IKKβ Regulates VEGF Expression and Is a Potential Therapeutic Target for Ovarian Cancer as an Antiangiogenic Treatment

VEGF expression correlates with poor prognosis in patients with ovarian cancer. Immunohistochemical staining of TMAs with malignant ovarian tissue sections (A). Representative areas of four different ovarian cancers stained using an anti-human VEGF-A antibody and scored as 1, 2, and 3. No positive signal was observed by nonimmune area. Original magnification, ×200. Bar, 100 μm. Kaplan–Meier curves of PFS (B) and OS (C) of patients with ovarian cancer (n = 94). High VEGF expression was significantly more frequent in tumors that showed phosphorylated IKK expression than in those that had negative phosphorylated IKK staining (D). Statistical differences were examined by χ² test (P = 0.037).

IMD-0354 inhibits NF-κB activation of ovarian cancer cells. Molecular formula of IMD-0354 (A). Culturing of ovarian cancer cells (left, SKOV3ip1; right, RMUG-S) with up to 1 μmol/L IMD-0354 had no effect on their viability (B). Data represent mean ± SEM, n = 5 from triplicate independent experiments. Western blot analysis (C). SKOV3ip1 and RMUG-S cells were incubated with IMD-0354 for 24 hours. Cell lysates were immunoblotted with anti-phosphorylated IKKα/β antibody (top). The membranes were stripped and rehybridized with antibodies detecting IκBα (middle) or β-actin (lower). IMD-0354 inhibits NF-κB nuclear translocation induced by TNFα (D). Representative confocal images of SKOV3ip1 cells stimulated by TNFα (+) (10 ng/mL, 30 minutes) with or without pretreatment of 0.1 μmol/L IMD-0354 are shown. Cells were immunostained with Alexa Fluor 555–labeled NF-κB p65 (red). In controls, NF-κB was dominantly located in the cytoplasm (top). TNFα stimulation (middle) induced the nuclear translocation of NF-κB p65. IMD-0354 inhibited its nuclear translocation (lower). Bar, 50 μm.

IMD-0354 causes significant inhibition of adhesion and invasion of ovarian cancer cell lines. In vitro adhesion assay (A). A total of 5 × 10⁴ ovarian cancer cells (left, SKOV3ip1; right, RMUG-S) were plated onto 50 μg/mL fibronectin- or collagen type 1–coated 96-well plates. After incubation for 45 minutes at 37°C, plates were washed to discard nonadherent cells and the number of adherent cells was counted under a light microscope. Data represent mean ± SEM, n = 5 from triplicate independent experiments. Representative image of in vitro adhesion assay of SKOV3ip1 cells is shown (B). Bar, 200 μm. Western blot analysis (C). SKOV3ip1, RMUG-S, and CaOV3 cells were incubated with 0.3 μmol/L of IMD-0354 or control for 24 hours. Cell lysates were immunoblotted with an antibody against integrin α1, α2, α5, β1, and β3. β-Actin was used as a loading control. Each sample was run in triplicate. Numbers show the ratio between integrins indicated and β-actin expression. In vitro invasion assay (D). A total 5 × 10⁴ SKOV3ip1 (left) or RMUS-S (right) cells were placed on the top chamber in serum-free medium with IMD-0354, and allowed to invade for 24 hours. Noninvading cells were removed using a cotton swab, and invading cells on the underside of the filter were enumerated. Representative images are shown. Bar, 100 μm. Data represent mean ± SEM, n = 5 from triplicate independent experiments. Western blot analysis (E). Cell lysates were immunoblotted with an antibody against MMP-2. β-Actin was used as a loading control. Each sample was run in triplicate. Numbers show the ratio between MMP2 and β-Actin expression. **, P < 0.01.

IMD-0354 inhibits VEGF production from ovarian cancer cells, which leads to the inhibition of HUVECs migration induced by cancer cells. Effect of IMD-0354 on VEGF-A transcription activation (A). pGL4-hVEGFA firefly luciferase vector and Renilla luciferase reporter, pRL-TK, were cotransfected into SKOV3ip1 cells. VEGF-A transcriptional activity was reduced in a dose-dependent manner by the treatment with IMD-0354. Data represent mean ± SEM, n = 5 from triplicate independent experiments. The schema of original and mutant pGL4-phVEGFA vector (mut pGL4-phVEGFA; B). Possible NF-κB binding sites of VEGFA promoter are shown. Luciferase activity was drastically abolished with mut pGL4-phVEGFA and IMD-0354 did not show any inhibitory effects (C). Data represent mean ± SEM, n = 4 from triplicate independent experiments. ELISA assay of VEGF-A (D). A total of 1 × 10⁵ SKOV3ip1 cells were cultured with 2 mL of 0.1% bovine serum albumin (BSA)/DMEM with or without IMD-0354 for 24 hours. Conditioned media were collected and the concentration of human VEGF-A was measured. Data represent mean ± SEM, n = 4 from triplicate independent experiments. Effect of IMD-0354 on HUVECs migration examined by a wound healing assay (E). Endothelial cell monolayers were wounded at time 0, and cultures were incubated with serum-free media (top) or conditioned media from SKOV3ip1 cells (lower). Forty-eight hours later, cells were fixed and representative pictures were taken. A total of 5 ng/mL of human VEGF-A was used as a positive control. Bar, 200 μm. Percentage wound recovery was measured and compared with that at time 0 (F). Experiments were repeated seven times and values are means ± SEM; n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.