Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-κB signaling pathway

Aim:

To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells.

Methods:

Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-κB-regulated gene products and TNF-α-induced activation of p65, IκBα, and IKK. The intracellular location of NF-κB p65 was detected using confocal microscopy.

Results:

Plumbagin (2.5–40 μmol/L) concentration-dependently reduced the viability of the GC cells. The IC₅₀ value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 μmol/L, respectively. The compound (5–20 μmol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-αand cisplatin. The compound (10 μmol/L) downregulated the expression of NF-κB-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-κB p65 nuclear translocation, the compound also suppressed TNF-α-induced phosphorylation of p65 and IKK, and the degradation of IκBα.

Conclusion:

Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-κB pathway.     

Thiocolchicoside suppresses osteoclastogenesis induced by RANKL and cancer cells through inhibition of inflammatory pathways: a new use for an old drug

BACKGROUND AND PURPOSE

Most patients with cancer die not because of the tumour in the primary site, but because it has spread to other sites. Common tumours, such as breast, multiple myeloma, and prostate tumours, frequently metastasize to the bone. To search for an inhibitor of cancer-induced bone loss, we investigated the effect of thiocolchicoside, a semi-synthetic colchicoside derived from the plant Gloriosa superba and clinically used as a muscle relaxant, on osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL) and tumour cells.

EXPERIMENTAL APPROACH

We used RAW 264.7 (murine macrophage) cells, a well-established system for osteoclastogenesis, and evaluated the effect of thiocolchicoside on RANKL-induced NF-κB signalling and osteoclastogenesis as well as on osteoclastogenesis induced by tumour cells.

KEY RESULTS

Thiocolchicoside suppressed osteoclastogenesis induced by RANKL, and by breast cancer and multiple myeloma cells. Inhibition of the NF-κB pathway was responsible for this effect since the colchicoside inhibited RANKL-induced NF-κB activation, activation of IκB kinase (IKK) and suppressed inhibitor of NF-κBα (IκBα) phosphorylation and degradation, an inhibitor of NF-κB. Furthermore, an inhibitor of the IκBα kinase γ or NF-κB essential modulator, the regulatory component of the IKK complex, demonstrated that the NF-κB signalling pathway is mandatory for osteoclastogenesis induced by RANKL.

CONCLUSIONS AND IMPLICATIONS

Together, these data suggest that thiocolchicoside significantly suppressed osteoclastogenesis induced by RANKL and tumour cells via the NF-κB signalling pathway. Thus, thiocolchicoside, a drug that has been used for almost half a century to treat muscle pain, may also be considered as a new treatment for bone loss.

LINKED ARTICLE

This article is commented on by Micheau et al., pp. 2124–2126 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01792.x     

α-Tocopheryl succinate induces apoptosis in erbB2-expressing breast cancer cell via NF-κB pathway

Aim:

To study the molecular mechanisms underlying α-tocopheryl succinate (α-TOS)-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand (hrTRAIL) act synergically to induce cell death of erbB2-expressing breast cancer cells.

Methods:

The annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAM^TM NF-κB p65 kit was used to assess NF-κB activation.

Results:

α-TOS (100 μmol/L) significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-κB. α-TOS (50 and 100 μmol/L) inhibited the expression of Flice-like inhibitory protein (FLIP) and cellular inhibitor of apoptosis protein 1 (c-IAP1) in erbB2-positive cells. α-TOS (100 μmol/L) inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2. α-TOS (50 μmol/L) and hrTRAIL (30 mg/mL) acted synergically to induce apoptosis in breast cancer cells. α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB .

Conclusion:

Our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.     

Berbamine, a novel nuclear factor κB inhibitor, inhibits growth and induces apoptosis in human myeloma cells

Aim:

We sought to investigate the effect of berbamine on the growth of human multiple myeloma cell line KM3 and elucidate the mechanism of its action.

Methods:

MTT assay was used to determine the inhibitory effect of berbamine alone or combined with chemotherapeutic drugs. Flow cytometry was performed to characterize cell cycle profile in response to berbamine treatment. Western blot was used to measure the protein levels of p65, IκB Kinase α (IKKα), TNFAIP3 (A20), IκBα, p-IκBα, cyclinD1, Bcl-2, BAX, Bcl-x_L, Bid, and survivin.

