Anti-IL-20 Monoclonal Antibody Suppresses Prostate Cancer Growth and Bone Osteolysis in Murine Models

Interleukin (IL)-20 is a proinflammatory cytokine in the IL–10 family. IL–20 is associated with tumor promotion in the pathogenesis of oral, bladder, and breast cancer. However, little is known about the role of IL–20 in prostate cancer. We hypothesize that IL–20 promotes the growth of prostate cancer cells. Immunohistochemical staining showed that IL–20 and its receptors were expressed in human PC–3 and LNCaP prostate cancer cell lines and in prostate tumor tissue from 40 patients. In vitro, IL–20 upregulated N-cadherin, STAT3, vimentin, fibronectin, RANKL, cathepsin G, and cathepsin K, and increased the migration and colony formation of prostate cancer cells via activated p38, ERK1/2, AKT, and NF-κB signals in PC–3 cells. We investigated the effects of anti-IL–20 monoclonal antibody 7E on prostate tumor growth in vivo using SCID mouse subcutaneous and intratibial xenograft tumor models. In vivo, 7E reduced tumor growth, suppressed tumor-mediated osteolysis, and protected bone mineral density after intratibial injection of prostate cancer cells. We conclude that IL–20 is involved in the cell migration, colony formation, and tumor-induced osteolysis of prostate cancer. Therefore, IL–20 might be a novel target for treating prostate cancer.     

MicroRNA-7 Inhibits Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Cells via Targeting FAK Expression

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix integrin signaling, cell motility, cell proliferation and cell survival. Increased FAK expression is observed in a variety of solid human tumors and increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Herein we identify miR-7 as a direct regulator of FAK expression. miR-7 expression is decreased in malignant versus normal breast tissue and its expression correlates inversely with metastasis in human breast cancer patients. Forced expression of miR-7 produced increased E-CADHERIN and decreased FIBRONECTIN and VIMENTIN expression in breast cancer cells. The levels of miR-7 expression was positively correlated with E-CADHERIN mRNA and negatively correlated with VIMENTIN mRNA levels in breast cancer samples. Forced expression of miR-7 in aggressive breast cancer cell lines suppressed tumor cell monolayer proliferation, anchorage independent growth, three-dimensional growth in Matrigel, migration and invasion. Conversely, inhibition of miR-7 in the HBL-100 mammary epithelial cell line promoted cell proliferation and anchorage independent growth. Rescue of FAK expression reversed miR-7 suppression of migration and invasion. miR-7 also inhibited primary breast tumor development, local invasion and metastatic colonization of breast cancer xenografts. Thus, miR-7 expression is decreased in metastatic breast cancer, correlates with the level of epithelial differentiation of the tumor and inhibits metastatic progression.     

Hesperetin suppresses RANKL-induced osteoclastogenesis and ameliorates lipopolysaccharide-induced bone loss

Destructive bone diseases caused by osteolysis are increasing in incidence. They are characterized by an excessive imbalance of osteoclast formation and activation. During osteolysis, the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are triggered by receptor activator of NF-κB ligand (RANKL), inflammatory factors, and oxidative stress. Previous studies have indicated that the common flavanone glycoside compound hesperetin exhibits anti-inflammatory and antioxidant activity by inhibition of NF-κB and MAPK signaling pathways. However, the direct relationship between hesperetin and osteolysis remain unclear. In the present study, we investigated the effects of hesperetin on lipopolysaccharide (LPS)-induced osteoporosis and elucidated the related mechanisms. Hesperetin effectively suppressed RANKL-induced osteoclastogenesis, osteoclastic bone resorption, and F-actin ring formation in a dose-dependent manner. It also significantly suppressed the expression of osteoclast-specific markers including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1. Furthermore, it inhibited osteoclastogenesis by inhibiting activation of NF-κB and MAPK signaling, scavenging reactive oxygen species, and activating the nuclear factor E2 p45-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway. Consistent with in vitro results, hesperetin effectively ameliorated LPS-induced bone loss, reduced osteoclast numbers, and decreased the RANKL/OPG ratio in vivo. As such, our results suggest that hesperetin may be a great candidate for developing a novel drug for destructive bone diseases such as periodontal disease, tumor bone metastasis, rheumatoid arthritis, and osteoporosis.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-κB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-κB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.     

