Sun Kyoung Lee

Secretion of IL-6 and IL-8 from lysophosphatidic acid-stimulated oral squamous cell carcinoma promotes osteoclastogenesis and bone resorption

Lysophosphatidic acid (LPA) is a bioactive lipid with a growth factor-like activity on a large range of cell types. Several pieces of evidence raise the possibility that LPA may play an important role in bone metastasis. Bone is a frequent metastatic site for oral cancer. However, the role of LPA in the progression of oral cancer metastasis to the bone is poorly understood. Here, we provide evidence for the role of LPA in the progression of oral cancer bone metastases and its regulatory mechanism. LPA induced the secretion of IL-6 and IL-8 in oral squamous cell carcinoma (OSCC). LPA-stimulated secretion of IL-6 and IL-8 is partly dependent on the LPA and EGF receptor (EGFR) pathways. ERK1/2 and Akt-mediated NF-κB and AP-1 were responsible for the LPA-induced IL-6 and IL-8 secretion. Moreover, conditioned medium (CM) derived from the LPA-stimulated OSCC supported osteoclast formation in bone marrow-derived macrophages (BMMs). Neutralization against both human IL-6 and IL-8 suppressed osteoclast formation induced by CM derived from the LPA-stimulated OSCC. Direct treatment with recombinant IL-6 (rIL-6) and/or soluble IL-6 receptor (sIL-6R), or IL-8 (rIL-8) reproduced the effect of the CM derived from the LPA-stimulated OSCC on osteoclast formation. In addition, CM derived from the LPA-stimulated OSCC induced receptor activator of nuclear factor (NF)-κB ligand (RANKL) expression in human osteoblasts and direct treatment with rIL-6 and/or sIL-6R or rIL-8 mimicked the effect of the CM derived from the LPA-stimulated OSCC for RANKL expression. Taken together, LPA may be a potent inducer of osteolytic factor IL-6 and IL-8 in OSCC. LPA-induced IL-6 and IL-8 exerted propound effects on RANKL expression in osteoblast and thereby promoted osteoclast formation from osteoclast precursors.

Betulinic acid, a bioactive pentacyclic triterpenoid, inhibits skeletal-related events induced by breast cancer bone metastases and treatment

Many breast cancer patients experience bone metastases and suffer skeletal complications. The present study provides evidence on the protective and therapeutic potential of betulinic acid on cancer-associated bone diseases. Betulinic acid is a naturally occurring triterpenoid with the beneficial activity to limit the progression and severity of cancer, diabetes, cardiovascular diseases, atherosclerosis, and obesity. We first investigated its effect on breast cancer cells, osteoblastic cells, and osteoclasts in the vicious cycle of osteolytic bone metastasis. Betulinic acid reduced cell viability and the production of parathyroid hormone-related protein (PTHrP), a major osteolytic factor, in MDA-MB-231 human metastatic breast cancer cells stimulated with or without tumor growth factor-β. Betulinic acid blocked an increase in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin ratio by downregulating RANKL protein expression in PTHrP-treated human osteoblastic cells. In addition, betulinic acid inhibited RANKL-induced osteoclastogenesis in murine bone marrow macrophages and decreased the production of resorbed area in plates with a bone biomimetic synthetic surface by suppressing the secretion of matrix metalloproteinase (MMP)-2, MMP-9, and cathepsin K in RANKL-induced osteoclasts. Furthermore, oral administration of betulinic acid inhibited bone loss in mice intra-tibially inoculated with breast cancer cells and in ovariectomized mice causing estrogen deprivation, as supported by the restored bone morphometric parameters and serum bone turnover markers. Taken together, these findings suggest that betulinic acid may have the potential to prevent bone loss in patients with bone metastases and cancer treatment-induced estrogen deficiency.

The inhibitory effect of roasted licorice extract on human metastatic breast cancer cell-induced bone destruction

The aim of this study was to determine whether the ethanol extract of roasted licorice (rLE) could inhibit breast cancer‐mediated bone destruction. rLE treatment reduced the viability of MDA‐MB‐231 human metastatic breast cancer cells but did not show any cytotoxicity in hFOB1.19 human osteoblastic cells and murine bone marrow‐derived macrophages (BMMs). rLE inhibited expression and secretion of receptor activator of nuclear factor κB ligand (RANKL) as well as the mRNA and protein expression of cyclooxygenase‐2 in osteoblastic cells exposed to the conditioned medium of breast cancer cells. rLE dramatically inhibited RANKL‐induced osteoclastogenesis in BMMs, thereby reducing osteoclast‐mediated pit formation. Moreover, treatment with licochalcone A and isoliquiritigenin as the active components, whose contents are increased by the roasting process, remarkably suppressed RANKL‐induced osteoclast formation in BMMs, respectively. Furthermore, orally administered rLE substantially blocked tumor growth and bone destruction in mice inoculated with breast cancer cells in the tibiae. Serum levels of tartrate‐resistant acid phosphatase and C‐terminal cross‐linking telopeptide of type I collagen and trabecular bone morphometric parameters were reversed to almost the same levels as the control mice by the rLE treatment. In conclusion, rLE may be a beneficial agent for preventing and treating bone destruction in patients with breast cancer. Copyright

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.

