Fig 1: IMD-4482 inhibits PAI-1 activation of PAI-1-positive ovarian cancer cells PAI-1 expression in 6 serous ovarian cell lines and 2 different primary cultures of ovarian surface epithelium (OSE) cells was analyzed by western blot(A). β-actin was used as a loading control. Real-time RT-PCR (B). Total RNAs from the six cell lines and one OSE were collected and subjected to RT-PCR. The relative abundance of PAI-1 with respect to GAPDH expression was calculated. Molecular formula of IMD-4482 (C). Western blot (D). SKOV3ip1, HeyA8, and OVCAR3 cells were incubated with or without IMD-4482 in serum-free medium for 24 hours. Cell lysates were immunoblotted with antibodies against PAI-1 and uPAR. β-actin was used as a loading control. Experiments were repeated three times and are expressed as mean ± SD. *; P < 0.05, **; P < 0.01.
Fig 2: IMD-4482 inhibits adhesion and invasion of PAI-1-positive ovarian cancer cells (SKOV3ip1 and HeyA8 cells) but not PAI-1-negative cells (OVCAR3)In vitro adhesion assay (A). A total of 1 × 105 ovarian cancer cells (left, SKOV3ip1; middle, HeyA8; right, OVCAR3) were plated onto vitronectin-, fibronectin-, collagen type I-, and laminin-coated 96-well plates. After incubation for 50 minutes at 37°C, plates were washed to discard non-adherent cells, and the relative number of attached cells was measured, Data represents mean ± SD, n = 5 from triplicate independent experiments. (B) Representative images of in vitro adhesion assay of SKOV3ip1 cells (B). Bar, 200 μm. In vitro invasion assay (C). A total of 4 × 104 SKOV3ip1 (left) and 8 × 103 HeyA8 (right) cells were plated on the top chamber in serum-free medium with IMD-4482, and allowed to invade for 48 hours. Invading cells on the underside of the filter were counted. Representative images are shown. Bar, 200 μm. Data represents mean ± SD, n = 5 from triplicate independent experiments. *; P < 0.05, **; P < 0.01, n.s.; not significant.
Fig 3: IMD-4482 inhibits the phosphorylation of FAK and ERK followed by the dissociation of PAI-1 and uPAR from αVβ3 integrin and FAKWestern blot (A). Cells were incubated with IMD-4482 for 24 hours. Cell lysates were immunoblotted with an antibody against integrin αV and integrin β3. β-actin was used as a loading control. Western blot (B). Cells were incubated with or without IMD-4482 for 24 hours. Cell lysates were immunoblotted with an antibody against PARP, p-FAK (Tyr-397), FAK, p-ERK, and ERK. β-actin was used as a loading control. (C and D) Immunoprecipitation. Ovarian cancer cells were incubated with IMD-4482 for 24 hours, and lysed in buffer containing 1% Triton X-100. Cell lysates were subjected to immunoprecipitation with anti-PAI-1 antibody (C) or anti-uPAR antibody (D), followed by immunoblotting to detect integrin αV, integrin β3, and FAK. Representative blots from three independent experiments are shown. Arrow indicates heavy chain of IgG band. Schema (E). PAI-1 inhibition with IMD-4482 dissociates the interaction between PAI-1/uPAR and αVβ3 integrin, which leads to the inhibition of FAK phosphorylation.
Fig 4: IMD-4482 inhibits peritoneal dissemination of ovarian cancer cells through inhibition of FAK phosphorylation and intratumoral vessel formationExperimental protocol (A). A total of 1 × 106 HeyA8 cells were injected intraperitoneally into female BALB/c nu/nu mice. Three days after the injection, IMD-4482 (50 mg/kg body weight) or an equal amount of 0.5% CMC-Na (control) was injected intraperitoneally daily for 11 days. Effect of IMD-4482 on intraperitoneal tumor weight (B) and number of metastases (C). Results are expressed as mean ± SD, each n = 5. Representative tumor areas were stained with H&E, and immunostained with antibodies against PAI-1 and p-FAK (D). Bar, 50 mm. Number of microvessels per field by CD31 staining (×200) (E, right). Results are expressed as mean ± SD, n = 5, each. The representative tumor areas immunostained with an antibody against mouse CD31 are shown (E, left). Bar, 50 μm. *, P < 0.05; **, P < 0.01; n.s., not significant.
Fig 5: IMD-4482 suppressed proliferation and induced G0/G1 cell cycle arrest and apoptosis in PAI-1-positive ovarian cancer cellsEffect of IMD-4482 on cell proliferation (A). SKOV3ip1, HeyA8, and OVCAR3 cells were plated onto 96-well plates and cultured in DMEM containing 2% FBS with or without IMD-4482. Effect of IMD-4482 on cell cycle distribution (B and C). Cells treated with or without IMD-4482 for 24 hours were stained with propidium iodine and analyzed by flow cytometry. Representative flow histograms (B), and percentages of cells in G0/G1, S, and G2/M phase (C) are shown. Western blot (D). Cells were incubated with or without IMD-4482 for 24 hours. Cell lysates were immunoblotted with antibodies against PARP, CDK2, cyclin D3, and p27kip1. β-actin was used as a loading control.
Supplier Page from Sino Biological, Inc. for Human PAI-1 / SerpinE1 Gene ORF cDNA clone expression plasmid