Osteoactivin promotes breast cancer metastasis to bone

The skeleton is a preferred site of metastasis in patients with disseminated breast cancer. We have used 4T1 mouse mammary carcinoma cells, which metastasize to bone from the mammary fat pads of immunocompetent mice, to identify novel genes involved in this process. In vivo selection of parental cells resulted in the isolation of independent, aggressively bone metastatic breast cancer populations with reduced metastasis to the lung. Gene expression profiling identified osteoactivin as a candidate that is highly and selectively expressed in aggressively bone metastatic breast cancer cells. These cells displayed enhanced migratory and invasive characteristics in vitro, the latter requiring sustained osteoactivin expression. Osteoactivin depletion in these cells, by small interfering RNA, also lead to a loss of matrix metalloproteinase-3 expression, whereas forced osteoactivin expression in parental 4T1 cells was sufficient to elevate matrix metalloproteinase-3 levels, suggesting that this matrix metalloproteinase may be an important mediator of osteoactivin function. Overexpression of osteoactivin in an independent, weakly bone metastatic breast cancer cell model significantly enhanced the formation of osteolytic bone metastases in vivo. Finally, high levels of osteoactivin expression in primary human breast cancers correlate with estrogen receptor-negative status and increasing tumor grade. Thus, we have identified osteoactivin as a protein that is expressed in aggressive human breast cancers and is capable of promoting breast cancer metastasis to bone.     

The Lysyl Oxidase Inhibitor, β-Aminopropionitrile,Diminishes the Metastatic Colonization Potential of Circulating Breast Cancer Cells

Lysyl oxidase (LOX), an extracellular matrix remodeling enzyme, appears to have a role in promoting breast cancer cell motility and invasiveness. In addition, increased LOX expression has been correlated with decreases in both metastases-free, and overall survival in breast cancer patients. With this background, we studied the ability of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, to regulate the metastatic colonization potential of the human breast cancer cell line, MDA-MB-231. BAPN was administered daily to mice starting either 1 day prior, on the same day as, or 7 days after intracardiac injection of luciferase expressing MDA-MB-231-Luc2 cells. Development of metastases was monitored by in vivo bioluminescence imaging, and tumor-induced osteolysis was assessed by micro-computed tomography (μCT). We found that BAPN administration was able to reduce the frequency of metastases. Thus, when BAPN treatment was initiated the day before, or on the same day as the intra-cardiac injection of tumor cells, the number of metastases was decreased by 44%, and 27%, and whole-body photon emission rates (reflective of total tumor burden) were diminished by 78%, and 45%, respectively. In contrast, BAPN had no effect on the growth of established metastases. Our findings suggest that LOX activity is required during extravasation and/or initial tissue colonization by circulating MDA-MB-231 cells, lending support to the idea that LOX inhibition might be useful in metastasis prevention.     

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease

Background

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.

Methods

Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.

Results

ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.

Conclusions

ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.

General significance

The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.     

Regulation of collagenolytic cysteine protease synthesis by estrogen in osteoclasts

In ovariectomized (Ovx) mice, collagenolytic cysteine protease (CCP) activity in calvaria significantly increased 7 days after ovariectomy and was about 50% of that observed in sham-operated (Sham) mice 3 weeks later. In Ovx mice, subcutaneously (s.c.) administered estradiol-17beta (E2) (10 microg/kg) for 2 weeks led to a decrease in CCP activity in calvaria to the level observed in Sham mice. In Ovx mice, though the amount of cathepsin L increased more than that of cathepsin K, cathepsin K and cathepsin L content increased by 200-400% compared with the Sham mice; cathepsin K was detected in larger amounts than cathepsin L in calvaria from both Sham and Ovx mice. The amounts of cathepsin K and cathepsin L in Ovx mice were reduced to the values seen with Sham mice after administration (s.c.) of E2 (10 microg/kg) for 2 weeks. In mouse calvarial organ culture, the increase of CCP activity and release of hydroxyproline, an indicator of degradation of type-I collagen, in the presence of 1alpha,25-(OH)(2)D(3), parathyroid hormone, interleukin (IL)-1alpha, IL-6, or tumor necrosis factor-alpha was suppressed by E2 (10(-9)-10(-7) M). In all cases, secretion of both cathepsin K and cathepsin L were suppressed by E2. In osteoclasts, expression of cathepsin K and cathepsin L was suppressed by E2 at the mRNA level. Cathepsin B was detected faintly or not at all. These results suggest that synthesis of cathepsin K and cathepsin L was negatively regulated by E2 at the mRNA level. In Ovx mice, deficiency of E2 resulted in an augmentation of cathepsin K and cathepsin L synthesis, and the cathepsins might share roles in bone resorption in vivo.     

