Effects of 4'-Demethylnobiletin and 4'-Demethyltangeretin on Osteoclast Differentiation In Vitro and in a Mouse Model of Estrogen-Deficient Bone Resorption.

Citrus nobiletin (NOB) and tangeretin (TAN) show protective effects against disease-related bone destruction. We achieved demethylation of NOB and TAN into 4'-demethylnobiletin (4'-DN) and 4'-demethyltangeretin (4'-DT) using enzyme-manufacturing methods. In this study, we examined the effects of 4'-DN and 4'-DT on in vitro osteoclast differentiation, and on in vivo osteoporotic bone loss in ovariectomized (OVX) mice. 4'-DN and 4'-DT clearly suppressed the osteoclast differentiation induced by interleukin IL-1 or RANKL treatment. 4'-DN and 4'-DT treatments resulted in higher inhibitory activity in osteoclasts in comparison to NOB or TAN treatments. RANKL induced the increased expression of its marker genes and the degradation of IκBα in osteoclasts, while these were perfectly attenuated by the treatment with 4'-MIX: a mixture of 4'-DN and 4'-DT. In an in silico docking analysis, 4'-DN and 4'-DT directly bound to the ATP-binding pocket of IKKß for functional inhibition. Finally, the intraperitoneal administration of 4'-MIX significantly protected against bone loss in OVX mice. In conclusion, 4'-DN, 4'-DT and 4'-MIX inhibited the differentiation and function of bone-resorbing osteoclasts via suppression of the NF-κB pathway. Novel 4'-DN, 4'-DT and 4'-MIX are candidates for maintaining bone health, which may be applied in the prevention of metabolic bone diseases, such as osteoporosis.

Prostate cancer expressing membrane-bound TGF-α induces bone formation mediated by the autocrine effect of prostaglandin E2 in osteoblasts.

Prostate cancer highly metastasizes to bone, and such cancer is associated with severe bone resorption and bone formation at the site of metastasis. Prostaglandin E2 (PGE2) promotes bone resorption in inflammatory diseases; however, the roles in prostate cancer-induced bone formation are still unclear. In the present study, we investigated the effects of membrane-bound TGF-α on prostate cancer-induced bone formation through autocrine PGE2 signaling in osteoblasts. In the prostate cancer explant experiment into tibiae, injected prostate cancer cells induced bone formation with the increased expression of osteogenic genes, such as Runx2 and Wnt5a, and prostaglandin synthase Ptgs2. In osteoblasts, PGE2 increased the number of calcified bone nodules with enhanced expression of Runx2 and Wnt5a. We also screened the factors involved in cancer progression, and 11 EGF family members were found to be expressed in the human prostate cancer cell line PC3. PC3 highly expressed amphiregulin, HB-EGF, and especially TGF-α. Treatment with recombinant TGF-α increased the Ptgs2 expression and PGE2 production in osteoblasts, which promoted the formation of calcified bone nodules, suggesting that the interaction between PC3 and osteoblasts promoted PGE2 production. In co-culture of osteoblasts and fixed PC3 cells, the phosphorylation of EGFR and ERK and subsequent Ptgs2 expression and PGE2 production were increased, an effect that was attenuated by treatment with inhibitors of EGFR and ERK. These results indicate that membrane-bound TGF-α enhances ERK signaling while also inducing PGE2-mediated bone formation in osteoblasts, thus suggesting that prostate cancer regulates both PGE2-mediated bone resorption and bone formation at the site of bone metastasis of prostate cancer.

A mouse model of lung cancer induced via intranasal injection for anticancer drug screening and evaluation of pathology.

