Role of Spm–Cer‐S1P signalling pathway in MMP‐2 mediated U46619‐induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin‐3‐gallate

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occur s , which contributes to pulmonary hypertension. Thromboxane A₂ (TxA₂) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin‐3‐gallate (EGCG) on the TxA₂ mimetic, U46619‐induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p³⁸MAPK, NF‐κB and MMP‐2 significantly inhibit U46619‐induced cell proliferation. EGCG markedly abrogate U46619‐induced p³⁸MAPK phosphorylation, NF‐κB activation, proMMP‐2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619‐induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP‐2 markedly abrogate U46619‐induced SMase activity and S1P level. EGCG markedly inhibit U46619‐induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline–Ceramide–Sphingosine‐1‐phosphate (Spm–Cer–S1P) signalling axis plays an important role in MMP‐2 mediated U46619‐induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP‐2 activation by modulating p³⁸MAPK–NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.     

Activated α2 macroglobulin induces matrix metalloproteinase 9 expression by low‐density lipoprotein receptor‐related protein 1 through MAPK‐ERK1/2 and NF‐κB activation in macrophage‐derived cell lines

Macrophages under certain stimuli induce matrix metalloproteinase 9 (MMP‐9) expression and protein secretion through the activation of MAPK‐ERK and NF‐κB signaling pathways. Previously, we demonstrated that activated α₂‐macroglulin (α₂M*) through the interaction with its receptor low‐density lipoprotein receptor‐related protein 1 (LRP1) induces macrophage proliferation mediated by the activation of MAPK‐ERK1/2. In the present work, we examined whether α₂M*/LRP1interaction could induce the MMP‐9 production in J774 and Raw264.7 macrophage‐derived cell lines. It was shown that α₂M* promoted MMP‐9 expression and protein secretion by LRP1 in both macrophage‐derived cell lines, which was mediated by the activation of MAPK‐ERK1/2 and NF‐κB. Both intracellular signaling pathways activated by α₂M* were effectively blocked by calphostin‐C, suggesting involvement of PKC. In addition, we demonstrate that α₂M* produced extracellular calcium influx via LRP1. However, when the intracellular calcium mobilization was inhibited by BAPTA‐AM, the α₂M*‐induced MAPK‐ER1/2 activation was fully blocked in both macrophage cell lines. Finally, using specific pharmacological inhibitors for PKC, Mek1, and NF‐κB, it was shown that the α₂M*‐induced MMP‐9 protein secretion was inhibited, indicating that the MMP production promoted by the α₂M*/LRP1 interaction required the activation of both signaling pathways. These findings may prove useful in the understanding of the macrophage LRP1 role in the vascular wall during atherogenic plaque progression. J. Cell. Biochem. 111: 607–617, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Valsartan independent of AT1 receptor inhibits tissue factor,TLR‐2 and ‐4 expression by regulation of Egr‐1 through activation of AMPK in diabetic conditions

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)‐1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT₁R) can reduce tissue factor (TF) and toll‐like receptor (TLR)‐2 and ‐4 by regulating Egr‐1 in THP‐1 cells and aorta in streptozotocin‐induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr‐1, TF, TLR‐2 and ‐4 which were significantly reduced by valsartan. HG increased Egr‐1 expression by activation of PKC and ERK1/2 in THP‐1 cells. Valsartan increased AMPK phosphorylation in a concentration and time‐dependent manner via activation of LKB1. Valsartan inhibited Egr‐1 without activation of PKC or ERK1/2. The reduced expression of Egr‐1 by valsartan was reversed by either silencing Egr‐1, or compound C, or DN‐AMPK‐transfected cells. Valsartan inhibited binding of NF‐κB and Egr‐1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF‐α, IL‐6 and IL‐1β) production and NF‐κB activity in HG‐activated THP‐1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT₁R in THP‐1 cells or CHO cells, which were devoid of AT₁R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr‐1, TLR‐2, ‐4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin‐induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr‐1 regulation.     

