Bradykinin enhances cell migration in human chondrosarcoma cells through BK receptor signaling pathways

Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. BK has recently been shown to be involved in carcinogenesis and cancer progression. In this study, we found that BK increased the migration and the expression of α2β1 integrin in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significantly higher expression of the B1 and B2 receptors comparing to normal cartilage. BK‐mediated migration and integrin up‐regulation was attenuated by B1 and B2 BK receptor siRNA or antagonist. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after BK treatment was demonstrated, and BK‐induced integrin expression and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. Taken together, our results indicated that BK enhances the migration of chondrosarcoma cells by increasing α2β1 integrin expression through the BK receptors/PLC/PKCδ/NF‐κB signal transduction pathway. J. Cell. Biochem. 109: 82–92, 2010. © 2009 Wiley‐Liss, Inc.     

CCL5/CCR5 axis promotes the motility of human oral cancer cells

CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 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.     

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     

12‐O‐tetradecanoylphorbol‐13‐acetate‐induced invasion/migration of glioblastoma cells through activating PKCα/ERK/NF‐κB‐dependent MMP‐9 expression

An increase in MMP‐9 gene expression and enzyme activity with stimulating the migration of GBM8401 glioma cells via wound healing assay by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was detected in glioblastoma cells GBM8401. TPA‐induced translocation of protein kinase C (PKC)α from the cytosol to membranes, and migration of GBM8401 elicited by TPA was suppressed by adding the PKCα inhibitors, GF109203X and H7. Activation of extracellular signal‐regulated kinase (ERK) and c‐Jun‐N‐terminal kinase (JNK) by TPA was identified, and TPA‐induced migration and MMP‐9 activity was significantly blocked by ERK inhibitor PD98059 and U0126, but not JNK inhibitor SP600125. Activation of NF‐κB protein p65 nuclear translocation and IκBα protein phosphorylation with increased NF‐κB‐directed luciferase activity by TPA were observed, and these were blocked by the PD98059 and IkB inhibitor BAY117082 accompanied by reducing migration and MMP‐9 activity induced by TPA in GBM8401 cells. Transfection of GBM8401 cells with PKCα siRNA specifically reduced PKCα protein expression with blocking TPA‐induced MMP‐9 activation and migration. Additionally, suppression of TPA‐induced PKCα/ERK/NK‐κB activation, migration, and MMP‐9 activation by flavonoids including kaempferol (Kae; 3,5,7,4′‐tetrahydroxyflavone), luteolin (Lut; 5,7,3′4′‐tetrahydroxyflavone), and wogonin (Wog; 5,7‐dihydroxy‐8‐methoxyflavone) was demonstrated, and structure–activity relationship (SAR) studies showed that hydroxyl (OH) groups at C4′ and C8 are critical for flavonoids' action against MMP‐9 enzyme activation and migration/invasion of glioblastoma cells elicited by TPA. Application of flavonoids to prevent the migration/invasion of glioblastoma cells through blocking PKCα/ERK/NF‐κB activation is first demonstrated herein. J. Cell. Physiol. 225: 472–481, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of integrin up‐regulation in RANKL/RANK pathway of chondrosarcomas migration

Invasion of tumor cells is the primary cause of therapeutic failure in malignant chondrosarcomas treatment. Receptor activator of nuclear factor‐κB ligand (RANKL) and its receptor, RANK, play a key roles in osteoclastogenesis and tumor metastasis. We found that the RANKL and RANK expression in human chondrosarcoma tissues was higher than that in normal cartilage. We also found that RANKL directed the migration and increased cell surface expression of β1 integrin in human chondrosarcoma cells (JJ012 cells). Pretreatment of JJ012 cells with MAPK kinase (MEK) inhibitors, PD98059 or U0126, inhibited the RANKL‐induced migration and integrin expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited RANKL‐induced cells migration and integrin up‐regulation. Taken together, these results suggest that the RANKL acts through MEK/ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activation of β1 integrin and contributing to the migration of human chondrosarcoma cells. J. Cell. Biochem. 111: 138–147, 2010. © 2010 Wiley‐Liss, Inc.     