Results:

Berbamine inhibits the proliferation of KM3 cells in a dose- and time-dependent manner. Combination of berbamine with dexamethasone (Dex), doxorubicin (Dox) or arsenic trioxide (ATO) resulted in enhanced inhibition of cell growth. Flow cytometric analysis revealed that KM3 cells were arrested at G₁ phase and apoptotic cells increased from 0.54% to 51.83% for 36 h. Morphological changes of cells undergoing apoptosis were observed under light microscope. Berbamine treatment led to increased expression of A20, down-regulation of IKKα, p-IκBα, and followed by inhibition of p65 nuclear localization. As a result, NF-κB downstream targets such as cyclinD1, Bcl-x_L, Bid and survivin were down-regulated.

Conclusion:

Berbamine inhibits the growth of KM3 cells by inducing G₁ arrest as well as apoptosis. Berbamine blocks NF-κB signaling pathway through up-regulating A20, down-regulating IKKα, p-IκBα, and then inhibiting p65 nuclear translocation, and resulting in decreased expression of the downstream targets of NF-κB. Our results suggest that berbamine is a novel inhibitor of NF-κB activity with remarkable anti-myeloma efficacy.     

Geniposide inhibits high glucose-induced cell adhesion through the NF-KB signaling pathway in human umbilical vein endothelial cells

Aim:

To investigate whether geniposide, an iridoid glucoside extracted from gardenia jasminoides ellis fruits, inhibits cell adhesion to human umbilical vein endothelial cells (HUVECs) induced by high glucose and its underlying mechanisms.

Methods:

HUVECs were isolated from human umbilical cords and cultured. The adhesion of monocytes to HUVECs was determined using fluorescence-labeled monocytes. The mRNA and protein levels of vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) were measured using real-time RT-PCR and ELISA. Reactive oxygen species (ROS) production was measured using a fluorescent probe. The amounts of nuclear factor-kappa B (NF-κB) and inhibitory factor of NF-κB (IκB) were determined using Western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined using immunofluorescence.

Results:

Geniposide (10–20 μmol/L) inhibited high glucose (33 mmol/L)-induced adhesion of monocytes to HUVECs in a dose-dependent manner. This compound (5–40 μmol/L) also inhibited high glucose-induced expression of VCAM-1 and E-selectin at the gene and protein levels. Furthermore, geniposide (5–20 μmol/L) decreased ROS production and prevented IκB degradation in the cytoplasm and NF-κB translocation from the cytoplasm to the nucleus in HUVECs.

Conclusion:

Geniposide inhibits the adhesion of monocytes to HUVECs and the expression of CAMs induced by high glucose, suggesting that the compound may represent a new treatment for diabetic vascular injury. The mechanism underlying this inhibitory effect may be related to the inhibition of ROS overproduction and NF-κB signaling pathway activation by geniposide.     

Novel role of curcumin in the prevention of cytokine-induced islet death in vitro and diabetogenesis in vivo

Background and purpose:

Oxidative stress caused by cytokine exposure is a major cause of pancreatic islet death in vitro and of diabetogenesis. Antioxidant compounds may prevent cytokine-induced damage to islet cells. Hence, we studied the potential of curcumin, an antioxidant and anti-inflammatory compound, in vitro to protect islets against pro-inflammatory cytokines and in vivo to prevent the progression of diabetes induced by multiple low doses of streptozotocin (MLD-STZ).

Experimental approach:

Pancreatic islets from C57/BL6J mice were pretreated with curcumin (10 μM) and then exposed to a combination of cytokines. Islet viability, reactive oxygen species (ROS), NO, inducible NO synthase and NF-κB translocation were studied. Curcumin pretreated (7.5 mg kg⁻¹ day⁻¹) C57/BL6J mice were given MLD-STZ (40 mg kg⁻¹), and various parameters of diabetes induction and progression were monitored.

Key results:

Curcumin protected islets from cytokine-induced islet death in vitro by scavenging ROS and normalized cytokine-induced NF-κB translocation by inhibiting phosphorylation of inhibitor of kappa B alpha (IκBα). In vivo, curcumin also prevented MLD-STZ, as revealed by sustained normoglycaemia, normal glucose clearance and maintained pancreatic GLUT2 levels. Pro-inflammatory cytokine concentrations in the serum and pancreas were raised in STZ-treated animals, but not in animals pretreated with curcumin before STZ.

Conclusions and implications:

Here, we have demonstrated for the first time that curcumin in vitro protects pancreatic islets against cytokine-induced death and dysfunction and in vivo prevents STZ-induced diabetes.     