Simvastatin abolishes nitric oxide‐ and reactive oxygen species‐induced cyclooxygenase‐2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO‐ or ROS‐induced cyclooxygenase‐2 (COX‐2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)‐ and interleukin 1 beta (IL‐1β)‐induced COX‐2 production. In addition, simvastatin attenuated SNP‐induced NO production and IL‐1β‐induced ROS generation. Treatment with simvastatin prevented SNP‐ and IL‐1β‐induced nuclear factor kappa B (NF‐κB) activity. Inhibiting NO production and ROS generation using N‐acetylcysteine (NAC) and NG‐monomethyl‐ l ‐arginine ( l ‐NMMA), respectively, accelerated the influence of simvastatin on NF‐κB activity. In addition, NAC blocked SNP and simvastatin‐mediated COX‐2 production and NF‐κB activity but did not alter IL‐1β and simvastatin‐mediated COX‐2 expression. l ‐NMMA treatment also abolished IL‐1β‐mediated COX‐2 expression and NF‐κB activation, whereas SNP and simvastatin‐mediated COX‐2 expression were not altered compared with the levels in the SNP and simvastatin‐treated cells. Our findings suggested that simvastatin blocks COX‐2 expression by inhibiting SNP‐induced NO production and IL‐1β‐induced ROS generation by blocking the NF‐κB pathway.     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

CTGF enhances migration and MMP‐13 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CTGF increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody (mAb) and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the CTGF‐induced increase of the migration and MMP‐13 up‐regulation of chondrosarcoma cells. CTGF stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited CTGF‐induced cell migration and MMP‐13 up‐regulation. Stimulation of JJ012 cells with CTGF also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The CTGF‐mediated increases in κB‐luciferase activities were inhibited by RGD, PD98059, U0126 or FAK, and ERK2 mutant. Taken together, our results indicated that CTGF enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3 integrin, FAK, ERK, and NF‐κB signal transduction pathway. J. Cell. Biochem. 107: 345–356, 2009. © 2009 Wiley‐Liss, Inc.     

Increased expression of bFGF is associated with carotid atherosclerotic plaques instability engaging the NF‐κB pathway

Unstable atherosclerotic plaques of the carotid arteries are at great risk for the development of ischemic cerebrovascular events. The degradation of the extracellular matrix by matrix metalloproteinases (MMPs) and nitric oxide induced apoptosis of vascular smooth muscle cells (VSMCs) contribute to the vulnerability of the atherosclerotic plaques. Basic fibroblast growth factor (bFGF) through its mitogenic and angiogenic properties has already been implicated in the pathogenesis of atherosclerosis. However, its role in plaque stability remains elusive. To address this issue, a panel of human carotid atherosclerotic plaques was analysed for bFGF, FGF‐receptors‐1 and ‐2 (FGFR‐1/‐2), inducible nitric oxide synthase (iNOS) and MMP‐9 expression. Our data revealed increased expression of bFGF and FGFR‐1 in VSMCs of unstable plaques, implying the existence of an autocrine loop, which significantly correlated with high iNOS and MMP‐9 levels. These results were recapitulated in vitro by treatment of VSMCs with bFGF. bFGF administration led to up‐regulation of both iNOS and MMP‐9 that was specifically mediated by nuclear factor‐κB (NF‐κB) activation. Collectively, our data demonstrate a novel NF‐κB‐mediated pathway linking bFGF with iNOS and MMP‐9 expression that is associated with carotid plaque vulnerability.     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

Effects of simvastatin and rosuvastatin on RAS protein,matrix metalloproteinases and NF‐κB in lung cancer and in normal pulmonary tissues

Objectives

In this study, we have evaluated effects of 24‐hour treatments with simvastatin or rosuvastatin on RAS protein, NF‐κB and MMP expression in LC tissues obtained from 12 patients undergoing thoracic surgery.

Materials and methods

Normal and lung tumour tissues obtained from each sample were exposed to simvastatin (2.5–30 μm ) or rosuvastatin (1.25–30 μm ) and western blot analysis was then performed.

Results

We documented increased expression of proteins, MMP‐2, MMP‐9 and NF‐κB‐p65 in LC tissues, with respect to normal tissues (P < 0.01). In the malignant tissues, simvastatin and rosuvastatin significantly (P < 0.01) and dose‐dependently reduced RAS protein, MMP‐2/9 and NF‐κB‐p65 expression.

Conclusions

In conclusion, our results suggest that simvastatin and rosuvastatin could play a role in LC treatment by modulation of RAS protein, MMP‐2/9 and NF‐κB‐p65.