Analysis of amphotericin B-induced cell signaling with chemical inhibitors of signaling molecules

Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor‐κB (NF‐κB), a well‐known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte‐like cell line. Among chemical inhibitors of signaling molecules, U‐73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA‐AM (calcium chelator), LFM‐A13 (Bruton's tyrosine kinase (Btk)‐specific inhibitor), and PP2 (c‐Src kinase inhibitor) suppressed AmB‐induced phosphorylation of p65 and translocation of p65 into the nucleus. U‐73122 and Gö6976 reduced AmB‐mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)‐α and interleukin (IL)‐6) in RAW264.7 cells. Furthermore, AmB‐induced activation of NF‐ κ B was observed in toll‐like receptor (TLR) 2‐expressed cells, and the activation of NF‐κB was inhibited by U‐73122, whereas peptidoglycan‐induced NF‐κB activation, which was also dependent on TLR2, was not inhibited by U‐73122. Finally, U‐73122 partially suppressed in vivo production of TNF‐α and IL‐6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c‐Src and NF‐κB. These signaling molecules may become a target for chemotherapy suppressing AmB‐induced proinflammatory cytokine production.     

Interleukin-11 increases cell motility and up-regulates intercellular adhesion molecule-1 expression in human chondrosarcoma cells

Interleukin‐11 (IL‐11) was originally identified as the cytokine that could induce the proliferation of human cells. Recent studies have shown that IL‐11 plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of IL‐11 on human chondrosarcoma cells are largely unknown. Here, we found that IL‐11 increased the migration and expression of intercellular adhesion molecule‐1 (ICAM)‐1 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of the IL‐11 which was higher than that in primary chondrocytes. The phosphatidylinositol 3‐kinase (PI3K), Akt, and NF‐κB pathways were activated by IL‐11 treatment, and the IL‐11‐induced expression of ICAM‐1 and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF‐κB cascades. Taken together, our results indicate that IL‐11 enhanced the migration of the chondrosarcoma cells by increasing ICAM‐1 expression through the IL‐11Rα receptor, PI3K, Akt, and NF‐κB signal transduction pathway. J. Cell. Biochem. 113: 3353–3362, 2012. © 2012 Wiley Periodicals, Inc.     

Glial cell‐derived neurotrophic factor increases migration of human chondrosarcoma cells via ERK and NF‐κB pathways

Invasion of tumor cells is the primary cause of therapeutic failure in the treatment of malignant chondrosarcomas. Glial cell‐derived neurotrophic factor (GDNF) plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that GDNF directed the migration and increased cell surface expression of αv and β3 integrin in human chondrosarcoma cells. Pretreated of JJ012 cells with MAPK kinase (MEK) inhibitors PD98059 or U0126 inhibited the GDNF‐mediated migration and integrin expression. Stimulation of cells with GDNF increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited GDNF‐mediated cells migration and integrin up‐regulation. Stimulation of cells with GDNF induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the GDNF‐mediated increasing of κB‐luciferase activity was inhibited by PD98059, U0126, PDTC and TPCK or MEK, ERK, IKKα, and IKKβ mutants. Taken together, these results suggest that the GDNF acts through MEK/ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrin and contributing the migration of human chondrosarcoma cells. J. Cell. Physiol. 220: 499–507, 2009. © 2009 Wiley‐Liss, Inc.     

CagA phosphorylation-dependent MMP-9 expression in gastric epithelial cells

Background: Infection of cagA‐positive Helicobacter pylori is associated with increased expression of MMPs in gastric epithelial cells. The role of phosphorylated CagA in the induction of MMP‐9, a protease‐degrading basement membrane, in gastric epithelial cells has not been clearly defined yet. The aim of this study is to analyze whether the presence of CagA and its phosphorylation status play a role in increased expression of MMP‐9 in gastric epithelial cells. Materials and Methods: Induction of MMP‐9 secretion was analyzed in gastric epithelial AGS cells harboring CagA with or without EPIYA motif, which is injected by H. pylori or ectopically expressed. In addition, signaling pathways involved in the CagA‐dependent MMP‐9 production have been studied. Results: The 147C strain of H. pylori expressing tyrosine‐phosphorylated CagA (EPIYA present) induced higher MMP‐9 secretion by AGS cells than the 147A strain expressing non‐tyrosine‐phosphorylated CagA (EPIYA absent). In addition, in bacteria‐free CagA‐inducible AGS cells, expression of wild‐type CagA induced more MMP‐9 secretion than phosphorylation‐resistant CagA. Inhibition of CagA phosphorylation by the Src family kinase inhibitor PP1 downregulated CagA‐mediated MMP‐9 secretion. Knockdown of SHP‐2 phosphatase dramatically reduced MMP‐9 secretion. ERK inhibitors, PD98059 and U0126, and NF‐κB pathway inhibitors, sulfasalazine and N‐acetyl‐l ‐cysteine, also inhibited MMP‐9 expression. Conclusion: These results support a model whereby the EPIYA motif of CagA is phosphorylated by Src family kinases in gastric epithelial cells, which initiates activation of SHP‐2. In addition, they suggest that the resultant activation of ERK pathway along with CagA‐dependent NF‐κB activation is critical for the induction of MMP‐9 secretion.     

