FCF decreases HIF-1α protein expression and HIF-1 transcriptional activity.

<p>PC-3 cells were treated with increasing concentrations of FCF for 6 h (<b>A</b>) or with 100 μM FCF for the indicated times (<b>B</b>) under normoxic and hypoxic conditions. Whole cell extracts were analyzed by SDS-PAGE and immunoblotted with antibodies to HIF-1α and tubulin. (<b>C</b>) PC-3 cells were transiently co-transfected with HRE-dependent firefly luciferase reporter and SV40-dependent renilla luciferase reporter plasmid. After 24 h of transfection, the cells were pretreated with vehicle or 100 µM FCF for 2 h and then grown overnight under normoxia or hypoxia. Whole cell extracts were analyzed by dual luciferase reporter assay. Relative luciferase units (RLU) represent arbitrary units of firefly luciferase activity normalized to renilla luciferase activity. Values were normalized to control vehicle at normoxia. <i>Columns</i>, mean (n = 3); <i>bars</i>, SD; *<i>P</i> < 0.01. (<b>D</b>) PC-3 cells were treated or not treated with 100 μM FCF for 2 h and then subjected overnight to normoxic or hypoxic conditions. Total RNA was isolated from the cells and analyzed by quantitative real-time PCR using primers for Glut-1, ET-1, and cyclophilin B as control. The results were normalized to cyclophilin B mRNA expression levels, and the mean induction of each gene was normalized to control untreated cells under normoxia. <i>Columns</i>, means (n=2); <i>bars</i>, SD; *<i>P</i> < 0.05. (<b>E</b>) PC-3 cells were treated with 0, 75 and 100 μM FCF for 2 h and then subjected to normoxia or hypoxia for an additional 4 h. Cellular extracts were subjected to immunoprecipitation (IP) using anti-HIF-1α antibodies and then immunoblotted (IB) with antibodies to SEPT9_i1 and HIF-1α. <i>None</i> refers to no IP, whole cell extracts only.</p

The effect of FCF on HIF-1α is general to cancer cells but specific only to HIF-1α.

<p>(<b>A</b>) The indicated cancer cells were treated with 100 μM FCF for 4 h under normoxia or hypoxia. Whole cellular extracts were subjected to Western blot analysis using anti-HIF-1α and anti-SEPT9_i1 antibodies. (<b>B</b>) PC-3 cells were treated with FCF as indicated and subjected to normoxia or hypoxia for 6 h. Whole cell extracts were analyzed by SDS-PAGE and immunoblotted with antibodies to HIF-1α, HIF-2α and tubulin. (<b>C</b>) PC-3 cells were transiently transfected with reporter plasmid expressing luciferase under the control of PTHrP P2 promoter (specific to HIF-2α). After 24 h of transfection, the cells were pretreated with FCF for 2 h and then subjected to normoxia or hypoxia for 48 h. Whole cell extracts were analyzed by luciferase luminescence assay. Arbitrary luciferase activity units were normalized to the amount of protein in each assay point. <i>Columns</i>, mean (n = 3); <i>bars</i>, SD. *<i>P</i> < 0.05.</p

FCF inhibits cell proliferation, migration and transformation.

<p>(<b>A</b>) PC-3 cells were treated with increasing concentrations of FCF and grown under normoxic conditions. Cells were analyzed for proliferation at the indicated times using XTT assay. Proliferation was expressed as increase in percentage of the initial absorbance that was measured 24 h after seeding (100%). Growth media and treatment were changed every other day. <i>Points</i>, mean (n=3 replicates); bars, SD; *<i>P</i> < 0.001. This is a representative experiment out of 3 independent repetitions. (<b>B</b>) PC-3 cells were treated with FCF and grown under normoxic conditions for 72 h and processed for SRB cytotoxicity assay. Cell survival was expressed as percentage of the initial absorbance measured in vehicle (0.08% DMSO) control cells (100%). <i>Points</i>, means (n=3); bars, SD. (<b>C</b>) PC-3 cells were grown in 6-well plates to reach 90% confluence. They were then treated with FCF and grown under normoxic conditions for 16 h. The cell monolayer was scratched using a sterile 200-μl pipette tip, and the wounded cultures were watched and photographed after 0, 2, 4 and 8 h (magnification x10). (<b>D</b>) Wound healing was calculated as percentage of the wound area in vehicle-treated cells at 8 h (100%). <i>Points</i>, mean (n = 3); <i>bars</i>, SD. *<i>P</i> < 0.01. (<b>E</b>) PC-3 cells were grown on soft agar and treated with 0, 75 or 100 µM FCF, under normoxic conditions for 3 weeks. A representative colony from each FCF treatment is shown (Magnification x20). (<b>F</b>) A quantitative analysis of colonies for each treatment. <i>Columns</i>, means (n=2); <i>bars</i>, SD. *<i>P</i> < 0.01.</p

Main characteristics of study participants.

<p>PSA, prostate-specific antigen; CI, confidence interval; NA, not available.</p><p>Main characteristics of study participants.</p

Characterization of SEPT9_i1 antibodies and scoring of SEPT9_i1 staining intensity.

<p>HEK-293T embryonic human kidney cells were transiently transfected with Flag-SEPT9_i1 construct or empty vector (EV). (A) Whole cellular extracts were prepared and analyzed by 4–20% SDS-PAGE and immunoblotting (IB) with antibodies to Flag (1:2000), SEPT9_i1 (1:3000), preimmune serum (1:3000) or SEPT9_i1 antibody (1:3000) pre-incubated with 10 μM of the immunogen peptide for 4 hours. (B) The same cellular extracts were subjected to immunoprecipitation (IP) using anti-Flag antibody and the immuneprecipitates were subjected to 4–15% SDS-PAGE and then immunoblotted with anti-Flag or anti-SEPT9_i1 antibodies. (C) Representative SEPT9_i1 staining in human prostate cancer specimens. Score 0: no SEPT9_i1 staining, 1: low SEPT9_i1 staining 2: medium SEPT9_i1 staining and 3: high SEPT9_i1 staining. Magnification x200, scale bar 50 μm.</p

Correlation between "patients' characteristics" and SEPT9_i1 staining.

<p>PSA, prostate-specific antigen.</p><p>Correlation between "patients' characteristics" and SEPT9_i1 staining.</p

SEPT9_i1 staining in prostate cancer metastases.

<p>Metastatic prostate cancer lesions from bone marrow, lymph node and bone were immunostained with SEPT9_i1. Left panels are low (x100) magnification (LM) (scale bar 100 μm) and right panel are high (x200) magnification (HM) (scale bar 50 μm). Note high level of SEPT9_i1 staining in all metastases.</p

Gleason score correlation with SEPT9_i1 staining intensity.

<p>Mean SEPT9_i1 staining intensity ± SE of each Gleason score group (score 5: 3 patients, score 6: 8 patients, score 7: 19 patients, score 8: 3 patients, score 9: 6 patients and score 10: 2 patients) was calculated using either visual scoring (A) or automated image analysis with the ARIOL-SL50 system (B).</p