Osteoprotegrin and the bone homing and colonization potential of breast cancer cells

Breast cancer cells preferentially metastasize to bone, leading to the formation of primarily osteolytic lesions. Osteoprotegerin (OPG) plays multifactorial roles in the development of osteolytic bone metastases. An increase in the ratio of receptor activator of nuclear factor kappaB ligand (RANKL) to OPG increases osteoclastogenesis within the bone microenvironment. OPG also acts as a survival factor for cancer cells by protecting them from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. This study compares OPG production in vitro in a number of breast cancer cell lines exhibiting both differences in metastatic capacity and in preferential metastasis to bone. Our studies demonstrated that OPG expression by MDA-231, MDA-MET, and MDA-231/K cancer cells was directly correlated with bone specific homing and colonization potential but not with metastasis of cancer cells to other organs; both in IL-1 beta stimulated and control cells. We also demonstrated expression of other bone-related markers including type I collagen, osteocalcin, osteopontin, and Runx2 in these cells. However, the generally lower expression of these markers in the bone selective cell line MDA-MET suggested that increased OPG expression in the bone specific variant was not merely a consequence of enhanced osteomimicry by these cells but that it has a significant role in the metastatic process. Co-culture of breast cancer cells with osteoblastic cells (hFOB 1.19) led to an overall downregulation in OPG production, which was not affected by the bone homing and colonization potential of the cell lines, suggesting that OPG alone is not indicative of osteolytic bone activity by breast cancer cells.     

TAK1-TAB2 signaling contributes to bone destruction by breast carcinoma cells

Advanced-stage breast cancers frequently metastasize to the bones and cause bone destruction, but the underlying mechanism is not fully understood. This study presents evidence that TGF-β-activated protein kinase 1 (TAK1) signaling in tumor cells promotes bone destruction by metastatic breast carcinoma cells, controlling expression of prometastatic factors including matrix metalloproteinase (MMP) 9 and COX2. Suppression of TAK1 signaling by dominant-negative TAK1 (dn-TAK1) in breast carcinoma MDA-MB-231 cells impairs bone colonization by carcinoma cells and bone osteolysis in the intracardiac injection model. Mechanistic studies showed that inhibition of TAK1 by dn-TAK1 or siRNA blocked expression of factors implicated in bone metastasis, such as MMP-9, COX2/PTGS2, parathyroid hormone-related protein (PTHrP) and interleukin 8 (IL-8), but did not affect activation of p38MAPK by TGF-β. TAK1 signaling is mediated by TAK1-binding partners TAB1, TAB2, and TAB3. Carcinoma cells express elevated mRNA levels of TAB2 and TAB3, whereas the TAB1 expression is noticeably low. Accordingly, depletion of TAB2 by siRNA reduced expression of MMP-9 and COX2. Together, these studies show that the TAK1-TAB2-TAB3 signaling axis is critical for carcinoma-induced bone lesions, mediating expression of proinvasive and osteolytic factors. These findings identify the TAK1-TAB2 axis as a potential therapeutic target in bone metastasis.     

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.     

The expression of matrix metalloproteinase 9 and cathepsin B in gastric carcinoma is associated with lymph node metastasis, but not with postoperative survival

Degradation components of basement membrane could be crucial for tumor invasion. A key role in this process has been assigned to cysteine proteases, i.e. cathepsins and matrix metalloproteinases. The purpose of this study was to investigate the relationship of the expression of MMP-9 and cathepsin B with tumor aggressiveness expressed by lymph node metastases and survival rates in gastric carcinoma patients. Slides of 5 mum-thick serial sections from 91 patients with primary gastric carcinoma were prepared and analyzed for MMP-9 and cathepsin B expression using anti-human monoclonal antibody (NCL-MMP-9 clone; dilution 1:40 and NCL-CATH-B clone; dilution 1:40). The patients were clinically monitored for 84 months. We found no association between the expression of MMP-9 and cathepsin B in main mass of tumor and patients' gender, tumor location, Lauren's classification or histological differentiation. Also no correlation was observed between the expression of MMP-9 in main mass of tumor and depth of invasion. A strong statistically significant association was found between the expression of MMP-9 and cathepsin B in main mass of tumor and lymph node involvement (p<0.001; p<0.001, respectively). However, we observed no correlation between the expression of MMP-9 and cathepsin B in main mass of tumor and lymph node involvement or 5-year overall survival. Our results may suggest that the expression of matrix metalloproteinase-9 (MMP-9) and cathepsin B is correlated with lymph node metastasis in advanced gastric carcinoma, but not with patients' postoperative survival.     