Loss of RUNX3 expression promotes cancer-associated bone destruction by regulating CCL5, CCL19 and CXCL11 in non-small cell lung cancer.

Non‐small cell lung cancer (NSCLC) frequently metastasizes to bone, which is associated with significant morbidity and a dismal prognosis. RUNX3 functions as a tumour suppressor in lung cancer and loss of expression occurs more frequently in invasive lung adenocarcinoma than in pre‐invasive lesions. Here, we show that RUNX3 and RUNX3‐regulated chemokines are linked to NSCLC‐mediated bone resorption. Notably, the receptor activator of nuclear factor‐κB ligand (RANKL)/osteoprotegerin (OPG) ratio, an index of osteoclastogenic stimulation, was significantly increased in human osteoblastic cells treated with conditioned media derived from RUNX3‐knockdown NSCLC cells. We aimed to identify RUNX3‐regulated factors that modify the osteoblastic RANKL/OPG ratio and found that RUNX3 knockdown led to CCL5 up‐regulation and down‐regulation of CCL19 and CXCL11 in NSCLC cells. Tumour size was noticeably increased and more severe osteolytic lesions were induced in the calvaria and tibiae of mice that received RUNX3‐knockdown cells. In response to RUNX3 knockdown, serum and tissue levels of CCL5 increased, whereas CCL19 and CXCL11 decreased. Furthermore, CCL5 increased the proliferation, migration, and invasion of lung cancer cells in a dose‐dependent manner; however, CCL19 and CXCL11 did not show any significant effects. The RANKL/OPG ratio in osteoblastic cells was increased by CCL5 but reduced by CCL19 and CXCL11. CCL5 promoted osteoclast differentiation, but CCL19 and CXCL11 reduced osteoclastogenesis in RANKL‐treated bone marrow macrophages. These findings suggest that RUNX3 and related chemokines are useful markers for the prediction and/or treatment of NSCLC‐induced bone destruction.

Isoliquiritigenin Inhibits Metastatic Breast Cancer Cell-induced Receptor Activator of Nuclear Factor Kappa-B Ligand/Osteoprotegerin Ratio in Human Osteoblastic Cells

Bone destruction induced by the metastasis of breast cancer cells is a frequent complication that is caused by the interaction between cancer cells and bone cells. Receptor activator of nuclear factor kappa-B ligand (RANKL) and the endogenous soluble RANKL inhibitor, osteoprotegerin (OPG), directly play critical roles in the differentiation, activity, and survival of osteoclasts. In patients with bone metastases, osteoclastic bone resorption promotes the majority of skeletal-related events and propagates bone metastases. Therefore, blocking osteoclast activity and differentiation via RANKL inhibition can be a promising therapeutic approach for cancer-associated bone diseases. We investigated the potential of isoliquiritigenin (ISL), which has anti-proliferative, anti-angiogenic, and anti-invasive effects, as a preventive and therapeutic agent for breast cancer cell-induced bone destruction. ISL at non-toxicity concentrations significantly inhibited the RANKL/OPG ratio by reducing the production of RANKL and restoring OPG production to control levels in hFOB1.19 cells stimulated with conditioned medium (CM) of MDA-MB-231 cells. In addition, ISL reduced the expression of cyclooxygenase-2 in hFOB1.19 cells stimulated by CM of MDA-MB-231 cells. Therefore, ISL may have inhibitory potential on breast cancer-induced bone destruction.

Platycodin D Blocks Breast Cancer-Induced Bone Destruction by Inhibiting Osteoclastogenesis and the Growth of Breast Cancer Cells

Background: Metastatic breast cancer cells are frequently associated with osteoclast-mediated bone resorption, resulting in severe bone destruction and increased mortality in patients. Platycodin D (PD) isolated from Platycodon grandiflorum is a triterpenoid saponin with anti-cancer and anti-angiogenic potential. Methods: The in vivo activity was determined in mice with the intratibial injection of human metastatic breast cancer cells. Osteoclast formation and activity were detected using tartrate-resistant acid phosphatase staining and calcium phosphate-coated plates. The expression of osteoclastogenesis-inducing molecules was detected by RT-PCR and western blotting in RANKL-treated bone marrow macrophages (BMMs). Cell viability and DNA synthesis were measured with MTT and BrdU incorporation assays. The induction of apoptosis was estimated using TUNEL staining and a caspase-3 activity assay. Results: The oral administration of PD inhibited MDA-MB-231 cell-induced osteolysis in an intratibial mouse model. PD treatment blocked RANKL-induced osteoclast formation by inhibiting the expression and nuclear translocation of NFATc1 and c-Fos in BMMs and consequently reduced osteoclast-mediated bone resorption. Furthermore, PD treatment induced apoptosis in osteoclasts and inhibited the growth of MDA-MB-231 cells. Conclusion: PD may block breast cancer-induced bone loss by suppressing the formation, activity, and survival of osteoclasts, as well as the growth of metastatic breast cancer cells.

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.