Cyclic anthraquinone derivatives, unique G-quadruplex binders, selectively induce cancer cell apoptosis and inhibit tumor growth.

Cyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder.

Substituent effects of cyclic naphthalene diimide on G-quadruplex binding and the inhibition of cancer cell growth.

Interaction of cyclic naphthalene diimide derivatives (cNDIs), 1-4, with TA-core and c-myc as G-quartet (G4) DNA was studied under dilute or molecular crowding condition. Binding study for TA-core based on an isothermal titration calorimetry showed that 1-4 has 106 M-1 order of binding affinity with the following order: 1 > 4 > 2 > 3 under both conditions. Meting temperature (Tm) of TA-core obtained from the temperature dependence of circular dichroism spectra shows that TA-core was most stabilized by 4, which is in agreement with the result of PCR stop assay and the stabilization effect for 1-3 was correlated with their binding affinity under dilute condition. 3 showed specific growth inhibition of cancer cell line Ca9-22 at <0.03 µM of IC50, with no inhibitory effect against normal bone marrow cells. 3, which has highest value of ΔH/ΔG, shows the highest inhibition ability for Ca9-22, carrying a highest expression level of telomerase mRNA.

Osteocalcin promotes proliferation, differentiation, and survival of PC12 cells.

Involvement of the bone matrix protein osteocalcin (OC) in the development of learning and memory, and the prevention of anxiety-like behaviors in mice. However, the direct effects of OC on neurons are still unknown comparing to the mechanism how OC affects systemic energy expenditure and glucose homeostasis. In this study, we investigated the effect of OC on proliferation, differentiation, and survival of neurons using the rat pheochromocytoma cell line PC12. RT-PCR analysis for OC receptor candidates revealed that Gpr158, but not Gprc6a, mRNA was expressed in PC12 cells. The growth of PC12 cells cultured in the presence of 5-50 ng/mL of either uncarboxylated (GluOC) or carboxylated (GlaOC) OC was increased compared to cells cultured in the absence of OC. In addition, NGF-induced neurite outgrowth was enhanced by OC, and H2O2-induced cell death was suppressed by pretreatment with OC. All of these results were observed for both GluOC and GlaOC at comparable levels, suggesting that OC may directly affect cell proliferation, differentiation, and survival by binding to its candidate receptor, GPR158.

Effect of aging on bone metabolism: the involvement of complement C1q.

Purpose Impairment of normal bone remodeling affects the successful osseointegration of dental implants. Recently, it has been reported that complement C1q level increases with age and delays wound healing by modulating Wnt signaling. As Wnt signaling is known to play an essential role in bone remodeling, we hypothesized that aging-dependent increases in C1q affect bone remodeling. In this study, we examined whether C1q affects the differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts, and investigated whether C1q could modify cellular signaling, including the Wnt/ß-catenin pathway in these cells.Methods Osteogenic differentiation of MC3T3-E1 cells was assessed using alkaline phosphatase staining. Differentiation of osteoclasts from mouse bone marrow cells was assessed using tartrate-resistant acid phosphatase staining. Activation of canonical Wnt signaling and protein phosphorylation was monitored using Western blotting.Results C1q, at 5-15 µg/mL promoted osteoclast fusion, whereas it did not affect the differentiation of osteoblasts. On the other hand, a higher concentration of C1q (50 µg/mL) suppressed both bone morphogenetic protein-2-induced osteogenic differentiation and osteoclast formation. C1q did not induce an obvious activation of Wnt/ ß-catenin signaling in either pre-osteoblasts or pre-osteoclasts, contrary to previous reports using other tissues. Instead, C1q upregulated the receptor activator of nuclear factor-kappa B ligand (RANKL)-induced phosphorylation of Akt.Conclusions C1q could affect cellular signaling and modify the differentiation of osteoblasts and osteoclasts, depending on the concentration. Therefore, an increase in C1q with age could be one of the factors that determine the prognosis of treatment of elderly patients.

Modification of TRPV4 activity by acetaminophen.

N-Acetyl-p-aminophenol (APAP/acetaminophen) is a widely used analgesic/antipyretic with weaker inhibitory effects on cyclooxygenase compared to those of non-steroidal anti-inflammatory drugs. The effect of APAP is mediated by its metabolites, N-arachidonoyl-phenolamine and N-acetyl-p-benzoquinone imine, which activate transient receptor potential (TRP) channels, including TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or cannabinoid receptor type 1. However, the exact molecular mechanism underlying the cellular actions of APAP remains unclear. Recently, we observed that APAP promotes cell migration through TRPV4; in this study, we examined the effect of APAP on Ca2+-channel activity of TRPV4. In the rat cell line PC12 expressing TRPV4, GSK1016790A (GSK), a TRPV4 agonist, stimulated an increase in [Ca2+]i; these effects were abrogated by HC-067047 treatment. This GSK-induced Ca2+ entry through TRPV4 was inhibited by APAP in a dose-dependent manner, whereas APAP alone did not affect [Ca2+]i. The specificity of the effect of APAP on TRPV4 was further confirmed using HeLa cells, which lack endogenous TRPV4 but stably express exogenous TRPV4 (HeLa-mTRPV4). GSK-induced [Ca2+]i elevation was only observed in HeLa-mTRPV4 cells compared to that in the control HeLa cells, indicating the specific action of GSK on TRPV4. APAP dose-dependently suppressed this GSK-induced Ca2+ entry in HeLa-mTRPV4. However, it is unlikely that the metabolites of APAP were involved in these effects as the reaction in this study was rapid. The results suggest that APAP suppresses the newly identified target TRPV4 without being metabolized and exerts antipyretic/analgesic and/or other effects on TRPV4-related phenomena in the body. The effect of APAP on TRPV4 was opposite to that on TRPV1 or TRPA1, as the latter is activated by APAP.

Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

BACKGROUND: N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. METHODS: Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. RESULTS: APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. CONCLUSION: APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions.

Celecoxib inhibits osteoblast differentiation independent of cyclooxygenase activity.

Non-steroidal anti-inflammatory drugs (NSAIDs) exert their effects primarily by inhibiting the activity of cyclooxygenase (COX), thus suppressing prostaglandin synthesis. Some NSAIDs are known to perform functions other than pain control, such as suppressing tumour cell growth, independent of their COX-inhibiting activity. To identify NSAIDs with COX-independent activity, we examined various NSAIDs for their ability to inhibit osteoblastic differentiation using the mouse pre-osteoblast cell line MC3T3-E1. Only celecoxib and valdecoxib strongly inhibited osteoblastic differentiation, and this effect was not correlated with COX-inhibiting activity. Moreover, 2,5-dimethyl (DM)-celecoxib, a celecoxib analogue that does not inhibit COX activity, also inhibited osteoblastic differentiation. Celecoxib and DM-celecoxib inhibited osteoblastic differentiation induced by bone morphogenetic protein (BMP)-2 in C2C12 mouse myoblast cell line. Although celecoxib suppresses the growth of some tumour cells, the viability and proliferation of MC3T3-E1 cells were not affected by celecoxib or DM-celecoxib. Instead, celecoxib and DM-celecoxib suppressed BMP-2-induced phosphorylation of Smad1/5, a major downstream target of BMP receptor. Although it is well known that COX plays important roles in osteoblastic differentiation, these results suggest that some NSAIDs, such as celecoxib, have targets other than COX and regulate phospho-dependent intracellular signalling, thereby modifying bone remodelling.

Promotion of insulin-induced glucose uptake in C2C12 myotubes by osteocalcin.

A close relationship between the bone and systemic glucose metabolism has recently been the center of attention, since the uncarboxylated form of osteocalcin (GluOC), a bone-derived protein, but not the γ-carboxylated form, is involved in glucose metabolism. However, the analysis of GluOC effect using isolated organs and related cell lines are required to understand its roles in a whole systemic metabolic status. In the present study, we examined the effect of GluOC on cell lines derived from skeletal muscle to explore the mechanisms by which GluOC regulates glucose uptake. In the differentiated C2C12 myotubes, GluOC dose-dependently induced the phosphorylation of ERK without affecting intracellular cAMP and Ca(2+) levels. This effect was inhibited by U0126, an inhibitor of ERK kinase (MEK). Additionally, U73122, an inhibitor of phospholipase C tended to inhibit it as well. Furthermore, cell treatment with GluOC for a long period promoted insulin-induced Akt phosphorylation and glucose uptake in the myotubes, which was abolished by ERK signaling inhibition. These results indicate that GluOC does not triggered Akt phosphorylation and glucose uptake by itself but promotes insulin-induced glucose uptake in myotubes, probably by up-regulating Akt signaling through ERK activation.

Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion.

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion and pancreatic ß-cell proliferation. We previously showed that the effect of GluOC on insulin secretion is mediated largely by glucagon-like peptide-1 (GLP-1) secreted from the intestine in response to GluOC exposure. We have now examined the effect of oral administration of GluOC on glucose utilization as well as the fate of such administered GluOC in mice. Long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level and improved glucose tolerance in mice without affecting insulin sensitivity. It also increased the fasting serum insulin concentration as well as the ß-cell area in the pancreas. A small proportion of orally administered GluOC reached the small intestine and remained there for at least 24h. GluOC also entered the general circulation, and the serum GLP-1 concentration was increased in association with the presence of GluOC in the intestine and systemic circulation. The putative GluOC receptor, GPRC6A was detected in intestinal cells, and was colocalized with GLP-1 in some of these cells. Our results suggest that orally administered GluOC improved glucose handling likely by acting from both the intestinal lumen and the general circulation, with this effect being mediated in part by stimulation of GLP-1 secretion. Oral administration of GluOC warrants further study as a safe and convenient option for the treatment or prevention of metabolic disorders.