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

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

Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

Septins are a family of cytoskeletal GTP-binding proteins that assemble into membrane-associated hetero-oligomers and organize scaffolds for recruitment of cytosolic proteins or stabilization of membrane proteins. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. The hypothesis tested here is that septins contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an important target for cancer treatment. Septins and ErbB2 were highly over-expressed in gastric cancer cells. Immunoprecipitation followed by mass-spectrometry analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2 or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of septin oligomerization, decreased surface and total levels of ErbB2. These treatments had no effect on EGFR, emphasizing the specificity and functionality of the septin-ErbB2 interaction. The level of ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with FCF, which was accompanied by a decrease in co-localization of ErbB2 with septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect ErbB2 from ubiquitylation, endocytosis, and lysosomal degradation. The FCF-induced degradation pathway is distinct from and additive with the degradation induced by inhibiting ErbB2 chaperone HSP90. These results identify septins as novel regulators of ErbB2 expression that contribute to the remarkable stabilization of the receptor at the plasma membrane of cancer cells and may provide a basis for the development of new ErbB2-targeting anti-cancer therapies