TNF‐α increases αvβ3 integrin expression and migration in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumour with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Tumour necrosis factor (TNF)‐α is a key cytokine involved in inflammation, immunity, cellular homeostasis and tumour progression. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. However, the effects of TNF‐α in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that TNF‐α increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of MAPK kinase (MEK), extracellular signal‐regulating kinase (ERK) and nuclear factor‐κB (NF‐κB) pathways after TNF‐α treatment were demonstrated, and TNF‐α‐induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK and NF‐κB cascades. Taken together, our results indicated that TNF‐α enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the MEK/ERK/NF‐κB signal transduction pathway. J. Cell. Physiol. 226: 792–799, 2011. © 2010 Wiley‐Liss, Inc.     

Thrombin stimulates RPE cell motility by PKC‐ζ‐ and NF‐κB‐dependent gene expression of MCP‐1 and CINC‐1/GRO chemokines

Retinal pigment epithelial cells (RPE) are the major cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR), which involves the epithelial‐mesenchymal transition, proliferation, and directional migration of transformed RPE cells to the vitreous upon RPE exposure to serum components, thrombin among them. Although the aqueous humor and vitreous of PVR patients contain high levels of chemokines, their possible involvement in PVR development has not been explored. We here analyzed the effect of thrombin on chemokine gene expression and its correlation with RPE cell migration using rat RPE cells in culture as a model system. We demonstrated that thrombin induces RPE cell migration through the dose‐dependent stimulation of MCP1 and GRO expression/release, and the autocrine activation of CXCR‐2 and CCR‐2 chemokine receptors. Whereas inhibition of CXCR2 by Sb‐225002 and of CCR2 by Rs‐504393 partially prevented hirudin‐sensitive cell migration, the joint inhibition of these receptors abolished thrombin effect, suggesting the contribution of distinct but coincident mechanisms. Thrombin effects were not modified by Ro‐32‐0432 inhibition of conventional/novel PKC isoenzymes or by the MAPkinase pathway inhibitor U0126. MCP1 and GRO expression/secretion, and cell migration were completely prevented by the inhibitory PKC‐ζ pseudosubstrate and by the nuclear factor‐kappa B (NF‐κB) inhibitor BAY11‐7082, but not by wortmannin inhibition of PI3K. Results show that signaling pathways leading to RPE cell migration differ from the MEK–ERK–PI3K‐mediated promotion RPE of cell proliferation, both of which concur at the activation of PKC‐ζ. J. Cell. Biochem. © 2010 Wiley‐Liss, Inc.     

Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis

The effects of the pleiotropic serine protease thrombin on tumor cells are commonly thought to be mediated by the thrombin receptor protease-activated receptor 1 (PAR1). We demonstrate here that PAR1 activation has a role in experimental metastasis using the anti-PAR1 antibodies ATAP2 and WEDE15, which block PAR1 cleavage and activation. Thrombin also stimulates chemokinesis of human melanoma cells toward fibroblast conditioned media and soluble matrix proteins. Thrombin-enhanced migration is abolished by anti-PAR1 antibodies, demonstrating that PAR1 cleavage and activation are required. The PAR1-specific agonist peptide TFLLRNPNDK, however, does not stimulate migration, indicating that PAR1 activation is not sufficient. In contrast, a combination of TFLLRNPNDK and the PAR2 agonist peptide SLIGRL mimics the thrombin effect on migration, whereas PAR2 agonist alone has no effect. Agonist peptides for the thrombin receptors PAR3 and PAR4 used alone or with PAR1 agonist also have no effect. Similarly, activation of PAR1 and PAR2 also enhances chemokinesis of prostate cancer cells. Desensitization with PAR2 agonist abolishes thrombin-enhanced cell motility, demonstrating that thrombin acts through PAR2. PAR2 is cleaved by proteases with trypsin-like specificity but not by thrombin. Thrombin enhances migration in the presence of a cleavage-blocking anti-PAR2 antibody, suggesting that thrombin activates PAR2 indirectly and independent of receptor cleavage. Treatment of melanoma cells with trypsin or PAR2 agonist peptide enhances experimental metastasis. Together, these data confirm a role for PAR1 in migration and metastasis and demonstrate an unexpected role for PAR2 in thrombin-dependent tumor cell migration and in metastasis.     

