Fig 1.
Effect of 15d-PGJ2 on the viability, migration, and invasion of MDA-MB-231 cells.
(A) Cells were incubated in serum-free media containing various concentrations of 15d-PGJ2 for 24 or 72 h. Cell viability was determined using the MTT assay. (B) Cells were grown to confluency in monolayers, scratched using a micropipette tip, and treated with the indicated concentrations of 15d-PGJ2 for 40 h. Scratched areas on cultured MDA-MB-231 cells were observed under a light microscope immediately and 40 h after scratching (40x magnification). Relative migrating distances of cells into scratched areas were measured using ImageJ software. Data are expressed as percentages of cell migrating distances at 40 h compared with 0 h. (C) Cells were stimulated with a 1% FBS attractant and treated with 15d-PGJ2 at the indicated concentrations for 24 h. Cells that traversed across the Matrigel matrix were stained with hematoxylin, and representative images were visualized using light microscopy (200x magnification). The numbers of invaded cells were counted in four random fields per membrane filter. Data are expressed as means ± SEM. *P<0.05, **P<0.001 vs. untreated cells.
Fig 2.
Effect of 15d-PGJ2 on PTHrP production in MDA-MB-231 cells.
The cells were treated with (A) various concentrations of 15d-PGJ2 or (B) TGF-β, 15d-PGJ2 and/or GW9662 for 24 h. PTHrP levels were measured in the cultured media of MDA-MB-231 cells using a commercial human PTHrP ELISA kit. Data are expressed as the means ± SEM. *P<0.05, **P<0.01 vs. untreated cells. #P<0.01 vs. TGF-β -treated cells. (C) The level of Smad2 in total lysates and nuclear and cytoplasmic fractions was determined using western blotting in MDA-MB-231 cells stimulated by TGF- β, 15d-PGJ2 and/or GW9662 PPARγ antagonist for 24 h. The cropped blots are representative of experiments that were repeated three times.
Fig 3.
Effect of 15d-PGJ2 on RANKL and OPG mRNA expression in hFOB1.19 human osteoblastic cells.
(A) hFOB1.19 cells were treated with the indicated concentrations of 15d-PGJ2 in DMEM/F12 for 6 h or 24 h. Cell viability was determined using the MTT assay. (B) hFOB1.19 cells were treated with the indicated concentrations of 15d-PGJ2 in DMEM/F12 containing 75% CM of MDA-MB-231 cells or PTHrP (100 ng) for 6 h. mRNA levels of RANKL and OPG were analyzed using real time-PCR. Graphs are expressed as the ratio of the densitometric intensity of RANKL to OPG after normalization to GAPDH. Data represent the means ± SEM. *P<0.05, **P<0.001 vs. untreated cells, #P<0.05, ##P<0.001 vs. CM- or PTHrP-treated cells.
Fig 4.
Effect of 15d-PGJ2 on RANKL-induced osteoclast differentiation and activation.
(A) BMMs isolated from ICR mice were treated with M-CSF (30 ng/ml), RANKL (100 ng/ml), 15d-PGJ2, and/or GW9662 for 5 days. TRAP staining was performed to detect osteoclast formation. TRAP-positive multinucleated cells (≥ 3 nuclei) as differentiated osteoclasts were observed (100x magnification) and counted under an inverted microscope. (B) The differentiated BMMs were treated with 15d-PGJ2 in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for an additional 10 days. The formed resorption pits were visualized using light microscopy (100x magnification). (C) The level of cathepsin K in the cultured media was measured using a commercially available ELISA kit. (D) The activities of MMPs were determined using gelatin zymograpy as clear bands against a blue background that corresponded to active MMP-2/9 (62/92 kDa) and pro-MMP-2/9 (72/105 kDa). The cropped gel image is representative of experiments that were repeated three times. Data are expressed as the means ± SEM. *P<0.001 vs. RANKL-untreated cells. #P<0.05, ##P<0.001 vs. RANKL-treated cells.
Fig 5.
Effect of 15d-PGJ2 on osteolytic bone metastasis in nude mice that received intracardiac injections of MDA-MB-231 cells.
MDA-MB-231/Luc+ cells were inoculated into the left ventricles of female nude mice. 15d-PGJ2 or zoledronic acid (ZA) was subcutaneously injected 3 times per week for 6 weeks at the indicated doses (n = 10). (A) Metastatic progression was detected by measuring bioluminescence in the same mice at 3 and 5 weeks after the injection of cancer cells. The formed metastases were quantified by measuring total photon flux per second. (B) Radiographic images of mandibles, distal femora, and proximal tibiae were scanned using μCT 6 weeks after the injection of cancer cells. Arrowheads indicate osteolytic lesions. (C) The mandibles of mice were analyzed using 3D-images. (D) Bone morphometric parameters, including BV/TV, Tb.N, Tb.Th, Tb.Sp, and SMI, were measured using μCT analysis of the proximal tibiae from mice. (E) Serum PTHrP levels were assayed using a commercially available ELISA kit. Data are expressed as the means ± SEM. #P<0.05, ##P<0.01 vs. control group.*P<0.05, **P<0.01 vs. vehicle-treated group inoculated with cancer cells.
Fig 6.
Effect of 15d-PGJ2 on ovariectomy-induced bone loss.
OVX mice were subcutaneously injected with vehicle, 15d-PGJ2, or E2 (10 μg/kg) for 10 weeks (n = 10). Sham-operated mice received vehicle alone (n = 10). (A) Bone morphometric parameters, including BMD, BV/TV, Tb.Th, Tb.N, Th.Sp, and SMI, were measured using μCT analysis of the femora from mice. (B) 3D images of distal femora of mice were obtained from the reconstruction of μCT data (upper). Sagittal sections of distal femora from mice were stained with Goldner's trichrome. Bone trabeculae appear green, and bone marrow appears red. Stained sections were photographed using a light microscope (100x magnification). (C) Body weights of all mice were measured, and blood sera were collected from all mice for analyses of biochemical parameters. Serum levels of calcium, ALP, osteocalcin, TRAP, and CTX were evaluated using the respective kits as described in Materials and Methods. (D) Serum levels of TNF-α and IL-1β were determined using specific ELISA kits. Data are expressed as the means ± SEM. #P< 0.05, ##P< 0.01 vs. sham group. *P<0.05, **P<0.01 vs. OVX group.