Forchlorfenuron disrupts SEPT9_i1 filaments and inhibits HIF-1

Abstract

Forchlorfenuron (FCF) is a synthetic plant cytokinin that has been shown to alter yeast and mammalian septin organization. Septins are a highly conserved family of GTP-binding cytoskeletal proteins. Mammalian septins are involved in diverse cellular processes including tumorigenesis. We have been studying the interaction between septin 9 isoform 1 (SEPT9_i1) and hypoxia inducible factor-1α (HIF-1α), the oxygen regulated subunit of HIF-1. HIF-1 is a key transcription factor in the hypoxic responses pathway, and its activation has been observed in carcinogenesis and numerous cancers. SEPT9_i1/HIF-1α interaction plays an important role in upregulation of HIF-1 transcriptional activity by preventing HIF-1α's ubiquitination and degradation leading to increased tumor growth and angiogenesis. We tested the hypothesis whether FCF affects SEPT9_i1 filamentous structures and consequently HIF-1 pathway in cancer cells. We showed that FCF suppresses tumorigenic properties, including proliferation, migration and transformation, in prostate cancer cells. FCF did not alter SEPT9_i1 steady state protein expression levels but it affected its filamentous structures and subcellular localization. FCF induced degradation of HIF-1α protein in a dose- and time-dependent manner. This inhibition was also shown in other common cancer types tested. Rapid degradation of HIF-1α protein levels was accompanied by respective inhibition in HIF-1α transcriptional activity. Moreover, HIF-1α protein half-life was markedly decreased in the presence of FCF compared with that in the absence of FCF. The FCF-induced degradation of HIF-1α was mediated in a significant part via the proteasome. To the best of our knowledge, this is the first demonstration of specific manipulation of septin filaments by pharmacological means having downstream inhibitory effects on the HIF-1 pathway.

Figure 1. FCF inhibits cell proliferation, migration and transformation.

(A) 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. Points, mean (n=3 replicates); bars, SD; *P < 0.001. This is a representative experiment out of 3 independent repetitions. (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%). Points, means (n=3); bars, SD. (C) 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). (D) Wound healing was calculated as percentage of the wound area in vehicle-treated cells at 8 h (100%). Points, mean (n = 3); bars, SD. *P < 0.01. (E) 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). (F) A quantitative analysis of colonies for each treatment. Columns, means (n=2); bars, SD. *P < 0.01.

Figure 2. FCF decreases HIF-1α protein expression and HIF-1 transcriptional activity.

PC-3 cells were treated with increasing concentrations of FCF for 6 h (A) or with 100 μM FCF for the indicated times (B) under normoxic and hypoxic conditions. Whole cell extracts were analyzed by SDS-PAGE and immunoblotted with antibodies to HIF-1α and tubulin. (C) 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. Columns, mean (n = 3); bars, SD; *P < 0.01. (D) 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. Columns, means (n=2); bars, SD; *P < 0.05. (E) 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. None refers to no IP, whole cell extracts only.

Figure 3. FCF affects HIF-1α expression on the posttranslational level.

(A) PC-3 cells were pretreated with 100 μM FCF for 2 h and then subjected overnight to normoxic or hypoxic conditions. Total RNA was isolated and reverse transcribed into cDNA. Quantitative real-time PCR analysis was done using primers for HIF-1α and cyclophilin B as control. Columns, means (n = 2); bars, SD. (B) PC-3 cells were treated with vehicle (control) and 100 μM FCF for 4 h under normoxia, and then CHX was added at a final concentration of 10 μg/ml for the indicated times. Whole cell extracts were analyzed by SDS-PAGE and immunoblotted with antibodies to HIF-1α and tubulin. (C) Densitometric quantification of HIF-1α levels in (B) normalized to tubulin. Fifty% decrease of HIF-1α levels by FCF is delineated in grey lines. This is a representative experiment out of 3 independent repetitions. PC-3 cells were treated with FCF in the presence of 10 µM MG-132 (D) or 1 µM epoxomicine (E) for 2 h and then grown under normoxia and hypoxia for 4 additional h. Whole cell extracts were prepared, analyzed by SDS-PAGE and immunoblotted with antibodies to HIF-1α and tubulin. Densitometric quantification of HIF-1α/tubulin levels normalized to control at each condition is outlined under the respective lanes.

Figure 4. FCF alters SEPT9_i1 filamentous organization and localization.

PC-3 cells were treated with FCF for 2 h and grown in normoxia (A) or hypoxia (B) for 4 additional h. They were fixed and processed for immunofluorescent labeling with anti-HIF-1α (red), anti-SEPT9_i1 (green) and DAPI (blue). Staining was analyzed by confocal laser-scanning microscope (magnification x63). (C) Representative cells from each treatment condition presented in (A) were further enlarged (zoom x2) for better visualization of SEPT9_i1 filaments (green).

Figure 5. The effect of FCF on HIF-1α is general to cancer cells but specific only to HIF-1α.

(A) 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) 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. (C) 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. Columns, mean (n = 3); bars, SD. *P < 0.05.