Selective tyrosine kinase inhibition of insulin‐like growth factor‐1 receptor inhibits human and mouse breast cancer–induced bone cell activity,bone remodeling,and osteolysis

Insulin‐like growth factor 1 (IGF‐1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF‐1 receptor tyrosine kinase inhibitor cis‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐a]pyrazin‐8‐ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA‐MB‐231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF‐1 and conditioned medium–induced osteoclast formation by preventing phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M‐CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF‐1 and conditioned medium and totally prevented conditioned medium–induced osteoclast formation in osteoblast–bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast–osteoclast coupling. PQIP also inhibited IGF‐1–induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA‐MB‐231 conditioned medium–induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer‐associated bone turnover. We conclude that inhibition of IGF‐1 receptor tyrosine kinase activity by PQIP suppresses breast cancer–induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer. © 2013 American Society for Bone and Mineral Research.     

Quantifying intra‐osseous growth of osteosarcoma in a murine model with radiographic analysis

The orthotopic murine osteosarcoma model is an excellent representation of the human condition as mice develop rapid growth of ‘primary’ tumor with subsequent lung metastasis. Currently, monitoring tumor growth relies on measuring pulmonary metastases occurring four weeks post injection. Studies show that amputation of the tumor‐bearing limb is required before pulmonary metastases are detectable due to rapid growth causing morbidity. Thus, a method measuring ‘primary’ tumor growth independent of metastasis is required. We hypothesized that serial radiography would allow for longitudinal quantification of ‘primary’ osteosarcoma growth and explored this idea by utilizing the tibial orthotopic model. Tumor growth was monitored weekly by radiography and calipers, and results were compared with µCT and histology. We found that radiographs demonstrate extra and intra‐osseous tumor growth by displaying lytic and blastic lesions and the surrounding radio‐opaque area enlarged significantly (p < 0.0001) allowing for quantification. Additionally, radiographs proved more precise than indirect caliper measurements (intra‐observer error ±6.64%: inter‐observer error ±15.84%). Therefore, we determined that radiography provides accurate, longitudinal quantification of ‘primary’ osteosarcoma tumor that can be performed serially in the same mouse, does not require introduction of bioluminescence to the host or cell, and is more precise than the current caliper method. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1957–1962, 2011     

Recombinant human platelet‐derived growth factor BB (rhPDGF‐BB) and beta‐tricalcium phosphate/collagen matrix enhance fracture healing in a diabetic rat model

Diabetes mellitus is a common systemic disease that has been associated with poor fracture healing outcomes. The mechanism through which diabetes impairs bone regeneration is unknown. One possible mechanism may be related to either decreased or uncoordinated release of local growth factors at the fracture site. Indeed, previous studies have found reduced platelet‐derived growth factor (PDGF) levels in the fracture callus of diabetic rats, suggesting that local application of PDGF may overcome the negative effects of diabetes and promote fracture healing. To test this hypothesis, low (22 µg) and high (75 ug) doses of recombinant human PDGF‐BB (rhPDGF‐BB) were applied directly to femur fracture sites in BB Wistar diabetic rats that were then compared to untreated or vehicle‐treated animals. rhPDGF‐BB treatment significantly increased early callus cell proliferation compared to that in control specimens. Low dose rhPDGF‐BB treatment significantly increased callus peak torque values (p < 0.05) at 8 weeks after fracture as compared to controls. High dose rhPDGF‐BB treatment increased callus bone area at 12 weeks postfracture. These data indicate that rhPDGF‐BB treatment ameliorates the effects of diabetes on fracture healing by promoting early cellular proliferation that ultimately leads to more bone formation. Local application of rhPDGF‐BB may be a new therapeutic approach to treat diabetes‐impaired fracture healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1074–1081, 2009     

Fourier transform infrared imaging of femoral neck bone: Reduced heterogeneity of mineral‐to‐matrix and carbonate‐to‐phosphate and more variable crystallinity in treatment‐naive fracture cases compared with fracture‐free controls