Glycyrrhetinic acid inhibits ICAM-1 expression via blocking JNK and NF-KB pathways in TNF-α-activated endothelial cells

Aim:

To investigate the effects of glycyrrhetinic acid (GA), an active component extracted from the root of Glycyrrhizae glabra, on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC).

Methods:

ICAM-1 mRNA and protein levels were detected using RT-PCR and cell enzyme-linked immunosorbent assays. The adherence of human monocytic THP-1 cells labeled with [³H]thymidine to HUVEC was determined by counting radioactivity with a scintillation counter. The activation of mitogen-activated protein kinases as well as the degradation of IκB and nuclear factor-κB (NF-κB) or phospho-c-Jun in the nucleus were detected by western blots. NF-κB binding activity was detected using electrophoretic mobility shift assay.

Results:

GA (50 and 100 μmol/L) significantly inhibits TNF-α-induced ICAM-1 mRNA and protein expressions, as well as THP-1 cell adhesiveness in HUVEC. GA selectively inhibited TNF-α-activated signal pathway of c-Jun N-terminal kinase (JNK), without affecting extracellular signal-regulated kinase 1/2 and p38. Furthermore, GA apparently inhibited IκB/NF-κB signaling system by preventing IκB degradation, NF-κB translocation, and NF-κB/DNA binding activity. Finally, pretreatment with GA or the inhibitors of NF-κB, JNK, and p38 reduced the ICAM-1 protein expression induced by TNF-α.

Conclusion:

GA inhibits TNF-α-stimulated ICAM-1 expression, leading to a decrease in adherent monocytes to HUVEC. This inhibition is attributed to GA interruption of both JNK/c-Jun and IκB/NF-κB signaling pathways, which decrease activator protein-1 (AP-1) and NF-κB mediated ICAM-1 expressions. The results suggest that GA may provide a beneficial effect in treating vascular diseases associated with inflammation, such as atherosclerosis.     

Okadaic acid induces matrix metalloproteinase-9 expression in fibroblasts: crosstalk between protein phosphatase inhibition and β-adrenoceptor signalling

BACKGROUND AND PURPOSE

Interactions between protein phosphatase inhibition and matrix metalloproteinase (MMP)-9 expression have implications for tissue remodelling after injury. Stimulation of β-adrenoceptors could affect such interactions as isoprenaline increases protein phosphatase 2A (PP2A) activity and MMP-9 abundance. We investigated the effect of okadaic acid (OA) on MMP-9 expression to assess interactions between phosphatase inhibition and β-adrenoceptor signalling in fibroblasts.

EXPERIMENTAL APPROACH

Fibroblasts were exposed to OA alone and in combination with isoprenaline. Effects on MMP-9 expression and intracellular signalling were studied using promoter assays, Western blot analysis and siRNA methodologies.

KEY RESULTS

Okadaic acid increased MMP-9 abundance in human cardiac ventricular fibroblasts, NIH3T3 fibroblasts and hepatic stellate cells. This effect was unaffected by PP2A knockdown in NIH3T3 cells. OA increased phosphorylation of NF-κB, but not NF-κB promoter activity, IκBα degradation, or nuclear translocation of p65-NF-κB. Exposure to SB202190 (p38 MAPK), U0126 (ERK1/2) and NF-κB III inhibitor revealed that OA induced MMP-9 activity through p38 MAPK. Isoprenaline inhibited OA-mediated MMP-9 expression in NIH3T3, in a β-arrestin 2- and PP2A-dependent manner. Mutation of the activator protein-1 (AP-1) and NF-κB binding sites demonstrated that OA-induced MMP-9 activity was mediated through the AP-1 but not NF-κB sites. The latter mediated the inhibitory effect of isoprenaline on OA-induced MMP-9 promoter activity.

CONCLUSION AND IMPLICATIONS

Okadaic acid induced MMP-9 activity through p38 MAPK and was inhibited by isoprenaline via a pathway involving β-arrestin 2, PP2A and an NF-κB binding motif. These findings elucidate how phosphoprotein phosphatases and adrenoceptors may modulate tissue remodelling by affecting fibroblast function.     