The pathologies and lethality of lung cancers are associated with smoking, lifestyle, and genomic factors. Several experimental mouse models of lung cancer, including those induced via intrapulmonary injection and intratracheal injection, have been reported for evaluating the pharmacological effect of drugs. However, these models are not sufficient for evaluating the efficacy of drugs during screening, as these direct injection models ignore the native processes of cancer progression in vivo, resulting in the inadequate pathological formation of lung cancer. In the present study, we developed a novel intranasal injection model of lung cancer simulating the native lung cancer pathology for anticancer drug screening. A mouse lung cancer cell line (Lewis lung carcinoma; LCC) was intranasally injected into mouse lungs, and injected cell number-dependent cancer proliferation was apparent in both the left and right lungs. Human non-small-cell lung cancer (NCI-H460) cells were also intranasally injected into nude mice and similarly showed injected cell number-dependent cancer growth. For the pharmacological evaluation of cisplatin, two different treatment frequencies were tested four times per month and twice a month. The intranasal injection model confirmed that cisplatin suppressed lung cancer progression to a greater extent under the more frequent treatment condition. In conclusion, these results indicated that our intranasal injection model is a powerful tool for investigating lung cancer pathology and may aid in the development of new anti-lung cancer agents.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Endosomal TLR3 signaling in stromal osteoblasts induces prostaglandin E2-mediated inflammatory periodontal bone resorption.

Toll-like receptors (TLRs) are pattern recognition receptors that play a critical role in innate immune diseases. TLR3, which is localized in the endosomal compartments of hematopoietic immune cells, is able to recognize double-stranded RNA (dsRNA) derived from viruses and bacteria and thereby induce innate immune responses. Inflammatory periodontal bone resorption is caused by bacterial infections, which initially is regulated by innate immunity; however, the roles of TLR3 signaling in bone resorption are still not known. We examined the roles of TLR3 signaling in bone resorption using poly(I:C), a synthetic dsRNA analog. In cocultures of mouse bone marrow cells and stromal osteoblasts, poly(I:C) clearly induced osteoclast differentiation. In osteoblasts, poly(I:C) increased PGE2 production and upregulated the mRNA expression of PGE2-related genes, Ptgs2 and Ptges, as well as that of a gene related to osteoclast differentiation, Tnfsf11. In addition, we found that indomethacin (a COX-2 inhibitor) or an antagonist of the PGE2 receptor EP4 attenuated the poly(I:C)-induced PGE2 production and subsequent Tnfsf11 expression. Poly(I:C) also prolonged the survival of the mature osteoclasts associated with the increased mRNA expression of osteoclast marker genes, Nfatc1 and Ctsk. In ex vivo organ cultures of periodontal alveolar bone, poly(I:C) induced bone-resorbing activity in a dose-dependent manner, which was attenuated by the simultaneous administration of either indomethacin or an EP4 antagonist. These data suggest that TLR3 signaling in osteoblasts controls PGE2 production and induces the subsequent differentiation and survival of mature osteoclasts. Endogenous TLR3 in stromal osteoblasts and osteoclasts synergistically induces inflammatory alveolar bone resorption in periodontitis.

Effects of Polymethoxyflavonoids on Bone Loss Induced by Estrogen Deficiency and by LPS-Dependent Inflammation in Mice.

Polymethoxyflavonoids (PMFs) are a family of the natural compounds that mainly compise nobiletin, tangeretin, heptamethoxyflavone (HMF), and tetramethoxyflavone (TMF) in citrus fruits. PMFs have shown various biological functions, including anti-oxidative effects. We previously showed that nobiletin, tangeretin, and HMF all inhibited interleukin (IL)-1-mediated osteoclast differentiation via the inhibition of prostaglandin E2 synthesis. In this study, we created an original mixture of PMFs (nobiletin, tangeretin, HMF, and TMF) and examined whether or not PMFs exhibit co-operative inhibitory effects on osteoclastogenesis and bone resorption. In a coculture of bone marrow cells and osteoblasts, PMFs dose-dependently inhibited IL-1-induced osteoclast differentiation and bone resorption. The optimum concentration of PMFs was lower than that of nobiletin alone in the suppression of osteoclast differentiation, suggesting that the potency of PMFs was stronger than that of nobiletin in vitro. The oral administration of PMFs recovered the femoral bone loss induced by estrogen deficiency in ovariectomized mice. We further tested the effects of PMFs on lipopolysaccharide-induced bone resorption in mouse alveolar bone. In an ex vivo experimental model for periodontitis, PMFs significantly suppressed the bone-resorbing activity in organ cultures of mouse alveolar bone. These results indicate that a mixture of purified nobiletin, tangeretin, HMF, and TMF exhibits a co-operative inhibitory effect for the protection against bone loss in a mouse model of bone disease, suggesting that PMFs may be potential candidates for the prevention of bone resorption diseases, such as osteoporosis and periodontitis.