After the age of 60 years, hip fracture risk strongly increases, but only a fifth of this increase is attributable to reduced bone mineral density (BMD, measured clinically). Changes in bone quality, specifically bone composition as measured by Fourier transform infrared spectroscopic imaging (FTIRI), also contribute to fracture risk. Here, FTIRI was applied to study the femoral neck and provide spatially derived information on its mineral and matrix properties in age‐matched fractured and nonfractured bones. Whole femoral neck cross sections, divided into quadrants along the neck's axis, from 10 women with hip fracture and 10 cadaveric controls were studied using FTIRI and micro‐computed tomography. Although 3‐dimensional micro‐CT bone mineral densities were similar, the mineral‐to‐matrix ratio was reduced in the cases of hip fracture, confirming previous reports. New findings were that the FTIRI microscopic variation (heterogeneity) of the mineral‐to‐matrix ratio was substantially reduced in the fracture group as was the heterogeneity of the carbonate‐to‐phosphate ratio. Conversely, the heterogeneity of crystallinity was increased. Increased variation of crystallinity was statistically associated with reduced variation of the carbonate‐to‐phosphate ratio. Anatomical variation in these properties between the different femoral neck quadrants was reduced in the fracture group compared with controls. Although our treatment‐naive patients had reduced rather than increased bending resistance, these changes in heterogeneity associated with hip fracture are in another way comparable to the effects of experimental bisphosphonate therapy, which decreases heterogeneity and other indicators of bone's toughness as a material. © 2013 American Society for Bone and Mineral Research     

4‐Methylcatechol inhibits cell growth and testosterone production in TM3 Leydig cells by reducing mitochondrial activity

4‐Methylcatechol (4‐MC) is a potential neuroprotective drug because it stimulates the synthesis of brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in neurons. The present study explored the effect of 4‐MC on cell growth and testosterone synthesis in the TM3 Leydig cells of mice. 4‐MC did not enhance expression of both BDNF and NGF in these cells. However, this compound significantly inhibited cell proliferation and increased the number of apoptotic cells in a dose‐dependent manner. The expression profile of Bax/Bcl‐2 gene was altered considerably, and mitochondrial activity was significantly decreased in cells. 4‐Methylcatechol also inhibited testosterone synthesis in TM3 Leydig cells. The inhibitory roles of this compound in relation to growth and testosterone synthesis in TM3 Leydig cells maybe associated with increased Bax gene expression and decreased mitochondrial activity. As a result, caspase cascade is activated.     

Utility of a human–mouse xenograft model and in vivo near‐infrared fluorescent imaging for studying wound healing

To study the complex cellular interactions involved in wound healing, it is essential to have an animal model that adequately mimics the human wound microenvironment. Currently available murine models are limited because wound contraction introduces bias into wound surface area measurements. The purpose of this study was to demonstrate utility of a human–mouse xenograft model for studying human wound healing. Normal human skin was harvested from elective abdominoplasty surgery, xenografted onto athymic nude (nu/nu) mice, and allowed to engraft for 3 months. The graft was then wounded using a 2‐mm punch biopsy. Wounds were harvested on sequential days to allow tissue‐based markers of wound healing to be followed sequentially. On the day of wound harvest, mice were injected with XenoLight RediJect cyclooxygenase‐2 (COX‐2) probe and imaged according to package instructions. Immunohistochemistry confirms that this human–mouse xenograft model is effective for studying human wound healing in vivo. Additionally, in vivo fluorescent imaging for inducible COX‐2 demonstrated upregulation from baseline to day 4 (P = 0·03) with return to baseline levels by day 10, paralleling the reepithelialisation of the wound. This human–mouse xenograft model, combined with in vivo fluorescent imaging provides a useful mechanism for studying molecular pathways of human wound healing.     

Hepatocyte growth factor/c‐met promotes proliferation,suppresses apoptosis,and improves matrix metabolism in rabbit nucleus pulposus cells in vitro

The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti‐apoptotic and anti‐inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H₂O₂ or TNF‐α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase‐2 and matrix metalloproteinase‐3 and ‐9 by TNF‐α was significantly suppressed by HGF treatment. Expression of c‐Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF‐α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c‐Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:709–716, 2016.     