Betulinic acid inhibits endotoxin-stimulated phosphorylation cascade and pro-inflammatory prostaglandin E(2) production in human peripheral blood mononuclear cells

BACKGROUND AND PURPOSE

Betulinic acid (BA) is a naturally occurring triterpenoid widely distributed throughout the plant kingdom. We previously reported that BA inhibits lipopolysaccharide (LPS)-induced interleukin-6 production through modulation of nuclear factor κB (NF-κB) in human peripheral blood mononuclear cells (hPBMCs). This study attempted to identify other mechanisms through which BA modulates LPS signalling in mononuclear cells. The effects of BA on signalling pathways downstream were focused on in this study.

EXPERIMENTAL APPROACH

We determined the ability of BA to interfere with p38 and extracellular regulated kinase (ERK) phosphorylation as well as Akt phosphorylation and nuclear factor-κB activation using LPS-activated hPBMCs as an in vitro model. LPS-induced endotoxin shock in mice was the in vivo model employed.

KEY RESULTS

BA inhibited LPS-induced COX-2 protein expression and prostaglandin E₂ production and also attenuated LPS-induced ERK and Akt phosphorylation, but not p38 in hPBMCs. BA abolished LPS-induced IκBα phosphorylation and thus normalized the levels of IκBα in cytosol. BA also inhibited LPS-induced reactive oxygen species formation and lactate dehydrogenase release. Interestingly, BA improved the life span of mice in endotoxin shock and also inhibited PGE₂ production and myeloperoxidase activity in vivo.

CONCLUSIONS AND IMPLICATIONS

BA modulates LPS-induced COX-2 expression in hPBMCs by inhibiting ERK and Akt pathways as well as by modulating IκBα phosphorylation. At the same time, no cell toxicity was observed. The effect of the drug was confirmed through in vivo experiments. The study gives an insight into the molecular mechanisms of BA.     

Icariin potentiates the antitumor activity of gemcitabine in gallbladder cancer by suppressing NF-κB

Aim:

Gemcitabine has been increasingly prescribed for the treatment of gallbladder cancer. However, the response rate is low. The aim of this study is to determine whether icariin, a flavonoid isolated from Epimedi herba, could potentiate the antitumor activity of gemcitabine in gallbladder cancer.

Methods:

Human gallbladder carcinoma cell lines GBC-SD and SGC-996 were tested. Cell proliferation and apoptosis were analyzed using MTT assay and flow cytometry, respectively. The expression of apoptosis- and proliferation-related molecules was detected with Western blotting. Caspase-3 activity was analyzed using colorimetric assay, and NF-κB activity was measured with ELISA. A gallbladder cancer xenograft model was established in female BALB/c (nu/nu) mice. The mice were intraperitoneally administered gemcitabine (125 mg/kg) in combination with icariin (40 mg/kg) for 2 weeks.

Results:

Icariin (40–160 μg/mL) dose-dependently suppressed cell proliferation and induced apoptosis in both GBC-SD and SGC-996 cells, with SGC-996 cells being less sensitive to the drug. Icariin (40 μg/mL) significantly enhanced the antitumor activity of gemcitabine (0.5 μmol/L) in both GBC-SD and SGC-996 cells. The mice bearing gallbladder cancer xenograft treated with gemcitabine in combination with icariin exhibited significantly smaller tumor size than the mice treated with either drug alone. In GBC-SD cells, icariin significantly inhibited both the constitutive and gemcitabine-induced NF-κB activity, enhanced caspase-3 activity, induced G₀-G₁ phase arrest, and suppressed the expression of Bcl-2, Bcl-xL and surviving proteins.

Conclusion:

Icariin, by suppressing NF-κB activity, exerts antitumor activity, and potentiates the antitumor activity of gemcitabine in gallbladder cancer. Combined administration of gemcitabine and icariin may offer a better therapeutic option for the patients with gallbladder cancer.     

Inhibiting the TLR4-MyD88 signalling cascade by genetic or pharmacological strategies reduces acute alcohol-induced sedation and motor impairment in mice

BACKGROUND AND PURPOSE

Emerging evidence implicates a role for toll-like receptor 4 (TLR4) in the CNS effects of alcohol. The aim of the current study was to determine whether TLR4–MyD88-dependent signalling is involved in the acute behavioural actions of alcohol and if alcohol can activate TLR4-downstream MAPK and NF-κB pathways.