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.     

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.     

c-Src/Jak2/PDGFR/PKCδ-Dependent MMP-9 Induction Is Required for Thrombin-Stimulated Rat Brain Astrocytes Migration

Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) were not completely understood. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 in RBA-1 cells and cells migration which were attenuated by pretreatment with the inhibitor of receptor tyrosine kinase (Genistein), c-Src (PP1), Jak2 (AG490), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), PKCs (Ro318220), PKCδ (Rottlerin), or NF-κB (Bay11-7082) and transfection with siRNA of c-Src, PDGFR, Akt, PKCδ, ATF2, p65, IKKα, or IKKβ. In addition, thrombin-stimulated c-Src, Jak2, or PDGFR phosphorylation was inhibited by a thrombin inhibitor (PPACK), PP1, AG490, or AG1296. Thrombin further stimulated c-Src and PDGFR complex formation in RBA-1 cells. Thrombin also stimulated Akt and PKCδ phosphorylation and PKCδ translocation which were reduced by PPACK, PP1, AG490, AG1296, or LY294002. We further observed that thrombin markedly stimulated ATF2 or IκBα phosphorylation and NF-κB p65 translocation which were inhibited by Rottlerin or LY294002. Finally, thrombin stimulated in vivo binding of p65 to the MMP-9 promoter, which was reduced by pretreatment with Rottlerin or LY294002. These results concluded that in RBA-1 cells, thrombin activated a c-Src/Jak2/PDGFR/PI3K/Akt/PKCδ pathway, which in turn triggered ATF2 and NF-κB activation and ultimately induced MMP-9 expression associated with cell migration.     

TNF‐α induces matrix metalloproteinase‐9 expression in A549 cells: Role of TNFR1/TRAF2/PKCα‐dependent signaling pathways

Matrix metalloproteinases (MMPs), in particular MMP‐9, have been shown to be induced by cytokines, including TNF‐α and contributes to airway inflammation. However, the mechanisms underlying TNF‐α‐induced MMP‐9 expression in human A549 cells remain unclear. Here, we report that TNF‐α‐induced MMP‐9 gene expression was mediated through the TNFR1/TRAF2/PKCα‐dependent signaling pathways in A549 cells, determined by zymographic, RT‐PCR, and Western blotting analyses. TNF‐α‐induced MMP‐9 expression was reduced by pretreatment with a TNFR Ab. Furthermore, TNF‐α‐induced TNFR1 and TRAF2 complex formation was revealed by immunoprecipitation using an anti‐TNFR1 Ab followed by Western blot analysis against an anti‐TRAF2 or anti‐TNFR1 Ab. In addition, TNF‐α‐induced MMP‐9 expression was also reduced by pretreatment with the inhibitor of PKCα (Gö6983), c‐Src (PP1), EGFR (AG1478), or PI3K (LY294002) or transfection with siRNAs of PKCα, Src, EGFR, Akt, p65, p300, and c‐Jun. On the other hand, TNF‐α stimulated the phosphorylation of c‐Src, EGFR, Akt, JNK1/2, and c‐Jun, which were inhibited by pretreatment with Gö6983. We also showed that TNF‐α induced Akt translocation and the formation of an Akt/p65/p300 complex. Pretreatment with the inhibitor of JNK1/2 (SP600125) but not the inhibitor of MEK1/2 (U0126), p38 MAPK (SB202190), or PI3K (LY294002), markedly inhibited TNF‐α‐induced c‐Jun mRNA levels. Taken together, these data suggest that in A549 cells, TNF‐α induces MMP‐9 expression via the TNFR1/TRAF2/PKCα‐dependent JNK1/2/c‐Jun and c‐Src/EGFR/PI3K/Akt pathways. J. Cell. Physiol. 454–464, 2010. © 2010 Wiley‐Liss, Inc.     