Systemic administration of a soluble betaglycan suppresses tumor growth, angiogenesis, and matrix metalloproteinase-9 expression in a human xenograft model of prostate cancer

BACKGROUND: Transforming growth factor beta (TGFbeta) over-expression in prostate cancer has been shown to promote tumor progression and neo-vascularization. In this study, we have investigated the efficacy and the potential mechanism of a TGFbeta antagonist, a recombinant soluble betaglycan (sBG), as a prostate cancer therapeutic agent after systemic administration in a xenograft model. METHODS: Recombinant sBG was delivered continuously via ALZET osmotic pumps or by daily bolus i.p. injection at 4.2 mg/kg/day for 14 days in human prostate cancer DU145 xenograft bearing nude mice. Tumors were analyzed for their size, blood volume by hemoglobin assay, microvessel density (MVD) by CD-31 immunostaining, and apoptosis by TUNEL assay. Matrix metalloproteinase-9 (MMP-9) activity and expression in the DU145 conditioned media were determined by gelatin zymography and Western blotting, respectively. Tissue sections were stained with a polyclonal antibody to MMP-9 using an immuno-fluorescence method. RESULTS: Continuous or bolus administration of sBG showed a similar significant inhibition of DU145 xenograft growth associated with a reduced tumor blood volume and MVD, and an enhanced intra-tumoral apoptosis. Treatment with sBG inhibited both endogenous and TGFbeta-induced MMP-9 activity and expression in a dose-dependent manner in vitro and reduced in vivo MMP-9 expression in DU145 xenografts. CONCLUSIONS: Our results for the first time indicate that TGFbeta blockade by systemic sBG administration can inhibit DU145 prostate xenograft growth and angiogenesis. The inhibition is likely in part mediated by the attenuation of TGFbeta-induced MMP-9 expression.     

Hyaluronan inhibits matrix metalloproteinase‐13 in human arthritic chondrocytes via CD44 and P38

We investigated the effects of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated matrix metalloproteinase (MMP)‐13 production in human chondrocytes from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Secreted levels of MMP‐13 in conditioned media were detected by immunoblotting, while intracellular MMP‐13 synthesis in articular cartilage was evaluated by immunofluorescence microscopic analysis. Mitogen‐activated protein kinases (MAPKs), p38, extracellular signal‐regulated kinases (ERK), and c‐jun NH2‐terminal kinase (JNK) were assessed by Western blotting. IL‐1β (2 ng/ml) stimulates the secretion of MMP‐13 in both OA and RA chondrocytes. Inhibition studies using specific MAPK inhibitors revealed that IL‐1β induced MMP‐13 via p38 in both OA and RA chondrocytes. HA down‐regulates IL‐1β‐stimulated MMP‐13 and phosphorylated p38 (p‐p38) in a dose‐dependent manner (0.1, 1, 2, and 4 mg/ml). When used at 4 mg/ml, HA inhibits p‐p38 phosphorylation by more than 60%. In response to IL‐1β, RA chondrocytes express a higher level of p‐p38 than that of OA chondrocytes. Inhibition of CD44, using a blocking antibody, significantly reversed the inhibitory effect of HA on both MMP‐13 and p‐p38. Our study clearly shows that HA inhibits IL‐1β‐induced MMP‐13 via its principal receptor, CD44, and subsequent intracellular p38 MAPK signaling in OA and RA chondrocytes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:258–264, 2011     

A single blunt impact on cartilage promotes fibronectin fragmentation and upregulates cartilage degrading stromelysin‐1/matrix metalloproteinase‐3 in a bovine ex vivo model