EXPERIMENTAL APPROACH

The TLR4 pathway was evaluated using the TLR4 antagonist (+)-naloxone (µ-opioid receptor-inactive isomer) and mice with null mutations in the TLR4 and MyD88 genes. Sedation and motor impairment induced by a single dose of alcohol were assessed by loss of righting reflex (LORR) and rotarod tests, separately. The phosphorylation of JNK, ERK and p38, and levels of IκBα were measured to determine the effects of acute alcohol exposure on MAPK and NF-κB signalling.

KEY RESULTS

After a single dose of alcohol, both pharmacological inhibition of TLR4 signalling with (+)-naloxone and genetic deficiency of TLR4 or MyD88 significantly (P < 0.0001) reduced the duration of LORR by 45–78% and significantly decreased motor impairment recovery time to 62–88% of controls. These behavioural actions were not due to changes in the peripheral or central alcohol pharmacokinetics. IκBα levels responded to alcohol by 30 min in mixed hippocampal cell samples, from wild-type mice, but not in cells from TLR4- or MyD88-deficient mice.

CONCLUSIONS AND IMPLICATIONS

These data provide new evidence that TLR4–MyD88 signalling is involved in the acute behavioural actions of alcohol in mice.

LINKED ARTICLE

This article is commented on by Pandey, pp. 1316–1318 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01695.x     

Delayed apoptosis of human monocytes exposed to immune complexes is reversed by oxaprozin: role of the Akt/I��B kinase/nuclear factor ��B pathway

Background and purpose

Monocytes-macrophages play a key role in the initiation and persistence of inflammatory reactions. Consequently, these cells represent an attractive therapeutic target for switching off overwhelming inflammatory responses. Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most common drugs for the symptomatic treatment of rheumatic diseases. Their effects have been explained on the basis of cyclooxygenase (COX) inhibition. However, some of the actions of these drugs are not related to inhibition of prostaglandin synthesis.

Experimental approach

We examined the effect of oxaprozin on apoptosis of immune complex-activated monocytes in comparison with drugs of the same class, and the signalling pathway that leads activated monocytes exposed to oxaprozin to apoptosis. In particular, we studied the activity of caspase-3, the involvement of IκB kinase (IKK)-nuclear factor κB (NF-κB) system and the activity of X-linked mammalian inhibitor of apoptosis protein (XIAP), Akt and mitogen-activated protein kinase (MAPK) in activated monocytes in the presence of oxaprozin.

Key results

Immune complexes caused the inhibition of monocyte apoptosis. Oxaprozin reversed in a dose-dependent manner immune complex-induced survival of monocytes, without affecting the apoptosis of resting cells. Other NSAIDs are ineffective. The activity of oxaprozin was related to inhibition of Akt activation that, in turn, prevented p38 MAPK, IKK and NF-κB activation. Consistently, the inhibition of NF-κB activation reduced the production of the anti-apoptotic molecule XIAP, leading to uncontrolled activity of caspase 3.

Conclusions and implications

These results suggest that oxaprozin exerts its anti-inflammatory activity also through COX-independent pathways. It is likely that oxaprozin-mediated inhibition of the Akt/IKK/NF-κB pathway contributes to its anti-inflammatory properties.     

Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro

Aim:

Epigallocatechin-3-gallate (EGCG) is the major polyphenolic constituent in green tea. The aim of this study is to investigate the effects of EGCG on proliferation and migration of the human colon cancer SW620 cells.

Methods:

Proliferation and migration of SW620 cells were induced by the protease-activated receptor 2-agonist peptide (PAR2-AP, 100 μmol/L) or factor VIIa (10 nmol/L), and analyzed using MTT and Transwell assays, respectively. The cellular cytoskeleton was stained with rhodamine-conjugated phalloidin and examined with a laser scanning confocal fluorescence microscope. The expression of caspase-7, tissue factor (TF) and matrix metalloproteinase (MMP)-9 in the cells was examined using QT-PCR, ELISA and Western blot assays. The activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and nuclear factor-kappa B (NF-κB) signaling pathways was analyzed with Western blot.

Results:

Both PAR2-AP and factor VIIa promoted SW620 cell proliferation and migration, and caused cytoskeleton reorganization (increased filopodia and pseudopodia). Pretreatment with EGCG (25, 50, 75, and 100 μg/mL) dose-dependently blocked the cell proliferation and migration induced by PAR2-AP or factor VIIa. EGCG (100 μg/mL) prevented the cytoskeleton changes induced by PAR2-AP or factor VIIa. EGCG (100 μg/mL) counteracted the down-regulation of caspase-7 expression and up-regulation of TF and MMP-9 expression in the cells treated with PAR2-AP or factor VIIa. Furthermore, it blocked the activation of ERK1/2 and NF-κB (p65/RelA) induced by PAR2-AP or factor VIIa.

