Hyungkeun Kim

Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma

High recurrence and lower survival rates in patients with oral squamous cell carcinoma (OSCC) are associated with its bone invasion. We identified the oncogenic role of RUNX3 during bone invasion by OSCC. Tumor growth and the generation of osteolytic lesions were significantly inhibited in mice that were subcutaneously inoculated with RUNX3-knockdown human OSCC cells. RUNX3 knockdown enhanced TGF-β-induced growth arrest and inhibited OSCC cell migration and invasion in the absence or presence of transforming growth factor-β (TGF-β), a major growth factor abundant in the bone microenvironment. RUNX3 knockdown induced cell cycle arrest at the G1 and G2 phases and promoted G2 arrest by TGF-β in Ca9.22 OSCC cells. RUNX3 knockdown also inhibited both the basal and TGF-β-induced epithelial-to-mesenchymal transition by increasing E-cadherin expression and suppressing the nuclear translocation of β-catenin. In addition, the expression and TGF-β-mediated induction of parathyroid hormone-related protein (PTHrP), one of key osteolytic factors, was blocked in RUNX3-knockdown OSCC cells. Furthermore, treating human osteoblastic cells with conditioned medium derived from RUNX3-knockdown OSCC cells reduced the receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin ratio compared with treatment with conditioned medium from RUNX3-expressing cells. These findings indicate that RUNX3 expression in OSCC cells contributes to their bone invasion and the resulting osteolysis by inducing their malignant behaviors and production of osteolytic factors. RUNX3 alone or in combination with TGF-β and PTHrP may be a useful predictive biomarker and therapeutic target for bone invasion by oral cancer.

Type I Saikosaponins A and D Inhibit Osteoclastogenesis in Bone Marrow-Derived Macrophages and Osteolytic Activity of Metastatic Breast Cancer Cells

Many osteopenic disorders, including a postmenopausal osteoporosis and lytic bone metastasis in breast and prostate cancers, are linked with a hyperosteoclast activity due to increased receptor activator of nuclear factor kappa-B ligand (RANKL) expression in osteoblastic/stromal cells. Therefore, inhibition of RANKL-induced osteoclastogenesis and osteoclast-induced bone resorption is an important approach in controlling pathophysiology of these skeletal diseases. We found that, of seven type I, II, and III saikosaponins isolated from Bupleurum falcatum, saikosaponins A and D, type I saikosaponins with an allyl oxide linkage between position 13 and 28 and two carbohydrate chains that are directly attached to the hydroxyl groups in position 3, exhibited the most potent inhibition on RANKL-induced osteoclast formation at noncytotoxic concentrations. The stereochemistry of the hydroxyl group at C16 did not affect their activity. Saikosaponins A and D inhibited the formation of resorptive pits by reducing the secreted levels of matrix metalloproteinase- (MMP-) 2, MMP-9, and cathepsin K in RANKL-induced osteoclasts. Additionally, saikosaponins A and D inhibited mRNA expression of parathyroid hormone-related protein as well as cell viability and invasion in metastatic human breast cancer cells. Thus, saikosaponins A and D can serve as a beneficial agent for the prevention and treatment of osteoporosis and cancer-induced bone loss.

Artemisia annua extract prevents ovariectomy-induced bone loss by blocking receptor activator of nuclear factor kappa-B ligand-induced differentiation of osteoclasts

The activities of osteoclasts and osteoblasts are balanced to maintain normal bone density. Many pathological conditions cause osteoclastic bone resorption in excess of osteoblastic bone formation, resulting in osteoporosis. We found that oral administration of Artemisia annua ethanol extract (AaE) or major components, artemisinin and arteannuin B, to ovariectomized (OVX) mice prevented bone loss, as verified by examining three-dimensional images and bone morphometric parameters derived from microcomputed tomography analysis, as well as serum levels of bone turnover markers and proinflammatory cytokines. The administered doses were not toxic to the liver or kidney and showed promising effects that were comparable to those of 17β-estradiol treatment. At non-cytotoxic concentrations, AaE and active components, artemisinin, artemisinic acid, and arteannuin B, potently inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis and the formation of osteoclast-mediated resorption pits. Furthermore, AaE, artemisinin, and arteannuin B remarkably reduced the expression of the c-Fos and NFATc1 transcription factors, which play critical roles in RANKL-induced osteoclast differentiation. Taken together, the in vivo anti-osteoporotic activity of AaE may be derived from the anti-osteoclastic and anti-bone resorptive activities of its active components. AaE has beneficial applications for the prevention and inhibition of osteoporosis and osteoclast-mediated bone diseases.

Abstract 1543: Wogonin suppresses the production of breast cancer-derived osteolytic factors

Breast cancer is the most frequent cancer in women and the main cause of its mortality is induced by metastasizing to distant organs. Breast cancer primarily metastasizes to the bone, lung, liver and brain, and the bone is the most susceptible to metastasis. When breast cancer metastasizes to the bone, the dominant lesion is osteolytic. This osteolytic bone metastasis is highly associated with the complex interaction between cancer cells and the bone microenvironment. Interleukin-1 beta (IL-1β) and IL-17 act as osteolytic factors and promote osteolytic lesions in breast cancer pateints by increasing osteoclastogenesis and decreasing osteoblastogenesis. Especially, both of IL-1β and IL-17 increase in patients with breast cancer and decrease disease-free survival in cancer patients. Wogonin, one of the major flavonoids in the roots of Scutellaria baicalensis Georgi, has been recognized as a potent anti-cancer agent through increased apoptosis and decreased proliferation. We investigated whether wogonin could reduce the cancer cell-induced osteolysis by controlling interaction between cancer cells and bone-related cells. Wogonin suppressed cell viability, DNA synthesis and migration in MDA-MB 231 cells. Wogonin reduced the secretion of IL-1β and IL-17 in MDA-MB 231 cells. Wogonin at non-cytotoxic concentrations inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, wogonin blocked an increase in RANKL/osteoprotegerin mRNA ratio in the osteoblastic hFOB1.19 cells exposed to MDA-MB 231 cells-derived conditioned medium. Finally, oral administration of wogonin significantly inhibited osteolytic lesions in MDA-MB 231 cells-injected mice. Taken together, these results indicate that wogonin is a promising agent for preventing and treating cancer-cell mediated bone loss. Citation Format: Hyungkeun Kim, Kwang-Kyun Park, Won-Yoon Chung. Wogonin suppresses the production of breast cancer-derived osteolytic factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1543. doi:10.1158/1538-7445.AM2015-1543