Post‐traumatic osteoarthritis (PTOA) is characterized by progressive cartilage degeneration in injured joints. Since fibronectin‐fragments (Fn‐fs) degrade cartilage mainly through up‐regulating matrix metalloproteinases (MMPs) and pro‐inflammatory cytokines, we hypothesized that Fn‐fs play a key role in PTOA by promoting chondrolysis in and around injured cartilage. To test this hypothesis, we profiled the catabolic events focusing on fibronectin fragmentation and proteinase expression in bovine osteochondral explants following a single blunt impact on cartilage with a drop tower device which created partial‐thickness tissue damage. Injured and control explants were cultured for up to 14 days. The presence of Fn‐fs, MMPs (‐1, ‐3, ‐13), ADAMTS‐5 in culture media and in cartilage was determined with immunoblotting. The daily proteoglycan (PG) depletion of cartilage matrix was assessed with DMMB assay. The effect of explant‐conditioned media on chondrocytes was also examined with immunoblotting. Impacted cartilage released significantly higher amount of native Fn, three chondrolytic Fn‐fs and PG than non‐impacted controls did. Those increases coincided with up‐regulation of MMP‐3 both in culture media and in impacted cartilage. These findings support our hypothesis that PTOA may be propelled by Fn‐fs which act as catabolic mediators through up‐regulating cartilage‐damaging proteinases. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:811–818, 2014.     

Decrease in cell proliferation by an matrix metalloproteinase inhibitor,doxycycline, in a model of immune‐complex nephritis

Aim: Renal expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) contribute to the development of tubulointerstitial fibrosis characteristic of progressive forms of primary glomerulonephritis (GN). The aim of this study was to investigate the therapeutic effect of MMP inhibitor, doxycycline, administration in an experimental rat model of immune‐complex nephritis (ICN). Methods: The induction of immune‐complex glomerulonephritis was carried out by the administration of an i.v. dose of 2 mg bovine serum albumin (BSA) daily for 28 days after 8 weeks of s.c. immunization with 1 mg of BSA in complete Freund's adjuvant. Doxycycline (30 mg/kg) was given daily (in groups 2 and 4) by gavage for 28 days. Results: Animals treated with doxycycline showed significant reduction in glomerular area and cell proliferation than non‐treated controls. Glomerular deposition of immunoglobulin (Ig)G and C3 was less intense in treated rats than non‐treated controls. Although not statistically significant, interstitial inflammation was less intense in treated rats than non‐treated controls. Glomerular expression of MMP‐9 by immunoflourescence was significantly inhibited in the treated group. In addition pro‐MMP‐2 on gelatin zymography was importantly suppressed by doxycycline in ICN. Conclusion: Doxycycline, in addition to its antibiotic property, may, following further investigation, provide a possible survival benefit in proliferative glomerulonephritis.     

The protective role of interleukin‐18 binding protein in a murine model of cardiac ischemia/reperfusion injury

IL‐18, a proinflammatory cytokine, is produced by macrophages, epithelial cells, T cells, neutrophils, NK‐T cells, and B cells, and has been implicated in the pathophysiology of a variety of inflammatory diseases including ischemia/reperfusion (IR) injury, transplant rejection, and autoimmune disease. Recent study indicated that neutralization of IL‐18 with anti‐IL‐18 antibody or IL‐18‐binding protein (IL‐18BP) ameliorates IR‐induced myocardial injury. However, the mechanism needs to be further investigated. In our current study, syngeneic heterotopic heart transplantation was performed by a modified non‐suture cuff technique. We found that IL‐18BP treatment ameliorated cardiomyocyte necrosis and infiltration of CD4⁺ T cells, neutrophils, and macrophages. IL‐18BP‐treated mice exhibited decreased expression of inflammatory cytokines including IL‐1β, IL‐23, IL‐18, and IL‐17. IL‐18BP treatment suppressed Th17 differentiation in vivo and in vitro. Adoptive transfer of T cells from IL‐18BP‐treated mice showed alleviated cardiac IR injury when compared with that transferred from control mice. Furthermore, the decreased infiltration of mononuclear cells and production of troponin T (TnT) induced by IL‐18BP treatment were both abrogated by additional administration of recombinant mouse IL‐17 (rmIL‐17). These data revealed a protective role of IL‐18BP in cardiac IR injury. Above all, IL‐18BP ameliorates cardiac IR injury in part through suppression of Th17 differentiation.     

Proteomic identification of 14‐3‐3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells

Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro‐apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14‐3‐3? protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14‐3‐3? mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14‐3‐3? using siRNA. The 14‐3‐3? gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14‐3‐3? is required for Bim expression and that it has an anti‐cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14‐3‐3? may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:848–854, 2014.