Proanthocyanidins from the American Cranberry (Vaccinium macrocarpon) inhibit matrix metalloproteinase‐2 and matrix metalloproteinase‐9 activity in human prostate cancer cells via alterations in multiple cellular signalling pathways

Prostate cancer is one of the most common cancers in the Western world, and it is believed that an individual's diet affects his risk of developing cancer. There has been an interest in examining phytochemicals, the secondary metabolites of plants, in order to determine their potential anti‐cancer activities in vitro and in vivo. In this study we document the effects of proanthocyanidins (PACs) from the American Cranberry (Vaccinium macrocarpon) on matrix metalloproteinase (MMP) activity in DU145 human prostate cancer cells. Cranberry PACs decreased cellular viability of DU145 cells at a concentration of 25 µg/ml by 30% after 6 h of treatment. Treatment of DU145 cells with PACs resulted in an inhibition of both MMPs 2 and 9 activity. PACs increased the expression of TIMP‐2, a known inhibitor of MMP activity, and decreased the expression of EMMPRIN, an inducer of MMP expression. PACs decreased the expression of PI‐3 kinase and AKT proteins, and increased the phosphorylation of both p38 and ERK1/2. Cranberry PACs also decreased the translocation of the NF‐κB p65 protein to the nucleus. Cranberry PACs increased c‐jun and decreased c‐fos protein levels. These results suggest that cranberry PACs decreases MMP activity through the induction and/or inhibition of specific temporal MMP regulators, and by affecting either the phosphorylation status and/or expression of MAP kinase, PI‐3 kinase, NF‐κB and AP‐1 pathway proteins. This study further demonstrates that cranberry PACs are a strong candidate for further research as novel anti‐cancer agents. J. Cell. Biochem. 111: 742–754, 2010. © 2010 Wiley‐Liss, Inc.     

c-Src mediates thrombin-induced NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells

In this study, we examined the regulation of NF-kappaB activation and IL-8/CXCL8 expression by thrombin in human lung epithelial cells (EC). Thrombin caused a concentration-dependent increase in IL-8/CXCL8 release in a human lung EC line (A549) and primary normal human bronchial EC. In A549 cells, thrombin, SFLLRN-NH2 (a protease-activated receptor 1 (PAR1) agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide), but not TFRGAP-NH2 (a PAR3 agonist peptide), induced an increase in IL-8/CXCL8-luciferase (Luc) activity. The thrombin-induced IL-8/CXCL8 release was attenuated by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (a thrombin inhibitor), U73122 (a phosphoinositide-phospholipase C inhibitor), Ro-32-0432 (a protein kinsase C alpha (PKC alpha) inhibitor), an NF-kappaB inhibitor peptide, and Bay 117082 (an IkappaB phosphorylation inhibitor). Thrombin-induced increase in IL-8/CXCL8-Luc activity was inhibited by the dominant-negative mutant of c-Src and the cells transfected with the kappaB site mutation of the IL-8/CXCL8 construct. Thrombin caused time-dependent increases in phosphorylation of c-Src at tyrosine 416 and c-Src activity. Thrombin-elicited c-Src activity was inhibited by Ro-32-0432. Stimulation of cells with thrombin activated IkappaB kinase alphabeta (IKK alphabeta), IkappaB alpha phosphorylation, IkappaB alpha degradation, p50 and p65 translocation from the cytosol to the nucleus, NF-kappaB-specific DNA-protein complex formation, and kappaB-Luc activity. Pretreatment of A549 cells with Ro-32-4032 and the dominant-negative mutant of c-Src DN inhibited thrombin-induced IKK alphabeta activity, kappaB-Luc activity, and NF-kappaB-specific DNA-protein complex formation. Further studies revealed that thrombin induced PKC alpha, c-Src, and IKK alphabeta complex formation. These results show for the first time that thrombin, acting through PAR1 and PAR4, activates the phosphoinositide-phospholipase C/PKC alpha/c-Src/IKK alphabeta signaling pathway to induce NF-kappaB activation, which in turn induces IL-8/CXCL8 expression and release in human lung EC.     

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.