Raloxifene reduces the risk of local alveolar bone destruction in a mouse model of periodontitis combined with systemic postmenopausal osteoporosis.

OBJECTIVE: Periodontitis is characterized by local inflammation leading to tooth loss and severe destruction of alveolar bone. Raloxifene is a selective estrogen receptor modulator (SERM) that halts estrogen deficiency-induced systemic bone loss in postmenopausal osteoporosis without the side effects of cancer in breast and uterus. In this study, we examined the effects of raloxifene on alveolar bone mass in a mouse model with estrogen deficiency-induced periodontitis. METHODS: Periodontitis was induced by the injection of lipopolysaccharide (LPS) into the lower gingiva in ovariectomized (OVX) mice, and the alveolar bone and femur bone mineral density (BMD) were analyzed by dual-energy X-ray absorptiometry. To explore the direct osteoclast inhibitory effect of raloxifene, a co-culture system for osteoclast formation and organ culture of alveolar bone was established. RESULTS: When OVX mice were treated with raloxifene, the bone loss in both alveolar bone and femur were abrogated. Interleukin 1 and/or LPS stimulated the osteoclast formation and bone-resorbing activity; however, raloxifene did not show any inhibitory effect on the osteoclast formation or function. In vivo local injection of raloxifene also did not prevent bone resorption in a mouse model of periodontitis. However, the systemic treatment of raloxifene using a mini-osmotic pump did prevent the loss of BMD of alveolar bone induced by LPS. CONCLUSION: These results suggest that the SERM raloxifene systemically maintain alveolar bone mass in a mouse model of periodontitis with osteoporosis. Increasing the alveolar bone mass by SERMs treatment in patients with postmenopausal osteoporosis may be a useful approach to preventing the destruction of alveolar bone in late-onset periodontitis.

Effects of O-methylated (-)-epigallocatechin gallate (EGCG) on LPS-induced osteoclastogenesis, bone resorption, and alveolar bone loss in mice.

(-)-Epigallocatechin-3-O-gallate (EGCG), present in green tea, exhibits antioxidant and antiallergy effects. EGCG3″Me, a 3-O-methylated derivative of EGCG, has been reported to show similar biological functions; the inhibitory activity of EGCG3″Me in a mouse allergy model was more potent than that of EGCG, probably due to the efficiency of absorption from the intestine. However, the functional potency of these EGCGs is controversial in each disease model. We previously observed that EGCG suppressed inflammatory bone resorption and prevented alveolar bone loss in a mouse model of periodontosis. In this study, we examined the role of EGCG3″Me in bone resorption using a mouse model of periodontitis. Lipopolysaccharide (LPS)-induced osteoclast formation was suppressed by adding EGCG3″Me to cocultures of osteoblasts and bone marrow cells, and LPS-induced bone resorption was also inhibited by EGCG3″Me in calvarial organ cultures. EGCG3″Me acted on osteoblasts and suppressed prostaglandin E (PGE) production, which is critical for inflammatory bone resorption, by inhibiting the expression of COX-2 and mPGES-1, key enzymes for PGE synthesis. In osteoclast precursor macrophages, EGCG3″Me suppressed RANKL-dependent differentiation into mature osteoclasts. In a mouse model of periodontitis, LPS-induced bone resorption was suppressed by EGCG3″Me in organ culture of mouse alveolar bone, and the alveolar bone loss was further attenuated by the treatment of EGCG3″Me in the lower gingiva in vivo. EGCG3″Me may be a potential natural compound for the protection of inflammatory bone loss in periodontitis.