Conclusion:

EGCG blocks the proliferation and migration of SW620 cells induced by PAR2-AP and factor VIIa via inhibition of the ERK1/2 and NF-κB pathways. The compound may serve as a preventive and therapeutic agent for colon cancers.     

Exendin-4 ameliorates oxidized-LDL-induced inhibition of macrophage migration in vitro via the NF-KB pathway

Aim： To investigate the effects of the glucagon-like peptide-1 （GLP-1） receptor agonist exendin-4 on oxidized low-density lipoprotein （ox- LDL）-induced inhibition of macrophage migration and the mechanisms underlying the effects of exendin-4. Methods： Primary peritoneal macrophages were extracted from the peritoneal cavity of mice treated with 3% thioglycollate （2 mL, ip）. Migration of the macrophages was examined using a cell migration assay. Macrophage migration-related factors including leptin-like ox-LDL receptor （LOX-1）, cyclooxygenase 2 （COX-2）, tumor necrosis factor （TNF）-a, interleukin-1 （IL-1）13, matrix metalloproteinase-2 （MMP-2）, intercellular adhesion molecule （ICAM）-I and macrophage migration inhibitory factor （MIF） were measured using semi quantitative RT-PCR. Expression of MIF and ICAM-1 proteins was examined with ELISA. Gelatin zymography was used to evaluate the activity of MMP-9. Activation of the NF-KB pathway was determined by confocal laser scanning microscopy. Results： Treatment of the macrophages with ox-LDL （50 pg/mL） markedly suppressed the macrophage migration. Furthermore, ox-LDL treatment substantially increased the expression of the macrophage migration-related factors, the activity of MMP-9 and the transloca-tion of the NF-KB p65 subunit. These effects of ox-LDL were significantly ameliorated by pretreatment with the specific NF-KB inhibitor ammonium pyrrolidine dithiocarbamate （100μmol/L）. These effects of ox-LDL were also significantly ameliorated by pretreatment with exendin-4 （25 and 50 nmoVL）. Conclusion： Exendin-4 ameliorates the inhibition of ox-LDL on macrophage migration in vitro, via suppressing ox-LDL-induced expres- sion of ICAM-1 and MIF, which is probably mediated by the NF-KB pathway.     

2-Methoxystypandrone represses RANKL-mediated osteoclastogenesis by down-regulating formation of TRAF6�CTAK1 signalling complexes

BACKGROUND AND PURPOSE

2-Methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum, a Chinese herb used to treat bone diseases. Here we have determined whether 2-MS antagonised osteoclast development and bone resorption.

EXPERIMENTAL APPROACH

RAW264.7 cells were treated with receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to induce differentiation into osteoclasts. RT-PCR and Western blot were used to analyse osteoclast-associated gene expression and signalling pathways.

KEY RESULTS

The number of multinuclear osteoclasts, actin rings and resorption pit formation were markedly inhibited by 2-MS, targeting osteoclast differentiation at an early stage and without significant cytotoxicity. The anti-resorption effect of 2-MS was accompanied by decreasing dendritic cell-specific transmembrane protein and matrix metalloproteinase-9 (MMP-9) mRNA expression. RANKL-increased MMP-9 gelatinolytic activity was also attenuated by concurrent, but not by subsequent addition of 2-MS. 2-MS markedly inhibited not only the RANKL-triggered nuclear translocations of NF-κB, c-Fos and nuclear factor of activated T cells c1 (NFATc1), but also the subsequent NFATc1 induction. Degradation of IκB and phosphorylation of mitogen-activated protein kinases were also suppressed. RANKL facilitated the formation of singaling complexes of tumour necrosis factor receptor-associated factor 6 and transforming growth factor β-activated kinase 1 (TRAF6–TAK1), important for osteoclastogenesis and formation of such signalling complexes was prevented by 2-MS.

CONCLUSIONS AND IMPLICATIONS

The anti-osteoclastogenic effects of 2-MS could reflect the block of RANKL-induced association of TRAF6–TAK1 complexes with consequent decrease of IκB-mediated NF-κB and mitogen-activated protein kinases-mediated c-Fos activation pathways and suppression of NFATc1 and other gene expression, essential for bone resorption.