     

Attenuation of NDEA‐induced hepatocarcinogenesis by naringenin in rats

Chemoprevention is one of the most promising and realistic approaches in the prevention of cancer. Several bioactive compounds present in fruits and vegetables have revealed their cancer curative potential on hepatocellular carcinoma. Naringenin is one such naturally occurring flavonoid widely found in citrus fruits. In this study, we examined the molecular mechanisms by which naringenin inhibited NDEA‐induced hepatocellular carcinoma in rats by analysing the expression patterns of proliferating cell nuclear antigen, Bcl‐2, NF‐κB, VEGF and MMP‐2/9. Enhanced cell proliferation and apoptotic evasion in NDEA‐induced hepatocarcinogenesis was associated with imbalance in pro‐apoptotic and anti‐apoptotic proteins together with upregulation of proliferating cell nuclear antigen (PCNA) and downregulation of caspase‐3. Administration of pretreatment and posttreatment of naringenin decreased the expression of PCNA and Bcl‐2 and increased the expression of Bax and caspase‐3, indicating antiproliferative and apoptotic effects, respectively. Administration of NDEA increased the tumour expression of NF‐κB, COX‐2, VEGF, MMP‐2 and MMP‐9 that was correlated with more aggressive lesions and tumour growth. Downregulation of NF‐κB, VEGF and MMPs by naringenin seen in the present study were correlated with the inhibition of liver tumour induced by NDEA. Our results suggest that naringenin could act as a legitimate agent by inhibiting cancer processes. Copyright © 2012 John Wiley & Sons, Ltd.     

Platycodin D,a triterpenoid saponin from Platycodon grandiflorum,suppresses the growth and invasion of human oral squamous cell carcinoma cells via the NF‐κB pathway

This work was undertaken to explore the effects of platycodin D, a triterpenoid saponin from Platycodon grandiflorum, on the growth and invasiveness of human oral squamous cell carcinoma (OSCC). Platycodin D caused a significant, concentration‐dependent inhibition of cell viability and induced significant apoptosis in OSCC cells. Moreover, platycodin D significantly inhibited OSCC cell invasion. At the molecular level, platycodin D increased the amounts of IκBα protein and reduced the expression of phosphorylated NF‐κB p65, MMP‐2, and MMP‐9. Ectopic expression of constitutively active NF‐κB p65 prevented platycodin D‐mediated induction of apoptosis and suppression of invasion in OSCC cells. In vivo studies confirmed that platycodin D retarded the growth of subcutaneous SCC‐4 xenograft tumors and reduced phosphorylation of NF‐κB p65. Altogether, platycodin D shows inhibitory activity on OSCC growth and invasion through inactivation of the NF‐κB pathway and might provide therapeutic benefits in the treatment of OSCC.     

Dauricine induces apoptosis,inhibits proliferation and invasion through inhibiting NF‐κB signaling pathway in colon cancer cells

Dauricine, a bioactive component of Asiatic Moonseed Rhizome, has been widely used to treat a large number of inflammatory diseases in traditional Chinese medicine. In our study, we demonstrated that dauricine inhibited colon cancer cell proliferation and invasion, and induced apoptosis by suppressing nuclear factor‐kappaB (NF‐κB) activation in a dose‐ and time‐dependent manner. Addition of dauricine inhibited the phosphorylation and degradation of IκBα, and the phosphorylation and translocation of p65. Moreover, dauricine down‐regulated the expression of various NF‐κB‐regulated genes, including genes involved cell proliferation (cyclinD1, COX2, and c‐Myc), anti‐apoptosis (survivin, Bcl‐2, XIAP, and IAP1), invasion (MMP‐9 and ICAM‐1), and angiogenesis (VEGF). In athymic nu/nu mouse model, we further demonstrated that dauricine significantly suppressed colonic tumor growth. Taken together, our results demonstrated that dauricine inhibited colon cancer cell proliferation, invasion, and induced cell apoptosis by suppressing NF‐κB activity and the expression profile of its downstream genes. These findings provide evidence for a novel role of dauricine in preventing or treating colon cancer through modulation of NF‐κB singling pathway. J. Cell. Physiol. 225: 266–275, 2010. © 2010 Wiley‐Liss, Inc.