ± SD (n = 3). *, P < 0.05.
- Figure 6.
Exogenous BCO2 expression inhibits NF-κB transcriptional activity in prostate cancer cells. A, DU145 cells were transiently transfected with a NF-κB luciferase reporter, β-gal (transfection efficiency control), and the indicated expression constructs for 24 hours followed by treatment with either 1 μmol/L lycopene or vehicle for 24 hours; cells were lysed and luciferase assays performed. Data represent mean ± SD (n = 3) normalized to β-gal activity (*, P < 0.05, BCO2, or BCO2-mt expression vs. control). B, C4-2 cells were transiently transfected with the NF-κB-luc reporter gene, β-gal, and the indicated constructs for 24 hours, and then treated with 1 μmol/L lycopene or DMSO for 20 hours. After stimulation with TNFα (10 ng/mL) for 3 hours, cells were assayed for luciferase activity. Data represent mean ± SD (n = 3) normalized to β-gal (*, P < 0.05, BCO2 or BCO2-mt expression vs. control).
- Figure 7.
BCO2 expression does not affect IκBα levels but reduces NF-κB nuclear translocation and DNA binding. A, C4-2 cells were transiently transfected with the indicated constructs for 24 hours and treated with TNFα (10 ng/mL) for 3 hours. Total cellular protein lysates were isolated and IκBα was detected by Western blot analysis using an IκBα-specific antibody and β-actin detection as a loading control. B, DU145 cells were transiently transfected with the indicated constructs for 24 hours. The cellular extracts were separated into nuclear and cytoplasmic fractions and probed for NF-κB p65; β-actin was used as a cytoplasmic marker and loading control; Histone H3 was used as nuclear marker (top). Bottom, quantification was performed using the results from three different transfections and immunoblots (*, P < 0.05, BCO2-wt or BCO2-mt expression vs. pcDNA3, respective subcellular fraction controls). C, DU145 cells were transiently transfected with the indicated constructs for 24 hours and nuclear protein fractions were extracted. OD, optical density. The NF-κB family protein DNA-binding assay is described in “Materials and Methods.”
Additional Files
Supplementary Data
- Supplemental Figure Legends - Figure S1 (Related to Figure 4A): Lycopene and lycopene metabolites inhibit cell growth in C4-2 prostate cancer cells. In these representative experiments, C4-2 cells (8 x 104) were seeded onto 6-well plates, then treated with 1 μM lycopene or apo-10-lycopenal, or vehicle (DMSO) for up to 3 days. Every 24 h, duplicate samples were trypsinized and counted. Data represent mean {plus minus} SD (n=3). Figure S2 (Related to Figure 4B): Lycopene and lycopene metabolites inhibit cell growth in PC3 prostate cancer cells. In these representative experiments, 8 x 104 PC3 cells were seeded onto 6-well plates, then treated with 1 μM lycopene or apo-10-lycopenal, or vehicle (DMSO) for up to 3 days. Every 24 h, duplicate samples were trypsinized and counted. Data represent mean {plus minus} SD (n=3). Figure S3: Sequence alignments of human BCO2, BCO1, RPE65, and bovine RPE65, showing conserved active site residues targeted for mutation (numbered as BCO2): Glu194, His226, His 286, His 387, Glu465, Glu499, His573). Figure S4: A. Active site structure of the human BCO2 enzyme showing the catalytic iron coordination site with four conserved histidine residues and second shell glutamate residues. B. Predicted 3D structure of human BCO2. Seven bladed β-propellers are indicated in yellow (I through VII) and the four α-helices in pink. Figure S5 (Related to Figure 5C,D): A. Representative colony formation assay for transfected C4-2 cells. B. Quantification of colonies per well from two 6-well plates of transfected C4-2 cells indicated in A. Data represent mean {plus minus} SD (n=4); (*, p< 0.05, BCO2 or BCO2-mt vs. control).
Volume 14, Issue 10
