Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts

Background/Aims: Bone metastasis of cancer cells decreases patient survival and quality of life. Hybridization via the covalent coupling of two bioactive natural products is a useful strategy for developing more potent anticancer agents by enhancing their bioavailability and avoiding drug resistance. Methods: The in vivo activities of artemisinin-daumone hybrid 15 (ARTD) were estimated in cancer cell-inoculated mice and ovariectomized mice. The viability, migration, and invasion of cancer cells were measured via MTT, wound-healing, and transwell invasion assays. ARTD-regulated transcription factors were detected with an RT2 profiler PCR array kit and Western blotting. Osteoclastogenesis and osteoclast activity were detected with tartrate-resistant acid phosphatase staining, a pit formation assay, gelatin zymography, and a cathepsin K ELISA assay. Results: ARTD blocked cancer-associated osteolysis more potently than artemisinin in mice with intratibially inoculated breast cancer or lung cancer cells. ARTD inhibited the viability, migration, and invasion of breast and lung cancer cells in the absence or presence of transforming growth factor-β1. ARTD treatment induced the expression of tumor suppressive activating transcription factor 3 and inhibited oncogenic E2F transcription factor 1 expression at the mRNA and protein levels. ARTD inhibited receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation and bone resorbing activity by reducing the secreted levels of matrix metalloproteinase-9 and cathepsin K. Furthermore, ARTD prevented estrogen deficiency-induced bone loss in ovariectomized mice. Conclusion: ARTD may be a promising candidate for inhibiting cancer-induced bone destruction. The application of ARTD may be extended to patients with chemotherapy-induced ovarian failure or postmenopausal osteoporosis

15-deoxy-δ12,14-prostaglandin j2 inhibits osteolytic breast cancer bone metastasis and estrogen deficiency-induced bone loss.

Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ2 on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ2 dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ2 suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ2 blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ2 exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ2 substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ2 prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ2 may be beneficial for the prevention and treatment of breast cancer-associated bone diseases

Effect of 15d-PGJ₂ on osteolytic bone metastasis in nude mice that received intracardiac injections of MDA-MB-231 cells.

<p>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 (<i>n</i> = 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. ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs. control group.*<i>P</i><0.05, **<i>P</i><0.01 vs. vehicle-treated group inoculated with cancer cells.</p

Effect of 15d-PGJ₂ on the viability, migration, and invasion of MDA-MB-231 cells.

<p>(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. *<i>P</i><0.05, **<i>P</i><0.001 vs. untreated cells.</p

Effect of 15d-PGJ₂ on ovariectomy-induced bone loss.

<p>OVX mice were subcutaneously injected with vehicle, 15d-PGJ₂, or E2 (10 μg/kg) for 10 weeks (<i>n</i> = 10). Sham-operated mice received vehicle alone (<i>n</i> = 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. ^#<i>P</i>< 0.05, ^##<i>P</i>< 0.01 vs. sham group. *<i>P</i><0.05, **<i>P</i><0.01 vs. OVX group.</p

Effect of 15d-PGJ₂ on PTHrP production in MDA-MB-231 cells.

<p>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. *<i>P</i><0.05, **<i>P</i><0.01 vs. untreated cells. ^#<i>P</i><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.</p