Role of prostaglandin E produced by osteoblasts in osteolysis due to bone metastasis

Prostaglandin E2 (PGE2) is produced in bone mainly by osteoblasts and stimulates bone resorption. Osteolytic bone metastasis of cancers is accompanied by bone resorption. In this study, we examined the roles of PGE2 in osteolysis due to bone metastasis of breast cancer. Injection of human breast cancer cells, MDA-MB-231 (MDA-231), into nude mice causes severe osteolysis in the femur and tibia. The expression of cyclo-oxygenase-2 (COX-2) and the receptor activator of NF-kappaB ligand (RANKL), a key molecule in osteoclast differentiation, mRNAs was markedly elevated in bone with metastasis. When MDA-231 cells were cocultured with mouse calvaria, COX-2-induced PGE2 production and bone resorption progressed. The contact with MDA-231 cells could induce the expression of COX-2 and RANKL in osteoblasts by mechanisms involving MAP kinase and NF-kappaB. The blockage of PGE2 signal by indomethacin and EP4 antagonist abrogated the osteoclast formation induced by the breast cancer cells. Here, we show a PGE-dependent mechanism of osteolysis due to bone metastasis.     

Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.     

Different effects of estradiol and various antiestrogens on TNF-alpha-induced changes of biochemical markers for growth and invasion of human breast cancer cells

In the field of estrogen therapy breast cancer risk is one of the most controversially discussed topic. Actually, the as yet largest placebo-controlled study, the Women's Health Initiative, rather showed a risk reduction, in contrast to observational studies. In the present study we have investigated the effect of estradiol on TNF-alpha-induced changes of various markers in human breast cancer cells and compare it with the effect of the antiestrogens tamoxifen and 2-methoxyestradiol. MCF-7 cells were used for the experiments, the incubation time was 96 h. TNF-alpha elicited a 3-4-fold increase of monocyte-attracting protein-1 (MCP-1) and interleukin-8 (IL-8) as compared to the control value, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) were enhanced by 30 to 40%. E2 alone had no effect on MCP-1, slightly reduced the synthesis of MMP-9 and increased VEGF concentrations by about 20%. In combination with TNF, E2 induced a further stimulation of MCP-1, IL-8 and VEGF, whereby the MMP-9 synthesis was not changed. Tamoxifen and the endogenous estradiol metabolite 2-methoxyestradiol seem to be able to partly inhibit the action of TNF-alpha and estradiol. Our results suggest that estrogens may slightly increase tumor growth and spreading beyond the effect of chemokines such as TNF-alpha. However, the magnitude of this E2 effect seems to be marginal as compared to the effect of TNF-alpha alone. The risk of recurrence of breast cancer in patients taking hormone therapy after breast cancer may be slightly enhanced by estrogens, but seems mainly to be driven by the potency of still existing tumor cells to secrete chemokines which can stimulate tumor growth and spreading.     

Inhibition of Sirtuin 3 prevents titanium particle-induced bone resorption and osteoclastsogenesis via suppressing ERK and JNK signaling

Implant-derived wear particles can be phagocytosed by local macrophages, triggering an inflammatory cascade that can drive the activation and recruitment of osteoclasts, thereby inducing peri-prosthetic osteolysis. Efforts to suppress pro-inflammatory cytokine release and osteoclastsogenesis thus represent primary approaches to treating and preventing such osteolysis. Sirtuin 3 (SIRT3) is a NAD⁺-dependent deacetylases that control diverse metabolic processes. However, whether SIRT3 could mitigate wear debris-induced osteolysis has not been reported. Herein we explored the impact of the SIRT3 on titanium particle-induced osteolysis. Tartrate resistant acid phosphatase (TRAP) staining revealed that the inhibition of SIRT3 suppressed nuclear factor-κB ligand (RANKL)-mediated osteoclasts activation in a dose-dependent fashion. Notably, inhibition of SIRT3 also suppressed matrix metallopeptidase 9 (MMP9) and nuclear factor of activated T‐cell cytoplasmic 1 (NFATc1) expression at the mRNA and protein levels, while also inhibiting the mRNA expression of dendritic cell-specific transmembrane protein (DC-STAMP), ATPase H⁺ Transporting V0 Subunit D2 (Atp6v0d2), TRAP and Cathepsin K (CTSK) . In addition, inhibition of SIRT3 suppressed titanium particle-induced tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) expression and prevented titanium particle-induced osteolysis and bone loss in vivo. This inhibition of osteoclasts differentiation was found to be linked to the downregulation and reduced phosphorylation of JNK and ERK. Taken together, inhibition of SIRT3 may be a potential target for titanium particle-induced bone loss.